Non-natural 2: H -azirine-2-carboxylic acids: An expedient synthesis and antimicrobial activity

Article abstract of DOI:10.1039/c9ra09345a

Non-natural 2H-azirine-2-carboxylic acids were obtained in high yields by FeCl₂-catalyzed isomerization of 5-chloroisoxazoles to azirine-2-carbonyl chlorides followed by their hydrolysis. The 3-aryl- and 3-heteroaryl-substituted acids are stable during prolonged storage, exhibit antibacterial activity against ESKAPE pathogens and show a low level of cytotoxicity.

Full text of DOI:10.1039/c9ra09345a

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Non-natural 2H-azirine-2-carboxylic acids: an
expedient synthesis and antimicrobial activity†
Cite this: RSC Adv., 2019, 9, 37901
a
a
a
Pavel A. Sakharov,
Alexander N. Koronatov,
Alexander F. Khlebnikov,
Mikhail S. Novikov,a Artem G. Glukharev,a Elizaveta V. Rogacheva,ab
Liudmila A. Kraeva,b Vladimir V. Sharoyko,a Tatiana B. Tennikova
a
a
*
and Nikolai V. Rostovskii
Received 10th November 2019
Accepted 14th November 2019
Non-natural 2H-azirine-2-carboxylic acids were obtained in high yields by FeCl2-catalyzed isomerization of
5-chloroisoxazoles to azirine-2-carbonyl chlorides followed by their hydrolysis. The 3-aryl- and 3-
heteroaryl-substituted acids are stable during prolonged storage, exhibit antibacterial activity against
ESKAPE pathogens and show a low level of cytotoxicity.
DOI: 10.1039/c9ra09345a
rsc.li/rsc-advances
chain 2H-azirine-2-carboxylic acids.5 Motualevic acid
F
Introduction
showed inhibitory activity against Staphylococcus aureus and
methicillin-resistant Staphylococcus aureus at low acid
loadings.5 According to observations, the presence of
a carboxyl group in the structure of azirine derivative was
important for displaying antimicrobial activity that was
conrmed by the fact that esters of azirinomycin4a and
motualevic acid F ((4E)-(R)-antazirine)5 showed much lower
antimicrobial activity.
This data shows that azirine-2-carboxylic acid derivatives are
perspective candidates in antibiotics development. Although
the compounds with a free carboxyl group showed high anti-
bacterial activity, further studies in this direction slowed down.
This was primarily due to the lack of methods for the synthesis
of azirine-2-carboxylic acids. The hydrolysis of motualevic acid F
methyl ester has long been the only example of the synthesis of
this class of carboxylic acids.6 Very recently another synthetic
acid, 3-phenyl-2H-azirine-2-carboxylic acid,7 was prepared by
the hydrolysis of its methyl ester. The poor availability of some
azirine-2-carboxylic esters and the strongly basic conditions of
the hydrolysis motivate the search for new approaches to
azirine-2-carboxylic acids as potential antibiotics.
Infectious diseases still remain among the ve main causes
of death in the World and cause 13 million deaths per year.1
At the same time, the effectiveness of existing antibacterial
drugs has been increasingly reduced during recent decades
due to the constant growth in the number of microorganisms
that are resistant to the drugs.2 Resistant microorganisms are
becoming clinically widespread and they pose a serious
threat to world health. Therefore, development of new
compounds with antibacterial activity is one of the most
signicant challenges in modern medicinal and pharma-
ceutical chemistry.3
In 1971, azirinomycin (3-methyl-2H-azirine-2-carboxylic
acid), the rst example of an azirine-2-carboxylic acid, was iso-
lated from the soil bacterial strain Streptomyces aureus (Fig. 1).4
It was found that azirinomycin exhibited a wide spectrum of
antibacterial activity in vitro against both Gram-positive and
Gram-negative bacteria.4a In vivo studies in mice have shown
rather high toxicity of azirinomycin of 50% purity isolated from
natural raw materials.4a As far as azirinomycin was very unstable
compound, it was not denitely established whether the
observed toxicity was related to azirinomycin, its decomposition
products or impurities present in the sample.
