Synthesis and antibacterial activities of enamine derivatives of dehydroacetic acid

Article abstract of DOI:10.1007/s00044-017-2110-8

Dehydroacetic acid is a common pyrone derivative used commercially as an antibacterial and antifungal agent. Based on the synthesis of dehydroacetic acid (1) from N-hydroxysuccinimdyl acetoacetate, a novel series of enamine-based derivatives were synthesised in order to improve the antibacterial activity of dehydroacetic acid. The antibacterial activities of the synthesised analogues were evaluated against Escherichia coli and Staphylococcus aureus. Derivative 4d (N-Ph) was identified as the most potent inhibitor of S. aureus growth. Overall, derivative 4b (N-Me) showed the best broad-spectrum activity with five-fold greater minimum inhibitory concentration and 11-fold greater minimum biocidal concentration against E. coli when compared to dehydroacetic acid, in addition to improved antibacterial activity against S. aureus.

Full text of DOI:10.1007/s00044-017-2110-8

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-017-2110-8
ORIGINAL RESEARCH
Synthesis and antibacterial activities of enamine derivatives of
dehydroacetic acid
Alex G. Baldwin1 Jonathan Bevan1 David Brough2 Ruth Ledder1
Sally Freeman
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●
1
Received: 17 August 2017 / Accepted: 2 November 2017
© The Author(s) 2017. This article is an open access publication
Abstract Dehydroacetic acid is a common pyrone deriva-
tive used commercially as an antibacterial and antifungal
agent. Based on the synthesis of dehydroacetic acid (1) from
N-hydroxysuccinimdyl acetoacetate, a novel series of
enamine-based derivatives were synthesised in order to
improve the antibacterial activity of dehydroacetic acid. The
antibacterial activities of the synthesised analogues were
evaluated against Escherichia coli and Staphylococcus
aureus. Derivative 4d (N-Ph) was identified as the most
potent inhibitor of S. aureus growth. Overall, derivative 4b
(N-Me) showed the best broad-spectrum activity with five-
fold greater minimum inhibitory concentration and 11-fold
greater minimum biocidal concentration against E. coli
when compared to dehydroacetic acid, in addition to
improved antibacterial activity against S. aureus.
Introduction
There is an urgent need for the development of new anti-
bacterial agents in order to overcome bacterial resistance. A
number of clinically important bacterial pathogens includ-
ing Staphylococcus aureus that were once susceptible to
antibiotic therapy are rapidly becoming multidrug-resistant
due to misuse of antibacterial agents within the medical and
agricultural field (Davies and Davies 2010; Blair et al.
2015). Recently, resistance to the last-resort antibiotic
colistin has emerged in animals and humans that have
spread globally from China, resulting in the potential for
pan-resistance among pathogenic bacteria (Liu et al. 2016).
3-Acetyl-4-hydroxy-6-methyl-2H-pyran-2-one
(dehy-
droacetic acid) (1), and its derivatives are known to possess
antibacterial, antifungal and phytotoxic activity (Nagawade
et al. 2005; Dias et al. 2009; Pulate et al. 2011; Nechak et al.
2015). 1 or its salt sodium dehydroacetate, are safe and are
widely found in cosmetics and personal care products as
well as additives in food and drink. 1 is also a versatile
starting material for the preparation of a variety of hetero-
cycles and cycloaddition products (Goel and Ram 2009;
Fadda and Elattar 2016).
Our intention was to synthesise acetoacetamide deriva-
tives (3) by reacting commercially available N-hydro-
xysuccinimidyl acetoacetate (2) with a range of amines (R
= H, Me, Et, Ph and substituted anilines, CH2Ph,
CH2CH2Ph, cyclohexyl and CH2C≡CH). These were
required as intermediates for a novel class of anti-
inflammatory agents that target the NLRP3 inflammasome
(Baldwin et al. 2017). However, surprisingly it soon became
apparent following characterisation of the products that the
desired compounds (3) were not obtained and that the
enamine analogues (4) were synthesised, arising from 1.
Herein, we discuss the novel synthesis of 1 from 2 and
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Keywords Dehydroacetic acid Pyran-2, 4-diones
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Staphylococcus aureus SAR Antibacterial activity
Electronic supplementary material The online version of this article
(https://doi.org/10.1007/s00044-017-2110-8) contains supplementary
material, which is available to authorised users.
