Exploration of piperazine-derived thioureas as antibacterial and anti-inflammatory agents. In vitro evaluation against clinical isolates of colistin-resistant Acinetobacter baumannii

Article abstract of DOI:10.1016/j.bmcl.2020.127411

A. baumannii is one of the most important multidrug-resistant microorganisms in hospital units. It is resistant to many classes of antibiotics and the development of new therapeutic strategies is necessary. The aim of this study was to evaluate the antiba

Full text of DOI:10.1016/j.bmcl.2020.127411

Journal Pre-proofs
Exploration of piperazine-derived thioureas as antibacterial and anti-inflam‐
matory agents. In vitro evaluation against clinical isolates of colistin-resistant
Acinetobacter baumannii
Sarah Mazzotta, Tania Cebrero-Cangueiro, Luca Frattaruolo, Margarita Vega-
Holm, Marta Carretero-Ledesma, Javier Sánchez-Céspedes, Anna Rita
Cappello, Francesca Aiello, Jerónimo Pachón, José Manuel Vega-Pérez,
Fernando Iglesias-Guerra, María Eugenia Pachón-Ibá ñez
PII:
S0960-894X(20)30522-9
DOI:
Reference:
https://doi.org/10.1016/j.bmcl.2020.127411
BMCL 127411
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Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
25 April 2020
6 July 2020
12 July 2020
Please cite this article as: Mazzotta, S., Cebrero-Cangueiro, T., Frattaruolo, L., Vega-Holm, M., Carretero-
Ledesma, M., Sánchez-Céspedes, J., Rita Cappello, A., Aiello, F., Pachón, J., Manuel Vega-Pérez, J., Iglesias-
Guerra, F., Eugenia Pachón-Ibá ñez, M., Exploration of piperazine-derived thioureas as antibacterial and anti-
inflammatory agents. In vitro evaluation against clinical isolates of colistin-resistant Acinetobacter baumannii,
Bioorganic & Medicinal Chemistry Letters (2020), doi: https://doi.org/10.1016/j.bmcl.2020.127411
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Exploration of piperazine-derived thioureas
as antibacterial and anti-inflammatory
agents. In vitro evaluation against colistin-
resistant Acinetobacter baumannii
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Sarah Mazzotta, Tania Cebrero-Cangueiro, Luca Frattaruolo, Margarita Vega-Holm, Marta Carretero-Ledesma,
Javier Sánchez-Céspedes, Anna Rita Cappello, Francesca Aiello, Jerónimo Pachón, José Manuel Vega-Pérez,
Fernando Iglesias-Guerra, Maria Eugenia Pachón-Ibáñez

Bioorganic & Medicinal Chemistry Letters
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Exploration of piperazine-derived thioureas as antibacterial and anti-inflammatory
agents. In vitro evaluation against clinical isolates of colistin-resistant Acinetobacter
baumannii.
Sarah Mazzottaa,d, Tania Cebrero-Cangueirob,c, Luca Frattaruolod, Margarita Vega-Holma*, Marta
Carretero-Ledesmab,c, Javier Sánchez-Céspedesb,c, Anna Rita Cappellod, Francesca Aiellod, Jerónimo
Pachónc,e, José Manuel Vega-Péreza*, Fernando Iglesias-Guerraa, María Eugenia Pachón-Ibáñezb,c
aDepartment of Organic and Medicinal Chemistry, Faculty of Pharmacy, University of Seville E-41071, Seville, Spain
bClinical Unit of Infectious Diseases, Microbiology, and Preventive Medicine, University Hospital Virgen del Rocío, CSIC, University of Seville. Seville, Spain
cInstitute of Biomedicine of Seville. University Hospital Virgen del Rocío, CSIC, University of Seville, Seville, Spain
dDepartment of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036 Arcavacata di Rende (CS) Italy
eDepartment of Medicine. University of Seville. Seville, Spain
S.M., T.C.C contributed equally to this work (joint first authors)
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Accepted
Available online
A. baumannii is one of the most important multidrug-resistant microorganisms in hospital units.
It is resistant to many classes of antibiotics and the development of new therapeutic strategies is
necessary. The aim of this study was to evaluate the antibacterial activity of a set of piperazine-
derived thioureas against 13 clinical strains of colistin-resistant A. baumannii. Six derivatives
were identified to inhibit bacterial growth of 46% of the A. baumannii strains at low micromolar
concentrations (Minimum Inhibitory Concentration from 1.56 to 6.25 μM). A common structural
feature in most active compounds was the presence of a 3,5-bis-trifluoromethyl phenyl ring at
the thiourea function. In addition, the ability of the compounds to inhibit production of nitric
oxide (NO) was examined in RAW 264.7 murine macrophages, highlighting the potential of
piperazine-derived thioureas as promising scaffolds for the design of new combined anti-
bacterial/anti-inflammatory agents.
Keywords:
Piperazine thiourea derivatives
Antibacterial agents
Multidrug-resistant
Acinetobacter baumannii
Anti-inflammatory
2009 Elsevier Ltd. All rights reserved.
Multidrug resistance (MDR) in bacteria is an emerging
problem for public health all over the world. The inappropriate
overuse of antibiotics for the treatment of infectious diseases
contributes to the insensitivity of bacteria strains to these drugs.1
Many nitrogen heterocycles derivatives with potential
antimicrobial activity against A. baumannii have been described
recently. Synthetic sets of pyrazole derivatives were prepared and
evaluated against A. baumannii. (1, Figure 1) Compounds with
halogen substituents on the N-aryl moiety as fluorine, chlorine or
bromine are the most active (lowest MIC value 1.56 μg/mL).8,9
Also, novel 2-(pyrrolidin-1-yl)-thiazole derived compounds (2,
Figure 1)10 and 1,2,5-oxadiazoles were identified as new potential
anti-Acinetobacter agents, reaching modest activity ( lowest MIC
0.5 mM) in the case of oxadiazole derivatives.11
Acinetobacter baumannii is a gram-negative bacillus (GNB)
of great clinical relevance for being a MDR and extensive drug
antimicrobial resistant bacterium (XDR), also carbapenems-
resistant (CRAb). It is an important healthcare-associated
microorganism that causes a high number of nosocomial
infections in hospitalized patients of intensive care units.2,3 Many
nosocomial infections caused by it involve organ systems
containing high levels of fluids, such as blood stream infections
(BSI), septicemia and pneumonia, with high mortality rates.4
During the years, the antimicrobial resistance of different species
of A. baumannii has increased dramatically. Colistin (COL) is
one of the last therapeutic options for the treatment of these
infections, though resistance to polymyxins has been also
reported.5,6 There is therefore a need to find novel active
compounds with growth inhibition properties against A.
baumannii strains.1,7
In the last few years, many compounds with piperazine core
have been assessed as new potential antibiotics. Compounds
having 4-aminoquinoline moiety and 1,3,5- triazine core linked
through a piperazine ring have been prepared (3, Figure 1) and
have shown activity against GNB (MIC from 3.12 to 6.25
μg/mL). A thiourea function was employed to connect the
piperazine ring with the other moieties.12 In other studies, the
piperazine ring is part of molecular hybrids. Recently, the
synthesis of new cumarine-piperazine derivatives with good
antimicrobial and anti-inflammatory properties has been
described.13 Moreover, some conjugates of adamantane and

piperazine/morpholine through an isothiourea bridge have been
reported as growth inhibitors of gram-positive Staphylococcus
aureus and Bacillus subtilis, (MIC from 0.25 to 8 μg/mL). The
most active compounds presented a 3,5-bis-trifluoromethyl group
on the phenyl ring (4, Figure 1).14 At present, we have found one
study that reports piperazine-derived compounds as potential
antimicrobial agents including anti-A. baumannii activity.15
Scheme 1. Synthetic route for the piperazine-derived thioureas.
Table 1. 4-Acyl-1-phenylaminothiocarbonyl-2-substituted-
piperazine derivatives 5-43.
O
O
R1
S
R1
N
O
N
H
H
In this letter we report the biological evaluation of a set of 39
N
N
R5
R4
N
N
R5
R4
4-N-acyl-1-phenylaminothiocarbonyl-2-substituted
piperazine
S
derivatives (34 of them previously described as new anti-
adenovirus compounds,16 and 5 described here) against 13
colistin-resistant A. baumannii clinical strains. We decided to
develop this preliminary screening based on the presence of those
privileged structures (the piperazine ring and the thiourea
function) of the general backbone of these molecules, that are
present in reported antimicrobial agents.17 As shown in Figure 1
the general scaffold consists on a piperazine ring as central core
with a phenylaminothiocarbonyl group at nitrogen 1 and different
acyl groups at nitrogen 4 (compounds 5-43).
B
A
R3
R5
R3
R5
R4
R3
R4
R3
S
S
N
N
N
N
H
H
N
N
R1
R1
O
O
C
D
Comp.
Structure
A
R1
R3
R4
R5
F
F
5
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
H
H
H
H
H
H
H
NO2
Cl
H
Cl
6
7
8
9
H
H
O
Ph
Ph
A
A
A
A
A
A
PhO2S
N
N
N
NH
O
S
N
CN
F
N
Br
Ph
O
CO2Me
H
1
OH
2
CF3
H
R1
S
R2
N
H
N
R5
R4
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
OCH3
CH3
H
H
N
H
H
N
R3
5-43
F3C
CF3
N
CF3
H
CF3
H
A
B
N
N
S
NO2
Cl
H2N
S
N
N
N
H
N
H
H
B
B
B
B
B
B
B
C
C
C
C
C
C
C
C
D
D
N
H
Cl
N
N
N
H
CN
F
H
3
4
N
H
H
H
CF3
OCH3
CH3
H
H
Figure 1. Some reported nitrogen heterocyclic compounds with
antibacterial activity (1-4) and general structure of piperazine-
derived anti-Acinetobacter agents (5-43).
H
H
H
H
The synthetic methodology was a brief and high yielded route,
previously described, that consists of two reactions.16 The first
CF3
H
CF3
H
NO2
Cl
step involved
a chemoselective N-acylation reaction of
commercial 2-substituted piperazines and provided the
introduction of an amide or urethane functions at the less
hindered nitrogen. From these monoacyl derivatives the thiourea
group was introduced at the other nitrogen by the reaction with
the appropriate phenylisothiocyanate (Scheme 1).18 Spectral
(NMR, mass spectrometry) and analytical data of the new
synthesized compounds (39-43)19 were in full agreement with the
proposed structures (Supplementary Information)
H
H
H
CN
F
H
H
H
H
CF3
OCH3
CH3
H
H
H
H
H
H
R1
O
R1
R1
R5
R4
O
H
i
N
R2
ii
N
CF3
H
CF3
H
N
NH
N
HN
NH
R2
S
NO2
Cl
R3
H
H
i: starting material 1 eq, acyl halyde or anhydride 1 eq, piridine 1.5 eq, dichloromethane
ii: monoamide 1 eq, isothiocyanate 1.2 eq, dichloromethane

39
40
-
3.12
12.5
-
12.5
6.25
-
-
-
127
3.12 124.6
108.6
6.25 162.1
Comp.
31
Structure
R1
CH3
CH3
CH3
CH3
CH3
CH3
Ph
R3
H
R4
CN
F
R5
H
-
6.25
-
-
6.25
-
3.12
6.25
6.25
6.25
1772
6.25
12.5
12.5
6.25
3545
D
D
D
D
D
D
B
D
A
C
A
B
B
41
-
32
H
H
42
33
H
CF3
OCH3
CH3
H
H
43
-
-
-
12.5
7
73.5
-
34
H
H
COL
14
56
> 111
35
H
H
a Cytotoxic concentration 50%.
36
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
Most of the monosubstituted derivatives at para position of
the phenyl ring, having electron-releasing groups (R3 = OCH3,
CH3) or electron withdrawing groups (R3 = NO2, Cl, CN, F, CF3)
exhibited MIC values from 25 to 50 µM (Supplementary
Information, Table S13). Only compound 25 (2-cyclohexylacetyl
derivative) with R3 = CF3 showed 6.25 and 3.12 µM against the
Ab22R and Ab24 strains, respectively.
37
H
38
Ph
H
39a
40a
41a
42a
43a
Ph
H
Ph
H
H
H
The most active compounds (lowest values of MIC) were
analogues 20, 28 and 36-38. The presence of a 3,5-bis-
trifluoromethyl phenyl ring at the thiourea function seems to be
crucial to improve the in vitro activity (reduced MIC values,
from 1.56 to 6.25 M). Depending on the acyl group at N-4, they
were active against different strains. Urethane derivative 12 was
active only against Ab 14 with low MIC (3.12 M) while amide
derivatives (20, 28 and 36) were very active against, mainly,
three different strains, Ab 21R, Ab 22P and Ab 24. 28 (2-
cyclohexylacetyl derivative) gave the lowest MIC (1.56 M
against Ab 22P) and 36 (2-phenylacetyl derivative) gave also low
MIC values (3.25 M and 6.25 M) with a broad spectrum of
activity (five strains). From the methyl pipeperazine-derived
thioureas collection, compounds 12, 28 and 36 were selected.
(R)-CH3
(S)-CH3
H
H
aNew compounds
The antibacterial activity of these piperazine derivatives was
evaluated through a growth inhibition screening, against 13
clinical colistin-resistant A. baumannii (Ab) strains from an
outbreak in 2002 in the University Hospital Virgen del Rocío of
Seville.20
Briefly, the inhibition activity at 50 µM of each compound
was carried out in 96-well plate to identify which compounds
produced growth inhibition of 5x105 Unit Forming Colonies
(UFC/ml) of the tested strains. Compounds were found to inhibit
the bacterial growth of 46% of strains.21 It is important to note
that our active compounds showed growth inhibition activity
against Ab 11, Ab 14, Ab 17, Ab 21R, Ab 22P and Ab 24
(Supplementary Information, Table S10). Then, the Minimal
Inhibitory Concentration (MIC) was determined for those
compounds that showed growth inhibition in the previous assay
by the standard broth microdilution method.22 MIC values of
COL were also determined. (Supplementary Information, Table
S12). Table 2 shows those compounds with MIC values ≤ 12.5
µM.
Keeping this substitution pattern at the phenyl ring, the 2-
methyl piperazine central core was exchanged with 2-
phenylpiperazine to give analogues 37-40. We focused not only
on trying to reduce MIC values but also on increasing the number
of strains that could be affected by these compounds. They were
active against the same strains than their methyl analogues.
Regarding the acyl group at N-4, 3,3-dimethylbutanoyl derivative
37 and 2-cyclohexylacetyl derivative 40 achieved lower MIC
values (3.12 µM) than their 2-methyl piperazine analogues
against most of the strains. However, compound 38, 2-
phenylacetyl derivative, did not improve the activity of 36.
Compound 41 (with an unsubstituted piperazine core) was
prepared as an achiral analogue of 12 (2-methylpiperazine core)
and 39 (2-phenylpiperazine core). It showed low MIC against
five strains, increasing the spectrum of activity compared to its
analogues.
Table 2. MIC values (µM) of the selected piperazine-derived
thioureas and COL against colistin-resistant A. baumannii
clinical strains and CC50 values (M).
MIC (µM) