Antibacterial action of (5-nitrofurfuryl)-derived aminophosphonates and their parent imines

Article abstract of DOI:10.1007/s11696-018-0597-1

Abstract: Ten aminophosphonates bearing 2-nitrofuran moiety and two N-aryl 5-nitro-furfuralaldimines, namely, N-(2-nitrofurfurylidene)-p-toluidine and N-(2-nitrofurfurylidene)-p-anisidine, were tested in aspect of their antibacterial action. O,O′-diphenyl

Full text of DOI:10.1007/s11696-018-0597-1

Chemical Papers
https://doi.org/10.1007/s11696-018-0597-1
ORIGINAL PAPER
Antibacterial action of (5‑nitrofurfuryl)‑derived aminophosphonates
and their parent imines
Jarosław Lewkowski1 · Marta Morawska · Aleksandra Kowalczyk
1
2
Received: 4 June 2018 / Accepted: 14 September 2018
©
The Author(s) 2018
Abstract
Ten aminophosphonates bearing 2-nitrofuran moiety and two N-aryl 5-nitro-furfuralaldimines, namely,
N-(2-nitrofurfurylidene)-p-toluidine and N-(2-nitrofurfurylidene)-p-anisidine, were tested in aspect of their antibacterial
action. O,O′-diphenyl derivatives were found inactive, while O,O′-dimethyl and O,O′-diethyl derivatives were revealed to act
moderately eꢀciently against clinical isolates of S. aureus, especially against methicillin-resistant (MRSA) strains. A high
activity against these strains was found for N-(4-methylphenyl)-5-nitrofurfuralaldimine and N-(4-methoxyphenyl)-5-nitro-
furfuralaldimine, but they showed the cytotoxicity at a dangerous level.
Graphical abstract
Keywords 5-Nitrofurfural-derived aminophosphonates · S. aureus · Antibacterial activity · MRSA strains · Cytotoxicity
Introduction
Electronic supplementary material The online version of this
Azomethine derivatives of 5-nitrofurfural are known for
article (https://doi.org/10.1007/s11696-018-0597-1) contains
their strong bactericidal action, and therefore, some of
supplementary material, which is available to authorized users.
them constitute active components of various drugs. Despite
Extended author information available on the last page of the article
some controversies around their safety (Goemaere et al.
Vol.:(0
1
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2
008; Huttner et al. 2015; Williams and Triller 2006; Tan
infections and it is a leading cause of death in hospitalized
patients (Hiramatsu et al. 2014). S. aureus causes disease
through tissue damage or through the action of toxins. The
multiplication of bacteria in the human body leads to the
formation of abscesses as well as the destruction of tissues.
More serious infections include, pneumonia, bacteremia,
bone and joint infections, toxic shock syndrome, and endo-
carditis. Moreover, staphylococci produce enterotoxin which
is not destroyed even at high temperature and its action is
as dangerous as the bacterial infection itself (Peacock and
Paterson 2015).
et al. 2012; Stricker et al. 1988), they are still authorized
to use in many countries, the most probably due to their
signiꢁcant usefulness. Nitrofurantoin (Garau 2008), nifu-
ratel (Grüneberg and Leakey 1976) as well as furazidine
are often applied in cases of various urinary tract infec-
tions, while nifuroxazide (Carron 1966; Cagliero 1978) and
furazolidine (Machado et al. 2008) for intestinal infections.
Although they are suspected to cause various toxicities, i.e.,
pulmonary toxicity (including pulmonary ꢁbrosis caused by
nitrofurantoin) (Goemaere et al. 2008; Huttner et al. 2015;
Williams and Triller 2006), hepatotoxicity (Tan et al. 2012)
and neurotoxicity (Stricker et al. 1988), they are still con-
sidered as eꢀcient antibacterial and antiprotozoal agents
Until recently, glycopeptides such as vancomycin have
been eꢂective in the treatment of MRSA. However, the
occurrence of VISA (vancomycin–intermediate S. aureus),
and VRSA (vancomycin-resistant S. aureus) strains (Hira-
matsu et al. 2014) caused possible variants of antibiotic
therapy in hospitals to be very limited. One of them is lin-
ezolid belonging to the family of oxazolidinones (Meka and
Gold 2004). Linezolid proved to be a suitable alternative to
vancomycin in the treatment of MRSA infection and reduced
the pressure on the use of vancomycin (Stevens et al. 2002).
Apart from it, oxadiazoles were recognized as a new class
of antibiotics, which inactivate PBP2a and inhibit cell-wall
biosynthesis of MRSA (O’Daniel et al. 2014). Moreover,
some newer cephalosporins, in particular, ceftaroline and
ceftobiprole are active against MRSA (Holmes and Howden
2014).
(
Jackson et al. 2010; Priotto et al. 2009) and are not with-
drawn from oꢀcial lists of pharmaceutical products (not in
all countries). It is not unreasonable action, because due to
the unique mode of action, nitrofurans together with nitro-
imidazole derivatives constitute an important and precious
group of antiprotozoal and antibacterial agents. Their most
important potential is related to their antiprotozoal action
against Trypanosoma brucei gambiense and Trypanosoma
cruzii causing the sleeping sickness and Chagas’ disease;
therefore, studies on action of nitrofuran derivatives on the
above parasites are performed continuously (Jackson et al.
