Synthesis and evaluation of biological activities of aziridine derivatives of urea and thiourea

Article abstract of DOI:10.3390/molecules23010045

In the present paper, we report the synthesis and evaluation of in vitro antimicrobial activities of aziridine-thiourea derivatives. A series of aziridines in reaction with isocyanates and isothiocyanates to obtain urea and thiourea derivatives were used.

Full text of DOI:10.3390/molecules23010045

molecules
Article
Synthesis and Evaluation of Biological Activities of
Aziridine Derivatives of Urea and Thiourea
Aleksandra Kowalczyk 1,†, Adam M. Pieczonka 2,*,†, Michał Rachwalski 2, Stanisław Les´niak 2
and Paweł Sta˛czek 1,
*
ID
1
Department of Microbial Genetics, Faculty of Biology and Environmental Protection, University of Lodz,
Banacha 12/16, 90-237 Lodz, Poland; aleksandra.strzelczyk@biol.uni.lodz.pl
Department of Organic and Applied Chemistry, Faculty of Chemistry, University of Lodz, Tamka 12,
91-403 Lodz, Poland; mrach14@wp.pl (M.R.); slesniak@chemia.uni.lodz.pl (S.L.)
Correspondence: adam.pieczonka@gmail.com (A.M.P.); pawel.staczek@biol.uni.lodz.pl (P.S.);
Tel.: +48-426-354-4466 (P.S.); Fax: +48-426-655-818 (P.S.)
2
*
†
These authors contributed equally to this work.
Received: 12 December 2017; Accepted: 21 December 2017; Published: 25 December 2017
Abstract: In the present paper, we report the synthesis and evaluation of in vitro antimicrobial
activities of aziridine-thiourea derivatives. A series of aziridines in reaction with isocyanates and
isothiocyanates to obtain urea and thiourea derivatives were used. The structures of all new products
were confirmed based on spectroscopic data (1H-NMR, 13C-NMR, HR-MS). These compounds
were screened for their in vitro antimicrobial activity against a panel of Gram-positive and
Gram-negative strains of bacteria. Six of the tested compounds appeared to be promising agents
against reference strains of Escherichia coli, Staphylococcus aureus and Staphylococcus epidermidis.
Subsequently, compounds exhibiting promising antibacterial activity were tested against twelve
clinical isolates of S. aureus from three different sources of infection. The most bactericidal compounds
(MIC = 16–32
µg/mL) showed better antibacterial activity against MRSA than ampicillin and
streptomycin. The in vitro cytotoxicity analysis on L929 murine fibroblast and HeLa human tumor
cell line using the MTT assay allowed us to select the least toxic compounds for future investigation.
Keywords: aziridines; thiourea derivatives; antimicrobial activity; cytotoxicity
1. Introduction
Aziridines are nitrogen-containing, three-membered ring heterocycles, which are widely known
as useful reactive intermediates in the synthesis of amino acid derivatives, azomethine ylides or
chiral amino alcohols [
1
–
3]. In addition, they are used as chiral auxiliaries and chiral ligands
in asymmetric synthesis [
4–
8
] or in fused heterocycles [ ]. Besides their importance as reactive
9
intermediates, aziridine-containing compounds possess many biological activities especially antitumor
and antibacterial ones, due to the presence of the aziridine ring [10]. Aziridines are powerful
alkylating agents and their in vivo potency is based primarily on toxicity rather than specific activity.
The toxicity of aziridine derivatives depends on their structure, and several important natural
products, such as mitomycin C [11], porfiromycin [12], and carzinophilin A [13] are well known
in the literature as biologically active agents. Physiological effect of mitomycin C relies on aziridine
ring opening and interaction with guanine nucleobase of DNA in the alkylation reaction. This
leads to covalent interstrand DNA–DNA crosslink formation, inhibition of replication and finally to
cell death. Aziridines with the amide function are currently of special interest with Imexon as the
well-known representative. Imexon is an anticancer agent active especially against human myeloma
cells where it binds to cellular thiols, reduces the amount of glutathione and cysteine in target cells
which leads to elevated levels of reactive oxygen species (ROS). As a result, mitochondria swell,
Molecules 2018, 23, 45; doi:10.3390/molecules23010045
www.mdpi.com/journal/molecules