* Sally Freeman
sally.freeman@manchester.ac.uk
1
Division of Pharmacy and Optometry, School of Health Sciences,
Faculty of Biology, Medicine and Health, Manchester Academic
Health Science Centre, The University of Manchester, Stopford
Building, Oxford Road, Manchester M13 9PT, UK
2
Division of Neuroscience and Experimental Psychology, School
of Biological Sciences, Faculty of Biology, Medicine and Health,
Manchester Academic Health Science Centre, The University of
Manchester, AV Hill Building, Oxford Road, Manchester M13
9PT, UK

Med Chem Res
given its known antibacterial activity, a number of enamine
analogues (4) were synthesised in order to improve its
antibacterial properties.
Materials and methods
Experimental
A batch of N-hydroxysuccinimidyl acetoacetate (purchased
from Acros Organics) was characterised as dehydroacetic
acid. Solvents were purchased from Fisher Scientific. All
other chemicals were purchased from Sigma-Aldrich. A
Bruker Avance 300/400 spectrometer was used to record 1H
and 13C NMR spectra at ambient temperature. Chemical
shifts are defined in ppm (δ) relative to CHCl3 (δ = 7.26
ppm). Electrospray ionisation mass spectrometry (ESI-MS)
and high resolution-mass spectrometry (HRMS) were car-
ried out on Waters SQD2 or Waters QTOF instruments with
lock spray. Infrared spectroscopy was conducted on a
JASCO FT/IR-4100 spectrophotometer using the Spectra
Manager II (JASCO) software package. Melting points
were taken using a Stuart SMP10 digital melting point
apparatus. Evaporation of solvents was conducted on a
Buchi Rotavapor R-200 equipped with Buchi heating bath
B-490. TLC was performed using silica gel 60 on alumi-
nium sheets with F254. All spots were visualised using a MV
Mineralight lamp (254/365) UVGL-58. Silica gel with
particle size 40–63 microns was used for column chroma-
tography. All purified products using the column chroma-
tography method were evaporated in vacuo to
completeness. Full characterisation data for the novel
1
compounds (4f–i and 4k–l) is given below and the H/13C
NMR data are shown in Table 1. Full characterisation data
for compounds that have been reported before (4a–e and 4j)
are given in the Supplementary Information (SI). All spectra
for compounds 4a–4l are included in the Supplementary
Information.
General procedure for synthesis of pyran-2,4(3H)-diones
Unless otherwise stated, dehydroacetic acid (1) (0.20 g, 1.19
mmol) in anhydrous dichloromethane (DCM) (5 mL) was
added dropwise to a solution containing the primary amine
(1.19 mmol) and Et3N (0.33 mL, 2.38 mmol) in anhydrous
DCM (5 mL) under N2. The reaction was stirred at room
temperature and monitored by TLC until completion. Upon
reaction completion, DCM (10 mL) was added to the reac-
tion mixture and washed with 5% HCl (2 × 10 mL). The
extracted organic layer was dried over MgSO4, filtered, and
the reaction mixture was purified by flash column chroma-
tography (EtOAc:n-hexane) to obtain the pure pyran-2,4-

Med Chem Res
dione derivative. Ratio of solvents used for flash column
chromatography is stated for each compound.
(E)-3-(1-((4-(tert-Butyl)phenyl)amino)ethylidene)-6-methyl-
2H-pyran-2,4(3H)-dione (4l)
The reaction was refluxed in DCE instead of DCM at room
temperature. Cream solid. EtOAc:n-hexane (1:4). Yield:
34%. mp 163–164 °C; MS (ESI+) (m/z): 300.2 [M+H,
100]+, 322.2 [M+Na, 5]+; HRMS (ESI+) (m/z): [M+H]+
calcd. for C18H22NO3, 300.1594, found: 300.1602, error:
2.7 ppm.
(E)-6-Methyl-3-(1-(phenethylamino)ethylidene)-2H-pyran-
2,4(3 H)-dione (4f)
Cream solid. EtOAc:n-hexane (1:3). Yield: 93%; mp 89–90
°C; IR (neat): νmax 1698, 1654, 1571, 1458, 1360, 1331,
997 cm−1; MS (ESI+) (m/z): 272.1 [M+H, 100]+, 294.1 [M
+Na, 10]+; HRMS (APCI+ TOF-MS) (m/z): [M+H]+
calcd. for C16H18NO3, 272.1287, found: 272.1277, error:
3.7 ppm.
