Synthetic studies on the reverse antibiotic natural products, the nybomycins

Article abstract of DOI:10.1039/c9md00207c

Antimicrobial resistance (AMR) is a serious issue that could have severe consequences if steps are not taken. The nybomycin natural products have the potential to extend the clinical efficacy of the marketed fluoroquinolone class of antibiotics through a 'reverse antibiotic' approach. However, only very limited structure-activity relationships are known for these fascinating compounds, in part due to challenges with their synthesis. Here we report a new scalable and robust synthetic route to the nybomycin natural products to aid in the development of this series. Through this synthesis, we report the antibiotic activity of novel analogues of this family confirming the selectivity for fluoroquinolone resistant bacteria and potential future opportunities for further optimisation.

Full text of DOI:10.1039/c9md00207c

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RESEARCH ARTICLE
Synthetic studies on the reverse antibiotic natural
products, the nybomycins†‡
ab
Cite this: DOI: 10.1039/c9md00207c
a
Oliver A. Bardell-Cox,
*
Andrew J. P. White,
a
*
Luis Aragón
b and Matthew J. Fuchter
Antimicrobial resistance (AMR) is a serious issue that could have severe consequences if steps are not
taken. The nybomycin natural products have the potential to extend the clinical efficacy of the marketed
fluoroquinolone class of antibiotics through a ‘reverse antibiotic’ approach. However, only very limited
structure–activity relationships are known for these fascinating compounds, in part due to challenges with
their synthesis. Here we report a new scalable and robust synthetic route to the nybomycin natural prod-
ucts to aid in the development of this series. Through this synthesis, we report the antibiotic activity of
novel analogues of this family confirming the selectivity for fluoroquinolone resistant bacteria and potential
future opportunities for further optimisation.
Received 7th April 2019,
Accepted 22nd May 2019
DOI: 10.1039/c9md00207c
rsc.li/medchemcomm
Antibiotic resistance is a worldwide issue. Globally, AMR
causes around 700 000 deaths per year at a low estimate.1 This
figure is likely to be an underestimate as incidences are hard
to attribute and sometimes not reported. However, with the
rise of AMR and the lack of new clinical options, this figure is
projected to rise to around 10 million deaths in 2050.1 That
equates to one death every 3 seconds.
The quinolone class of antibiotics, which include cipro-
floxacin and levofloxacin, are used to treat a variety of bacte-
rial infections including from both Gram-positive and Gram-
negative organisms.2 They are one of the most prescribed
classes of antibiotics.3 The targets for these antibiotics are
the homologous type II topoisomerase enzymes, DNA gyrase
and topoisomerase IV.4,5 Both of these enzymes relieve the
strain (or amount of supercoiling) generated in DNA as a con-
sequence of its unwinding during DNA replication.6–8 Fluoro-
quinolones function by stabilising the cleaved complex of
DNA gyrase (or topoisomerase IV) and DNA, leading to frag-
mentation of the DNA and death of the bacterial cells.8–10
The main cause of resistance to this class of antibiotics oc-
curs when there is a mutation in one of the key residues in
the quinolone-resistance determining region (QRDR) of the
enzyme.9,11 These mutations disrupt a water-mediated mag-
nesium bridge linking the quinolone to the protein via the
non-catalytic Mg2+ ion, resulting in a significant loss of affin-
ity of the fluoroquinolone to the enzyme.6,9,12,13
Resistance to fluoroquinolones was noted rapidly upon
initial clinical usage and the resistance rate continues to in-
crease in most bacteria species.14 Given the success of these
antibiotics, new approaches are needed to re-establish their
clinical effectiveness. One emerging approach is the use of a
‘reverse antibiotic’,15,16 a concept experimentally verified
in vitro with the fluoroquinolone ciprofloxacin and the natu-
ral product deoxynybomycin.16 In this approach (Fig. 1),
deoxynybomycin was identified to be active against
fluoroquinolone-resistant bacteria through binding to QRDR
mutant type II topoisomerase enzymes, S84L gyrA and E84K
parC. Importantly however, resistance to deoxynybomycin
occured by a back-mutation to the wild-type type II topoisom-
erase enzymes, thus re-sensitising the bacteria to fluoro-
quinolones. In theory, the combined usage of fluoro-
quinolones and nybomycin natural products could trap
bacteria in an ‘evolutionary loop’ where they will always be
sensitive to one of the antibiotics. This approach breaks the
cycle of needing to constantly develop new antibiotics for re-
sistant bacteria and may be a powerful way to tackle AMR, es-
pecially once further developed. Furthermore, development
of the nybomycin series may provide alternative antibiotics
against this validated bacterial target class, given some of the
side effects observed for the fluoroquinolones.17,18
a Department of Chemistry, Imperial College London, Molecular Sciences Research
Hub, White City Campus, Wood Lane, London, W12 OBZ, UK.