Molecules 2018, 23, 45
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cytochrome c is released, caspase 3 and 9 are activated and the cells enter an apoptotic pathway [14,15].
Imexon is also known to disrupt the redox balance of the endoplasmic reticulum which inhibits
protein translation and arrests cell growth [16]. In combination with docetaxel it has been successfully
applied in the trial treatment of different cancers [17]. Other work related its activity to suppression of
B-lymphocyte activation which suggested Imexon to be useful in the treatment of B-cell or plasma
cell lymphomas or neoplasias, certain autoimmune disorders and infection with Rauscher leukemia
virus [18,19]. Natural aziridine alkaloids, as well as their lipophilic semi-synthetic, and synthetic
analogs, in addition to antitumor activity, have also a strong antibacterial activity [20]. Well known
mitomycin A, C and mitosane compounds show antimicrobial activity primarily against Gram-positive
bacteria and Klebsiella pneumoniae [10,20]. Azirinomycin (3-methyl-2H-azirine-2-carboxylic acid)
is most active against Staphylococcus aureus followed by Streptococcus faecalis, Proteus vulgaris and
Bacillus subtilis. The methyl ester of azirinomycin exhibited broad spectrum antibiotic activity in vitro
against both Gram-positive and Gram-negative bacteria [21,22]. The alkaloidal antibiotic ficellomycin
produced by Streptomyces ficellus inhibited growth of Gram-positive bacteria in vitro and in vivo during
treatment of experimental Staphylococcus aureus infections in mice [23]. Some naturally occurring
peptides containing an aziridine ring, for example madurastatin A1 and B1, consisting of Ser and
salicylic acid moieties exhibit antibacterial activity against Micrococcus luteus [24]. Anticancer drugs,
azinomycin A and B are active against both Gram-positive and Gram-negative bacteria. Two other
aziridine derivatives, azicemicin A and B demonstrate strong antibacterial activity mainly against
Mycobacterium smegmatis, Escherichia coli NIHJ, Corynebacterium bovis and Micrococcus luteus [25].
A chromoprotein antitumor drug maduropeptin exhibits inhibitory activity against Gram-positive
bacteria [26]. There are also known some aziridine, 2-aminoethylaziridine and azirine complexes
of copper(II) and palladium(II) with potent antimicrobial properties against Gram-positive bacteria
(S. aureus, S. epidermidis, E. faecalis) [27]. Moreover, one derivative of diaziridinyl quinone isoxazole
hybrid showed good antibacterial and anti-biofilm activities with very low MIC values against S. aureus
and B. subtilis and it also exhibited antifungal activity against Candida albicans [28].
Many other natural or synthesized compounds with different structures like lipids, steroids,
amino acids and peptides containing the aziridine moiety have also shown biological activity and are
promising candidates for the development of new drugs against several diseases [10,27,29,30].
In the present paper, we focused on the design and synthesis of structurally novel urea and
thiourea aziridine derivatives and evaluation of their biological activity based on the fact that many
aziridine ring containing compounds have demonstrated antibacterial and cytotoxic activities.
2. Results and Discussion
2.1. Chemistry
In continuation of the studies on the synthesis and application of chiral aziridines [1,8,10,29,31–33]
we used chiral aminoalcohols to convert them into NH aziridines via a modified Wenker
synthesis [34]. Aminoalcohols 1a in the presence of chlorosulfonic acid form sulfonic esters under
,
1
2
–b
mild conditions in quantitative yields. These esters under strong basic conditions were converted into
the corresponding aziridines 2a–c at elevated temperature (Figure 1).
i
1a-c
2a-c
Figure 1. Reagents and conditions: (i) 1. ClSO3H, MeCN; 2. 5 M NaOH, reflux.

Molecules 2018, 23, 45
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Non-protected aziridines are stable compounds under basic conditions and they can easily react
with diverse electrophiles. In our case, we used a series of aziridines in reactions with isothiocyanates
2
to obtain urea and thiourea derivatives. This methodology was previously used in 1987 to obtain
mitomycin C derivatives [35], but none of the presented compounds exhibited significant activity
against leukemia cells.
In the course of our studies, three aziridines 2a–c bearing different substituents ((S)-2-methyl,
(S)-2-isopropyl, 2,2-dimethyl), were reacted with a series of isothiocyanates yielding expected products
in high yields after 16 h (Figure 2, Table 1) [30].
i
3a-h
2a-c
Figure 2. Reagents and conditions: (i) CH2Cl2, 30 min to 16 h.
Table 1. Yields of the new urea and thiourea derivatives 3a–3h.
Compound
R1
R2
R3
Yield (%)
iPr
iPr
iPr
Me
Me
Me
Me
iPr
3a
3b
3c
3d
3e
3f
Bu
Me
cHex
Me
H
H
H
H
Me
H
92
90
89
89
94
97
96
76
Me
cHex
cHex
Allyl
3g
3h
Me
H
Structures of all new products were confirmed based on spectroscopic data (1H-NMR, 13C-NMR,
HR-MS). For example, the 1H-NMR spectrum of thiourea 3a revealed the presence of two characteristic
doublets at 2.17 and 2.51 ppm attributed to CH2 group of aziridine ring and broad signal at 6.61 ppm
attributed to NH group. In the 13C-NMR spectra absorbance of a C=S group was found at 197.9 ppm.
Due to the fact that many aziridine alkaloids exhibit good antimicrobial activity against selected
phatogens [10], we decided to test preliminary the antibacterial and cytotoxic activity of our aziridine
derivatives. Based on the results obtained from biological analysis of 3a–3h compounds, we decided
to confirm our preliminary conclusions regarding the biological role of some atoms and conformations
in analyzed aziridine-containing agents by designing and synthesizing the next set of compounds.
In pursuit of the second goal, we synthesized aziridine-thiourea derivatives containing sterically
crowded substituents, like benzyl (3i
,
3j) and benzhydryl (3k, 3l). We also decided to include additional
amine function in the aliphatic chain (compounds 3m
–
3p) as a modification of compound 3a. We also
replaced sulfur atom in compounds 3a and 3c to check the influence of the oxygen atom in the new urea
derivatives, and we also synthesized an analog of compound 3o with (R)-isopropylaziridine (Table 2)
to test if the configuration of the aziridine subunit had any significance to the biological activity.
All new compounds were obtained according to a previously described procedure, only the reaction of
(S)-2-isopropylaziridine 2b butylisocyanate and cyclohexylisocyanate gave the corresponding urea
derivatives 3r, 3s in excellent yields after 30 min.

