Synthesis and Structure-Activity Relationship of Xenocoumacin 1 and Analogues as Inhibitors of Ribosomal Protein Synthesis

Article abstract of DOI:10.1002/cmdc.202000793

Ribosomal protein synthesis is an important target in antibacterial drug discovery. Numerous natural products have served as starting points for the development of antibiotics. We report here the total synthesis of xenocoumacin 1, a natural product that binds to 16S ribosomal RNA at a highly conserved region, as well as analogues thereof. Preliminary structure–activity relationship studies were aimed at understanding and modulating the selectivity between eukaryotic and prokaryotic ribosomes. Modifications were mainly tolerated in the aromatic region. Whole-cell activity against Gram-negative bacteria is limited by efflux and penetration, as demonstrated in genetically modified strains of E. coli. Analogues with high selectivity for eukaryotic ribosomes were identified, but it was not possible to obtain inhibitors selective for bacterial protein synthesis. Achieving high selectivity (albeit not the desired one) was thus possible despite the high homology between eukaryotic and prokaryotic ribosomes in the binding region.

Full text of DOI:10.1002/cmdc.202000793

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Synthesis and Structure-Activity Relationship of
Xenocoumacin 1and Analogues as Inhibitors of Ribosomal
Protein Synthesis
Cornelia Zumbrunn,*[a] Daniela Krüsi,[a] Christina Stamm,[a] Patrick Caspers,[a] Daniel Ritz,[a]
and Georg Rueedi[a]
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Ribosomal protein synthesis is an important target in antibacte-
rial drug discovery. Numerous natural products have served as
starting points for the development of antibiotics. We report
here the total synthesis of xenocoumacin 1, a natural product
that binds to 16S ribosomal RNA at a highly conserved region,
as well as analogues thereof. Preliminary structure–activity
relationship studies were aimed at understanding and modulat-
ing the selectivity between eukaryotic and prokaryotic ribo-
somes. Modifications were mainly tolerated in the aromatic
region. Whole-cell activity against Gram-negative bacteria is
limited by efflux and penetration, as demonstrated in genet-
ically modified strains of E. coli. Analogues with high selectivity
for eukaryotic ribosomes were identified, but it was not possible
to obtain inhibitors selective for bacterial protein synthesis.
Achieving high selectivity (albeit not the desired one) was thus
possible despite the high homology between eukaryotic and
prokaryotic ribosomes in the binding region.
Introduction
overlap the binding sites of any of the clinically used antibiotics,
it is reasonable to assume that the coumacin analogues might
overcome pre-existing resistance to other antibiotics targeting
protein synthesis. Compounds 1[12] and 2,[13] with reported
antibacterial activity in whole-cell screens, were both discov-
ered more than 25 years ago. Several total syntheses of
amicoumacin-related natural products were published,[14] as
well as the lack of selectivity for bacterial over eukaryotic
ribosomes.[15]
A recent analysis of interactions of small molecules with
RNA has shown that π-stacking as well as H-bonds are the most
important type of bonding interactions, but in contrast to
interactions in protein–inhibitor complexes, interactions with
RNA are much less understood.[16] X-ray structures of 1 with the
ribosomes of Thermus thermophilus (PDB ID: 4W2F) and
Saccharomyces cerevisiae (PDB ID: 5I4L) were solved.[17] The
binding of 1 to the bacterial ribosome is shown in Figure 1.
Main features of the interactions are the π-stacking of the
aromatic coumacin moiety with G693, the H-bond network of
the aminodiol linker bridging between rRNA and mRNA and the
intercalation of the isobutyl moiety between residues C795 and
C796. Unfortunately, in the S. cerevisiae structure mRNA is not
visible (or absent) and the aminodiol moiety of amicoumacin
adopts a slightly different conformation. Due to these differ-
ences and the relatively high flexibility of RNA, an approach
using docking was rated too speculative. We resorted to a
traditional medicinal chemistry approach for our structure–
activity relationship studies for which in turn a robust synthetic
access to derivatives of 2 was essential.
The need for novel antibacterial drugs is undisputed in the
scientific community,[1–3] and numerous initiatives aim to foster
research in this important domain.[4,5] In particular, hard-to-kill
Gram-negative pathogens such as Pseudomonas aeruginosa,
Acinetobacter baumannii and Klebsiella pneumoniae accumulate
resistance at high rates, and some species are also naturally
unaffected by many antibiotic classes. Although the majority of
commercially used antibacterials act via inhibition of i) cell-wall
biosynthesis, ii) protein synthesis or iii) DNA metabolism, mining
bacterial genomes to identify conserved essential functions has
failed to provide useful novel targets.[2] Addressing old targets
with novel chemical matter acting by a different mode[6] could
be one way to identify inhibitors without cross-resistance.