Assay for antibacterial activity
The stock solutions of all test compounds (10.2–22.6 mg)
were prepared by dissolving in absolute ethanol (1 mL).
Following this they were filtered sterilised (0.2 micron pore
size) to remove potential contaminants. Minimum inhibi-
tory concentrations (MICs) and minimum biocidal con-
centrations (MBCs) were carried out against Escherichia
coli ATCC 25922 and Staphylococcus aureus ATCC 6538.
Testing was performed in 96-well microtitre plates (Becton
Dickinson, New Jersey USA). To determine the MIC for
test compounds, overnight cultures were diluted 1:100 and
delivered to each test well (100 µL). Stock solutions of test
compounds (100 µL) were added to the first column of test
organism and mixed. Twofold serial dilutions were then
carried out across the plate using a multi-channel pipette,
changing the tips at each dilution step. The plates were then
incubated for 48 h in a standard incubator at 37 °C. Growth
was detected as turbidity (495 nm), relative to an unin-
oculated well using a microtitre plate reader. MICs were
expressed as the lowest concentration of synthesised ana-
logue at which growth did not occur, i.e., that which
inhibited continued growth in the presence of the com-
pound. Each MIC determination was carried out in quad-
ruplicate. Negative (sterile broth) and positive (overnight
culture with no added dentifrice) controls were also
included.
(E)-3-(1-(Cyclohexylamino)ethylidene)-6-methyl-2H-pyran-
2,4(3H)-dione (4g)
Cream solid. EtOAc:n-hexane (1:3). Yield: 56%; mp
109–110 °C; IR (neat): νmax 2933, 2857, 1696, 1668, 1559,
1473, 1353, 1001 cm−1; MS (ESI+) (m/z): 250.2 [M+H,
100]+, 272.2 [M+Na, 38]+; HRMS (APCI+ TOF-MS)
(m/z): [M+H]+ calcd. for C14H20NO3, 250.1443, found:
250.1434, error: 3.6 ppm.
(E)-6-Methyl-3-(1-(prop-2-yn-1-ylamino)ethylidene)-2H-
pyran-2,4(3H)-dione (4h)
Cream solid. EtOAc:n-hexane (2:3). Yield: 50%; mp
114–115 °C; IR (neat): νmax 3241, 1694, 1661, 1581, 1466,
1357, 1321, 1000 cm−1; MS (ESI+) (m/z): 206.0 [M+H,
100]+; HRMS(APCI+ TOF-MS) (m/z): [M+H]+ calcd. for
C11H12NO3, 206.0817, found: 206.0824, error: 3.4 ppm.
(E)-3-(1-(3-Chlorophenylamino)ethylidene)-6-methyl-2H-
pyran-2,4(3H)-dione (4i)
The reaction was refluxed in 1,2-dichloroethane (DCE)
instead of DCM at room temperature. Orange solid. EtOAc:
n-hexane (1:4). Yield: 21%. mp 133–134 °C; MS (ESI+)
(m/z): 278.1 [M+H, 35Cl, 100]+, 280.1 [M+H, 37Cl, 33]+,
300.1 [M+Na, 35Cl, 12]+, 302.0 [M+Na, 37Cl, 5]+; HRMS
(ESI+) (m/z): [M+H]+ calcd. for C14H1335ClNO3,
278.0578, found: 278.0578, error: 0.0 ppm.
MBCs were determined using the microtitre plates set-up
for the MIC determinations. Aliquots (1 µL) taken from
each well up to and including the MIC endpoint were
transferred and spot-plated onto the appropriate agar. MBCs
were expressed as the lowest concentration of synthesised
analogue at which growth was not observed after 5 days of
incubation, i.e., that which inactivated all bacteria in the
original inoculum.
(E)-6-Methyl-3-(1-((3-(trifluoromethyl)phenyl)amino)
ethylidene)-2H-pyran-2,4(3H)-dione (4k)
The reaction was refluxed in DCE instead of DCM at room
temperature. Cream solid. EtOAc:n-hexane (1:4). Yield:
35%. mp 145–146 °C; MS (ESI+) (m/z): 312.2 [M+H,
82]+, 334.1 [M+Na, 12]+; HRMS (ESI+) (m/z): [M+H]+
calcd. for C15H13F3NO3, 312.0842, found: 312.0839, error:
1.0 ppm.
