Pronounced activity of aromatic selenocyanates against multidrug resistant ESKAPE bacteria

Article abstract of DOI:10.1039/C9NJ00563C

Selenocyanates represent an interesting class of organic selenium compounds. Due to their similarity with better known natural (iso-)thiocyanates, they promise high biological activity and may also be metabolized to other Reactive Selenium Species (RSeS),

Full text of DOI:10.1039/C9NJ00563C

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K. Kononowicz, J. Handzlik and C. Jacob, New J. Chem., 2019, DOI: 10.1039/C9NJ00563C.
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Pronounced activity of aromatic selenocyanates against multidrug
resistant ESKAPE bacteria
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Muhammad Jawad Nasima,b Karolina Witek b,c, Annamária Kincsesd, Ahmad Yaman Abdina, Ewa
Żesławskae, Małgorzata Anna Marćb, Márió Gajdácsd, Gabriella Spenglerd, Wojciech NitekF,
Gniewomir Lataczb, Elżbieta Karczewskac, Katarzyna Kieć-Kononowiczb, Jadwiga Handzlikb,* and
Claus Jacoba,*
www.rsc.org/
Selenocyanates represent an interesting class of organic selenium compounds. Due to their similarity with better known
natural (iso-)thiocyanates, they promise high biological activity and may also be metabolized to other Reactive Selenium
Species, such as selenols, diselenides and seleninic acids. Thirteen arylmethyl selenocyanates (1-13) have been synthesized
and evaluated for potential antimicrobial, nematicidal and cytotoxic activity. The compounds exhibit pronounced
antimicrobial activity against various strains of Gram-positive and Gram-negative bacteria and yeasts, including multidrug
resistant strains. The results obtained so far demonstrate that these arylmethyl selenocyantes are also non-mutagenic and
with limited cytotoxicity against human cells. Here, benzylselenocyanate (1) represents the most active anti-ESKAPE agent,
with potent activity against multidrug resistant MRSA strains (HEMSA 5) - with a competitive MIC value of just 0.76 µg mL-1
(3.88 µM) whereas it exhibits low(er) cytotoxicity (IC50 = 31 µM) and no mutagenicity against mammalian cells. Because of
this selective antimicrobial activity, aromatic selenocyanates may provide an interesting lead in the development of
antimicrobial agents, particularly in the context of drug resistance.
.
one of the biggest threats and challenges facing humanity.1 The
phenomenon of resistance to available antibiotics has attracted the
attention of scientists for over two decades now, and has led to a
considerable demand for the development of new types of
Introduction
antibiotics against such resistant strains of bacteria and fungi.
Since the discovery of the first modern antibiotics almost one
hundred years ago, substances such as the penicillin have served as
important and effective weapons in the prevention and treatment
of a wide spectrum of infectious diseases. Nonetheless, over- and
misuse of antibiotics, among various other reasons, have led to a
surge of resistance in pathogenic bacteria, which has now become
Fortunately, Nature itself is an affluent source of antibiotics. Natural
substances acquired from medicinal and culinary plants, such as
garlic and mustard, have been employed extensively as antibiotics
throughout History.2-4 More recently, such secondary metabolites
have become important leads in the development of new and
effective medicines.5-7 These phytochemicals include alkaloids,
flavonoids, terpenes, polyphenols and, particularly, organosulfur
compounds (OSCs).8-11 OSCs are distributed widely in Nature, for
instance as allicin in garlic, ergothioneine in mushrooms, and
thiocyanates and isothiocyanates in cruciferous vegetables, to
name just a few (Figure 1).12-14 Isothiocyanates and thiocyanates are
reactive, electrophilic substances belonging to the class of natural
Reactive Sulfur Species (RSS) and are cherished for their ability to
randomly modify cysteine proteins and enzymes of the cellular
thiolstat.15 Such a wider “oxidative onslaught” in microorganisms
frequently results in pronounced toxicity, even in otherwise
resistant organisms, and RSS are often seen as promising
candidates in the battle against resistant bacteria and fungi.14-18
a.Division of Bioorganic Chemistry, School of Pharmacy, Saarland University,
Campus B2.1, D-66123 Saarbruecken, Saarland, Germany
Department of Technology and Biotechnology of Drugs, Faculty of Pharmacy,
b.
Jagiellonian University, Medical College, ul. Medyczna 9, 30-688 Cracow, Poland
c. Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Jagiellonian
University, Medical College, ul. Medyczna 9, 30-688 Cracow, Poland
d.Department of Medical Microbiology and Immunobiology, Faculty of Medicine,
University of Szeged, Dóm tér 10, H-6720 Szeged, Hungary
e. Department of Chemistry, Institute of Biology, Pedagogical University,
Podchorążych 2, 30-084 Kraków, Poland
f. Faculty of Chemistry, Jagiellonian University, ul. Gronostajowa 2, 30-387 Kraków,
Poland
† Footnotes relating to the title and/or authors should appear here.