In this work, a new effective method for the synthesis of
azirine-2-carboxylic acids has been developed involving FeCl2-
Motualevic acid F, isolated from the marine sponge Sili-
quariaspongia (Fig. 1), was the rst reported example of long
aInstitute of Chemistry, Saint Petersburg State University, 7/9 Universitetskaya nab.,
Saint Petersburg, 199034, Russia. E-mail: n.rostovskiy@spbu.ru
bPasteur Institute of Epidemiology and Microbiology, 14 Mira Street, Saint Petersburg,
197101, Russia
† Electronic supplementary information (ESI) available: Experimental procedure
for the preparation of new compounds and the biological studies, spectroscopic
data for all new compounds, TGA and DSC data. CCDC 1956437 (3a). For ESI
and crystallographic data in CIF or other electronic format see DOI:
10.1039/c9ra09345a
Fig. 1 2H-Azirine-2-carboxylic acids found in nature.
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catalyzed isomerization of 5-chloroisoxazoles to azirine-2-
carbonyl chlorides followed by their hydrolysis (Scheme 1).
Results and discussion
Scheme 2 Synthesis of acid 3a.
The catalytic isomerization of isoxazoles, bearing a heteroatom
substituent at the C5 position, to 2H-azirine-2-carboxylic acid
derivatives has been intensively studied and widely used in
recent years.8,9 In particular, it has been demonstrated that 5-
chloroisoxazoles can be transformed to 2H-azirine-2-carbonyl
chlorides under treatment with anhydrous FeCl2 in
acetonitrile.10
Keeping this fact in mind we started searching for a catalyst
suitable for the implementation of the two step reaction sequence
“isoxazole isomerization – azirinecarbonyl chloride hydrolysis” in
one synthetic operation. At rst we tested FeCl2$4H2O in acetoni-
trile using isoxazole 1a as a model compound (Scheme 2, Table 1,
entry 1). However, only traces of azirinecarboxylic acid 3a were
detected even aer addition of water. The use of anhydrous FeCl2
in acetonitrile for isomerization of 1a and addition of water aer
full conversion of the isoxazole into chloride 2a allowed to obtain
azirine-2-carboxylic acid 3a in almost quantitative yield (entry 2).
FeCl2 catalysis of 2a isomerization in other solvents (acetone,
tetrahydrofuran, toluene) turned out to be less effective (entries 3–
5). We also tested several other salts as catalysts for isomerization
of chloroisoxazoles. Among Fe(NTf2)2,9d,i CuI and Rh2(Piv)4 (ref. 9a)
only the latter allowed to synthesize acid 3a in moderate yield
(entry 8).
strong band of azirine C]N bond in IR spectra lies in 1763–
1781 cmꢀ1 range which is characteristic of C3-substituted
azirines.11
rac-Azirinomycin was found to be unstable in pure form
without solvent: it underwent spontaneous explosive decom-
position. On the contrary, other synthesized acids containing an
aryl or heteroaryl substituent at the C3 atom are quite stable and
can be stored at ꢀ20 ꢁC for months without notable decom-
position. It was noticed that the solid acids melted with
vigorous decomposition. We studied this process in detail for
acid 3a using thermogravimetric analysis (TGA) and differential
scanning calorimetry (DSC). According to the TGA data, weight
loss of the sample of acid 3a under heating is 27.1% that
corresponds to the loss of CO2 (27.3%) (ESI†). The DSC analysis
showed that the acid melted rst (a small endothermic region)
and aerwards the exothermic decomposition of the sample
occurred (ESI†).