2
2
010; Priotto et al. 2009; Stewart et al. 2004; Zhou et al.
013).
Their mechanism of action is complex; nitroreductases
Interesting scope of action of nitrofuran derivatives
prompted us to perform tests with 5-nitrofurfural-derived
aminophosphonates 2b–c, 2f, 3b–c, 3f, 4a–c, 4f and some
of their parent Schiꢂ bases (1c and 1f) in aspect of their
action against S. aureus clinical strains including MRSA.
In the course of the ecotoxicological evaluation of 5-nitro-
furan-derived aminophosphonates, their harmful action on
Aliivibrio fscheri bacteria was observed and it was a driving
force for performing studies described herein. The choice of
nitrofuran derivatives was driven also by results obtained
for nitrofuran- and nitrothiophene-derived benzoic acid
hydrazones (Osório et al. 2012), which showed antibacte-
rial potential of these compounds.
(
particularly nitrofuran reductase) catalyze reduction of
their nitro groups inside the bacterial cell to multiple reac-
tive intermediates, which react with ribosomal proteins,
DNA, and other macromolecules within the cell, and aꢂect
pyruvate metabolism (Tu and McCalla 1975). The complex
mechanism of action is the most probably responsible for the
low development of resistance to their eꢂects, as drugs aꢂect
many diꢂerent processes important to the bacterial cell.
Microbial resistance to antibiotics comprises a signiꢁcant
problem in the treatment of bacterial infections. Although
antibiotics are an eꢂective way to control such infections,
their excessive use can lead to faster spread of resistant
strains in the population. Methicillin-resistant Staphylococ-
cus aureus (MRSA) is the most common and widespread
antibiotic-resistant species. Until the mid-1990s, infections
caused by MRSA were limited to hospitals (healthcare-asso-
ciated MRSA, HA-MRSA); however, over the past 20 years,
MRSA has been reported for healthy individuals who were
not subject to prior hospitalization and were not in contact
with the medical staꢂ. These community-associated MRSA
Experimental
General
All solvents (POCh, Gliwice, Poland) were routinely dis-
tilled and dried prior to use. Amines, diphenyl phosphite,
dimethyl phosphite, diethyl phosphite, and 5-nitrofurfural
(Aldrich, Poznań, Poland) were used as received. Melting
points were measured on a MelTemp II apparatus, and NMR
spectra were recorded on a Bruker Avance III 600 MHz
(
CA-MRSA) strains have methicillin resistance as well as
enhanced virulence and ꢁtness (Otto 2013; Peacock and
Paterson 2015).
Staphylococcus aureus colonizes about a third of the
population and may cause moderately severe-to-severe
1
13
operating at 600 MHz ( H NMR), 150 MHz ( C NMR),
1
3

Chemical Papers
3
1
3
4
and 243 MHz ( P NMR) or on a Varian Gemini 2000 BB
6.63 (d, J =7.5 Hz, H , 1H); 6.60 (t, J =3.5 Hz, m-
HH fur HH
3
1
3
operating at 81 MHz ( P NMR). IR spectra were recorded
on Nexus FT-IR (Thermo Nicolet). Elemental analyses were
carried out at the Laboratory of Microanalysis, Faculty of
Chemistry, University of Łódź, Poland.
C H , 1H); 6.50–6.49 (m, m-C H , 1H); 6.46 (dd, J =8.1
6
4
6
4
HH
4
2
and J = 2.4 Hz, m-C H , 1H); 4.97 (d, J = 25.0 Hz,
HH
6
4
PH
3
CHP, 1H); 3.88 (d, J = 10.8 Hz, POCH , 1H); 3.77 (d,
PH
3
3
13
J
=10.8 Hz, POCH , 1H); 2.26 (s, CH , 1H). C NMR
PH
3 3
Imines were synthesized following the previously pub-
lished procedure (Csaszar 1984; Matusiak et al. 2013; Sai-
kachi and Shimamura 1960).
(150 MHz, CDCl ): δ 153.8 (C ); 152.0 (C ); 145.1
3 ar fur
2
(d, J = 12.6 Hz, C ); 139.4 (C ); 129.4 (C ); 120.8
PC
fur
ar
ar
4
(C ); 114.8 (C ); 112.6 (d, J = 3.1 Hz, C ); 111.9 (d,
ar
ar
PC
fur
3
3
J
= 6.6 Hz, C ); 110.9 (C ); 54.6 (d, J = 6.7 Hz,
PC
fur ar PC
3
1
Synthesis of Schiꢀ bases 1a–c and 1f
POC); 54.0 (d, J =7.2 Hz, POC); 50.3 (d, J =157.3 Hz,
PC
PC
31
PC); 21.5 (C -C). P NMR (CDCl , 600 MHz): δ 20.25. IR
ar
3
Furfural derivative (0.24 g, 2.5 mmol) was dissolved in
methanol (15 mL), and to this solution, amine (2.5 mmol)
was added. The mixture was stirred at room temperature for
(KBr): 3298 (νNH); 1533, 1388 (νArNO ); 3108 (νCH );
2 arom
); 1237 (νP-O); 872
1609, 1592, 1585, 1490 (νCC
arom
(CH ). Elem. anal. Calcd. for C H N O P: C, 49.42; H,
arom
14 17
2
6
2
4 h, and then, solvent was evaporated and residue dried on
5.04; N, 8.23. Found: C, 49.54; H, 5.12; N, 8.21.
vacuum to obtain pure Schiꢂ base 1c and 1f. N-methylphe-
nyl derivative 1c was compared to a previously obtained
sample (Matusiak et al. 2013) and was found to be identi-
cal. Its data are given in our previous paper (Matusiak et al.
rac‑Diethyl N‑(3‑methylphenyl)amino(5‑nitro‑2‑furyl)
methylphosphonate 3b
2
013). Imines 1a and 1b, after isolation as above, and con-
Y = 1.4 g (46%). mp = 111–112 °C (yellow crystals).