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
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greatVeierw AarbticilleitOynlitnoe
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Compared to RSS, Reactive Selenium Species (RSeS) are target several cellular proteins, possess
a
considerably less common in Biology. Despite the overarching modulate the redox cycle in the cells and alsDoOinI:d1u0.c1e03e9le/Cv9aNteJd00le5v6e3Cls
importance of this trace element in humans, we actually find only a of apoptosis as compared to isothiocyanates.28 Since
handful of selenium secondary metabolites in higher organisms, isoselenocyanates have been explored already for biological
such as the ergothioneine-analogue selenoneine in blue tuna, applications, for instance against several kinds of cancers (e.g. lung,
whereas most selenium in higher organisms is incorporated in the colon, liver, prostate and breast) and infective diseases (e.g.
amino acids selenocysteine and selenomethioneine and forms part malaria, tuberculosis and leishmaniasis) we have shifted our focus
of a range of selenoproteins and selenoenzymes.19-21
to aromatic selenocyanates, which have received less attention and
also promise greatly improved physico-chemical properties.29-32
These compounds are intriguing as they are not only active on their
own, they are also often metabolized to a range of other RSeS, such
as selenols, diselenides and seleninic acids.33-37 Once again, such
organoselenium compounds are not “exotic” and - either directly or
as metabolites - play significant roles in Biology.38 Although
aromatic selenocyanates so far have mostly been studied when
attached to some other “bioactive” scaffolds which may have
influenced or even dominated their biological activity, they have
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The lack of a wider spectrum of selenium containing secondary
metabolites is rather surprising and somewhat disappointing. A
remarkable upsurge in activity and reactivity is often observed for
the selenium analogues of organosulfur compounds, and “going for
selenium” in form of selenium analogues of naturally occurring RSS
is quite attractive from the perspective of drug design, especially at
a time when certain selenium-containing organic compounds are
receiving renewed attention. Selenazole compounds, such as
ebselen (2-Phenyl-1,2-benzoselenazol-3-one), not only mimic the
activity of the selenium enzyme glutathione peroxidase, they have
already entered clinical trials in the context of mania and
hypomania.22-25 Moreover, naturally occurring (iso-)thiocyanates,
such as allyl isocyanate in mustard oil, and selenium analogues
thereof, e.g. primarily (iso-)selenocyanates, represent a particularly
promising class of compounds. Unfortunately, the most obvious
analogues of such natural antibiotics are either not stable
35, 39, 40
also been reported to exhibit leishmanicidal activity.
Moreover, the dietary intake of benzyl selenocyanate has been
reported to inhibit the incidence of small intestinal and colon
adenocarcinoma.41 Since hardly any wider investigations of such -
chemically quite stable - benzylselenocyanates are found in the
more biological literature, we have turned our attention to this
class of compounds first. Here we report the synthesis of aromatic
selenocyanate compounds, including four novel compounds (2, 8, 9
and 13), a pronounced and even somewhat selective antimicrobial
activity against pathogenic and resistant microorganisms and a set
of relevant pharmacological parameters which indicate a low(er)
risk to human cells.
chemically or extraordinarily difficult to synthesise and to handle
because of intense odour and inherent toxicity.26,
This is
27
particularly the case for the respective, chemically simple
isoselenocyanates, whilst replacement of sulfur with selenium in
aromatic isothiocyanate has been reported to enhance the
reactivity of compounds towards thiols in proteins.28 Additionally,
isoselenocynates demonstrate a higher reactivity towards GSH,
Figure 1. An “isosteric replacement” of sulfur for selenium is found in Nature where it leads from Reactive Sulfur Species (RSS), such as
ergothioneine, to Reactive Selenium Species (RSeS), such as selenoneine. In pharmaceutical research, this strategy encompasses a range of
(iso-)selenocyanates based on naturally occurring allyl(iso-)thiocyanates. Notably, the direct analogues of allyl(iso-)thiocyanates are rather
unpleasant substances. The aromatic counterparts (1-13), shown here with their synthesis and respective percentage yields, are more
promising candidates in the context of modern drug development.
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drawn at 30 % probability level. Partial packing views,ViinewdiAcratictilenOgntlhinee
intermolecular interactions in a layer of D(Oc)I: 110.1a0n3d9/C(d9)NJ1020.56T3hCe
intermolecular interactions are depicted as dashed lines.
Results and discussion
Chemical Synthesis. Thirteen arylmethyl selenocyanates (1-13,
Figure 1) were designed and synthesized based on basic
pharmacokinetic and pharmacodynamic considerations.42-44 The
compounds were produced employing appropriate commercially
available arylmethyl halides and potassium selenocyanate (KSeCN),
according to the general procedure described by Wheeler and
Merriam, and with minor modifications (Figure 1).45 All of the
compounds - of which four are novel - were obtained in good yields
6
7
8
9
Antimicrobial Activity. Once available, compounds 1-13 were pre-
screened for potential antimicrobial activity against selected Gram-
positive and Gram-negative bacteria, fungi and multicellular
nematodes. The antimicrobial activity was evaluated in terms of
Minimum Inhibitory Concentrations (MICs) and values were
compared to standard reference antimicrobial agents (Table 1).47-52
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1
(62–88 %) and structures of compounds 1-13 were confirmed by H
Overall, a significant and on occasion astonishingly selective
antimicrobial activity against highly pathogenic organisms has been
observed, frequently overcoming the kind of multidrug resistance
traditional antibiotics are faced with. The impact on cultured
human cells was considerably more modest, pointing towards a
possible selectivity against some - particularly nasty - pathogens.
Indeed, most of compounds displayed significant activities against
and 13C-NMR. Molecular mass and purity were determined by LC-
MS. (see Experimental Section and Electronic Supplementary
Information (ESI)).
Crystallographic Studies. In order to provide a deeper insight into
the structural properties, two selected compounds (1 and 12) were
studied by X-ray crystallographic analysis (Figure 2 and Table S1 in
ESI). In both crystal structures, the unit cells consisted of four
molecules. The values of bond lengths formed by the selenium
atom for C(sp)-Se were 1.850 Å and 1.837 Å for compound 1 and
12, respectively, whereas for C(sp3)-Se the bond length was 1.991 Å
for both structures. Similar values were observed in other crystal
structures with selenium atoms. The search of the CSD
demonstrated that the geometry of the methyleneselenocyanate
moiety in 1 and 12 is not exceptional among structures containing
this fragment. The crystal structure of 1 has been determined
earlier lacking hydrogen atoms.46
both, Gram-positive and Gram-negative members of
a most
obnoxious family of bacteria from the perspective of resistance, i.e.