2H-Azirine-2-carboxylic acids 3a–t are soluble in water and
common organic solvents except aliphatic hydrocarbons. The
measured solubility of acid 3a in water is ca. 1.5 mg mLꢀ1. The
acidity of compound 3a (pKa z 3.2) was also estimated using
the potentiometric method (ESI†).
Acids 3 can be easily converted to the corresponding potas-
sium salts 4 in high yield (95% for compound 3a) using potas-
sium tert-butoxide in methanol. The salt is much more soluble
in water (ca. 35 mg mLꢀ1) and more thermally stable than the
acid. Thus, the preparation of the salts is especially useful as
a solution to the problem of long-term storage of thermally
unstable azirine-2-carboxylic acids.
Next, we examined the scope of acids that could be prepared
by the method using anhydrous FeCl2 (Scheme 3). The most
starting materials, 5-chloroisoxazoles, were obtained from the
corresponding isoxazol-5-ones and POCl3 (see ESI†). Azir-
inecarboxylic acids 3 bearing halogen, alkyl, and methoxy
groups in the aryl ring were synthesized under these conditions
in good to excellent yields (compounds 3a–k). Moreover, azir-
inecarboxylic acids containing heterocyclic substituent (furan,
thiophene, pyrrole) at the C3 atom were also obtained but in
slightly lower yields (compounds 3l–n). This method was
applied to the synthesis of racemic form of natural azir-
inomycin (compound 3o). It was synthesized in high yield under
the standard reaction conditions. Finally, 4-aryl- and 4-alkyl-
substituted isoxazoles gave acids with quaternary azirine C2
atom in good yields (compounds 3p–t). It is important that all
reactions were carried out at room temperature and in most
cases there was no need to perform chromatographic purica-
tion in order to obtain pure products.
The structure of acid 3a was also studied by X-ray diffraction
analysis (Fig. 2). In crystal phase, the acid 3a does not exist in
form of zwitter-ion unlike amino acids: according to the
ꢀ
carbon–oxygen bond distances (1.21 and 1.33 A), there is
Table 1 Optimization of acid 3a synthesisa
Entry
Solvent
Catalyst (20 mol%)
Time, h
Yield of 3ab (%)
1
1
2
3
4
5
6
7
8
MeCN
MeCN
Acetone
THF
PhMe
MeCN
MeCN
PhMe
FeCl2$4H2O
FeCl2
FeCl2
FeCl2
FeCl2
Fe(NTf2)2
CuI
Rh2(Piv)4
100
2
24
Trace
98
60
35
10
The structures of acids were established on the basis of H,
13C NMR and IR spectroscopy as well as the data of HRMS. The
48
100
100
100
2
0
Trace
51c
a
NTf2 ¼ bis(triuoromethylsulfonyl)imide, Piv ¼ trimethylacetate.
b
c
Isolated yields. Isomerization was carried out at 110 ꢁC with
5 mol% of Rh2(Piv)4.
Scheme 1 Retrosynthetic scheme to 2H-azirine-2-carboxylic acids.
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Table 2 Antimicrobial activity of compounds 3a,b,d,e,m against ESKAPE pathogens evaluated by determining the minimum inhibitory
concentration (MIC)a
Acid
E. faecium
S. aureus
K. pneumoniae
A. baumannii
P. aeruginosa
E. aerogenes
3a
3b
3d
3e
56
50
304
152
>864
16
56
50
19
38
864
64
225
200
76
76
864
64
450
600
608
608
216
128
56
600
608
608
54
56
600
304
608
>864
32
3m
Sulfamethoxazole
32
a
MIC values are reported as mM.
developing resistance to drugs)12 was carried out initially by the
disk diffusion method (DDM; see ESI† for details). Subse-
quently, for the lead compounds 3a,b,d,e,m antibacterial
activities were evaluated by determining the minimum inhibi-
tory concentration (MIC) and compared to Sulfamethoxazole
used as a positive control (Table 2). In many cases, MICs of 2H-
azirine-2-carboxylic acids toward ESKAPE pathogens were
similar to that of Sulfamethoxazole. Moreover, compounds
3a,b,d,e inhibited growth of Staphylococcus aureus at concen-
trations even lower than Sulfamethoxazole. Notably, methyl
ester of acid 3a (compound 3a0) displayed activity only against
Klebsiella pneumoniae (DDM; see ESI† for details). This fact
indicates that a free carboxylic group is essential for displaying
antibacterial activity of azirine-2-carboxylic acids.