Inhibition of protein synthesis is one of the most important
modes of action of antibiotics we currently have at our
disposal.[7] A large variety of structural classes, including
tetracyclines, aminoglycosides, macrolides, lincosamides, oxazo-
lidinones as well as fusidic acid inhibit ribosomal protein
synthesis at different stages of the process by binding at various
sites of the 70S bacterial ribosome. Recently, several groups
have been able to elucidate the binding sites of a number of
natural products in complete ribosomal complex X-ray
crystallography.[8–10] One of those natural inhibitors is amicou-
macin A (1),[11] a close analogue of xenocoumacin 1 (2), which
binds to the E-site of the ribosome. As this region does not
[a] Dr. C. Zumbrunn, D. Krüsi, C. Stamm, Dr. P. Caspers, Dr. D. Ritz,
Dr. G. Rueedi
Idorsia Pharmaceuticals Ltd.
Hegenheimermattweg 91, 4123 Allschwil (Switzerland)
E-mail: Cornelia.Zumbrunn@idorsia.com
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cmdc.202000793
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on the binding mode and the knowledge on synthetic
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accessibility of 1 we devised the strategy depicted in Figure 2.
We were mainly interested in three regions of the molecule:
the π-stacking aromatic moiety, the RNA-intercalating residue,
which in the natural product originates from a lysine amino
acid, and the right hand side chain (RHS) differing between 1
and 2. Changes in the aminodiol moiety in the linker were of
secondary interest because of its engagement in a neatly
defined H-bond network which is clearly visible in the X-ray
structure.
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In a first step we devised the synthesis of the RHS of
xenocoumacin as described in Scheme 1 and based on the
published synthesis of 1.[14] Starting form orthogonally pro-
tected (S)-ornithine (4) we set up the diol moiety as previously
described.[18] The first step consisted of a coupling with
Meldrum’s acid (3), followed by cyclisation under concomitant
decarboxylation. The double bond in 6 was installed via a
three-step sequence (reduction, mesylation, elimination). Dihy-
droxylation of 6 proceeded stereoselectively and was followed
by protection as a cyclic acetal (7). Hydrogenation of the Cbz
group and introduction of the guanidine group using N,N’-bis-
Boc-1-guanylpyrazole was performed in a one-pot procedure.
Mild hydrolysis of the Boc-protected lactam ring with LiOH
finally gave carboxylic acid 8 in good yields.
With the RHS ready for coupling, we turned our interest to
the aromatic moiety (Figure 3). Our aim was to not only
synthesise the methoxy isochromanone moiety 9 necessary for
the synthesis of the natural product (and well precedented in
the literature[14]), but to also investigate the replacement of the
hydroxy group by hydrogen (10) and the corresponding
isochromenones (11–13).
Figure 1. Representation of the X-ray crystal structure 4W2F showing the
binding mode of amicoumacin A (1; adapted from ref. [11]).
Results and Discussion
We chose 2 as starting point for our investigations to attempt
to improve potency and increase the selectivity window. Based
The synthesis of isochromenones by Sonogashira coupling
of an acetylene followed by cyclisation is well documented in
the literature.[19,20] The synthesis of the unsubstituted isochro-
menone 13 was achieved as depicted in Scheme 2.
Figure 2. Strategy envisaged for SAR studies of 2: Modifications in four parts
of the molecule (colour coded) can be accessed by formation of the bonds
indicated with a dashed line.
Figure 3. conservative modifications of coumacin left hand side (LHS).
Scheme 1. Synthesis of the RHS of xenocoumacin. a) DCC, DMAP, CH2Cl2, RT,
°
4 h; b) MeOH, reflux, 1 h, 58% over 2 steps; c) NaBH4, HOAc, CH2Cl2, 0 C,
Scheme 2. Synthesis of unsubstituted isochromenone 13. a) (S)-(À )-2-Meth-
°
80%; d) MsCl, NEt3, CH2Cl2, 0 C–RT, then DBU, RT, 1 h, 86%; e) OsO4, NMO,
yl-2-propanesulfinamide, Ti(OEt)4, THF, RT, 2 h, 84%; b) TMS acetylene,
°
MeCN/water/acetone, RT, 1 h, 80%; f) acetone, CSA, 2,2-dimethoxypropane,
RT, 1 h, 77% ; g) Pd/C, H2, N,N’-bis-Boc-1-guanylpyrazole, EA, RT; h) LiOH,
THF/H2O, RT, 15 min, 85% over 2 steps.
iPrMgCl, THF, À 78 C, 65%; c) TBAF, THF, RT, 85%; d) 2-iodobenzoic acid,
°
trans-4-hydroxy-(l)-proline, CuI, K2CO3, DMSO, 75 C, 1 h, 33%; e) HCl, MeOH/
THF, RT, 99%.