1
1
4
3
ꢁ
rming their identity (Saikachi and Shimamura 1960) ( H
H NMR (CDCl , 600 MHz): δ 7.24(dd, J = 3.7 and
3
HH
3
NMR spectra) were used for further conversion without any
J
=0.8 Hz, H , 1H); 7.06 (t, J =7.8 Hz, m-C H , 1H);
HH fur HH 6 4
1
3
4
puriꢁcation. Routine H NMR spectra of crude imines 1a–b
6.62 (d, J =7.5 Hz, H , 1H); 6.59 (t, J =3.4 Hz, m-
HH fur HH
3
are in Supplementary Material, Figs. S6a and S6b.
N‑(2‑Nitrofurfurylidene)‑p‑anisidine 1f
C H , 1H); 6.49–6.48 (m, m-C H , 1H); 6.44 (dd, J =8.0
6
4
6
4
HH
4
2
and J = 2.4 Hz, m-C H , 1H); 4.93 (d, J = 25.0 Hz,
HH
6
4
PH
CHP, 1H); 4.27–4.22 (m, POCH CH , 2H); 4.18–4.14
2
3
(
m, POCH CH , 1H); 4.10–4.05 (m, POCH CH , 1H);
2 3 2 3
3
Quantitative yield (0.61 g), mp=124–125 °C, lit (Csaszar
2.26 (s, CH , 3H); 1.34 (t, J = 7.1 Hz, POCH CH ,
3 PH 2 3
3
13
1
984) 124–126 °C
3H); 1.28 (t, J = 7.1 Hz, POCH CH , 3H). C NMR
PH 2 3
1
4
H NMR (CDCl , 600 MHz): δ 8.42 (s, CH=N, 1H); 7.41
(150 MHz, CDCl ): δ 154.4 (d, J = 1.7 Hz, C ); 151.9
3 PC ar
3
3
5
fur
2
(
dd, J =3.8 and J =0.6 Hz, H , 1H); 7.32 (A part of
(C ); 145.3 (d, J = 12.6 Hz, C ); 139.5 (C ); 129.4
fur PC fur ar
HH
HH
4
3
3
4
4
AA’XX’ system, J =10.1, J =10.1, J =2.8 Hz, p-
(C ); 120.7 (C ); 114.9 (C ); 112.6 (d, J = 3.2 Hz,
HH
HH
HH
ar
ar
ar
PC
3
4
fur
3
C H , 2H); 7.16 (dd, J =3.8 and J =0.5 Hz, H , 1H);
C
); 111.8 (d, J = 6.4 Hz, C ); 111.0 (C ); 64.3 (d,
6
4
HH
HH
3
fur
PC
fur
ar
3
3
3
3
6
.96 (X part of AA’XX’ system, J = 10.1, J = 10.1,
J
=7.0 Hz, POC); 63.8 (d, J =7.2 Hz, POC); 50.7 (d,
HH
HH
PC
PC
4
1
3
J
=2.8 Hz, p-C H , 2H); 3.85 (s, OCH , 3H).
J
= 156.0 Hz, PC); 21.6 (C -C); 16.4 (d, J = 5.6 Hz,
HH
6
4
3
PC
ar
PC
3
31
POCC); 16.3 (d, J =5.9 Hz, POCC). P NMR (CDCl ,
PC
3
Preparation of aminophosphonates 2a–f, 3a–f, and 4a–f
600 MHz): δ 17.64. IR (KBr): 3316 (νNH); 3110, 3050,
2
982, 2910 (νCH ); 1530, 1353 (νArNO ); 1607,1589,
arom
2
To a mixture of imine (5 mmol) and phosphite (5 mmol)
in THF, BF •OEt (1 mmol) was added via syringe. The
1492 (νCC ), 1237 (νP-O); 768 (δCH ). Elem. anal.
arom arom
Calcd. for C H N O P: C, 52.17; H, 5.75; N, 7.61. Found:
3
2
16 21
2
6
mixture was stirred at room temperature over 24 h under a
nitrogen atmosphere. A solution was puriꢁed by extraction
and crystallization. Detailed description is to be found in
our recent paper (Lewkowski et al. 2017) as well as detailed
data concerning aminophosphonates 2c, 2f, 3c, 3f, 4c, and
C, 52.12; H, 5.91; N, 7.65.
rac‑Diphenyl N‑(2‑methylphenyl)amino(5‑nitro‑2‑furyl)
methylphosphonate 4a
1
4
f (Lewkowski et al. 2017).