ESKAPE pathogens, which comprise of Enterococcus faecium,
Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter
baumannii, Pseudomonas aeruginosa, and Enterobacter species.53
In the case of Gram-positive bacteria, the compounds were
examined against the reference strain (ATCC 25923) and the
multidrug resistant (MDR) clinical isolate of Staphylococcus aureus
(S. aureus, HEMSA 5). It is noticeable that all compounds, except
compound 11, displayed MIC values against the MDR strain which
were considerably lower than the ones for the reference antibiotic
oxacillin.47, 48 In the case of the most active compound, i.e. benzyl
selenocyanate (1) an excellent antimicrobial activity was observed
against HEMSA 5. Amazingly, the potency of this compound against
the resistant strain (MIC 0.76 µg mL-1 or 3.88 µM) was almost one
hundred fold higher than the reference drug oxacillin (MIC 128 µg
mL-1 or 318.86 µM) (Table 1).
Other compounds (2, 3, 6, 8, 9, 12 and 13) also demonstrated anti-
Staphylococcal activity with MIC values below 10 µg mL-1(62.5 µM).
Once more, these selenocyanates did not discriminate between
reference and resistant MDR strains of S. aureus, displaying similar
antibacterial potency against both, with an activity comparable or
even slightly higher against the MDR strain (12). Hence,
selenocyanates appear to overcome bacterial MDR, most likely by
circumventing the components responsible for resistance. Still, the
exact biochemical causes for this “resistance busting activity” are
unclear, and, as mentioned earlier, may also involve reactive
metabolic products of selenocyanates, such as selenols, diselenides
and seleninic acids, which together with the original selenocyanate
may interfere with microbial targets to overcome resistance.
Notably, most of these compounds were considerably less active
against the harmless Staphylococcus strain S. carnosus, with an
almost twenty to thirty-fold selectivity for HEMSA 5 over S.
carnosus observed for compounds 12 and 1, respectively. Except for
compounds 3, 6 and 13 which inhibited the growth of S. carnosus at
the concentrations of 13.19, 6.69 and 15.39 µg mL-1 (62.5µM, 31.25
µM and 62.5µM), respectively, no significant activity against this
reference strain could be found.
Figure 2. The molecular structure of (a) 1 and (b) 12, with the
appropriate atomic numbering scheme. Displacement ellipsoids are
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Table 1. Antimicrobial activity of arylmethyl selenocyanates against selected bacteria from non-ESKAPE and
ESKAPE families of bacteria and yeasts (1-13).
MICs (µg mL-1)
Compounds
6
7
8
9
S. aureus
ATCC25923
≤0.76
6.57
6.59
16.51
≥26.76
MRSA *
HEMSA 5
0.76
6.57
6.59
33.02
>26.76
6.69
28.8
7.2–14.4
8.28
A. baumannii
4184/2/5
1.53
6.57–13.14
13.19
33.01
26.76
13.38
14.4–28.8
7.2–14.4
8.28–16.56
34.38
>482
15.44
P. aeruginosa
4600
S. carnosus
C. albicans
S. cerevisiae
1
2
3
4
5
6
7
8
9
10
11
12
13
Ref.
24.48
26.27
13.19
264.08
53.52
6.69
230.4
115.28
33.13
275.04
30.125
123.09
15.39
0.03 a
6.12
52.54
26.37
33.01
12.24
52.54
13.19
264.08
6.69
6.69
7.4
28.82
33.13
275.04
7.53
61.55
30.78
0.09–4 d
24.48
52.54
6.59
66.04
53.52
6.69
460.8
28.82
66.25
68.76
60.25
123.09
61.56
0.03–2 e
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26.76
6.69
107.05–210.10
28.8
14.4–28.8
7.2–14.4
8.28
34.38
482
115.28
66.25
34.38
>34.38
482
7.72
>482
15.44
246.17
246.16
≤16 c
7.69
7.69
15.39
0.45 b
128 b
<4 b
* MRSA; methicillin-resistant S. aureus; MIC values for reference antibiotics: a ampicillin, b oxacillin, c piperacillin d fluconazole and e itraconazole.48-53. Bold
denotes significant values.
The antibacterial activity of the selenocyanates was not limited to bacteria, also possessed some activity against unicellular fungi. It
Gram-positive bacteria. Compound 1 also demonstrated excellent exhibited slightly higher fungicidal activity against pathogenic C.
antimicrobial activity against pathogenic Acinetobacter baumannii albicans (MIC 12.24 µg mL-1 (62.5µM)) compared to S. cerevisiae
(A. baumannii) and Pseudomonas aeruginosa (P. aeruginosa). Once (MIC 24.48 µg mL-1 (62.5-125 µM)). Such outcomes are promising in
again, compound 1, with a MIC of 1.53 µg mL-1( 7.8 µM) against A. terms of utilizing these compounds as antifungal agents, for
baumannii and 6.12 µg mL-1 (31.25 µM) against P. aeruginosa, was instance in the treatment of fungal infections, such as cutaneous,
more potent against these Gram-negative pathogens when oropharyngeal and vulvovaginal candidiasis etc.56-58
compared to the respective reference drug, in this case piperacillin
Nematicidal Activity. Although often ignored in developed
50
with a MIC of 16 µg mL-1 (30.92 µM) against P. aeruginosa.49,
countries, multicellular microorganisms, such as parasitic
Compounds 2, 3, 6, 9, 12 and 13 also demonstrated a significant,
nematodes, still represent important targets in drug design which
are particularly difficult to attack. In order to extend the scope of
our preliminary studies, the series of aromatic selenocyanates (1-
13) was evaluated for nematicidal activity against the agricultural
albeit slightly lower activity against A. baumannii (MIC values 6.57-
15.44 µg mL-1, i.e. 31.25-62.5 µM). Although the compounds exhibit
selectivity for both Gram-positive and Gram-negative bacteria, the
selectivity of compounds against pathogenic strains of Gram-
nematode S. feltiae, which often serves as a simple and reliably
negative bacteria is particularly stimulating from a pharmaceutical
representative model of a multicellular organism (Figure 3).