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3-Methylbutanal 14 was condensed with (S)-2-meth-
with TFA or HCl to obtain the methoxy as well as the
corresponding unsubstituted analogues thereof (Scheme 5).
All new compounds were tested in vitro for their ability to
inhibit bacterial (Escherichia coli) or eukaryotic (rabbit reticulo-
cyte) protein synthesis (in vitro transcription translation assay
(IVTT)).[17] The results are summarised in Table 1.
Novel xenocoumacin analogues were identified that inhibit
protein synthesis in vitro at concentrations between 10 nM and
>100 μM and showed very different potencies against eukary-
otic or bacterial ribosomes. Surprisingly, despite virtually
identical RNA sequence at the binding sites, the ribosome of
eukaryotic systems is much less susceptible to structural
changes of the inhibitors. While even small modifications
drastically affected the inhibition of the ribosome of E. coli, the
compounds still remained potent inhibitors of eukaryotic
protein synthesis.
Conservative modifications on the aromatic portion of 2,
such as presence or absence of hydroxy or methoxy group,
presence or absence of double bond does not have a profound
effect on the activity (cf. compounds 2, 19–23) in vitro. Bigger
losses of potency were observed for the R,R analogue 24 with a
~100-fold higher IC50 value in the micromolar range.
We chose the unsubstituted isochromenone (R=H) inter-
mediate 13 as suitable simplified aromatic moiety for most of
our investigations.
Modifications of the isobutyl group (intercalating moiety,
compounds 25–30) led to much bigger loss of activity in the E.
coli IVTT assay (1.27!100 μM), whereas in the reticulocyte assay
sub-micromolar activities were generally still measured.
Variations in the linker region affected activity less than
expected. In the X-ray structure of 1, the two hydroxy groups
and the amine are tightly bound in a H-bond network and were
therefore expected to be essential for inhibitory activity. The
two analogues with one inverted hydroxy group (31 and 32,
absolute configuration of diol not assigned) as compared to 23
still retained some activity and showed IC50’s in the double-digit
micromolar range (about 100-fold less potent) in the bacterial
assay and are about 15 to 150 times more active in the
reticulocyte IVTT assay. The same range of activity was observed
1
2
3
4
5
6
7
8
9
ylpropane-2-sulfinamide[21] and treated with the anion of
trimethylsilylacetylene (generated in situ by treatment with
iPrMgCl) to give in good yield and with good diastereoselectiv-
ity the required intermediate which after cleavage of the
trimethylsilyl group with tetrabutylammonium fluoride (TBAF)
led to acetylene 15. Copper catalysed coupling of 15 with 2-
iodobenzoic acid gave the desired enantiomer of isochrome-
none 16 in modest yields, along with some phthalide by-
product. Deprotection with HCl in MeOH yielded the desired
unsubstituted isochromenone 13.
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All our attempts to synthesise the analogous methoxy
derivative 11 via the same route failed, as cyclisation of the
Sonogashira coupling product only led to the 5-membered
phthalide derivative via formal 5-exo-dig process (data not
shown). This methoxylated analogue 11 had to be accessed via
a different route.[17] To obtain hydroxy analogue 12 (Scheme 3)
we had to modify the sequence and use acetal protected
salicylic acid derivative 17 for the Sonogashira coupling which,
after hydrolysis of the acetal, was cyclised under Pd catalysed
conditions.[22]
The employed sulfinimide-based chemistry proved versatile
for the exploration of the intercalating moiety (Scheme 4, top),
as well as for the variation of the aromatic moiety in general
(Scheme 4, bottom) when using the opposite enantiomer of the
chiral auxiliary. Yields of this unoptimised sequence were rather
low but led rapidly to the desired enantiopure building blocks.
A range of additional side-chain analogues of 8 were
synthesised varying the stereochemistry of the aminodiol part
as well as the terminal moiety.[17] With side chains and aromatic
moieties in hand, we proceeded to the final coupling followed
by deprotection with BBr3 to access hydroxychromanones or
Scheme 3. Synthesis of hydroxyisochromenone 12. a) CuI, Pd(dppf), NEt3,
overnight, 70%; b) LiOH, THF/H2O, RT, 2.5 h, 100%; c) PdCl2(MeCN)2, NEt3,
°
THF, 50 C, 1 h, 43%; d) HCl/MeOH, 30 min, 70%.