Molecules 2018, 23, 45
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Table 2. Yields of the urea and thiourea derivatives 3i–3s.
3i-s
Compound
R1
R2
X
Yield (%)
iPr
Me
iPr
Me
iPr
Me
iPr
iPr
Me
iPr
iPr
3i
3j
3k
3l
3m
3n
3o
(R)-3o
3p
3r
3s
CH2Ph
CH2Ph
CHPh2
S
S
S
S
S
S
S
S
S
90
89
88
85
92
93
95
90
91
96
98
CHPh2
2-(4-morpholino)ethyl
2-(4-morpholino)ethyl
2-piperidinoethyl
2-piperidinoethyl
2-piperidinoethyl
Bu
O
O
cHex
2.2. Biology
All compounds were tested for their antimicrobial activity against a representative panel of
bacteria i.e., Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Staphylococcus epidermidis,
Enterococcus faecalis, Proteus vulgaris and Proteus mirabilis using nitrofurantoin, ampicillin and
streptomycin as reference drugs. The in vitro antimicrobial activities of the compounds 3a
concentrations ranging from 1 to 512 g/mL were screened using the microdilution method. The results
showed that two compounds 3g 3h were inactive against all tested bacteria in analyzed concentrations.
Six compounds, 3a 3f, showed antibacterial activity against Gram-positive strains (MIC value ranging
from 16 to 512 g/mL), however, lower than the reference drugs, and were inactive against P. aeruginosa
–3h at
µ
,
–
µ
and Proteus strains. The in vitro results of antibacterial activity of these compounds are presented in
Table 3 as a minimal inhibitory concentration (MIC) and a minimal bactericidal concentration (MBC).
Table 3. In vitro antibacterial activity of 3a
concentration (MIC) ( g/mL) and a minimal bactericidal concentration (MBC) (
ampicillin, NTF: nitrofurantoin, STR: streptomycin.
,
3b
,
3c
,
3d
,
3e
,
3f expressed as a minimal inhibitory
µ
µg/mL). AMP:
E. coli NCTC 8196
S. aureus ATCC 6538
S. aureus ATCC 29213
S. epidermidis ATCC 12228
Tested Strain
Compound
NCTC 8196
ATCC 6538
ATCC 29213
ATCC 12228
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
3a
3b
3c
3d
3e
>512
32
nd
32
nd
128
nd
512
8
32
32
32
256
128
16
16
1
128
64
64
512
128
128
32
32
32
32
256
128
16
16
2
128
64
64
512
128
128
32
32
16
32
256
128
16
64
16
64
512
128
16
>512
128
>512
256
8
3f
NTF
AMP
STR
8
1
8
1
4
1
4
2
1
1
4
2
1
1
>512
>512
Note: nd—not determined.
Among Gram-positive species, the most sensitive to all of the active compounds were two
strains of S. aureus and S. epidermidis. The most effective against these strains were compounds
3a, 3b, 3c, 3f (MIC = 16–32 µg/mL) although their activities were lower than those exhibited by
ampicillin and streptomycin which are the antibiotics commonly used in the therapy of staphylococcal
infections. Among six tested compounds, 3b was the only effective agent against E. coli reference strain

Molecules 2018, 23, 45
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(MIC = 32 µg/mL). 3f was the most active compound especially against Gram-positive strains with
activity similar to nitrofurantoin (MIC = 16
was more bacteriostatic than bactericidal with high MBC value (MBC = 128
µ
g/mL), however, in case of S. aureus strains, its activity
g/mL) while other agents
showed bactericidal activity (MBC/MIC = 2 or 3, respectively). Compounds 3d and 3e showed lower
antibacterial activity than other compounds with MIC= 128–256 g/mL. Our observations suggest that
µ
µ
2,2-dimethyl substituent at R2 position of aziridine significantly reduces antibacterial activity (Table 3,
see data 3e vs. 3b and 3g vs. 3c). The most potent derivatives were aziridines with (S)-2-methyl
or (S)-2-isopropyl moiety at R2 however their activity also depended on the type of substituent at
R1 position. For R1-methyl derivatives the replacement of (S)-2-methyl with (S)-2-isopropyl group
highly increased antibacterial activity (4–8 fold) (Table 2, see data 3d vs. 3b). On the other hand, for
R1-cyclohexyl derivatives the replacement of (S)-2-methyl with (S)-2-isopropyl did not have such
significant consequences, however, compound 3f with (S)-2-methyl group showed two-fold stronger
antimicrobial activity than 3c.
Considering good activity of the tested compounds, especially 3a, 3b, 3c, 3f, against
Staphylococcus spp., a set of 12 S. aureus clinical strains including the ones isolated from two typical
sources such as naso-pharynx (carrier state) and ulcers/furuncles (skin and soft tissue infections),
but also those from infected bones (invasive infections) were tested against these agents. S. aureus
D15 and D17 strains were characterized as MRSA due to their resistance to high concentrations of
oxacillin. Similarly to the reference Staphylococcus spp. Strains, clinical isolates displayed high level of
susceptibility to the analyzed compounds (MIC ranging from 16 to 64 µg/mL, Table 4).
Again, compound 3f showed the strongest activity, with MIC being equal to 8–16 µg/mL for all
of the tested clinical isolates, which for most of the strains was a better result than in case of ampicillin
and nitrofurantoin (used as positive control drugs). On the other hand, the other common antibiotics
oxacillin and streptomycin were the most active in almost all cases, except for the two MRSA strains
isolated from bones (S. aureus D15, and D17, which were 16–32 times more sensitive to 3f compound
and 8–16 times to 3a, 3b, 3c compounds. For all four tested compounds, the MBC values were in a
similar range, suggesting their bactericidal activity (MBC/MIC = 2).
Many aziridine-containing compounds are known to demonstrate strong anticancer activity.
The cytotoxic activities of 3a–3f were assessed using L929 murine cell line (recommended by the
International Standard ISO 10993:2009 for evaluation of cytotoxic activities) as well as HeLa human
tumor cell line. The percentage of viability inhibition compared to the negative control in which cells
were grown in the absence of tested compounds was estimated for concentrations ranging from 2 to
256 µg/mL of the compound. A common antitumor drug, cisplatin, was used as a positive control.
Compounds 3d and 3e showed weak cytotoxic effect on both tested cell lines and their activity was
~30-fold lower than for cisplatin (Table 5, Figures 3 and 4). The most toxic were compounds which
were the most bactericidal as well (3b
, 3f) with IC30 = 20–28 µg/mL which is only nearly three-fold
lower activity than in case of cisplatin however, they showed no selectivity against tumor cells.