Y = 2.1 g (70%). mp = 131–133 °C (yellow crystals). H
NMR (CDCl , 600 MHz): δ 7.33–7.29 (m, C H , 4H);
3
6
5
rac‑Dimethyl N‑(3‑methylphenyl)amino(5‑nitro‑2‑furyl)
methylphosphonate 2b
7.22-7.15 (m, H , H , 7H); 7.12–7.07 (m, o-C H , 2H);
fur Ph 6 5
3
6.80–6.77 (m, o-C H , 1H); 6.65 (t, J =3.6 Hz, H , 1H);
6
5
HH
fur
3
2
6
.56 (d, J =7.9 Hz, o-C H , 1H); 5.33 (d, J =25.3 Hz,
HH 6 5 PH
1
13
Y = 1.7 g (56%). mp = 124–126 °C (yellow crystals). H
CHP, 1H); 4.54 (s, NH, 1H); 2.19 (s, CH , 3H). C NMR
3
3
NMR (CDCl , 600 MHz): δ 7.24 (dd, J = 3.8 and
(150 MHz, CDCl ): δ 152.7 (C ); 152.1 (C ); 150.1 (d,
3
HH
3
ar
fur
4
3
2
2
J
=0.9 Hz, H , 1H), 7.07 (t, J =7.8 Hz, m-C H , 1H);
J
=9.4 Hz, C ); 150.0 (d, J =9.2 Hz, C ); 142.8 (d,
HH
fur
HH
6
4
CP ar CP ar
1
3

Chemical Papers
2
4
J
ar
= 12.6 Hz, C ); 130.8 (C ); 129.9 (d, J = 6.6 Hz,
of glycerol until testing. Before using, S. aureus strains
were subcultured on blood agar and identiꢁed by routine
methods (catalase, coagulase and clumping factor). Minimal
inhibitory concentration (MIC) was determined as the low-
est concentration of the compound preventing growth of the
tested microorganism using microdilution method according
to EUCAST guidelines [ISO 20776-1 (2006)]. The 96-well
microplates were used; 50 µl of recommended Mueller–Hin-
ton broth with a series of twofold dilutions of the tested
compound in the range of the ꢁnal concentrations from 2 to
CP
fur
ar
CP
C ); 127.2 (C ); 125.9 (C ); 125.7 (C ); 123.9 (C ); 120.4
ar
ar
ar
ar
3
3
(
(
d, J =4.1 Hz, C ); 120.0 (d, J =4.7 Hz, C ); 119.9
CP ar CP ar
3
4
C ); 112.5 (d, J =6.7 Hz, C ); 112.43 (d, J =3.3 Hz,
ar
CP
fur
CP
1
C ); 111.3 (C ); 50.7 (d, J =159.0 Hz, PC); 17.3 (C -C).
fur
ar
CP
ar
3
1
P NMR (CDCl , 600 MHz): δ 10.09. IR (KBr): 3387
3
(
νNH); 3129 (νCH ); 1530, 1355 (νArNO ); 1588, 1489,
arom
2
1
452 (νCC
); 1199 (νP-O); 953 (CH
). Elem. anal.
arom
arom
Calcd. for C H N O P: C, 62.07; H, 4.56; N, 6.03. Found:
2
4
21
2
6
C, 62.10; H, 4.70; N, 6.03.
1
28 µg/mL was inoculated with 50 µl of microbial suspen-
rac‑Diphenyl N‑(3‑methylphenyl)amino(5‑nitro‑2‑furyl)
methylphosphonate 4b
sion with a ꢁnal cell number concentration approximately 5
5
× 10 CFU/mL. All of the tested compounds were dissolved
in dimethyl sulfoxide (DMSO) and its ꢁnal concentration
on plate (1%) had no inꢃuence on growth of microorgan-
isms. The incubation was carried out at 37 °C for 18 h and
Y = 1.9 g (66%). mp = 131–133 °C (pale yellow powder).
1
H NMR (CDCl , 600 MHz): δ 7.33–7.28 (m, C H , 4H);
3
6
5
3
7
.22–7.13 (m, H , H , 7H); 7.09 (t, J = 7.9 Hz, m-
optical density (OD ) was measured. Ampicillin, oxacil-
fur
Ph
HH
600
C H , 1H); 6.67–6.65 (m, o-C H , 2H); 6.51 (m, H , 1H);
lin, nitrofurantoin, and streptomycin were used as control
6
5
6
5
fur
3
4
6
.48 (dd, J = 2.5 Hz, J = 8.0 Hz, o-C H , 1H); 5.29
antimicrobials. All evaluations were performed in triplicates.