Considering initially documented toxicity of azirinomycin,4a
the in vitro data on cytotoxicity of synthetic azirine-2-carboxylic
acids 3 is of obvious interest. Compounds 3a,b,d,e,m were
tested at 1–100 mM concentrations for their ability to affect the
cell culture viability of non-cancerous human retinal pigment
epithelial cell line ARPE-19 (ref. 13) and human epithelial
kidney cell line HEK293.14 As followed from the MTT assay data
(Fig. 3; see ESI† for details), the studied compounds displayed
no appreciable cytotoxicity within the concentration range
tested on both the cell lines. The pattern of cell viability in the
presence of tested compounds was practically the similar in
ARPE-19 and HEK293 cell lines (Fig. 3). Overall HEK293 cell line
was less sensitive to cytotoxic effect of the tested compounds
compared to ARPE-19 cell line. The lowest cell viability was
detected for compounds 3b,d at 100 mM concentrations in
ARPE-19 cell line.
Scheme 3 Scope of 2H-azirine-2-carboxylic acids.
a carboxylic group in the structure. However, chains which are
formed by intermolecular hydrogen bonding between azirine
nitrogen atom of one molecule and the hydrogen atom of the
carboxylic group of another azirine molecule are observed in the
crystal structure.
We also obtained primary data on antibacterial activity of the
non-natural 2H-azirine-2-carboxylic acids. A screening of the
antibacterial activity of synthesized acids against of the so-
called ESKAPE pathogens (i.e. the ones most prone to
Fig.
2 X-ray structure of azirine-2-carboxylic acid 3a indicating
hydrogen bond.
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Research Centre, Centre for X-ray Diffraction Studies, Ther-
mogravimetric and Calorimetric Research Centre and Chem-
istry Educational Centre of the Research Park of St. Petersburg
State University.
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Fig. 3 Cell viability MTT assay results for compounds 3a,b,d,e,m (1 mM,
10 mM, 50 mM and 100 mM) against (A) APRE-19 cell line and (B) HEK293
cell line (values are expressed as the mean ꢂ SEM of three experi-
ments): (*) P < 0.05 and (**) P < 0.01 in comparison to control.
Conclusions
In conclusion, a high-yield synthesis of 3-mono- and 2,3-
disubstituted 2H-azirine-2-carboxylic acids from readily acces-
sible 5-chloroisoxazoles has been developed. The method is
based on FeCl2-catalyzed isomerization of 5-chloroisoxazoles
followed by hydrolysis of formed azirine-2-carbonyl chlorides. 3-
Aryl-2H-azirine-2-carboxylic acids exist as OH form in crystal,
are stable during prolonged storage at ꢀ20 ꢁC, but undergo
decarboxylation aer melting. These compounds exhibit anti-
bacterial activity against ESKAPE pathogens comparable to that
of Sulfamethoxazole and show low-level cytotoxicity.
Conflicts of interest
There are no conicts to declare.
Acknowledgements
We gratefully acknowledge the nancial support of the Scien-
tic Council of the President of the Russian Federation (MK- 10 (a) K. I. Mikhailov, E. E. Galenko, A. V. Galenko,
2698.2019.3). This research used resources of the Magnetic
Resonance Research Centre, Chemical Analysis and Materials
M. S. Novikov, A. Yu. Ivanov, G. L. Starova and
A. F. Khlebnikov, J. Org. Chem., 2018, 83, 3177–3187; (b)
37904 | RSC Adv., 2019, 9, 37901–37905
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