Molecules 2018, 23, 45
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Table 4. In vitro activity of 3a, 3b, 3c, 3f against clinical isolates of S. aureus expressed as the minimal inhibitory concentration (MIC) (µg/mL) and minimal bactericidal
concentration (MBC) (µg/mL). OX: oxacillin, AMP: ampicillin, NTF: nitrofurantoin, STR: streptomycin.
Compound
3a
3b
3c
3f
OX
AMP
NTF
STR
Tested Strain
MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC MIC MBC
naso-pharynx isolates
S. aureus
C4
C7
C8
32
32
32
32
64
64
64
64
32
32
32
32
64
32
32
32
32
32
32
32
64
64
64
64
16
16
16
16
16
16
16
16
0.25
0.25
0.25
0.5
0.25
0.25
0.25
0.5
512
64
512
64
512
128
>512
128
16
16
16
32
16
16
16
32
8
8
8
8
8
16
16
8
C19
ulcers/furuncles isolates
D12
F1
F7
32
32
32
32
64
64
64
64
16
32
32
16
32
128
128
32
32
64
32
32
128
128
64
8
16
16
8
16
64
64
16
0.5
0.5
256
128
4
256
128
4
32
32
16
16
32
32
16
32
8
8
8
8
16
8
16
32
0.25
0.25
0.25
0.25
0.25
0.25
F12
64
64
128
bone isolates
D14
32
32
32
32
64
64
64
64
32
32
16
32
32
32
16
32
32
32
32
64
64
64
64
16
16
8
16
16
32
64
0.5
>512
>512
0.5
0.5
>512
>512
0.5
256
>512
>512
128
512
>512
>512
256
32
32
32
16
32
32
32
32
8
16
>512
>512
8
D15 (MRSA)
D17 (MRSA)
D20
256
256
8
128
16
Table 5. Cytotoxic activity data.
IC30 (µg/mL)/(µM) L929 Cells
Compound
HeLa Cells
3a
3b
68/340
27/171
83/388
28/177
3c
3d
3e
34/147
69/304
>250/>1922
214/1485
20/102
>250/1922
250/1735
22/111
3f
Cisplatin
7.24/24
6.53/22

Molecules 2018, 23, 45
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140
130
120
110
100
90
3a
3b
3c
3d
3e
3f
80
70
60
50
40
30
20
10
0
2
4
8
16
32
64
128
256
concentration [µg/mL]
Figure 3. Concentration-dependent cytotoxic activity of 3a, 3b, 3c, 3d, 3e, 3f for L929 cell line.
140
130
120
110
100
90
3a
80
70
60
50
40
30
20
10
0
3b
3c
3d
3e
3f
2
4
8
16
32
64
128
256
concentration [µg/mL]
Figure 4. Concentration-dependent cytotoxic activity of 3a, 3b, 3c, 3d, 3e, 3f for HeLa cell line.
Eleven new compounds, 3i
reference panel of bacteria and again using nitrofurantoin, ampicillin and streptomycin as reference
drugs. The in vitro antimicrobial activities of the compounds 3i 3s at concentrations ranging from 1 to
512 g/mL were screened using the microdilution method. The results showed that five compounds
3i 3n 3r 3s and (R)-3o were completely inactive against all tested bacteria in analyzed concentrations.
–3s, were tested for their antimicrobial activity against the same
–
µ
,
,
,
Interestingly, both urea derivatives had no biological activity compared to their thiourea analogs
(Tables 2 and 6, see data 3r vs. 3a and 3s vs. 3c) which suggests that the sulphur atom is a principal
factor for antibacterial properties. Moreover, an analog of compound 3o with (R)-isopropylaziridine
also had no significant biological activity which confirmed that configuration of the aziridine plays an
important role for evolving antimicrobial profiles. Six compounds 3j
–3o and 3p showed antibacterial
activity mainly against Gram-positive strains (MIC value ranging from 4 to 256
µg/mL) and were
inactive against P. aeruginosa and Proteus strains. The in vitro results of antibacterial activity of
these compounds are presented in Table 6 as a minimal inhibitory concentration (MIC). For the
two most active compounds 3o and 3p minimal bactericidal concentration was also determined
(Table 7). For both compounds, the MBC values were in the similar range as MIC, suggesting their
bactericidal activity (MBC/MIC = 2 or 3).

