Synthesis of essramycin and comparison of its antibacterial activity

Article abstract of DOI:10.1021/np100648q

The triazolopyrimidine natural product essramycin (1) was synthesized without the use of protecting groups via a two-step reaction scheme involving a 3-amino-1,2,4-triazole intermediate, and its structure was unequivocally determined. However, in contrast

Full text of DOI:10.1021/np100648q

1940
J. Nat. Prod. 2010, 73, 1940–1942
Synthesis of Essramycin and Comparison of Its Antibacterial Activity
Ernest H. L. Tee, Tomislav Karoli, Soumya Ramu, Johnny X. Huang, Mark S. Butler, and Matthew A. Cooper*
Institute for Molecular Bioscience, UniVersity of Queensland, St Lucia, Queensland 4072, Australia
ReceiVed September 15, 2010
The triazolopyrimidine natural product essramycin (1) was synthesized without the use of protecting groups via a two-
step reaction scheme involving a 3-amino-1,2,4-triazole intermediate, and its structure was unequivocally determined.
However, in contrast to the natural product, the synthetic essramycin (1) did not display any antibacterial activity.
Essramycin (1) is a triazolopyrimidine that was isolated from a
marine actinomycete (Streptomyces sp. Merv8102) obtained from
sediment samples collected in the Egyptian Mediterranean Sea.1
the strains used in the original study: P. aeruginosa (ATCC 10145)
and B. subtilis (ATCC 6051).1 However, no antibacterial activity
was found for synthetically derived 1 at a maximum concentration
of 64 µg/mL. Negative results were also obtained using the disk
diffusion method and when 1 was assayed as the sodium salt and
formulated with carboxymethylcellulose. To check whether 1 had
been degraded or was only partially soluble during the antibacterial
assays, its concentration in the assay plates was quantified by HPLC,
and it was found to be present at an appropriate level. Furthermore,
the binding of 1 to proteins in the LB assay broth (above 30 kDa)
was determined by ultrafiltration to be only 7%. The kinetic
solubility of 1 in water and pH 7.4 phosphate buffer was determined
to be greater than 150 µg/mL, while its thermodynamic solubilities
in water and pH 7.4 phosphate buffer were 175 and 898 µg/mL
respectively. These data demonstrated that at a maximum concentration
of 64 µg/mL the antibacterial results of 1 were not compromised by
high protein binding and/or aqueous solubility limitations. Incidentally,
1 was not cytotoxic against HEK293 and HepG2 cell lines at a
maximum concentration of 200 µM.
In conclusion, we have performed the total synthesis of essra-
mycin (1) in two steps via a 3-amino-1,2,4-triazole intermediate.
While the structure assignation was confirmed, the synthetically
derived material did not possess any antibacterial activity as reported
for the natural product.1 After submission of this paper, Battaglia
and Moody also published the synthesis of 1 using a similar
synthetic methodology, confirming the structure assignation, but
in the absence of associated biological data.22 Unfortunately, the
1
Spectroscopic data (IR, UV, HRMS, H, 13C, HSQC, and HMBC
NMR) used in the initial structure elucidation of 1 could not
unequivocally distinguish between the isomers 1 and 2 (Figure 1).
However, comparison of the 13C NMR chemical shifts with
synthetic triazolopyrimidine analogues2 allowed the structure of the
isolated essramycin to be assigned as isomer 1. Triazolopyrimidines
display multifaceted biological properties, including antibacterial,3,4
antimalarial,5-8 leishmanicidal,9 bronchodilatory,10 vasodilatory,11,12
anticancer,13,14 immunosuppressive,15 and anxiolytic16 activities.
Essramycin (1), which was claimed to be the first isolated
triazolopyrimidine natural product, was reported to be active against
Escherichia coli, Pseudomonas aeruginosa, Bacillus subtilis, Sta-
phylococcus aureus, and Micrococcus luteus.1 The broad-spectrum
antibacterial properties reported for 1 and the opportunity to explore
a new antibacterial pharmacophore led us to undertake its synthesis.
Herein, we present the total synthesis of 1 and the confirmation of
its structure. We also report that the synthetically derived 1 displayed
no antibacterial activity.
The synthesis of 1 (Scheme 1) proceeded via a 3-amino-1,2,4-
triazole intermediate 3, which was isolated as a minor product from
the reaction between aminoguanidine and ethyl benzoylacetate.17
Compound 3 and ethyl acetoacetate were then heated under acidic
conditions18 followed by recrystallization from methanol to yield
compound 1 with 98% purity. The spectroscopic data of compound
1 were consistent with those previously reported.1 The melting point
of 1 was 22 °C higher than that reported for its naturally isolated
counterpart, which most likely reflects the crystalline nature of the
synthetic material against the amorphous nature of the natural
product, which was isolated by column chromatography.
1
Analysis of the H NMR (d6-DMSO) of 1 and 3 revealed the
presence of keto-enol tautomerism of the methylene protons
adjacent to the ketone group.19 Treatment of compound 1 with a
tertiary base such as DIPEA (diisopropylethylamine) shifted the
equilibrium in favor of the enol tautomer (from 6% to 20%), which
Figure 1. Structure of essramycin (1) and its isomer (2).
Scheme 1. Synthesis of Essramycin (1)a
1
assisted with the assignment of H and 13C NMR resonances of
the enol form of 1 (Figure 2). Further evidence for the structure
1
assignment of essramycin was obtained by analyzing the H-15N
gHMBC NMR spectrum of 1 (Figure 3). Correlations between the
methyl protons and the enaminone nitrogen (δN -253 ppm) and
between the olefinic methine H-6 and the bridgehead hydrazine
nitrogen (δN -155 ppm) unequivocally supported the structure
assignment of essramycin as isomer 1.
The antibacterial activity of synthetically derived 1 was evaluated
against S. aureus (ATCC 25923), E. faecalis (ATCC 51299), E.
coli (ATCC 25922), K. pneumoniae (ATCC 700603), P. aeruginosa
(Meropenem-resistant), A. baumannii (Meropenem-resistant), and
a Reagents and conditions: (a) n-butanol, reflux, 130 °C, 3 h, 5%; (b) glacial
CH3CO2H, heat, 114 °C, 4 h, 18%.
* To whom correspondence should be addressed. Phone: +61 7
33462044. E-mail: m.cooper@uq.edu.au.
10.1021/np100648q  2010 American Chemical Society and American Society of Pharmacognosy
Published on Web 11/04/2010