perspective, as Gram-negative bacteria are generally more difficult
to target, in part due to the specific structure and limited
permeability of their cell wall.54, 55
When evaluated against pathogenic yeast Candida albicans (C.
albicans), compounds 1, 3, 5, 7 and 11 revealed an encouraging
growth inhibitory activity at concentrations below 20 µg mL-1 (62.5
µM). Compounds 5, 7 and 11 prevailed with MIC values even below
10 µg mL-1 (31.25 µM). The fungistatic effect, however, is lower
compared to clinically relevant
antifungal drugs, such as
fluconazole and itraconazole ((MIC values 0.09-4 µg mL-1 (0.29-
13.06 µM) and 0.03-2 µg mL-1 (0.04-2.83 µM), respectively) (Table
1).46, 47 When compared to a non-pathogenic yeast, the fungicidal
activity of compound 7 exhibited a high level of selectivity towards
C. albicans, where it was more than 60-fold more active (MIC of 7.4
µg mL-1 (31.25 µM)) compared to baker’s yeast Saccharomyces
cerevisiae (S. cerevisiae)(MIC 460.8 µg mL-1 (2 mM)). Compounds 5
and 11 exhibited similar activities, although their selectivity
declined noticeably when compared to compound 7. Nonetheless,
these two compounds still maintained remarkably low MIC values
against the pathogenic yeast (6.69 µg mL-1 (31.25 µM) and 7.53 µg
mL-1 (31.25 µM), respectively) compared to non-pathogenic S.
cerevisiae (MIC 53.52 and 60.25 µg mL-1 (31.25-250 µM),
respectively). Compound 1, which was extraordinarily active against
Figure 3. Concentration-dependent activities of the most active
selenocyanates (from left 1, 9 and 11) against S. feltiae. PBS buffer
and ethanol (70 % v/v) and were employed as negative and positive
controls, respectively. Values represent mean ± S.D. *** p < 0.001
and ** p < 0.01.
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Figure 4. Different targets affected by a typical aromatic selenocyanate and relevant concentrations required to affect for these targets.
One may note the distinct differences in concentrations affecting resistant and non-resistant pathogens, normal and cancer cell lines (see
text for details).
After a pre-screen to determine the concentration range relevant 11 and 12 were more cytotoxic - and also selective. They displayed
for this organism, compounds were evaluated at four different significant cytotoxic activities against the parental and multidrug-
concentrations, i.e. at 3.75, 7.5, 15 and 30 µM. A remarkable, resistant sublines of mouse T-lymphoma cells and were less
concentration-dependent decrease in the viability of the cytotoxic against the non-cancerous NIH/3T3 cell line, pointing
nematodes was observed for all compounds, which was towards a slight, 3 to 10-fold selectivity against the cancer cells.
pronounced even at the lowest concentrations employed.
It is also notable, that the concentrations required for cytotoxicity
Compounds 9 and 11 exhibited the most significant nematicidal
in non-cancerous NIH/3T3 cells are generally two to three-fold
activity and decreased the viability of nematodes to less than 40 %
lower compared to the concentrations required for antimicrobial
at the concentration of 3.75 and 15 µM. These compounds
activity (see below).
exhibited LD50 values of 0.28 µM and 4.90 µM against S. feltiae,
Table 2. Cytotoxicity of arylmethyl selenocyanates against non-
cancerous and cancer cells.
respectively. Compound 1, which has shown considerable
antibacterial and antifungal activity (see above), also exhibited
significant nematicidal activity (LD50 = 6.85 µM) and decreased the
viability of nematodes to less than 40 % at the concentration of 15
µM. Other selenocyanates were less active.
Cytotoxicity against Mammalian Cell Lines
Mouse T-Lymphoma Cells
Non-
Cancerous
NIH/3T3
IC50
PAR
MDR
Cpd
SI
Although practical applications are more speculative at this time,
these results indicate that the aromatic selenocyanates may also
serve as excellent nematicidal agents, possibly against pathogenic
nematodes affecting plants, animals and humans - clearly a matter
of further investigation.
IC50
IC50
(µM)
7.69
9.72
SD ±
SD ±
SD ±
(µM)
5.84
5.33
9.03
89.18
>100
7.93
>100
8.58
8.53
>100
7.26
8.47
(µM)
31.00
55.12
29.58
>100
>100
59.94
>100
44.73
42.17
>100
70.08
24.18
0.45
0.41
0
2.33
-
0.36
-
0.18
0.39
-
0.66
1.1
0.18
-
0.76
0.55
1.52
≤0.89
n.d.
0.98
n.d.
0.89
0.83
n.d.
3.24
2.81
0.95
-
1
2
3
4
5
6
7
8
9
10
11
12
DOX
5.95
>100
>100
8.08
>100
9.64
10.32
>100
14.09
7.05
2.64
>2 %
v/v
Cytotoxicity of Arylmethyl Selenocyanates against Mammalian
Cells. In medicine, selenocyanates have been associated frequently
with outright toxicity. In order to rule out any major cytotoxicity
against mammalian cells and to investigate if there may be any
selectivity against microorganisms, compounds 1-12 were
investigated for their activity towards two cancer cell lines of mouse
T-lymphoma, i.e., the sensitive (PAR) and the multidrug resistant
cell line (MDR) transfected with the human MDR1 (ABCB1) gene
which codes for the ABC transporter, and a normal NIH/3T3 mouse
embryonic fibroblast cell line as control.