Molecules 2018, 23, 45
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Table 6. In vitro antibacterial activity of 3j, 3k, 3l, 3m, 3o, 3p expressed as a minimal inhibitory
concentration (MIC) (µg/mL). AMP: ampicillin, NTF: nitrofurantoin, STR: streptomycin.
E. coli NCTC 8196
S. aureus ATCC 6538
S. aureus ATCC 29213
S. epidermidis ATCC 12228
Tested Strain
Compound
NCTC 8196
ATCC 6538
ATCC 29213
ATCC 12228
MIC (µg/mL)
3j
3k
3l
3m
3o
3p
NTF
AMP
STR
256
256
256
na
64
256
8
128
na*
256
128
8
64
16
1
128
Na
256
256
8
64
16
2
128
256
256
128
4
64
8
1
4
1
1
1
>512
na*—no activity.
Table 7. In vitro antibacterial activity of 3o and 3p expressed as a minimal bactericidal concentration
(MBC) (µg/mL).
E. coli NCTC 8196
S. aureus ATCC 6538
S. aureus ATCC 29213
S. epidermidis ATCC 12228
Tested Strain
Compound
NCTC 8196
ATCC 6538
ATCC 29213
ATCC 12228
MBC (µg/mL)
3o
3p
128
256
16
128
16
128
8
128
Considering good activity of these two compounds against S. aureus reference strains, we used
a set of 12 S. aureus clinical strains to test antibacterial activity of 3o and 3p. Our results revealed
satisfactory activity only in case of compound 3o with MIC being equal to 32 µg/mL for all of the
tested clinical isolates, which, for most of the strains, was a better result than in case of ampicillin.
Moreover, two MRSA strains isolated from bones (S. aureus D15, and D17 were 8–16 times more
sensitive to 3o compound. For both tested compounds, the MBC values were in the similar range,
suggesting their bactericidal activity (MBC/MIC = 2, Table 8).
Table 8. In vitro activity of 3o and 3p against clinical isolates of S. aureus expressed as a minimal
inhibitory concentration (MIC) (
µg/mL) and a minimal bactericidal concentration (MBC) (
µg/mL).
OX: oxacillin, AMP: ampicillin, NTF: nitrofurantoin, STR: streptomycin.
Compound
3o
3p
OX
MBC
AMP
NTF
STR
Tested Strain
MIC
MBC
MIC
MBC
MIC
MIC
MBC
MIC
MBC
MIC
MBC
naso-pharynx isolates
S. aureus
C4
C7
C8
32
32
32
32
64
64
64
64
256
256
256
256
256
256
256
256
0.25
0.25
0.25
0.5
0.25
0.25
0.25
0.25
512
64
512
64
512
128
>512
128
16
16
16
32
16
16
16
32
8
8
8
8
8
16
16
8
C19
ulcers/furuncles isolates
D12
F1
F7
32
32
32
32
64
64
64
64
256
256
256
256
256
256
256
256
0.5
0.5
256
128
4
256
128
4
32
32
16
16
32
32
16
32
8
8
8
8
16
8
16
32
0.25
0.25
0.25
0.25
0.25
0.25
F12
64
128
bone isolates
D14
32
32
32
32
64
64
64
64
256
256
256
256
256
512
512
512
0.5
>512
>512
0.5
0.5
>512
>512
0.5
256
>512
>512
128
512
>512
>512
256
32
32
32
16
32
32
32
32
8
16
>512
>512
8
D15 (MRSA)
D17 (MRSA)
D20
256
256
8

Molecules 2018, 23, 45
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As in the case of previous compounds, the cytotoxic activities of 3o
–
3p were assessed using
L929 murine cell line as well as HeLa human tumor cell line. The percentage of viability inhibition
compared to the negative control in which cells were grown in the absence of tested compounds was
estimated for concentrations ranging from 2 to 128 µg/mL of the compound. Compound 3o was more
toxic (IC30 = 42–45 µg/mL), with cytotoxicity about two-fold higher than 3p (Table 9, Figures 5 and 6).
However, 3o seemed to be harmless for cells in a concentration corresponding to its antibacterial
activity. Both compounds showed no selectivity against tumor cells.
Table 9. Cytotoxic activity data.
Compound
IC30 (µg/mL)/(µM) L929 Cells
HeLa Cells
3o
3p
Cisplatin
45/187
103/483
7.24/24
42/174
71/333
6.53/22
140
130
120
110
100
90
80
70
3o
3p
60
50
40
30
20
10
0
2
4
8
16
32
64
128
concentration [µg/mL]
Figure 5. Concentration-dependent cytotoxic activity of 3o and 3p for L929 cell line.
140
130
120
110
100
90
80
70
3o
3p
60
50
40
30
20
10
0
2
4
8
16
32
64
128
concentration [µg/mL]
Figure 6. Concentration-dependent cytotoxic activity of 3o and 3p for HeLa cell line.