Notes
Journal of Natural Products, 2010, Vol. 73, No. 11 1941
1
Figure 2. H-13C gHMBC spectrum of 1 and its enol tautomer (1e) after the addition of 2 molar equiv of DIPEA.
on a Bruker MicrOTOF mass spectrometer, and the data obtained were
analyzed on the Bruker DataAnalysis 4.0 software. The melting point
determinations were measured using the Stuart SMP11 melting point
apparatus, which was calibrated against benzoic acid. Thin-layer
chromatography was performed on Merck silica gel 60 F254 plates and
visualized using a UV lamp at 254 nm or 2% ninhydrin/ethanol. Flash
chromatography employed the use of Merck silica gel 60, 40-63 µm.
All commercial chemical reagents were used as received.
1
Figure 3. H-15N gHMBC correlations and 15N chemical shifts
for 1 (δN externally referenced to CH3NO2 at 0 ppm)20,21 with
Synthesis of 2-(5-Amino-4H-1,2,4-triazol-3-yl)-1-phenylethanone
(3).17 A mixture of aminoguanidine bicarbonate (21.9073 g, 160.95
mmol), n-butanol (28 mL), and ethyl benzoylacetate (28 mL, 161.70
mmol) was stirred at 130 °C for 3 h under an inert nitrogen atmosphere.
A dark brown solution was formed, which produced a yellowish-brown
suspension upon cooling to room temperature. The product was collected
by filtration, washed sequentially with ice-cold n-butanol (100 mL)
and water (100 mL), and then dried in vacuo overnight (7.1 g, yield
22% with 54% purity at 254 nm by HPLC). The compound was further
purified by stirring the product as a slurry in DMF (50 mL) at room
temperature for 40 min. The resulting creamy white suspension was
then filtered under vacuum to produce compound 3 as a white solid
(1.78 g, 5% overall yield with 97% purity at 254 nm by HPLC). Further
recrystallizations in DMF, THF, and ethanol did not increase the final
product purity as analyzed by 1H NMR and HPLC. Analysis of the 1H
NMR of 3 revealed keto-enol tautomerism in a 7:3 ratio. HPLC: tR )
4.44 min using method 1; 1H and 13C NMR, see Supporting Information,
Table S1; HRESMS (+ve) m/z 203.0922 (calcd for C10H11N4O [M +
H]+, 203.0927).
Synthesis of Essramycin (1). A solution of 3 (400 mg, 1.978 mmol),
ethyl acetoacetate (250 µL, 1.975 mmol), and glacial acetic acid (1.2
mL) was stirred at 114 °C for 4 h, at which time a light brown
precipitate had formed. Toluene was added, and the mixture was
evaporated to dryness in vacuo (60 mbar, 40 °C water bath) to give a
brown-white solid (517 mg, yield 98%). The product was recrystallized
from methanol to give compound 1, mp 241-243 °C (98% purity at
254 nm by HPLC) as colorless needles (96.4 mg, overall yield 18%).
Further efforts to purify 1 by flash chromatography and preparative
HPLC were unsuccessful: UV (MeOH) λmax (log ε) 246 (4.68), 275
published 15N chemical shifts of the triazolopyrimidine core.2
originally naturally occurring material was not available for a direct
comparison with the synthetic material. A recent review of the
originally published experimental work leading to the first paper1
suggests that the activity reported for the natural product was based
upon testing of a crude sample, rather than purified material
(personal communication, Prof. H. Laatsch). In our hands essra-
mycin is not an antibiotic.
Experimental Section
General Experimental Procedures. NMR data were collected in
d6-DMSO at 298 K using either a Varian Unity 400 MHz or a Bruker
Avance 600 MHz spectrometer as noted, and spectra calibrated to
residual solvent signals and TMS. LC-MS was conducted using an
Agilent 1200 system with UV, ELSD, and ESIMS detection (Agilent
6110 quadropole). HPLC columns used were Agilent Zorbax SB-C18
columns [(2.1 × 100 mm) or (9.4 × 100 mm)] and Agilent Zorbax
Eclipse Plus Phenyl Hexyl columns (2.1 × 100 mm). Solvent A:
CH3CN with 0.1% (v/v) TFA; solvent B: H2O with 0.1% (v/v) TFA;
solvent C: CH3CN with 0.05% (v/v) formic acid; solvent D: H2O with
0.05% (v/v) formic acid. HPLC method 1: Agilent Zorbax SB-C18 (9.4 ×
100 mm). One minute hold with A:B (2:98), linear gradient of A:B
(2:98) to A:B (100:0) over 9 min, followed by 5 min hold with A:B
(100:0). HPLC method 2: Agilent Zorbax Eclipse Plus Phenyl Hexyl
column (2.1 × 100 mm). One minute hold with C:D (5:95), gradient
of C:D (5:95) to C:D (100:0) over 8 min, followed by 2 min hold at
C:D (100:0). IR spectra were scanned using a Perkin-Elmer Spectrum
2000 FTIR spectrometer as KBr disks, while UV spectra were obtained
using a BMG Labtech PolarStar Omega microplate reader. The path
length correction was determined by the microplate reader. High-
resolution electrospray mass spectrometry (HRESMS) was performed
(4.50) nm; IR (KBr disk) ν
3061, 2934, 2818, 1684, 1660, 1606,
max
1568, 1518, 1426, 1375, 1219, 760, 692 cm-1; full assignments of 1H
and 13C NMR in Supporting Information, Table S3; HRESMS (+ve)
m/z 269.1070 (calcd for C14H13N4O2 [M + H]+, 269.1033), 559.1811
(calcd for C28H24N8O4Na [2M + Na]+, 559.1813).

