Epoxy Isonitriles, A Unique Class of Antibiotics: Synthesis of Their Metabolites and Biological Investigations

Article abstract of DOI:10.1002/cbic.201800550

Epoxy isonitrile containing natural products often possess specific and potent antibacterial activity against Gram-positive pathogens. This scaffold, however, is extremely labile under acidic and basic conditions, undergoing a Payne rearrangement to produce a stable epoxy ketone metabolite and releasing hydrogen cyanide. We synthesized and performed biological assays with epoxy ketone containing metabolites and identified that the epoxy isonitrile moiety is pertinent for biological activity. Serendipitously, we discovered an α,β-unsaturated epoxy ketone analogue that exhibited moderate activity against Staphylococcus aureus.

Full text of DOI:10.1002/cbic.201800550

Accepted Article
Title: Epoxy isonitriles, a unique class of antibiotics – Synthesis of their
metabolites and biological investigations
Authors: Guillaume Ernouf, Ingrid Wilt, Sara Zahim, and William M.
Wuest
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10.1002/cbic.201800550
ChemBioChem
COMMUNICATION
Epoxy isonitriles, a unique class of antibiotics – Synthesis of
their metabolites and biological investigations
Guillaume Ernouf,[a] Ingrid K. Wilt,[a] Sara Zahim,[a] and William M. Wuest*[a][b]
Abstract: Epoxy isonitrile containing natural products often possess
specific and potent antibacterial activity against Gram-positive
pathogens. This scaffold, however, is extremely labile under acidic
and basic conditions, undergoing a Payne rearrangement to produce
a stable epoxy ketone metabolite and releasing hydrogen cyanide.
We synthesized and performed biological assays with epoxy ketone
containing metabolites and identified that the epoxy isonitrile moiety
is pertinent for biological activity. Serendipitously, we discovered an
α,β-unsaturated epoxy ketone analogue that exhibited moderate
activity against S. aureus.
synthesis of cyclic epoxy isonitrile trichoviridin in 1996,7 attempts
by Baldwin et al. to produce aerocyanidin were fruitless, resulting
in only the desepoxy form of the natural product.8
Natural products play an important role in the
pharmaceutical industry, especially for antimicrobial drug
discovery.1 Due to the widespread resistance of antibiotics,2 there
is an urgent need for new and improved antimicrobials against
multi-resistant bacteria. Selective or narrow-spectrum antibiotics
may act as a means of combating emerging bacterial resistance.
Ideally, these treatments would only target pathogenic bacteria
and allow for the commensal bacteria to survive, which would both
aid a host in fighting off infection and prevent rapid evolution of
resistance. Despite their highly potent and selective antimicrobial
activity, epoxy isonitrile natural products have attracted the
attention of few synthetic chemists and pharmacologists, mainly
due to their complex frameworks. Isolated by Sykes et al. in 1987,
the epoxy isonitrile containing aerocyanidin3 exhibits a highly
potent activity against Gram-positive pathogens. A decade later,
an elongated analogue of aerocyanidin, YM-47515,4 was isolated,
and very recently Clardy et al., identified amycomicin through
bacterial co-culturing with Streptomyces 84 coelicolor M145,
which displays a very selective profile for S. aureus.5
In all cases, the epoxy isonitrile moiety proves to be labile to
mildly basic and acidic conditions causing considerable difficulty
during the isolation and purification process. Exposure to pH less
than 4 and greater than 8 triggers a Payne rearrangement of the
natural compound to produce a stable epoxy ketone metabolite
(Scheme 1). Due to the lability of the epoxy isonitrile moiety,
synthesis of such scaffolds are particularly challenging and
examples are scarce in the literature.6 Despite the successful
Scheme 1. Under mildly basic or acidic conditions, epoxy isonitrile scaffolds of
aerocyanidin, YM-47515, and amycomicin undergo a Payne rearrangement to
produce stable epoxy ketone metabolites. MICs reported for S. aureus.3,5
The specific bioactivity exhibited by this class of natural
products stimulated our desire to better understand its mode of
action. While these potent antibacterial agents exhibit
tremendous activity and selectivity against S. aureus (MIC < 0.05
µM), the instability of the epoxy isonitrile scaffold and release of
toxic hydrogen cyanide upon exposure to mildly acidic or basic
conditions render it an unusable antibiotic.3,9 Upon comparison to
inorganic sodium cyanide, however, aerocyanidin was shown to
be much more potent,3 leading us to believe it might be acting as
a prodrug. If the epoxy isonitrile is acting as a prodrug, the epoxy
ketone metabolite resulting from the Payne rearrangement would
therefore retain activity and simultaneously decrease its toxicity
associated with the release of hydrogen cyanide.
[a]
[b]
Dr. G. Ernouf, I. K. Wilt, Dr. S. Zahim, Dr. W. M. Wuest
Department of Chemistry
Emory University
Atlanta, Georgia 30322, USA
E-mail: wwuest@emory.edu
Dr. W. M. Wuest
Emory Antibiotic Resistance Center
Emory University
Atlanta, Georgia 30322, USA
Figure 1. Known inhibitors of fatty acid biosynthesis.