Results and discussion
Reaction of commercially available 2 with a range of
amines failed to give the expected acetoacetamides 3 based
on NMR spectroscopy. This led to an analysis of the
commercially available material
2
by 1H NMR

Med Chem Res
Scheme 1 Synthesis of (E)-3-(1-aminoethylidene)-6-methyl-2H-
pyran-2,4(3H)-diones 4 from 1 as opposed to intended acetoacetamide
derivatives 3 from 2. (i) RNH2, Et3N, DCM/DCE, r.t.−70 °C, 3–16 h
spectroscopy to confirm its structure. However, the 1H
NMR spectrum showed that 2 had cyclised into dehy-
droacetic acid 1 (Scheme 1), confirmed by comparing the
1H NMR and mass spectra with commercially available 1.
Compared to the original synthesis of 1 using ethyl acet-
oacetate and sodium carbonate (Arndt et al. 1940), the
cyclisation of 2 into 1 represents a novel method for the
preparation of 1. We propose that two equivalents of 2
undergo Claisen condensation, followed by intramolecular
nucleophilic substitution and subsequent loss of two
equivalents of N-hydroxysuccinimide in support of the
proposed reaction mechanism (Scheme 2).
Scheme 2 Proposed synthesis of 1 from 2 via Claisen condensation
and intramolecular cyclisation
Table 2 Antibacterial activity of 1 and amino-substituted analogues
4a–4l against one Gram-positive (S. aureus) and one Gram-negative
bacterium (E. coli)
Compounds MW
R
E. coli
S. aureus
MIC MBC
MIC MBC
1
168.15
167.16
—
0.42 1.69
0.17 0.66
0.08 0.15
0.38 1.51
0.35 1.4
0.84 1.69
0.17 1.33
4a
4b
4c
4d
4e
4f
H
Based on the unexpected synthesis of 1 and its known
antibacterial activity, 12 enamine analogues of 1 were
prepared by reacting equimolar equivalents of 1 with a
selection of primary amines to afford the corresponding (E)-
181.19 CH3
195.22 CH2CH3
243.26 Ph
0.3
0.6
0.38 0.38
0.18 0.35
1
enaminopyran-2,4-diones (4a–4l, Scheme (1)). The H/13C
257.29 CH2Ph
0.38 2.05 (0.9) 0.38 0.38
1.2 0.3 1.2
271.32 CH2CH2Ph 0.3
NMR data obtained for novel analogues 4f–i and 4k–l along
with their assignments are presented in Table 1. It has been
previously shown that equimolar equivalents of 1 with
ammonia or alkylamines favourably form (E)-enam-
inopyran-2,4-diones (Garratt 1963) while primary aromatic
amines give the corresponding secondary enamines
(Edwards et al. 1964). The characterisation data obtained
for compounds 4a–e and 4j are in agreement with literature
values (Garratt 1963; Edwards et al. 1964; Dias et al. 2009),
whereas derivatives 4f–i and 4k–l are novel.
4g
4h
4i
249.31 Cyclohexyl >6.5 1.63
205.21 CH2C≡CH 0.32 1.87(1)
0.41 1.63
0.64 2.58
0.40 1.60
0.50 >2.00
1.00 2.55
NA NT
277.70 3-Cl-Ph
277.70 4-Cl-Ph
311.26 3-CF3-Ph
299.37 4-tBu-Ph
0.80 >1.60
1.00 >2.00
0.32 >2.55
NA NT
4j
4k
4l
Data were determined by broth dilution endpoint (n = 4). Units are mg
mL−1. Where data varied between replicates, standard deviations are
given in parenthesis
The antibacterial activities of 1 and analogues 4a–4l
were then examined on Gram-negative Escherichia coli and
Gram-positive Staphylococcus aureus bacteria (Table 2).
All synthesised derivatives except 4k–l showed improved
MIC values against S. aureus compared to 1, while com-
pounds 4a–f, 4h and 4k demonstrated improved MIC
values against E. coli when compared to 1. The MBC of
each derivative was also measured against E. coli and S.
NA not active, NT not tested
aureus and the results showed that derivatives 4a–4d, 4f
and 4g have greater bactericidal activities against both
bacterial strains than 1.