HH HH 6 5
2
13
(
d, J =25.5 Hz, CHP, 1H); 2.27 (s, CH , 3H). C NMR
PH 3
(
150 MHz, CDCl ): δ 152.8 (C ); 152.1 (C ); 150.1 (d,
Cell viability assay
3
arr
fur
2
2
J
J
=9.8 Hz, C ); 150.0 (d, J =9.4 Hz, C ); 144.8 (d,
CP
ar CP ar
2
4
= 13,2 Hz, C ); 139.5 (C ); 129.9 (d, J = 7.6 Hz,
The eꢂect of tested compounds on host cells was detected
by determining cellular viability using MTT reduction
CP
fur
ar
CP
C ); 129.4 (C ); 125.9 (C ); 125.7 (C ); 121.1 (C ); 120.6
ar
ar
ar
ar
ar
3
3
®
(
d, J =4.3 Hz, C ); 120.0 (d, J =4.7 Hz, C ); 115.1
assay. Murine fibroblasts L929 cells (ATTC —CCL-1,
CP
ar
CP
ar
3
4
®
(
C ); 112.7 (d, J =6.8 Hz, C ); 112.5 (d, J =3,2 Hz,
mouse ꢁbroblasts) or human tumor HeLa cells (ATTC —
ar
CP
fur
CP
1
C
); 111.1 (C ); 50.53 (d, J = 160.4 Hz, PC); 21.5
CCL-2™, human epithelial cells) were plated in 96-well
fur
ar
CP
3
1
4
(
C -C). P NMR (CDCl , 600 MHz): δ 10.10. IR (KBr):
microplates at density of 1×10 cells/100 µL according to
ar
3
3
1
321 (νNH); 3141 (νCH ); 1531, 1358 (νArNO ); 1605,
international standards: ISO 10993-5:2009(E) (American
National Standard 2009) and cultivated in Iscove’s modiꢁed
Dulbecco’s medium (IMDM), supplemented with 10% fetal
bovine serum (FBS). 100 U/mL of penicillin and 100 μg/mL
of streptomycin were added to the media. The cell cultures
were incubated at 37 °C in a humidiꢁed atmosphere with
arom
2
588, 1489 (νCC ); 1176 (νP-O); 939 (CH ). Elem.
arom
arom
anal. Calcd. for C H N O P: C, 62.07; H, 4.56; N, 6.03.
2
4
21
2
6
Found: C, 62.19; H, 4.70; N, 6.08.
Antibacterial assay
1
0% CO . After overnight incubation, the growth medium
2
The in vitro antimicrobial activity of tested compounds was
evaluated against the reference strains of Gram-negative
was removed and 100 μL of medium supplemented with
twofold dilutions of tested compounds in the range of con-
centration 2–128 µg/mL were added. All of the tested com-
pounds were dissolved in dimethyl sulfoxide (DMSO), in
which the ꢁnal concentration on plate (1%) had no inꢃuence
on cells viability. Cisplatin was used as known, toxic, control
antitumor agent, and nitrofurantoin as control antibacterial
drug. After 24 h incubation, the medium was removed and
MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) was added to each well at a ꢁnal concentration
of 0.5 mg/mL and plates were incubated for the next 2 h
(
Escherichia coli NCTC 8196, Proteus vulgaris ATCC
4
9990, Proteus mirabilis ATCC 29906, Pseudomonas aer-
uginosa NCTC 6749), and Gram-positive (Staphylococcus
aureus ATCC 6538, Staphylococcus aureus ATCC 29213,
and Staphylococcus epidermidis ATCC 12228) bacterial
species. Due to satisfying results for S. aureus, selected com-
pounds were also examined against a set of twelve clinical
isolates of S. aureus (including two MRSA) from the col-
lection of the Chair of Immunology and Infectious Biology,
University of Łódź. These strains were isolated from the
following three sources: naso-pharynx of young patients
hospitalized at Children’s Hospital in Łódź (n =4), ulcers
and furuncles from adult patients of Dermatological Clinic
in Łódź (n=4), and from infected bones of patients hospi-
talized at Oncological Hospital in Łódź (n=4). All strains
were kept frozen at −80 °C on Tryptic Soy Broth with 15%
at 37 °C, 10% CO . Then, formazan crystals were solubi-
2
lized in 150 μL DMSO. The optical density was measured at
550 nm. The results of the experiments were shown as mean
arithmetic values from 3 repeats in each of two independ-
ent experiments and the percentage of viability inhibition in
comparison with untreated control was calculated for each
concentration of the tested compounds and IC values were
5
0
1
3

Chemical Papers
determined with the Prism GraphPad 7 software using non-
linear regression.
2017) and their identities were conꢁrmed by comparison to
authentic samples.
Spectra of new aminophosphonates 2b, 3b, 4a, and 4b
1
13
31
1
(
FT-IR, H, C, and P NMR) as well as H NMR spectrum
of a Schiꢂ base 1f are collected in Supplemental Materials.
Results and discussion
Evaluation of antibacterial action of studied
compounds
Synthesis of imines 1 and aminophosphonates 2, 3,
and 4
Ten nitrofuran-derived racemic aminophosphonates (2b,
2c, 2f, 3b, 3c, 3f, 4a, 4b, 4c, 4f) and some of their parent
imines 1c and 1f (Fig. 1) were examined for its antimicro-
bial activity against a representative panel of bacteria, i.e.,
Escherichia coli, Staphylococcus aureus, Staphylococcus
epidermidis, Proteus vulgaris, Proteus mirabilis, and Pseu-
domonas aeruginosa using ampicillin, nitrofurantoin, and
streptomycin as control antibacterial drugs. The in vitro
antimicrobial activity of the tested compounds at concen-
trations ranging from 0.5 to 128 μg/mL was screened using
the microdilution method. The results showed that diphenyl
aminophosphonic acid esters 4a–c, 4f were inactive against
all tested strains and, therefore, were excluded from further
studies. The remaining eight derivatives were only active
against S. aureus and S. epidermidis. Aminophosphonates
2b, 2c, 2f, 3b, 3c, and 3f showed weak antimicrobial activity
against both strains (MIC = 128 μg/mL). The most eꢂec-
tive against these strains were Schiꢂ bases 1c and 1f with
MIC values of 16 µg/mL. The strong antibacterial activity
of 1c and 1f against S. aureus was equal to nitrofurantoin;
however, their activity was lower than exhibited by ampi-
cillin and streptomycin—antibiotics commonly used in the
therapy of staphylococcal infections. In addition, 1c and 1f
showed good activity against E. coli (MIC=64 µg/mL). The
in vitro results of antibacterial activity of these compounds
are presented in Table 1 as a minimal inhibitory concentra-
tion (MIC).