Supporting information for this article is given via a link at the end of
the document.
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10.1002/cbic.201800550
ChemBioChem
COMMUNICATION
Additionally Clardy et al. recently demonstrated that
amycomicin inhibits fatty acid biosynthesis through inhibition of
FabH.5 The chemotype similarities between epoxy ketones (+)-4,
(+)-5, (–)-6 and cerulenin, a known FabB/F inhibitor, led us to
hypothesize that epoxy isonitrile metabolites may be targeting β-
ketoacyl-ACP synthases through a covalent adduct mechanism
akin to cerulenin.16 Indeed lipid biosynthesis is a target of interest
for antibacterial agents as metabolism of fatty acids is often
unique to each species of bacteria.17 Despite controversial
interest of targeting such a biological pathway,18 several natural
products including cerulenin and phomallenic acid C19 are
encountered in the literature. Other synthetic inhibitors are also
currently in development to fight MRSA infections including
CG40054920 and Afabicin21 (FabI) confirming the potential of this
pathway for developing narrow-spectrum antibiotics. (Figure 1).18
Motivated by the potential of this approach, we began
synthetic investigations of epoxy ketone metabolites of isonitrile
containing natural products. We sought to determine if the epoxy
were obtained by rapid treatment with lithium hydroxide (THF:H2O
(1/1), rt, 30 min). It is worth noting that the utilization of sodium
hydroxide led to slower reaction time and partial degradation of
the epoxy ketone. To test the feasibility of our approach to access
post-amycomicin (–)-6, we envisaged transforming the methyl
ketone (±)-11 to the enone (±)-14. After aldolization of (±)-11 and
acetaldehyde (LiHMDS, THF, –78 °C), the resulting
diastereoisomeric mixture of alcohols (1:1) was treated with
methanesulfonyl chloride in CH2Cl2 in the presence of Et3N to
afford the enone (±)-13 in 85% yield over two steps containing the
α,β-unsaturated ketone epoxy moiety found in amycomicin.
Attempts to deprotect methyl ester (±)-13 with hydroxide sources
were unfruitful and lead to complete degradation of the substrate.
However, hydrolysis in the presence of pig liver esterase (PLE)
smoothly proceeded at 37 °C giving rise to the desired product
(±)-14 (75%) (Scheme 2).14 With model product (±)-14 in hand,
we set about the coupling reaction toward the synthesis of post-
amycomicin (±)-6. Suberic acid monomethyl ester 15 was
converted to the corresponding acyl chloride and treated with a
solution of hex-5-enyl zinc bromide in the presence of
tetrakis(triphenylphosphine) palladium to provide the ketone 16 in
85% yield over 2 steps.15 Subsequent cross-metathesis and
stereoselective epoxidation afforded the desired methyl ketone
(±)-18. Enone (±)-19 was then obtained following the same
sequence and final enzymatic hydrolysis resulted in desired post-
amycomicin (±)-6 (Scheme 3).14
isonitrile scaffold was acting as
a prodrug, focusing on
aerocyanidin and its elongated analogue as well as amycomicin,
with the goal of creating new, potent, selective, and less toxic
antibiotics.
1) (COCl)2 (1.05 equiv)
DMF cat.
CH2Cl2, 0 °C
O
O
OMe
OMe
HO
5
ZnBr
O
2)
O
15
16
Pd(PPh3)4 (5 mol %)
THF, -78 °C to rt
(85%)
MVK (3 equiv)
Grubbs II (2 mol %)
CuI (3 mol %)
Et2O, 50 °C
(65%)
t-BuNH2 (30 mol %)
H2O2 (1.5 equiv)
MeOH, rt
O
O
Me
OMe
Me
OMe
5
5
O
O
O
(72%)
O
O
(±)-18
17
1) LiHMDS (1.5 equiv)
CH3CHO (3 equiv)
THF, -78 °C
2) MsCl (1.5 equiv)
Et3N (3 equiv)
CH2Cl2, rt
O
Me
OR
O
O
(55%)
O
R = Me, (±)-19
Pig Liver
Esterase
R = H, post-amycomicin, (±)-6 (45%)
Scheme 2. Synthesis of post-aerocyanidin, post-YM-47515, and post-
amycomicin prototype. [a] Condition A: Alkene (1 equiv), H2O2 in H2O 30% (w/w)
(1.5 equiv), t-BuNH2 (30 mol %), MeOH, rt, 16 h; Condition B: Alkene (1 equiv),
H2O2 in H2O 30% (w/w) (1.5 equiv), cat.1 (30 mol %), dioxane, 50 °C, 16 h. [b]
(–)-11 was obtained using the same conditions with cat.2 (55%); [b] PLE was
used since LiOH hydrolysis gave lower yield.
Scheme 3. Synthesis of post-amycomicin (±)-6.
Having obtained all rearranged natural products, we began
biological assays with S. aureus to test our prodrug hypothesis.