E-mail: m.fuchter@imperial.ac.uk
b Cell Cycle Group, MRC London Institute of Medical Sciences, Du Cane Road,
London W12 0NN, UK. E-mail: luis.aragon@lms.mrc.ac.uk
† Raw data files are openly available DOI: 10.14469/hpc/5498
‡ Electronic supplementary information (ESI) available: Experimental proce-
dures, characterisation data, NMR spectra, further X-ray data. CCDC 1908213.
For ESI and crystallographic data in CIF or other electronic format see DOI:
10.1039/c9md00207c
Deoxynybomycin belongs to the nybomycin series of natu-
ral products. Both nybomycin and deoxynybomycin are
reported to be highly active against the fluoroquinolone resis-
tant bacteria strains (FQR) but inactive against the
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the scaffold can lead to broad spectrum activity.19 Despite
the key importance this places upon this natural product se-
ries, very limited medicinal chemistry exploration of the
nybomycin scaffold has been reported.16,19 Such medicinal
chemistry studies are clearly needed: both nybomycin and
deoxynybomycin have poor aqueous solubility and selectivity
issues over the human topoisomerase isoforms.20 Optimisa-
tion will be needed to alleviate these liabilities, in order for
this class of antibiotics to progress into clinical exploration.
Here we report our synthetic studies on the nybomycins,
aiming to develop a reliable and reproducible synthesis that
is amenable to analogue synthesis. Through this route, we re-
port some initial structure activity relationships (SAR) of the
nybomycin series', identifying novel structural determinants
for their antibiotic activity.
Although nybomycin and deoxynybomycin are natural
products that were first reported in 1955,21 their isolation is
far from straight-forward22 and the fermentation process is
inefficient (1000 L is reported to give 350–200 mg of
nybomycin).23 The previously reported total syntheses are
also relatively low yielding (13%,24 11%,16 0.84%25,26 overall
yield). An overview of the prior synthetic approaches is
shown in Fig. 2. Route 1, developed by Rinehart and
Forbis,25,26 disregards the symmetry of the molecule and
constructs the central framework sequentially. Given this, in
addition to some low yielding steps, an overall yield of 0.84%
is obtained for the synthesis of deoxynybomycin, which is
clearly not ideal for analogue synthesis. Routes 2 and 3, de-
veloped by Nussbaum and co-workers24 and Hergenrother
and co-workers16,27,28 respectively, better exploit the symmet-
rical nature of the molecule, reducing the overall number of
linear steps.
Fig.
1 Diagrammatic representation of the ‘reverse antibiotic’
concept. Ciprofloxacin (a fluoroquinolone antibiotic) is an effective
treatment of wild type S. aureus shown in blue. Target mutations lead
to a mutant strain, shown in red, that is resistant to ciprofloxacin.