Molecules 2018, 23, 45
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To date, many anticancer agents have been found to show antimicrobial activity against several
bacterial pathogens. Among them, mitomycin C as well as other aziridine derivatives have been
tested. It is believed that, like in case of their anticancer activity, these compounds act mainly as
alkylating agents also in bacterial cells. For example, mitomycin C which was found to be passively
transported into the cells, may be used not only for killing metabolically active bacteria but also act
against dormant presister cells, leading to formation of DNA crosslinks [36]. However, despite this
typical crosslinking activity of aziridine derivatives, other possible modes of antibacterial action cannot
be excluded. For example, naturally occurring aziridine derivative (2S,3S)-aziridine-2,3-dicarboxylic
acid was found to inhibit the activity of E. coli aspartase [37]. We presume that the mechanism of action
of the aziridine derivatives described in this paper may be typical as for the other members of this
group of compounds, however, on the basis of this study, it is too early to predict it.
3. Experimental Section
3.1. Chemistry
General: Melting points were determined in a capillary using a STUART SMP30 and were
uncorrected. The 1H- (600 MHz), 13C{1H}- (150 MHz) spectra were measured on a Bruker Avance III
instrument (Bruker, Billerica, MA, USA) using solvent signals as reference. Chemical shifts (δ) are
given in ppm and coupling constants J in Hz. Assignments of signals in13C-NMR spectra were made
on the basis of HMQC experiments. HR-MS: Bruker Esquire LC spectrometers (Bruker Daltonics).
All solvents are commercially available reagents and were used as received. Aziridines 2a–c were
obtained according to a published procedure [34].
3.2. General Procedure for Synthesis of Urea and Thiourea Derivatives 3
To a stirred solution of an aziridine
of the isocyanate or isothiocyanate was slowly added. The mixture was stirred for 16 h at room
temperature (3a ), or 30 min (3r 3s), the solution was concentrated and the resulting mixture was
2
(1 mmol) in CH2Cl2 (5 mL) at 20 ◦C, an equimolar quantity
–
p
,
purified by flash chromatography (SiO2/CH2Cl2).
(2S)-N-Butyl-2-isopropyl-aziridine-1-carbothioamide (3a): colorless crystals; yield: 92%; m.p. 71–72 ◦C
(MeOH). 1H-NMR (600 MHz, CDCl3,
δ, ppm): 6.61 (1H, br. s, NH); 3.61–3.57 (2H, m, N-CH2); 2.51 (1H,
d, J = 6.6 Hz, CH2); 2.31–2.28 (1H, m, aziridine CH); 2.17 (1H, d, J = 4.2 Hz, CH2); 1.63–1.35 (5H, m);
1.07 (3H, d, J = 6.6 Hz, CH3); 0.98 (3H, d, J = 7.2 Hz, CH3); 0.95 (3H, t, J = 1.8 Hz, butyl CH3). 13C-NMR
(150 MHz, CDCl3, δ, ppm): 197.9 (C=S); 48.9 (aziridine CH); 45.8 (N-CH2); 34.8 (aziridine CH2); 30.8
(CH); 30.6, 20.1 (2 CH2); 20.0, 19.3 (2 CH3); 13.7 (butyl CH3). HR-EI-MS: 200.1350 (M+, C10H20N2S+;
calcd. 200.1347).
(2S)-2-Isopropyl-N-methyl-aziridine-1-carbothioamide (3b): colorless crystals; yield: 90%; m.p. 65–66 ◦C
(MeOH). 1H-NMR (600 MHz, CDCl3,
δ, ppm): 6.67 (1H, br. s, NH); 3.16 (3H, d, J = 5.4 Hz, N-CH3);
2.55 (1H, d, J = 6.6 Hz, CH2); 2.37–2.33 (1H, m, aziridine CH); 2.21 (1H, d, J = 4.2 Hz, CH2); 1.62–1.55
(1H, m, isopropyl CH); 1.10 (3H, d, J = 6.6 Hz, CH3); 1.01 (3H, d, J = 7.2 Hz, CH3). 13C-NMR (150 MHz,
CDCl3,
δ, ppm): 198.2 (C=S); 48.9 (aziridine CH); 34.9 (aziridine CH2); 33.2 (N-CH3); 30.8 (CH); 20.0,
19.4 (2 CH3). HR-EI-MS: 158.0879 (M+, C7H14N2S+; calcd. 158.0878).
◦
(2S)-N-Cyclohexyl-2-isopropyl-aziridine-1-carbothioamide (3c): colorless crystals; yield: 89%; m.p. 75–76 C
(MeOH). 1H-NMR (600 MHz, CDCl3,
, ppm): 6.60 (1H, br. s, NH); 4.18–4.16 (1H, m, N-CH); 2.49
δ
(1H, d, J = 6.6 Hz, aziridine CH2); 2.29–2.26 (1H, m, aziridine CH); 2.07 (1H, d, J = 4.2 Hz, aziridine
CH2); 2.06–2.04 (2H, m, cyclohexyl CH2); 1.72–1.37 (6H, m); 1.24–1.17 (3H, m); 1.07 (3H, d, J = 6.6 Hz,
CH3); 0.98 (3H, d, J = 7.2 Hz, CH3). 13C-NMR (150 MHz, CDCl3,
δ, ppm): 197.7 (C=S); 54.1 (cyclohexyl
CH); 48.9 (aziridine CH); 34.9 (aziridine CH2); 33.6, 33.4, 25.6, 25.5, 24.7 (5 CH2); 30.9 (CH); 20.0, 19.3
(2 CH3). HR-EI-MS: 226.1500 (M+, C12H22N2S+; calcd. 226.1504).