Scheme 5. Coupling and final deprotection. a) ((Benzotriazol-1-yloxy)tris
(dimethylamino)phosphonium hexafluorophosphate or HBTU, NEt3, DMF/
°
°
CH2Cl2, 0 C–RT, overnight, 56–93%; b) BBr3 in CH2Cl2, À 78 C, 1 h, 11–55%; c)
°
Scheme 4. Synthetic access to various aromatic and intercalating moieties.
aq. TFA, or HCl in MeOH, 50 C, overnight, 19–56%.
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Table 1. Summary of IVTT of xenocoumacin 1 analogues.
1
2
3
Structure / compound no.
IVTT [μM]
Structure / compound no.
IVTT [μM]
E. coli[a]
reticulocytes
E. coli[a]
reticulocytes
4
5
6
7
0.029
0.076
0.073
0.014
0.018
0.014
0.076
0.200
0.200
0.017
0.030
0.013
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
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57
6.8
1.50
>100
>100
19.10
0.48
1.27
0.025
83
0.28
>100
0.14
1.10
7.2
0.021
16.4
18.2
0.13
27.8
0.64
2.00
6.10
4.46
0.029
0.014
0.055
>100
2.90
0.26
0.035
0.068
72.0
71.0
3.90
0.20
15.2
3.0
0.027
0.039
0.011
1.7
0.53
0.10
0.047
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Table 1. continued
1
2
Structure / compound no.
IVTT [μM]
Structure / compound no.
IVTT [μM]
E. coli[a]
reticulocytes
0.18
E. coli[a]
reticulocytes
18.0
3
4
5
6
7
8
9
0.58
34.4
36.4
3.3
>100
16.7
48.7
0.76
38.1
9.7
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
0.042
>100
1.60
0.029
1.70
0.026
>100
>100
>100
>100
[a] IC50 are means of several independent experiments.[17]
when one or the other hydroxy group in the linker were
completely removed (compounds 33 (mixture of 2 isomers) and
34).
More dramatic changes in the π-stacking region (com-
pounds 35–43) led to compounds with micromolar affinities in
E. coli IVTT and roughly 100-fold better activity in the
reticulocyte IVTT assay.
Modifications in the RHS also mostly led to loss of activity.
The activity of amicoumacin (1) and the methoxylated analogue
45 showed the expected sub-micromolar activity in both assays,
whereas the three degradation products thereof (lactones 44
and 48, acid 46) showed much weaker activity. The presence of
the phenol, however, seems to be of importance in this series.
Compound 44 only loses a factor of 10 as compared to 1, while
48 loses dramatically more (additional 20-fold compared to 44).
The presence of a substituted amide in 47 does not lead to
improvement of activity compared to 48. Neither does the
substitution with a non-basic group (49). The addition of an
additional primary amine in compound 50 does improve the
activity against E. coli ribosomes tenfold, but 50 is still much
less active than compound 20, where the basicity is added
through a guanidinium group. Similarly, replacement of that
basic group by a tertiary amine in compound 51 leads to a
drastic reduction of the activity when compared to 23.
Surprisingly, shortening the carbon linker from three to two
atoms led to compounds 52 and 53 which showed 100-fold
lower activity against E. coli, but kept good affinity in the
reticulocyte assay compared to the corresponding longer
homologues 2 and 50, respectively.
Finally, the basic amine present in the linker of 23 was
replaced by an amide functionality (compounds 54 and 55,
absolute stereochemistry not determined) which however was
detrimental for activity in both assays.
In addition, compounds were tested in a panel of Gram-
positive and Gram-negative bacteria.[17] The panel consisted of
wild-type reference strains of Staphylococcus aureus, S. pneumo-
niae, Enterococcus faecalis, Moraxella catarrhalis, Haemophilus
influenzae, E. coli, and P. aeruginosa, as well as multi drug-
resistant (MDR) clinical isolates of Enterococcus faecium, A.
baumannii, and K. pneumoniae. Antibacterial activity was only
observed against S. aureus, S. pneumoniae, M. catarrhalis and H.
influenzae (minimal inhibitory concentrations (MIC) of 0.063–
16 μg/mL, Table 2 and the Supporting Information) but were
inactive on all other species (data not shown). Activity in the
IVTT assay of E. coli correlated well with antibacterial activity on
M. catarrhalis: IC50 <10 μM translated to MIC <8 μg/mL on this
strain.