Deoxynybomycin (a reverse antibiotic) can then be used to treat the
mutant S. aureus. Importantly, the mutation required to gain resistance
to deoxynybomycin reverts back to the wild-type target, which re-
sensitises the S. aureus to fluoroquinolone antibiotics. This could be an
important approach in breaking the constant need to discover new
antibiotics with novel modes of action.15,16 Image created with
BioRender.com.
fluoroquinolone sensitive strains (FQS) and are, thus far, the
only example of
nybomycins show a lack of activity against Gram-negative
bacteria,19 it has been shown that structural modification of
a
‘reverse antibiotic’.15,16 Whilst the
Fig. 2 Previously reported routes to deoxynybomycin.
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Route 3 was identified as the preferred route for further
medicinal chemistry as it had the best overall yield and of-
fered the most reliable synthesis of deoxynybomycin. The cis-
iodo-alkene 1 and borylated core 2 intermediates, shown in
Scheme 1, were synthesised in accordance with the literature
protocols.27–29 However, the reported Suzuki-Miyaura cross-
coupling failed to yield any of the desired compound 3 in our
hands. A screen for alternative reaction conditions was there-
fore carried out; altering the catalyst, ligand, base and sol-
vent. Regrettably, all conditions surveyed failed to yield any
of the desired cross-coupled product. Due to the relatively
unactivated nature of aryl boronic ester 2 and the base insta-
bility of the iodo-alkene 1, we proposed that swapping the
coupling partners would improve the robustness of this ca-
pricious cross-coupling reaction.
Borylated-alkene 4 was synthesised as shown in Scheme 2.
This was achieved in a high yield in accordance with the
work of Aggarwal and co-workers from ethyl but-2-ynoate
using bisIJpinacolato)diboron, sodium tert-butoxide and
copperIJII) chloride.30 Determination of the stereoisomer
formed in this reaction was challenging, but mechanistically
it was more likely to be the Z-isomer following syn-addition:
incorrect stereochemistry for the latter cyclisation. This mate-
rial was progressed in the hope that in situ isomerisation
would occur, either heat or transition metal mediated, during
palladium catalysed amination. Pleasingly, cross-coupling of
the borylated alkene 4 and the iodo core 5 progressed in an
excellent yield (97%). This validated the approach to ex-
change to coupling partners in order to further optimise this
chemistry. However, prior to cyclisation, the ethyl ester 6
needed to be converted to the amide 8, via the carboxylic acid
7. Unfortunately, base-mediated hydrolysis of the di-ester 6
proved problematic and the desired product could not be
obtained due to degradation.
In order to avoid such degradation, the chemistry was re-
peated using N-(4-methoxybenzyl)but-2-ynamide (9). The for-
mation of the alkyne amide 9 was achieved from but-2-ynoic
acid using propylphosphonic anhydride (T3P) in good yields
(Scheme 3). Alkyne amide 9 then underwent hydroboration
as before, to yield the (presumably) syn-hydroborated alkene
amide 10.30 Product 10 was then successfully coupled with
the iodo core 5 using the previous conditions, again in an ex-
cellent yield (86%). The next step was to cyclise the
Scheme 1 Unsuccessful Suzuki-Miyaura cross-coupling of 1 and 2 to
form 3 using the previously reported conditions.27–29 (a) PdIJdppf)Cl2
·CH2Cl2, K2CO3, DME, H2O, 85 °C.
Scheme 2 Attempted formation of amide 8. (a) B2pin2, NaOtBu, CuIJII)Cl2, xantphos, MeOH, THF, rt, 77%, (b) PdIJdppf)Cl2·CH2Cl2, K2CO3, DME, H2O,
85 °C, 97%. (c) NaOH, H2O, THF.
Scheme 3 Attempted synthesis of 11. (a) NH2PMB, T3P, NEt3, THF, rt, 84%, (b) B2pin2, NatOBu, CuIJII)Cl2, xantphos, MeOH, THF, rt, 97%, (c)
PdIJdppf)Cl2·CH2Cl2, K2CO3, DME, H2O, 85 °C, 86%, (d) Pd G2 Xphos, Xphos, K2CO3, i-PrOH, 90 °C.