-
-
0.75
-
0.88
1.13
-
1.58
0.15
0.09
0.35
-
1.79
3.08
-
1.55
1.97
0.98
0.74
0.1
0.17
0.52
1.20
0.16
0.42
>2 %
v/v
13.38
>2 %
v/v
-
-
n.d.
-
DMSO
Compounds 1–12 exhibited some cytotoxicity against the non-
cancerous NIH/3T3 mouse fibroblast cell line at concentrations
ranging from 24 to above 100 µM, which was around two to five-
fold lower when compared to the anticancer reference drug,
doxorubicin and also significantly lower when pitched against the
activity of these selenocyanates affecting various microorganisms
(Table 2). Compounds 4, 5, 7 and 10 were the selenocyanates with
the lowest cytotoxicity against both T-lymphoma cell lines,
compared to doxorubicin. Intriguingly, compounds 1, 2, 3, 6, 8, 9,
PAR: parental T-lymphoma cells; MDR: multidrug resistant T-
lymphoma cells overproducing efflux pump ABCB1; NIH/3T3: non-
cancerous mouse embryonic fibroblast cells; DOX: doxorubicin;
DMSO: dimethyl sulfoxide; SD: standard deviation; SI: selectivity
index; n.d.: not determined.
The substantial activity of compounds 1, 3 and 12 against MDR cells
is worth noticing, despite the lack of any selectivity for particular
cell lines. Aromatic selenocyanates 1-3, 6, 8, 9, 11 and 12 may not
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only be of interest in the context of antimicrobial action - as characterized by a permeability coefficient (Kp). Since some of the
anticipated initially - they may also represent a starting point in the aromatic selenocyanates seem to enter cells readily and also appear
search for anticancer agents with MDR-reversing properties. Such to circumvent resistance based on efflux transporters, their
anti-cancer activity is a very complex issue and requires further transport properties were investigated employing a pre-coated
View Article Online
DOI: 10.1039/C9NJ00563C
investigations.
PAMPA Plate System Gentest™ (Corning, Tewksbury, MA, USA),
which provides good predictability and correlation of data for
absorption in the human Caco-2 cell line. The concentrations of the
compounds tested in the donor and acceptor compartments were
estimated by capillary electrophoresis (CE) as described
previously.60-62 The data on permeability obtained was compared to
the one for selected reference drugs, i.e., highly permeable caffeine
and lowly permeable norfloxacin (see ESI for details).
Pharmacological parameters associated with activity
Since the series of selenocyanates under investigation
demonstrated pronounced antimicrobial and nematicidal activities,
it was considered important to investigate their “drug-likeness”
profile. Four compounds (1, 2, 4 and 13) were selected for further
investigations employing in vitro assays indicative of safety and
absorption properties. The mutagenic potential and membrane
permeability of compounds were evaluated employing a modified
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60
Notably, all compounds investigated, i.e. 1, 2, 4 and 13, exhibited
good permeability with Kp values above the threshold for highly
permeable compounds (i.e. > 1.5 × 10−6 cm s-1).62 In this context, the
highest permeability was observed for compound 2 (Kp = 3.17 × 10-6
cm s-1) and compound 1 (Kp = 2.69 × 10-6 cm s-1) which are
comparable to the permeability of the reference drug caffeine (kP
=3.61 × 10-6 cm s-1).
Ames fluctuation assay and
a Parallel Artificial Membrane
Permeability Assay (PAMPA) (see ESI for details).53, 59-62
Ames fluctuation assay. Since inorganic sodium selenite (Na2SeO3)
is reputed for its toxic and mutagenic actions, the selenocyanates
were evaluated for potential mutagenic potential. The Ames
fluctuation assay was employed to calculate the mutagenic index
(MI) and binomial B-values according to the method described in
the literature.53 Compounds are generally considered mutagenic if
the MI is above 2.0 and B is equal to or above 0.99.53, 59, 60 Neither
the selenocyanates (1, 2, 4 and 13), nor the reference selenium
compound ebselen displayed any mutagenic potential at
concentrations of 1 µM and 10 µM (details in ESI), therefore ruling
out any major mutagenic potential. Compound 1 exhibited an MI
value of 1.15 (B= 0.74) and 1.20 (B= 0.81) at 1 µM and 10 µM
concentrations, respectively.
Whilst permeability may explain the ability of the selenocyanates
investigated to enter - and to remain inside - target cells, the
mode(s) of action underlying the biological activities associated
with these compounds are still elusive. Here, one may speculate
that selenocyanates may act as strong electrophiles, hence widely
modifying cysteine thiols and possibly also amine groups in proteins
and enzymes (Figure 5).63 Such redox modulating interactions with
proteins and enzymes of the cellular thiolstat may also explain, in
part, some of the selectivity observed for certain bacteria and cell
lines. Other activities, possibly exerted by the various metabolic and
breakdown products of the selenocyanates, may also be possible
and clearly require further investigation.
In Vitro PAMPA Permeability. The PAMPA permeability screening
test imitates the structural and biological conditions of the cell
membrane and allows for a rapid and simple determination of the
passive transport of a compound through biological membranes,
Figure 5. Selenocyanates and their metabolites exhibit a wide spectrum of redox activity, which includes electrophilic attacks, oxidative
modifications of thiol and selenol functions in proteins and enzymes, catalysis metal binding. The considerable impact on the cellular redox
homeostasis in general, and on the cellular thiolstat, in particular, may explain the pronounced biological activities observed as part of this
study.