It has been hypothesised that microbial natural products
with antibiotic activity have evolved their ability to reach their
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Table 2. cellular activities of selected xenocoumacin analogues.[a]
1
2
3
4
5
6
7
8
9
Cpd
Microbiology panel (MIC [mg LÀ 1])
Permeability panel E. coli (MIC [mg LÀ 1])
MG1655
(wt)
Cytotoxicity (IC50 [μM])
S aureus
S pneum.
wt
M catar.
A894
H infl.
A921
2031
(ΔtolC)
2052
(ΔacrAB)
2081
(ΔrfaC)
2085
Δ(tolCrfaC)
CHOÀ K1
wt
48 h
1
2
2
1
4
2
16
�16
>32
16
0.5
1
16
2
8
>32
32
16
0.25
2
2
8
2
2
8
2
32
>16
16
>32
32
16
>32
>16
>32
0.5
0.5
8
2
2
1
16
8
32
8
1
0.25
2
0.5
0.5
8
1.2
0.3
3.6
0.7
0.6
2.4
0.7
26
�0.063
0.5
16
2
19
20
21
22
23
45
0.25
0.125
�0.063
0.25
0.25
4
8
16
�16
�16
>32
>16
>16
4
8
2
4
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
16
>8
[a] MIC (minimal inhibitory concentration), S aureus wt (ATCC29213), S pneumoniae wt (ATCC49619). MIC and IC50 are means of several independent
experiments.[17]
ization (PDF), and a summary table of biological data (IC50, MIC,
cytotoxicity) (PDF)) are available in the Supporting Information.
intracellular targets.[23] Therefore, selected compounds (Table 2)
were also tested on a set of genetically modified E. coli strains
lacking either the main efflux pumps (ΔtolC or ΔacrAB) or with
a modified lipopolysaccharide (LPS) layer (ΔrfaC). The results
allowed to estimate the extent to which compounds are Acknowledgements
affected by efflux or have penetration issues.
The analysis revealed that 2 and its analogues are substrates
for efflux pumps and, to a lesser extent, are prevented to
penetrate through the outer membrane LPS layer. Intracellular
concentrations are thus not high enough to exert reasonable
The authors would like to thank Daniel Bur and Christoph P. Sager
for modelling support.
antibacterial activity especially against Gram-negative Conflict of Interest
bacteria.[17] A few compounds were also evaluated for cytotox-
icity in eukaryotic cells (CHO cells) showing significant toxicity
after 48 h. Generally, cytotoxicity tracked in parallel to protein
synthesis inhibition measured with rabbit reticulocytes and thus
confirmed on-target activity of these protein synthesis inhib-
itors.
The authors declare no conflict of interest.
Keywords: antibiotics · natural products · protein synthesis
inhibition · RNA recognition · structure-activity relationships
Conclusion
[1] E. D. Brown, G. D. Wright, Nature 2016, 529, 336–343.
[2] R. Tommasi, D. G. Brown, G. K. Walkup, J. L. Manchester, A. A. Miller, Nat.
Rev. Drug. Discovery 2015, 14, 529–542.
We report here the first total synthesis of xenocoumacin 1 and
several structurally diverse analogues which were tested for
inhibition of prokaryotic and eukaryotic ribosomal protein
synthesis. Although the binding site lies in a conserved region
of the ribosome, surprising differences are observed between
eukaryotic and prokaryotic systems. Some of the novel
analogues were potent and selective inhibitors of eukaryotic
protein synthesis with much reduced activity on bacterial
systems. The reasons for this difference are not obvious from a
structural point of view. Further investigations would be
needed to formulate a hypothesis on the origin of these
differences. Whole cell activity of xenocoumacin analogues was
furthermore limited by efflux in many bacterial species. Due to
all these findings we turned away from the coumacin natural
products as starting point for novel antibiotics.
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Experimental Section
Experimental Details (experimental methods, detailed description
of the synthesis of all novel compounds as well as their character-
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Manuscript received: October 9, 2020
Accepted manuscript online: November 25, 2020
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Different ribosome=different prop-
erties: Xenocoumacin 1 is an
Dr. C. Zumbrunn*, D. Krüsi, C. Stamm,
Dr. P. Caspers, Dr. D. Ritz, Dr. G. Rueedi
inhibitor of bacterial and eukaryotic
ribosomal protein synthesis. For this
first total synthesis, a convergent
approach allowed the rapid assembly
of structurally diverse analogues for
SAR investigations. Despite the high
homology between prokaryotic and
eukaryotic ribosomes, the
compounds showed surprising differ-
ences in potency and selectivity
assessed in enzymatic and whole-cell
assays.
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Synthesis and Structure-Activity Re-
lationship of Xenocoumacin 1and
Analogues as Inhibitors of
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Ribosomal Protein Synthesis