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compound to form the core of deoxynybomycin 11. Using the
Buchwald-Hartwig conditions reported by Hergenrother and
co-workers,28,29 no evidence of cyclised material was observed
and only the starting amide remained. A screen of alternative
reaction conditions was carried out varying the catalyst, base
and solvent, as well as alternative pathways such as nucleo-
philic aromatic substitution (SNAr), but none of these condi-
tions successfully delivered the product. We attribute the fail-
ure of these reactions to the incorrect stereochemistry of the
alkene and lack of in situ isomerisation. We therefore instead
explored alternative methods to access the correct stereo-
chemistry to facilitate cyclisation.
Scheme 4 Attempted synthesis of amide coupling partner 12. (a)
HBpin, PIJBu)3, 60 °C, MeCN, 47%.
in MeOH – yielded small amounts of the di-acid 16, but purify-
ing this compound proved problematic and telescoping the
crude material into the amide formation using T3P as the
coupling agent or via the formation of the acyl chloride did
not yield any of the desired di-amide 3. Direct amination of
ester 15 using trimethylaluminum also failed.
Hydroboration of an alkyne typically occurs to provide the
anti-Markovnikov adduct with syn-selectivity via
a four-
membered transition state. Although such regiochemistry is
required for our coupling intermediate, the stereochemistry
is
incorrect.
Given
our
failed
attempt
above,
E-stereochemistry of the hydroboration product would be re-
quired in order for the resultant coupled product to be in the
correct stereochemistry to cyclise. Sawamura and co-workers
have previously reported an anti-hydroboration of alkynyl es-
ters to give hydroborated alkenes with the desired regio- and
stereochemistry.31 Given the previous challenges with ester
hydrolysis once attached to the core, synthesis of the amide
coupling partner 12 was attempted (Scheme 4). Unfortunately
the anti-hydroboration chemistry developed by Sawamura and
co-workers did not work for substrate 9. Instead of hydro-
boration, reduction was the preferred pathway and the major
isolated product was (E)-N-(4-methoxybenzyl)but-2-enamide
(13).
Given the apparent base lability of the intermediate 15, we
changed to the use of a tert-butyl ester, which would allow
formation of carboxylic acid 16 through acid-mediated
tert-butyl cleavage (Scheme 6). Alkyne 17 was synthesised as
reported previously in the literature,32 via an elimination and
deprotonation of a mixture of cis/trans 1-bromopropene with
nBuLi, then trapping the lithiated alkyne anion formed with
di-tert-butyl dicarbonate. Pleasingly, the hydroboration devel-
oped by Sawamura and co-workers31 worked well on alkyne
17 to give alkene 18 in an acceptable yield (52%). This mate-
rial was purified by distillation and crystals were grown from
a mixture of pentane and diethyl ether to allow for determi-
nation of the geometry across the double bond by a single-
crystal X-ray diffraction. As evident from the X-ray structure
of alkene 18 (Fig. 3), the desired anti-addition product was in-
deed obtained. This information compared to the 1H NMR
data let us unequivocally assign the stereochemistry of alkene
4 as Z.
Therefore, we returned to an ester coupling partner,
shown in Scheme 5. Pleasingly, ethyl but-2-ynoate was hydro-
borated in a reasonable yield (60%) and ∼9 : 1 anti:syn. Evi-
dence for the alternative stereochemistry of this reaction
1
came from the H NMR data. There was a downfield shift of
0.43 ppm in the alkene proton and 0.22 ppm in the alkene
methyl group when compared to the previously synthesised
addition product, assumed to be syn. Coupling of this material
14 to iodo core 5 progressed in excellent yield (98%). However,
base-mediated hydrolysis once again proved be problematic.
Conditions using different hydroxide sources, reaction times,
temperatures, solvents and solvent ratios were screened,
shown in the ESI.‡ The best conditions – using NaOH (3M)
The key coupling step proceeded cleanly and efficiently to
yield the di-ester 20 in a good yield (75%) and proved to be
robust and suitable to gram scale synthesis (Scheme 6).