Unfortunately, none of the epoxy ketone metabolites exhibited
notable activity against S. aureus (Table 1 and Table S4)
undermining our postulate. Notably, during the course of our study,
Clardy observed complete loss of activity of amycomicin at pH >
8, which hints at the formation of an inactive rearranged product
(–)-6.5 We then tested the intermediates in our synthesis of the
metabolites and serendipitously found that the amycomicin model
substrate, enone (±)-13, was active against hospital-acquired
methicillin-resistant S. aureus (HA-MRSA) and community-
acquired methicillin-resistant S. aureus (CA-MRSA) albeit at
modest concentrations.
The synthesis of post-aerocyanidin (+)-4 and post-YM-
47515 (+)-5 commenced from terminal alkenes 7 and 8
respectively prepared from commercially available 10-undecenoic
acid10 and 11-bromo-undecan-1-ol11 in six steps. Subsequent
cross-metathesis with the methyl vinyl ketone (MVK) afforded the
α,β-unsaturated ketones
9
and 10.12 Organocatalyzed
asymmetric epoxidation13 of the α,β-unsaturated ketones 9 and
10 in the presence of cinchona primary amine catalyst yielded
optically enriched epoxides (+)-11 and (+)-12 in moderate yields.
From there, post-aerocyanidin (+)-4 and post-YM-47515 (+)-5
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COMMUNICATION
Table 1. Growth inhibition of amycomicin 3, post-amycomicin 6 and enone 13.
IC50 (µg/mL)[a],[b],[c]
S. aureus
(CA-MRSA)
S. aureus
(HA-MRSA)
S. aureus
(MSSA)
E. faecalis
P. aeruginosa
compound
(USA300)
0.011[d]
(ATCC 33592)
(Newman)
-
(ATCC 51575)
(PAO1)
-
>1[d]
>1[d]
>160
29
-
-
-
-
43
46
36
32
35
27
53
52
50
51
55
34
32
20
[a] Quaternary ammonium cation (12,3,2,3,12) was included as a positive control.22 [b] No inhibition observed against B. subtilis (ATCC 6633) and
E. coli (MC4100). [c] These assays were realized in triplicate and over 16 h. [d] MICs reported in ref. 5
Interestingly, methyl ester and the α,β-unsaturated ketone were
essential to preserve the bioactivity since neither carboxylic acid
(±)-14 nor methyl ketone (±)-11 were active (Table S4). To
Experimental Section
identify if a single enantiomer was more active, we then prepared
the two optically enriched enantiomers (–)-13 and (+)-13 from
enone 9 using both cinchona primary amine catalysts.13
We then measured the IC50 of 13 against various strains of
Gram-positive (S. aureus, E. faecalis, B subtilis) and Gram-
A full description of the experimental details can be found in the supporting
information.
Acknowledgements
negative (E. coli, P. aeruginosa) pathogens and observed
compound (–)-13 was in all cases the most active enantiomer.
The increased activity observed by only enantiomer (–)-13 may
indicate growth inhibition is occurring through substrate
We gratefully acknowledge funding from the National Institute of
General Medical Sciences (GM119426) and the National Science
Foundation (CHE1755698). The NMR instrumentation for this
work was funded by the NSF (CHE1531620). We also want to
thank Prof. Jon Clardy (Harvard Medical School) for helpful
discussions.
interaction with an enzyme active site. Although possessing
similar chemotypes, further studies are necessary to determine if
enone (–)-13 bioactivity is occurring through a novel mechanism
or if it acts in a similar manner to cerulenin targeting FabB/F.
In conclusion, we herein describe the first total synthesis of
epoxy ketone natural product metabolites and demonstrate that
they are inactive as prodrugs. These findings suggest that the
epoxy isonitrile moiety is crucial for maintaining potency and
selectivity of aerocyanidin, YM-47515, and amycomicin against S.
aureus. Through these studies, we serendipitously discovered an
enantiopure α,β-unsaturated epoxy ketone (–)-13 that possessed
modest antibacterial activity against S. aureus.
Keywords: Staphylococcus aureus • epoxy isonitrile • natural
product
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COMMUNICATION
Entry for the Table of Contents (Please choose one layout)
COMMUNICATION
O
Epoxy isonitrile containing natural
products often possess specific and
potent antibacterial activity against
G. Ernouf, I. K. Wilt, S. Zahim, and W.
M. Wuest*
Me
OH
NC OH
O
aerocyanidin
Page No. – Page No.
Gram-positive
pathogens.
This
• Highly active
against S. aureus
pH < 4 or pH > 8
H2O, minutes
scaffold, however, is extremely labile
under acidic and basic conditions,
undergoing a Payne rearrangement to
BUT
Epoxy isonitriles, a unique class of
antibiotics – Synthesis of their
metabolites and biological
investigations
HCN
• Toxicity comparable to NaCN
Me
OH
O
produce
a
stable epoxy ketone
O
O
metabolite. We synthesized and
performed biological assays with
epoxy ketone containing metabolites
and identified that the epoxy isonitrile
moiety is pertinent for biological activity
Rearranged product is active?
Natural compounds act as a prodrug?
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