1942 Journal of Natural Products, 2010, Vol. 73, No. 11
Notes
Broth Dilution Antibacterial Assays. MIC assays were performed
in triplicates on methicillin-susceptible S. aureus (ATCC 25923), E. faecalis
VanB (ATCC 51299), E. coli (ATCC 25922), K. pneumoniae (ATCC
700603), and Meropenem-resistant P. aeruginosa and A. baumannii
cultured in Luria-Bertani (LB) (Ambresco) and Mueller-Hinton (MH)
broths. Overnight bacterial cultures in LB broth were diluted 40-fold
into fresh LB and MH broths and shaken at 37 °C for 1 h. The resultant
assay, solubility determination, and cytotoxicity assay. This material
is available free of charge via the Internet at http://pubs.acs.org.
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mid-log phase cultures (OD600
≈ 0.6) were diluted to a final
nm
concentration of 5 × 105 cfu/mL and were then added to assay wells
containing 2-fold serial dilutions of the synthetic essramycin (1) from
64 to 0.03 µg/mL in a maximum DMSO concentration of 1.28%. Plates
were incubated at 37 °C for 24 h, and the MIC was recorded as the
lowest concentration of essramycin showing no visible bacterial growth.
Tetracycline, vancomycin, and colistin were used as positive controls.
Disk Diffusion Antibacterial Assays. MIC assays were performed
on methicillin-susceptible S. aureus (ATCC 25923), E. faecalis VanB
(ATCC 51299), E. coli (ATCC 25922), K. pneumoniae (ATCC
700603), and Meropenem-resistant P. aeruginosa and A. baumannii
cultured in LB broth. Bacterial cultures were prepared following the
experimental procedures for the MIC determination of synthetic
essramycin. The mid-log phase bacterial cultures (OD600 nm ≈ 0.6) were
streaked as a bacterial lawn on LB agar plates. Autoclaved Whatman
filter paper discs were impregnated with 10 µL of 2-fold serial dilutions
of essramycin from 32 to 1 µg/mL. The disks were placed onto the
inoculated LB agar plates, and the zone of inhibition was measured
after incubating the plates at 37 °C for 24 h. Tetracycline (64 µg/mL)
was used as a positive control.
Essramycin Sodium Salt and Carboxymethylcellulose Formulation.
Essramycin (1) was formulated with carboxymethylcellulose (CMC)
by adding a 2% (w/v) aqueous CMC stock solution to an essramycin
stock solution (4 mg/mL in DMSO) to give a final concentration of
1.28 mg/mL. The sodium salt of 1 was prepared by titrating the
compound with NaOH (18.64 mM) to a point just after the buffer
region. MIC assays were performed in triplicates on LB broth cultures
of methicillin-susceptible S. aureus (ATCC 25923), E. coli (ATCC
25922), P. aeruginosa (ATCC 10145), and B. subtilis (ATCC 6051)
following the experimental protocols for the MIC determination of
synthetic 1.
Acknowledgment. Financial support for this project was provided
by the National Health and Medical Research Council (NHMRC) grant
AF 511105.
Supporting Information Available: NMR data and spectra of 3,
1, and 1 with DIPEA, comparison of the physicochemical and NMR
data of the naturally occurring and synthetic essramycin, protein-binding
NP100648Q