Molecules 2018, 23, 45
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(2S)-N-Methyl-2-methyl-aziridine-1-carbothioamide (3d): colorless crystals; yield: 89%; m.p. 70–71 ◦C
(MeOH). 1H-NMR (600 MHz, CDCl3,
, ppm): 6.81 (1H, br. s, NH); 3.07 (3H, s, N-CH3); 2.54–2.52 (1H,
m, CH); 2.46 (1H, d, J = 6.6 Hz, CH2); 2.07 (1H, d, J = 4.2 Hz, CH2); 1.25 (3H, d, J = 5.4 Hz, aziridine
CH3). 13C-NMR (150 MHz, CDCl3,
, ppm): 198.5 (C=S); 38.2 (aziridine CH); 37.0 (aziridine CH2); 33.2
(N-CH3); 17.6 (aziridine CH3). HR-EI-MS: 130.0569 (M+, C5H10N2S+; calcd. 130.0565).
δ
δ
◦
(2S)-N-Methyl-2,2-dimethyl-aziridine-1-carbothioamide (3e): colorless crystals; yield: 94%; m.p. 77–78 C
(MeOH). 1H-NMR (600 MHz, CDCl3,
, ppm): 6.39 (1H, br. s, NH); 3.10 (3H, d, J = 5.4 Hz, N-CH3); 2.40
δ
(2H, s, CH2); 1.23 (6H, s, 2 aziridine CH3). 13C-NMR (150 MHz, CDCl3,
δ, ppm): 195.8 (C=S); 42.5 (aziridine
CH2); 33.2 (N-CH3); 22.4 (aziridine CH3). HR-EI-MS: 144.0722 (M+, C6H12N2S+; calcd. 144.0721).
(2S)-N-Cyclohexyl-2-methyl-aziridine-1-carbothioamide (3f): colorless crystals; yield: 97%; m.p. 68–67 ◦C
(MeOH). 1H-NMR (600 MHz, CDCl3,
δ
, ppm): 6.52 (1H, br. s, NH); 4.12–4.08 (1H, m, N-CH); 2.54–2.51
(1H, m, aziridine CH); 2.42 (1H, d, J = 6.6 Hz, aziridine CH2); 2.07 (1H, d, J = 4.2 Hz, aziridine CH2);
2.06–1.20 (10H, m); 1.25 (3H, d, J = 6.0 Hz, CH3). 13C-NMR (150 MHz, CDCl3,
, ppm): 198.7 (C=S);
δ
54.2 (cyclohexyl CH); 38.0 (aziridine CH); 36.9 (aziridine CH2); 33.6, 33.4, 25.6, 25.5, 24.7 (5 CH2); 17.5
(aziridine CH3). HR-EI-MS: 198.1198 (M+, C10H18N2S+; calcd. 198.1191).
(2S)-N-Cyclohexyl-2,2-dimethyl-aziridine-1-carbothioamide (3g): colorless crystals; yield: 96%; m.p.
◦
1
76–77 C (MeOH). H-NMR (600 MHz, CDCl3,
δ, ppm): 6.24 (1H, br. s, NH); 4.19–4.13 (1H, m,
N-CH); 2.38 (2H, s, aziridine CH2); 2.03–1.23 (10H, m); 1.15 (6H, s, 2 CH3). 13C-NMR (150 MHz, CDCl3,
δ
, ppm): 192.8 (C=S); 54.5 (cyclohexyl CH); 42.1 (aziridine CH2); 33.3, 32.5, 25.5, 25.2, 24.8 (5 CH2); 22.0
(aziridine CH3). HR-EI-MS: 212.1351 (M+, C11H20N2S+; calcd. 212.1347).
(2S)-N-Allyl-2-isopropyl-aziridine-1-carbothioamide (3h): colorless crystals; yield: 76%; m.p. 65–66 ◦C
(MeOH). 1H-NMR (600 MHz, CDCl3,
δ, ppm): 6.56 (1H, br. s, NH); 5.86–5.80 (2H, m, CH2=); 5.20–5.16
(1H, m, CH=); 4.19–4.17 (allyl CH2); 2.46 (1H, d, J = 6.6 Hz, CH2); 2.28–2.25 (1H, m, CH); 2.14 (1H,
d, J = 4.2 Hz, CH2); 1.53–1.47 (1H, m); 1.01 (3H, d, J = 6.6 Hz, CH3); 0.92 (3H, d, J = 7.2 Hz, CH3).
13C-NMR (150 MHz, CDCl3,
δ, ppm): 198.1 (C=S); 132.7 (CH2=); 117.6 (CH=); 48.9 (aziridine CH); 48.3
(CH2); 34.9 (aziridine CH2); 30.8 (isopropyl CH); 20.0, 19.0 (2 aziridine CH3). HR-EI-MS: 184.1039 (M+,
C9H16N2S+; calcd. 184.1034).
(2S)-N-Cyclohexyl-2-isopropyl-aziridine-1-carboxamide (3s): colorless crystals; yield: 96%; m.p. 74–75 ◦C
(MeOH). 1H-NMR (600 MHz, CDCl3,
δ, ppm): 5.16 (1H, br. s, NH); 3.57–3.55 (1H, m, N-CH); 2.30 (1H,
d, J = 6.6 Hz, aziridine CH2); 2.10–2.07 (1H, m, aziridine CH); 1.92–1.85 (2H, m, cyclohexyl CH2); 1.82
(1H, d, J = 4.2 Hz, aziridine CH2); 1.70–1.67, 1.61–1.57 (4H, 2 m, 2 cyclohexyl CH2); 1.42–1.31 (3H, m,
isopropyl CH, cyclohexyl CH2); 1.18–1.12 (2H, m, cyclohexyl CH2);1.04 (3H, d, J = 6.6 Hz, CH3); 0.96
(3H, d, J = 7.2 Hz, CH3). 13C-NMR (150 MHz, CDCl3,
δ, ppm): 163.1 (C=O); 54.5 (cyclohexyl CH); 45.9
(aziridine CH); 33.6, 33.4, 25.6, 25.5, 24.7 (5 CH2); 31.8 (aziridine CH2); 30.8 (CH); 20.0, 19.3 (2 CH3).
HR-EI-MS: 210.1734 (M+, C12H22N2O+; calcd. 210.1732).
3.3. Biology
3.3.1. Antibacterial Assay
The in vitro antimicrobial activity of newly synthesized compounds was evaluated against
the reference strains of Gram-negative (Escherichia coli NCTC 8196, Proteus vulgaris ATCC
49990, Proteus mirabilis ATCC 29906, Pseudomonas aeruginosa NCTC 6249), and Gram-positive
(Staphylococcus aureus ATCC 6538, Staphylococcus aureus ATCC 29213, Enterococcus faecalis ATCC 29212,
Staphylococcus epidermidis ATCC 12228) bacterial species. Moreover, the most active (against the
reference strains) compounds were examined against a set of twelve clinical isolates of S. aureus
received from the collection of the Chair of Immunology and Infectious Biology, University of
Łódz´. These strains were isolated from the following three sources: naso-pharynx of young patients
hospitalized at Children’s Hospital in Łódz´ (n = 4), ulcers and furuncles from adult patients of