Treatment of 20 with trifluoroacetic acid afforded the desired
di-acid 16 in an excellent yield (95%), which was cleanly
converted to the di para-methoxybenzyl (PMB) amide 3 by
first formation of the di-acyl chloride and then treatment
with excess PMB amine. Amide 3 was cyclised using the
Scheme 5 Attempted synthesis of amide 3. (a) HBpin, PIJMe)3, THF, rt, 60%, (b) PdIJdppf)Cl2·CH2Cl2, K2CO3, DME, H2O, 85 °C, 98%, (c) NaOH, H2O,
MeOH, rt, (d) (i) (COCl)2, rt, (ii) NH2PMB, NEt3, DCM, rt.
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Scheme 6 Synthesis of ethoxy-pyridine analogues of 24, 25 and 26. (a) n-BuLi, Boc2O, THF, −78 °C, 98%, (b) HBpin, PIJMe)3, THF, rt, 52%, (c) LDA,
TMSCl, THF, −78 °C, quant, (d) ICl, DCM, rt, 86%, (e) PdIJdppf)Cl2·CH2Cl2, K2CO3, DME, H2O, 85 °C, 75%, (f) TFA, DCM, rt, 95%, (g) (i) (COCl)2, rt, (ii) NH2-
PMB, NEt3, DCM, rt, 96%, (h) Pd G2 Xphos, Xphos, K2CO3, i-PrOH, 90 °C, 99%, (i) TFA, rt, 98%, (j) EtI, K2CO3, DMSO, 70 °C, 59%, (k) BBr3, DCM, rt, 41%,
(l) CH2Br2, K2CO3, DMSO, 85 °C, 14% for 24, BrCH2CH2Br, K2CO3, DMSO, 85 °C, 13% for 25, BrCH2CH3CH2Br, K2CO3, DMF, 110 °C, 18% for 26.
Buchwald–Hartwig amination conditions used by Hergenrother
and co-workers27,28 to yield 11. Deprotection of the PMB
protecting groups was achieved using trifluoroacetic acid,
resulting in 21. The solubility of compound 21 was very poor in
a range of organic solvents, but ethylation using iodoethane in
DMSO at 85 °C with potassium carbonate as the base gave a
more soluble product. After analysis of the product by 13C NMR
and comparison with similar compounds in the literature,16
this reaction was found to have afforded the O-alkylated
ethoxy-pyridine 22 as the major reaction product. The
chemoselectivity of this alkylation was contradictory to the liter-
ature, which suggested N-alkylation to be preferred.33–35 How-
ever, we took this as an opportunity to explore the antibacterial
effect of these novel nybomycin-like analogues and probe novel
structure–activity relationships. Chemoselective demethylation
of anisole 22 was achieved using BBr3 and the product 23 could
be isolated via precipitation with water and filtration. The
resulting off-white solid 23 was cyclised using 1,1-
dibromomethane for 24, 1,2-dibromoethane for 25 and 1,3-
dibromopropane for 26, to access three novel nybomycin-like
analogues for anti-bacterial testing.
column chromatography affording the desired amine 27 in a
42% yield and the undesired di-PMB amide 3 which could be
recycled for the synthesis of the ethoxy-pyridines above. The
desired amide 27 was cyclised under Buchwald–Hartwig
amination conditions using Xphos Pd G2.36 This gave good
conversion (85%) to the cyclic product 28 which was then
globally deprotected using HBr at reflux. Treatment of the re-
action mixture with water gave an off-white precipitate which
was isolated via filtration to give 29. This material could then
Attention then returned to the synthesis of 30. To avoid
O-ethylation the N-ethyl N-PMB amide 27 was prepared, prior
to cyclisation, as shown in Scheme 7. Di-acid 16 underwent
an unsymmetrical amide coupling with a 1 : 1 mixture of
ethylamine and PMB amine to give the statistical mixture of
the amide products. These products could be separated by
Fig. 3 Structural conformation of 18. X-ray structure of 18, confirming
anti-hydroboration had occurred and the coupling partner had the de-
sired conformation (E).