This work is to some extend a continuation of our previ-
ous study, where we reported the synthesis of new ami-
nophosphonates bearing a 5-nitrofuran moiety. This group
of compounds has never been described before our recent
paper (Lewkowski et al. 2017) (except for N-benzylamino-(5
nitrofurfuryl)methylphosphonic acid, which was reported by
Boduszek 2001; Boduszek et al. 2001).
Schiꢂ bases 1a–c and 1f were synthesized following
the previously published procedure and their identity was
1
conꢁrmed by melting point measurement and the H NMR
spectroscopy and comparing the obtained data with litera-
ture reports (Csaszar 1984; Matusiak et al. 2013; Saikachi
and Shimamura 1960). Imines 1c and 1f were isolated and
puriꢁed before undergoing the biological evaluation, while
1
a and 1b were used for further conversion without any
puriꢁcation. Antibacterial action of imines 1a–b was not
evaluated.
N-arylamino(5-nitrofurfuryl)methylphosphonic acid
esters (2b–c, 2f, 3b–c, 3f, 4a–c, 4f) were prepared based on
the recently published procedure (Lewkowski et al. 2017)
by the aza-Pudovik reaction as it was described (Lewkowski
et al. 2017). (Scheme 1) The aminophosphonates 2b, 3b, 4a,
and 4b were synthesized for the ꢁrst time and have never
been described in the literature. Their identities were con-
1
13
31
ꢁ
rmed by elemental analysis as well as by H, C, and P
NMR spectroscopy. NMR spectra demonstrated diagnostic
peaks, which were discussed previously (Lewkowski et al.
Such properties are closely related to structures of
investigated compounds. Diphenyl N-arylamino(5-nitro-
2-furyl)methylphosphonates 4a–c, 4f having a phosphonic
group substituted with phenyl rings is undoubtedly more
2
017).
Aminophosphonates 2c, 2f, 3c, 3f, 4c, and 4f were
obtained following the reported procedure (Lewkowski et al.
Scheme 1 Synthesis of studied
aminophosphonates 2b–c, 2f,
O
P
OR2
OR
HP(O)(OR2)2
Et O BF
2
3
b–c, 3f, 4a–c, 4f
O N
O N
2
2
O
O
2
3
R1
N
R1 THF, rt
HN
1a-c and 1f
2b-c, 2f, 3b-c, 3f, 4a-c and 4f
1
1
1
1
a: R = 2-CH ; b: R = 3-CH ; c: R = 4-CH ; d: R = 2-OCH ;
3
3
3
3
1
1
e: R = 3-OCH ; f: R = 4-OCH ;
3
3
2
2
2
2
: R = CH ; 3: R = CH CH ; 4: R = Ph
3 2 3
1
3

Chemical Papers
O
P
O
P
OCH3
OCH3
O
P
OCH3
OCH3
OCH3
OCH3
O N
2
O N
O
2
O N
CH3
O
2
O
HN
HN
HN
OCH3
2b
2c
CH3
2f
O
P
O
P
OCH CH
OCH
OCH
2
CH
CH
3
O
P
2
3
OCH CH3
2
OCH CH
2
3
2
3
OCH CH3
O N
CH
O
2
N
O N
2
2
2
O
3
O
O
HN
HN
HN
CH3
OCH3
3
b
3c
3f
O
P
OPh
OPh
O
P
O
P
OPh
OPh
OPh
OPh
O N
2
O
O N
2
CH3
O N
2
O
O
HN
HN
HN
4a
4
b
4c
CH3
H C
3
O
P
OPh
OPh
O N
O N
2
2
O
O
O N
2
O
HN
N
N
1
c
OCH3
CH3
1f
4f
OCH3
Fig. 1 Structures of aminophosphonates and imines under study
Table 1 In vitro antibacterial activity of 2b–c, 2f, 3b–c, 3f, 1c, and 1f expressed as a minimal inhibitory concentration (MIC) [µg/mL]
Compound
MIC [µg/mL]
E. coli NCTC S. aureus
S. aureus ATCC P. vulgaris
P. mirabilis
ATCC 29906
S. epidermidis
ATCC 12228
P. aerugi-
8
196
ATCC 6538
29213
ATCC 49990
nosa NCTC
6
749
3
3
3
2
2
2
1
1
b
c
f
b
c
f
na
na
na
na
na
na
64
64
4
128
128
128
128
128
128
16
16
1
16
1
128
128
128
128
128
128
16
16
2
16
1
na
na
na
na
na
na
na
na
nd
nd
nd
na
na
na
na
na
na
na
na
nd
nd
nd
128
128
128
128
128
128
16
16
1
8
na
na
na
na
na
na
na
na
na
nd
nd
nd
c
f
AMP
NFT
STR
8
1
AMP ampicillin, NFT nitrofurantoin, STR streptomycin, na no activity, nd not determined
hydrophobic than all other aminophosphonates 2 and 3,
which may decrease the ability of their molecules to pen-
etrate bacterial cells. Apart from this, the presence of two
large, electron-rich phenyl rings does not improve it either.