Structure–activity relationship (SAR) analysis revealed a
number of interesting features. It was found that increasing

Med Chem Res
the alkyl chain length negatively affected antibacterial
activity (R = H > Me > Et, 4a–4c) (Table 2). Substitution
with a phenyl ring (4d) improved activity, but similar to
alkyl derivatives, increasing the alkyl linker between the N
atom and phenyl ring (derivatives 4e–4f) generally
decreases antibacterial activity. The presence of an unsa-
turated phenyl ring was essential as saturation of the ring to
the cyclohexyl derivative (4g) significantly impacted on
activity, particularly against E. coli. Given that compound
4d was the most effective compound against S. aureus, we
then designed and synthesised derivatives 4i–l in order to
investigate the effect of ring substitutions on antibacterial
activity. Although none of the synthesised analogues were
more potent than 4d, it was interesting to note that electron-
withdrawing substituents reduced antibacterial activity
against both E. coli and S. aureus. In particular, the activ-
ities of derivatives 4i (R = 3-Cl-Ph) and 4j (R = 4-Cl-Ph)
were very similar, indicating the requirement for the phenyl
ring to be electron-rich. Derivative 4l (R = 4-tBu-Ph) was
inactive against both E. coli and S. aureus, most likely due
to steric reasons. Overall, 4b was the best derivative iden-
tified in the series, with an MIC of 80 µg mL−1 and an MBC
of 150 µg mL−1 against E. coli and an MIC of 300 µg mL−1
and an MBC of 600 µg mL−1 against S. aureus.
Conclusions
A novel synthesis of 1 is proposed based on the dimerisa-
tion of 2. A number of (E)-enaminopyran-2,4-diones based
on the structure of 1 were prepared and evaluated for
antibacterial activity against Gram-positive and Gram-
negative bacteria and demonstrate broad-spectrum activ-
ities. We identified 4b (R = Me) and 4d (R = Ph) as
improved antibacterial agents that inhibited and prevented
the growth of both E. coli and S. aureus to a greater extent
than 1. Derivative 4b significantly inhibited the growth of
E. coli compared to S. aureus while derivative 4d had
greater potency against S. aureus than E. coli. Further
research is necessary to investigate their proposed
mechanism of action as antagonists of bacterial commu-
nication as a potential avenue for antimicrobial drug
development. Given the rise in antibiotic resistance, anti-
bacterial agents based on the lead compounds 4b/4d may be
of use in the search for broad-spectrum antibiotics.
Acknowledgements We thank the Division of Pharmacy and
Optometry, The University of Manchester and the Presidential Doc-
toral Scholar award for providing funding to AGB.
Compliance with ethical standards
The ability of compounds 4b and 4d to inhibit and
prevent the growth of both Gram-positive and Gram-
negative bacteria at much lower concentrations than the lead
compound 1 is promising. This cross-species antibacterial
effect suggests a mode of action common among bacteria
essential for its survival and growth. α-Pyrones have
recently been identified as important endogenous signalling
molecules in bacterial cell–cell communication (Brachmann
et al. 2013) and it has been suggested that α-pyrones could
have antagonist effects by disrupting quorum-like signalling
in pyrone-producing bacteria, similar to the antimicrobial
effects of synthetic analogues of N-acyl homoserine lactone
(AHL) autoinducers on quorum sensing (Swem et al. 2009).
The results of this study support the notion that some α-
pyrone derivatives exhibit antibacterial properties and that
the potential mechanism of action of pyrone derivatives 4b
and 4d may involve interference with cell–cell bacterial
communication. It is interesting to note that the α-pyrone
signalling molecules described by Brachmann et al. had
long fatty acid side chains and future analogues of 4 with
these R groups may enhance their antibacterial activity.
There are also reports that derivatives of 1 bind to and
cleave DNA (Chitrapriya et al. 2008) and are suggested to
target the essential bacterial enzyme DNA gyrase (Pal et al.
2014). Chemoproteomic studies using 4h (R = propargyl)
as a reagent for the ‘Click-chemistry’ method could be
undertaken to elucidate the mechanism of action of these α-
pyrone derivatives and potentially give rise to a new class of
antibacterial agents.
Conflict of interest The authors declare that they have no competing
interests.
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://crea
tivecommons.org/licenses/by/4.0/), which permits unrestricted use,
distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
link to the Creative Commons license, and indicate if changes were
made.
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