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Scheme 7 Synthesis of deoxynybomycin analogues 30, 31 and 32. (a) (i) (COCl)2, rt, (ii) NH2Et, NH2PMB, NEt3, DCM, rt, 42%, (b) Pd G2 Xphos,
Xphos, K2CO3, i-PrOH, 85 °C, 85%, (c) HBr, 110 °C, 91%, (d) CH2Br2, K2CO3, DMSO, 85 °C, 33% for 30, BrCH2CH2Br, K2CO3, DMSO, 110 °C, 28% for
31, BrCH2CH3CH2Br, K2CO3, DMF, 110 °C, 44% for 32.
be cyclised using 1,1-dibromomethane for 30, 1,2-
dibromoethane for 31 and 1,3-dibromopropane for 32, to two
novel nybomycin analogues for anti-bacterial testing and
comparison with the previously described compound 30.16
The antibiotic activity of 30 and novel analogues (24, 25,
26, 31 and 32) were determined using the Kirby–Bauer disc
diffusion assay37 using the protocol described by Sridhar
and co-workers,38 with the fluoroquinolone sensitive (FQS)
S. aureus strain SH1000 and the fluoroquinolone resistant
(FQR) S. aureus strain USA300 JE2 (Table 1). S. aureus
USA300 JE2 has a mutated DNA gyrase (S84L) and topo-
isomerase IV (S80Y) which causes resistance to ciprofloxacin
as well as additional mutations that confer resistance to
other antibiotic classes.39
As previously reported,15,16 compound 30 showed the de-
sired mutant selectivity, resulting in effective killing of
USA300 JE2. Ring expanded analogues 31 and 32 gave compa-
rable activity, with the 6 membered ring (31) tentatively the
most active. This initial result will have to be confirmed
using an additional susceptibility testing method.
Interestingly, compounds 24, 25, and 26, where the oxygen
of the pyridone was alkylated rather than the nitrogen,
showed greatly reduced activity compared to 30, 31, and 32.
24 was inactive at a concentration of 32 mmol in both the
FQR strain and FQS strain (data not shown). However, com-
pound 25 with the 6-membered ring, showed some activity
against the FQR strain at 10 mmol. This novel SAR is in-
triguing and suggests that further investigation may be
warranted. Such work is ongoing and will be reported in
due course.
Table 1 Zone of inhibitions (in cm) of known compounds ciprofloxacin
and 30 and novel analogues 31, 32, 24, 25 and 26. SH1000 was used as
the FQS strain and USA300 JE2 was used as the FQR strain. Compounds
were tested at 1 and 10 mmol using the Kirby–Bauer disc diffusion assay
with 7 replicates
Conclusions
In summary we have developed a robust and scalable route
to the nybomycin scaffold which can facilitate the synthesis
diverse array of analogues. The key differentiating step of our
approach was to switch the coupling partners of the capri-
cious Suzuki-Miyaura cross-coupling, resulting in scalable
coupling yields up to 98%. Our route only involves the use of
column chromatography three times which further enables
scalability. We believe this route should allow for much
needed medicinal chemistry exploration of the nybomycin
‘reverse antibiotics’. Indeed, we have initially surveyed five
novel analogues which indicate key structure determinants
for antibiotic activity and opportunities for further
optimisation.
Conflicts of interest
There are no conflicts to declare.
Acknowledgements
MJF would like to thank the EPSRC for an Established Career
Fellowship (EP/R00188X/1). The Aragon laboratory is
supported by the UK Medical Research Council. OBC was
supported by a Cross Faculty Collaborative studentship from
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the London Institute of Medical Research (LMS), which re-
ceives its core funding from the UK Medical Research Coun-
cil. The authors would like to give thanks Dr Andrew Edwards
for providing the SH1000 and USA300 JE2 bacterial strains,
Peter Haycock for NMR support, Adam Jarmuz for microbiol-
ogy support and Dr. Lisa Haigh for mass spectrometry.
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