Molecules 2018, 23, 45
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Dermatological Clinic in Łódz´ (n = 4), and from infected bones of patients hospitalized at Oncological
Hospital in Łódz´ (n = 4). All strains were kept frozen at
80 ◦C on Tryptic Soy Broth with 15% of
−
glycerol until testing. Before using, S. aureus strains were subcultured on blood agar and identified
by routine methods (catalase, coagulase and clumping factor). Minimal inhibitory concentration
(MIC) was determined as the lowest concentration of the compound preventing growth of the tested
microorganism using microdilution method according to EUCAST guidelines. The inoculum density
was adjusted to 0.5 McFarland standard. All of the tested compounds were dissolved in dimethyl
sulfoxide (DMSO). Concentration of the agents evaluated in Mueller-Hinton broth ranged from 1
to 512
µg/mL. DMSO at the final concentration in the medium had no influence on growth of the
tested microorganisms. The incubation was carried out at 37 ◦C for 18 h and optical density (OD600
)
measurements were determined for bacterial cultures in the presence and absence of the tested
compounds. Ampicillin, nitrofurantoin, and streptomycin widely used in the treatment of infectious
diseases were used as positive control antimicrobial agents. Minimal bactericidal concentration (MBC),
defined as the lowest concentration of a compound that resulted in >99.9% reduction in CFU of the
initial inocula (2
×
108 cfu) was assessed only for compounds 3c
–3h and 3o, 3p. MBC was determined
by a broth microdilution technique followed by plating out the contents of the ◦wells that showed no
visible growth of bacteria onto Mueller-Hinton agar plates and incubating at 35 C for 18 h. Both MIC
and MBC evaluations were performed in triplicates and are given in µg/mL.
3.3.2. Cytotoxicity Assay
Cytotoxic effect of compounds 3c
viability using MTT reduction assay. Murine fibroblasts L929 cells (ATTC® catalog No. CCL-1, mouse
fibroblasts) or human tumor HeLa cells (ATTC® catalog No. CCL-2
, human epithelial cells) were
plated in 96-well microplates at density of 1 L per well) and cultivated in Iscove’s
104 cells/mL (100
modified Dulbecco’s medium (IMDM), supplemented with 100.0 U/mL penicillin and streptomycin,
10−5 M 2-mercaptoethanol and enriched with 10% fetal bovine serum (FBS). After overnight
incubation at 37 ◦C, the growth medium was removed and 100
L of medium supplemented with
different concentrations of synthetic compounds in the range of 1–300 g/mL were added. Cells were
–3h and 3o, 3p on host cells was detected by determining cellular
™
×
µ
5
×
µ
µ
further incubated for 24 h with tested agents. At the end of the incubation time, the medium was
removed and MTT was added to ◦each well at a final concentration of 0.5 mg/mL and plates were
incubated for the next 2 h at 37 C. Then, formazan crystals were solubilized in 150 µL DMSO.
The optical density was measured at 550 nm. The results of experiments were shown as mean arithmetic
values of eight repeats (two experiments) and the percentage of inhibition of viability compared to
control wells was calculated for each concentration of the tested compounds and IC30 value (which is
considered to be save for tested cells) was determined in each case. The results of the experiments
were shown as mean arithmetic values from 3 repeats in each of two independent experiments.
4. Conclusions
In conclusion, we have reported the synthesis and preliminary evaluation of the biological
activity of 19 novel urea and thiourea aziridine derivatives. While comparing the structure of tested
compounds with the antibacterial and cytotoxic activity, we could already draw some substantial
conclusions. The presence of sulphur atom is a principal factor for antibacterial properties of aziridine
derivatives. The (S)-configuration of the aziridine plays an important role for conferring antimicrobial
activity. All tested (S)-isopropylaziridine derivatives with 1-methyl (3b), 1-butyl (3a), 1-cyclohexyl
(3c) and 1-(2-piperidinoethyl) (3o) substituents, showed similar satisfactory antibacterial results.
The only exception was compound 3h with a 1-allyl moiety which can suggest that it decreases
the antimicrobial activity. We have also observed that a 2,2-dimethyl moiety at C2 position of aziridine
(3e, 3g) significantly reduces antibacterial activity. The most bactericidal aziridine derivatives of
thiourea also showed the highest toxic activity against both cell lines with no selectivity for tumor
cells. It can be explained by the alkylating activity of aziridine ring possessing agents and their

Molecules 2018, 23, 45
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in vivo potency based primarily on toxicity rather than specific activity. We selected five of the tested
compounds 3a 3b 3c 3f 3o which showed the best activity against clinical S. aureus strains, and
,
,
,
,
in two cases of invasive infections of MRSA, these agents exceeded the activity of commonly used
antibiotics such as ampicillin, streptomycin and oxacillin by 16-fold. To conclude, all the collected data
could provide valuable information for further modifications of these compounds in order to select
those with stronger antibacterial activity which would be less harmful. Simultaneously, our attention
will be also focused on the improvements leading to the increase of their selectivity against tumor cells,
bearing in mind their potential usage in antitumor therapy.
Acknowledgments: We are grateful to Beata Sadowska from the Chair of Immunology and Infectious Biology,
University of Łódz´ for providing us with a set of S. aureus clinical isolates. Financial support by the Ministry
of Science and Higher Education in Poland, Grant Iuventus Plus nr IP2014–035873 for A.M.P., is gratefully
acknowledged. This research was in part conducted using the equipment of the Laboratory of Microscopic Imaging
and Specialized Biological Techniques, Faculty of Biology and Environmental Protection, University of Łódz´.
Author Contributions: A.M.P. and A.K. performed the experiments. S.L. and M.R. analyzed the data. A.M.P.,
A.K. and P.S. wrote the paper.
Conflicts of Interest: Authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 3a–3s are available from the authors.
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