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140.24, 131.29 (4 C), 129.67, 105.19 (Se-CN), 31.23. LC–MS:
View Article Online
DOI: 10.1039/C9NJ00563C
purity 99.49 %, tR = 6.86, (ESI) m/z: calculated for C8H5Cl2NSe
Experimental
[M + H]+: 158.98, found: 158.97.
Chemical Synthesis. 1H NMR and 13C NMR spectra were
recorded on a Varian Mercury-VX 300 MHz PFG instrument in
DMSO-d6 at ambient temperature employing the solvent signal
as an internal standard. The values of the chemical shifts are
expressed in δ values and the coupling constants (J) in Hz.
Mass spectra were recorded on a UPLC–MS/MS system
consisting of a Waters ACQUITY® UPLC® (Waters Corporation,
1-(Selenocyanatomethyl)naphthalene (13). 1-Chloromethyl
naphthalene (3.533 g, 20 mmol), KSeCN (3.6 g. 25 mmol) and
ethanol (20 mL) were employed. Compound 13 was obtained
as yellow crystals. Yield 75.5 % (3.71 g, 15.1 mmol); m.p.= 92-
1
94 °C, TLC Rf (DCM, 100 %): 0.64. H NMR (DMSO-d6, ppm): δ
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8.27 (d, J = 8.21 Hz, 1H, CH), 7.96 (dd, J1 = 7.03 Hz, J2 = 7.03
Hz, 2H, CH), 7.49 (m, 4H, CH), 4.78 (t, J = 9.38 Hz, 2H, CH2). 13
C
Milford, MA, USA) coupled to
a Waters TQD mass
NMR (DMSO-d6, ppm): δ 134.02, 130.87, 129.21, 128.61,
126.77, 125.73, 124.54, 105.33 (Se-CN), 31.09. LC–MS: purity
94.69 %, tR = 6.59, (ESI) m/z: calculated for C12H9NSe [M + H]+:
141.07, found: 141.03.
spectrometer (electrospray ionization mode ESI-tandem
quadrupole). The purity of final products was confirmed by
UPLC/MS to be higher than 95 %. Retention times (tR) are
provided in min. Thin-layer chromatography (TLC) was
performed on pre-coated Merck silica gel 60 F254 aluminium
sheets.
General Procedure for the Synthesis of Selenocyanates.
Selenocyanates were synthesized following the general
protocol described by Wheeler and Merriam with some
modifications.45 Alkyl halides (10-20 mmol) were treated with
KSeCN (12-25 mmol) in ethanol (10-20 mL). The reaction
mixture was refluxed for 6 h and progress of the reaction was
monitored periodically by TLC. After completion, the inorganic
salt was separated by filtration and the filtrate was purified
with charcoal, condensed and crystallized with ethanol, to
X-Ray Crystallography. Single crystals suitable for X-ray
analysis were obtained in ethanol for
12 by slow evaporation of the solvent at room temperature.
Intensity data of was collected on the Bruker-Nonius Kappa
1 and in butan-2-ol for
1
CCD four circle diffractometer, whereas data for 12 was
collected on an Oxford Diffraction SuperNova Diffractometer
equipped with a Mo (0.71069 Å) Kα radiation source. The
positions of non-hydrogen atoms were determined by the
direct method employing the SIR-2014 programme.66
Hydrogen atoms bonded to carbons atoms were included at
idealized positions and were refined utilising a riding model.
The aryl hydrogen atoms were constrained with C-H 0.93 Å,
the methylene groups with C-H 0.97 Å and Uiso(H) = 1.2 Ueq.
The final refinements were performed by the SHELXL
programme. ORTEP and MERCURY programmes were
employed for molecular graphics.67-69
Compound 1: C8H7NSe, Mr = 196.11, crystal size 0.08 × 0.16 ×
0.46 mm3, monoclinic, space group P21/c, a = 5.9880(1) Å, b =
7.4440(2) Å, c = 17.4880(5) Å, β = 96.277(2)°, V = 774.85 Å3, Z
= 4, T = 100(2) K, 6844 reflections collected, 1786 unique
reflections [RINT = 0.0326], R1 = 0.0226, wR2 = 0.0521 [I >
2σ(I)], R1 = 0.0226, wR2 = 0.0536 [all data].
Compound 12: C12H9NSe, Mr = 246.16, crystal size 0.25 × 0.48
× 0.60 mm3, monoclinic, space group Ia, a = 8.2486(2) Å, b =
5.9838(1) Å, c = 20.4158(7) Å, β = 93.097(3)°, V = 1006.23 Å3, Z
= 4, T = 130(2) K, 4415 reflections collected, 2048 unique
reflections [RINT = 0.0300], R1 = 0.0368, wR2 = 0.0921 [I >
2σ(I)], R1 = 0.0397, wR2 = 0.0947 [all data].
CCDC 1819893-1819894 contains the Supplementary
Crystallographic Data for this manuscript. This data can be
obtained free of charge from The Cambridge Crystallographic
Data Centre via www.ccdc.cam.ac.uk/data_request/cif.
yield crystals of arylmethylselenocyanates (
1-13).
Compounds and 10 12 have been described in the
1
,
3
-
7
-
literature before and analytical data for these compounds is in
agreement with the values reported (see ESI).40, 64, 65
4-Methylbenzyl Selenocyanate (2). 4-Methylbenzyl chloride
(1.4 g, 10 mmol), KSeCN (1.73 g, 12 mmol) and ethanol (10 mL)
were employed. Compound
2 was obtained as light crystals.
Yield 83.6 % (1.76 g, 8.36 mmol); m.p.= 43-45 °C, TLC Rf (DCM,
1
100 %): 0.51. H NMR (DMSO-d6, ppm): δ 7.23 (d, J = 7.62 Hz,
2H, 2 C-H), 7.17 (d, J = 8.21 Hz, 2H, 2 C-H), 4.27 (s, 2H, CH2),
2.28 (t, J = 9.10 Hz, 3H, CH3). 13C NMR (DMSO-d6, ppm): δ
137.62,135.65, 129.61,129.22,105.39 (Se-CN), 33.08, 21.22.
LC–MS: purity 100 %, tR = 6.25, (ESI) m/z: calculated for
C9H9NSe [M + H]+: 105.07, found: 105.02.
3-Chlorobenzyl Selenocyanate (8). 3-Chlorobenzyl chloride
(3.22 g, 20 mmol), KSeCN (3.60 g. 25 mmol) and ethanol (20
mL) were employed. Compound
crystals. Yield 82.55 % (3.81 g, 16.51 mmol); m.p.= 40-42 °C,
TLC Rf (DCM, 100 %): 0.75. 1H NMR (DMSO-d6, ppm): δ 7.42(m,
1H, CH), 7.37 (m, 2H, CH), 7.33 (m, 1H, CH), 4.28 (t, J = 9.15 Hz,
2H, CH2). 13C NMR (DMSO-d6, ppm): δ 141.45, 133.38, 130.95,
129.04, 128.17, 128.00, 105.23 (Se-CN), 31.96. LC–MS: purity
98.95 %, tR = 6.24, (ESI) m/z: calculated for C8H6ClNSe [M + H]+:
125.02, found: 125.02.
3,4-Dichlorobenzyl Selenocyanate (9). 3,4-Dichlorobenzyl
chloride (3.91 g, 20 mmol), KSeCN (3.6 g. 25 mmol) and
ethanol (20 mL) were employed. Compound
8 was obtained as light
Antimicrobial Activity. The minimal inhibitory concentrations
(MICs) were determined by the standard microdilution method
in cation-adjusted Mueller-Hinton II Broth (MHB, Becton-
Dickinson, Germany) according to the recommendations of the
Clinical and Laboratory Standard Institute (CLSI).70 The
compounds (1-13) were evaluated for their antimicrobial
9 was obtained as
activity against a broad spectrum of microorganisms, including
Gram-positive bacteria (S. carnosus and S. aureus), Gram-
negative bacteria (A. baumannii and P. aeruginosa) and yeasts
(C. albicans and S. cerevisiae). The values of MIC were
recorded after 20 h and 24 h incubation of compounds for
yellow crystals. Yield 88.2 % (4.673 g, 17.64 mmol); m.p.= 76-
79 °C, TLC Rf (DCM, 100 %): 0.60. H NMR (DMSO-d6, ppm): δ
1
7.62 (m, 2H, CH), 7.37 (dd, J1 = 2.12 Hz, J2 = 2.09 Hz, 1H, CH),
4.28 (t, J = 9.15 Hz, 2H, CH2). 13C NMR (DMSO-d6, ppm): δ
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concentrations comparable to or even below the ones of
bacteria and yeasts, respectively. Experiments were performed
in triplicate and on three different occasions (i.e., a total of
nine repeats for each individual measurement).
Nematicidal Activity. S. feltiae was obtained from Sautter and
Stepper GmbH (Ammerbuch, Germany). The assay was
performed according to an established literature protocol.71, 72
Results are provided as means ± SD. GraphPad Prism 5 was
employed to perform the statistical analysis. Statistical
significances were calculated by employing one-way ANOVA,
with p < 0.05 considered to be statistically significant.
Cytotoxicity Assays. L5178 mouse T-cell lymphoma cells (PAR)
(ECACC Cat. No. 87111908, obtained from FDA, Silver Spring,
MD, USA) were transfected with pHa MDR1/A retrovirus, as
described previously by Cornwell et al..73 The NIH/3T3 mouse
embryonic fibroblast cell line (ATCC CRL-1658) was purchased
from LGC Promochem, Teddington, UK. The cell line was
cultured in Dulbecco’s Modified Eagle’s Medium (DMEM,
containing 4.5 g L-1 glucose) supplemented with 10 % heat-
inactivated fetal bovine serum. The cell line was incubated at
37 °C in a 5 % CO2, 95 % air atmosphere. Cytotoxicity assays
were performed following the procedure described in the
literature.74, 75
Mutagenicity assay. The Salmonella typhimurium TA100 strain
with base pair substitution (hisG46 mutation, whose target is
GGG) was purchased from Xenometrix, Allschwil, Switzerland,
and employed in the Ames 384-well microtiter assay.76 Prior to
the experiment, the Salmonella typhimurium TA100 strain was
cultivated overnight (NB-2 liquid medium in the presence of 25
µg mL-1 ampicillin). Then, all of the compounds were assayed
according to the microtitre liquid Ames fluctuation protocol
described in the literature.76 NQNO was utilized as a positive
control in the mutagenicity assays. This reagent causes point
mutations in the genome as it induces G:C→A:T transitions in
the Salmonella typhimurium TA-100 strain.76
View Article Online
traditional antibiotics, such as ampicillin, oxacillin and
DOI: 10.1039/C9NJ00563C
piperacillin. In the case of the most aggressive drug-resistant
strains of S. aureus, the activity of some of these
selenocyanates even seems to supersede the one of oxacillin,
which is not active against these dangerous pathogens.
Similarly, an excellent activity has been noted against Gram-
negative organisms, such as A. baumannii and P. aeruginosa,
and against a pathogenic yeast, C. albicans. In the case of
yeasts, a surprisingly high activity against infectious C. albicans
and a surprisingly low activity against baker’s yeast has been
observed for several compounds, which besides an astonishing
antibacterial action also promises some interesting selectivity
within the fungal kingdom.
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Additional studies are now required to elucidate the
underlying mode(s) of action and to enhance the activity and
selectivity of these agents. Here, the notion of a random attack
of such selenocyanates - and their respective metabolites -
against a range of redox-sensitive cysteine proteins and
enzymes composing the cellular thiolstat of the target
organisms may serve as a first hypothesis (Figure 5). Such a
more random attack is not uncommon within the realm of
chalcogen redox chemistry and may also explain the ability of
such electrophiles to overcome the traditional, more focussed
mechanisms of drug resistance.
Here, it should be emphasized that such seemingly
indiscriminate modifications of certain proteins and enzymes
are not necessarily preventing selectivity, as the defence
mechanisms against such oxidative onslaughts tend to differ
dramatically between different target organisms, and also
between targets and healthy human cells. Notably, our initial
studies de facto hint at a low(er) (cyto-)toxicity against human
cells, and relevant toxicity studies in higher organisms are
clearly warranted now. As for other redox modulating drugs,
such a low(er) activity may be due to the presence of a
pronounced antioxidant defence in human cells, which is often
lacking in smaller organisms. Still, this is speculative at this
time and provides space for further studies, also addressing
questions concerning any indiscriminatory activity and toxicity.
In Vitro PAMPA Permeability Assay. Compounds 1, 2, 4, and
13 and reference substances were dissolved in PBS buffer (pH
= 7.4) from 10 mM DMSO stocks, according to a protocol
described previously.60 The concentrations of compounds and
references drugs - in this case caffeine, and norfloxacin - were
estimated in the donor and acceptor compartments employing
capillary electrophoresis (CE), and calibration curves were
determined accordingly. Finally, the permeability coefficients
(Kp, (cm s-1)) of the compounds tested were calculated
employing the formula provided by the PAMPA Plate System
manufacturer.60, 62
Furthermore, sulfur and selenium compounds are notorious
for their unpleasant odour and one may indeed “sniff a rat”
here when considering the smell of several naturally occurring
compounds, such as polysulfanes from garlic and allyl
isothiocyanate in mustard oil.26, 77, 78 Since such a smell is due
to high volatility of compounds, and aromatic selenocyanates
are fairly stable solids, odour, and any apparent toxicity which
may traditionally be associated with it, are not of any major
concern. In fact, this aspect has been considered carefully as
part of the selection of suitable compounds, as depicted in
Figure 1.
Conclusions
In any case, selenocyanates and their closely related
isoselenocyanates represent an interesting addition to the
menu of selenium agents able to break through the resistance
mechanisms of dangerous pathogens.47 Once developed and
studied in more detail, they may spice up the search for the
next generation of effective antibiotics with a specific culinary
note of matured broccoli-mustard.
The comprehensive studies presented in the previous sections
have provided new insights into the chemistry and biological
activity of small aromatic selenocyanates, which may be
valuable in the search for new antimicrobial agents. It is
particularly noteworthy that several of the compounds
investigated, in particular benzyl selenocyanate (
1
), exhibit
considerable activity against Gram-positive and Gram-negative
bacteria at concentrations below
1
μg mL-1, i.e. at
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14. M. O. Ko, M. B. Kim and S. B. Lim, J Microbiol BiVoiteewcAhrtnic,le2O0n1lin6e,
26, 2036-2042.
15. C. Jacob, Biochem Soc T, 2011, 39, 1247-1253.
Conflicts of interest
There are no conflicts to declare
DOI: 10.1039/C9NJ00563C
16. V. Dufour, M. Stahl and C. Baysse, Microbiology+, 2015, 161
,
Author Contributions: M.J.N., K.W., G.S., J.H. and C.J. conceived
and designed the experiments; M.J.N. synthesized the compounds;
E.Ż. and W.N. performed crystallographic studies; M.J.N. and K.W.
performed the experiments with microbes; A.K., M.G. and G.S.
performed the experiments with mammalian cells; M.A.M. and
M.J.N. performed ADMET studies in vitro; G.L. and K.K.-K.
supervised ADMET studies in vitro; E.K. supervised microbiological
studies; M.J.N., K.W., A.Y.A, C.J. and J.H. wrote the paper.
229-243.
17. E. L. Thomas and T. M. Aune, Infect immun, 1978, 20, 456-
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18. J. D. Oram and B. Reiter, Biochem J, 1966, 100, 382-388.
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University,
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(project
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K/ZDS/005593), University of Saarland, Saarbruecken,
Germany and INTERREGVAGR program (BIOVAL, Grant No. 4-
09-21). We also acknowledge the support of Erasmus +
mobility programme 2016–2017. Special thanks go to many
other colleagues from the Academics International network
(www.academiacs.eu) and “Pharmasophy” for their helpful
discussions and advice. Gabriella Spengler was supported by
the János Bolyai Research Scholarship of the Hungarian
Academy of Sciences. Annamária Kincses and Márió Gajdács
were supported by the UNKP-17-3 New National Excellence
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Pronounced activity of aromatic selenocyanates against multidrug
resistant ESKAPE bacteria
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
Muhammad Jawad Nasima,b Karolina Witek b,c, Annamária Kincsesd, Ahmad Yaman Abdina Ewa
Żesławskae, Małgorzata Anna Marćb, Márió Gajdácsd, Gabriella Spenglerd, Wojciech NitekF,
Gniewomir Lataczb, Elżbieta Karczewskac, Katarzyna Kieć-Kononowiczb, Jadwiga Handzlikb,* and
Claus Jacoba,*
www.rsc.org/
.
Selenocyanates demonstrate pronounced activity against bacteria of ESKAPE family, yeast and nematodes with limited cytotoxicity against
human cells
This journal is © The Royal Society of Chemistry 20xx
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