Comparison of antibacterial action of aminophospho-
nates 2b–c, 2f, 3b–c, and 3f with Schiꢂ bases 1c and 1f,
shows that although an active center (NO group) is in
2
each tested compound, the presence of an azomethine bond
1
3

Chemical Papers
is a necessary condition for a molecule to penetrate eꢀ-
ciently bacterial cells. It is not surprising, since all approved
nitrofuran-derived drugs bear azomethine groups in their
molecules.
important role in toxicity. These results coincide well with
our recently reported observations (Lewkowski et al. 2017),
where we demonstrated that N-methoxyphenyl aminophos-
phonates are less harmful for Aliivibrio fscheri bacteria than
N-methylphenyl derivatives.
Considering good antibacterial activity of aminophos-
phonates 2b–c, 2f, 3b–c, and 3f, and especially 1c and 1f,
against Staphylococcus spp., a set of 12 S. aureus clinical
strains were used for further antimicrobial assays. These
strains were isolated from patients from two typical sources
such as naso-pharynx (carrier state) and ulcers/furuncles
In addition to study antibacterial activity of tested com-
pounds, we also evaluated their safety for mammalian cell
lines. The cytotoxic activity was assessed using L929 murine
cell line (recommended by the International Standard ISO
10993:2009 for evaluation of in vitro cytotoxicity) as well
as HeLa human tumor cell line. The percentage of cells
viability inhibition compared to the untreated control was
estimated for concentrations of compounds ranging from 2
to 128 µg/mL and IC values were determined. Cisplatin,
(
skin and soft tissue infections), as well as those from
infected bones (invasive infections) with two representatives
of MRSA (S. aureus D15 and D17). Similar to the reference
S. aureus strains, all clinical isolates displayed high level of
susceptibility to Schiꢂ bases 1c and 1f (MIC ranging from
5
0
as known cytotoxic, antitumor agent, and nitrofurantoin as
control antibiotic was used for comparison. Our results dem-
onstrated that Schiꢂ bases 1c and 1f showing the highest
antibacterial activity were the most toxic as well with IC
1
6 to 64 µg/mL, Table 2). In most cases, except F7 strain,
they had better activity than ampicillin. Especially, these
two imines showed higher antibacterial activity against
both MRSA strains than all control antibiotics, with MIC
being equal to 16 µg/mL. Other derivatives also displayed
good activity especially against MRSA strains, better than
oxacillin, ampicillin, and streptomycin (MIC=64–128 µg/
mL); however, some clinical isolates were not sensitive to
all tested compounds in analyzed concentrations. It is to
note that dimethyl aminophosphonates 2b–c, 2f were gen-
erally more eꢀcient against both MRSA strains than diethyl
derivatives 3b–c and 3f (MIC =64 µg/mL vs. 128 µg/mL,
respectively).
5
0
approximately 12 µg/mL, which was only twofold higher
than IC for cisplatin. N-methoxyphenylamino(5-nitrofur-
5
0
furyl)methylphosphonates 2f and 3f were found to have the
lowest cytotoxicity, but inhibitory concentrations for all ana-
lyzed compounds were lower than MIC values. The HeLa
cells seemed to be a little less sensitive to tested compounds
than L929 cells. The in vitro results of viability assay are
presented in Table 3 and Fig. 2.
This results coincide with some extent with our observa-
tion described in our recent work (Lewkowski et al. 2017),
where we found that N-methoxyphenylamino(5-nitrofur-
furyl)methylphosphonates were signiꢁcantly less toxic for
Aliivibrio fscheri bacteria and for Heterocypris incongruens
crustaceans than N-methylphenylamino derivatives. It is to
state, however, that methoxyphenyl-substituted nitrofurfuryl
N-(2-nitrofurfurylidene)-p-anisidine 1f turned out to be
less toxic than N-(2-nitrofurfurylidene)-p-toluidine 1c for
seven of S. aureus strains. It suggests that the presence of
a methyl group increases a harmful action of the imine 1c
in comparison with a methoxy group in 1f and plays an
Table 2 In vitro antibacterial
S. aureus
MIC [µg/mL]
activity of 2b–c, 2f, 3b–c,
3
f, 1c, 1f against clinical
naso-pharynx isolates
ulcers/furuncles isolates
bone isolates
isolates of S. aureus expressed
as the minimal inhibitory
Compound C4
C7
C8
C19
F1
F7
F12
D12 D14 D15 D17 D20
concentration (MIC) [µg/mL]
3
3
3
2
2
2
b
c
f
128
na
na
128
128
na
128
128
na
na
na
na
32
64
128
128
na
128
128
128
16
128
128
na
128
128
na
128
128
na
128
128
na
128
128
na
128
128
128
16
128
128
na
na
na
na
32
64
128
128
128
128
128
128
64
128
128
128
64
64
64
128
128
128
64
64
64
16
16
na
na
128
128
na
b
c
f
128
128
128
32
128
128
128
32
na
128
128
32
1
c
f
32
32
16
16
1
OX
32
64
64
64
64
32
64
16
0.25 0.25 0.25 0.25 0.25 0.25 0.25 0.5
0.5
256
32
na
na
32
256
0.5
256
32
AMP
NFT
STR
512
16
8
64
16
8
512
16
8
64
32
8
128
32
8
4
16
8
64
16
8
256
32
8
32
256
8
8
OX oxacillin, AMP ampicillin, NFT nitrofurantoin, STR streptomycin, na no activity
1
3

Chemical Papers
Table 3 Eꢂect of 2b–c, 2f,
IC50 [µg/mL]
3
b–c, 3f, 1c, 1f on viability
of L929 and HeLa cell lines
expressed as compound
3b
3c
3f
2b
2c
2f
1c
1f
CIS
NFT
44.32
150.90
concentration inhibiting cell
growth by 50% (IC ) [µg/mL]
L929
HeLa
20.14
28.12
14.40
25.96
44.25
71.66
31.68
33.86
31.97
48.67
44.00
77.04
12.01
11.32
11.93
16.23
6.48
6.80
50
CIS cisplatin, NFT nitrofurantoin
Fig. 2 Concentration-dependent cytotoxic activity of 2b–c, 2f, 3b–c,
centrations in logarithmic scale with nonlinear regression used for
IC50 determination
3
f, 1c, 1f. Figures show the viability of L929 (a, b) and HeLa cells (c,
d) as a function of the compound concentration [µg/mL]. b, d Con-
aminophosphonates showed higher phytotoxicity than
methylphenyl derivatives. There is no possibility to com-
pare cytotoxicological behavior of amino(5-nitrofurfuryl)-
methylphosphonates to literature data, because our team syn-
thesized this group of compounds for the ꢁrst time (except
for N-benzylamino(5-nitrofurfuryl)methylphosphonic acid,
which was reported by Boduszek 2001 and Boduszek et al.
Conclusions
N-arylamino(5-nitrofurfuryl)methylphosphonic acid esters
(2b–c, 2f, 3b–c, 3f, 4a–c, 4f) were tested in aspect of
their antibacterial action. Synthesis of p-phenyl deriva-
tives (2c, 2f, 3c, 3f, 4c, and 4f) was recently published by
us (Lewkowski et al. 2017), while the preparation of the
rest (2b, 3b, 4a–b) is reported herein for the ꢁrst time.
Diphenyl aminophosphonic esters (4a–c, 4f) were found
to be inactive for tested bacteria, while the rest was mod-
erately active against Staphylococcus aureus. Dimethyl
esters (2b–c, 2f) revealed their interesting activity against
methicillin-resistant strains of S. aureus (MIC = 64 μg/
mL), and this activity was much greater than its values
for oxacillin, ampicillin, and streptomycin. Unfortunately,
measured cytotoxicity against L929 and HeLa cell lines
2
001, and which was not biologically tested).
Cytotoxicity of 5-nitrofuraldimines 1c and 1f was found
to be significant. These data coincide with previously
reported observations, where we found the Schiꢂ base 1c to
be signiꢁcantly phytotoxic (Matusiak et al. 2013).
However, it is to stress that such high values of antibacte-
rial action of 5-nitrofuraldimines 1c and 1f against MRSA
strains should be an impulse for testing various azome-
thine derivatives of 5-nitrofuraldehyde in this aspect to ꢁnd
another eꢀcient anti-MRSA agents.
1
3

Chemical Papers
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5
0
(
Table 3). Such activity and toxicity of nitro compounds
has been recently reported by del Casino et al. (2018) in
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very diꢂerent. The unacceptably close concentrations for
antibacterial eꢀcacy and for cytotoxicity might diverge fol-
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tract infection with nifuratel. Br Med J 2:908–910. https://doi.
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which was found higher than four tested commercial anti-
biotics (MIC=16 μg/mL), their cytotoxicity against above-
mentioned cell lines was dangerously high (ca. 12–13 μg/
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Acknowledgements The part of this work was funded by the Polish
National Centre of Science (NCN) in the framework of the Grant No.
Huttner A, Verhaegh EM, Harbarth S, Muller AE, Theuretzbacher U,
Mouton JW (2015) Nitrofurantoin revisited: a systematic review
and meta-analysis of controlled trials. J Antimicrob Chemother
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014/13/B/NZ9/02418. We are grateful to Beata Sadowska from the
Chair of Immunology and Infectious Biology, University of Łódź for
providing us with a set of S. aureus clinical isolates. This research was
in part conducted using the equipment of the Laboratory of Micro-
scopic Imaging and Specialized Biological Techniques, Faculty of
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Aꢀliations
Jarosław Lewkowski1 · Marta Morawska · Aleksandra Kowalczyk
1
2
2
*
*
Jarosław Lewkowski
Department of Microbial Genetics, Faculty of Biology
and Environmental Protection, University of Łódź, Banacha
jaroslaw.lewkowski@chemia.uni.lodz.pl
1
2/16, 90-237 Łódź, Poland
Aleksandra Kowalczyk
aleksandra.strzelczyk@biol.uni.lodz.pl
1
Department of Organic Chemistry, Faculty of Chemistry,
University of Łódź, Tamka 12, 91-403 Łódź, Poland
1
3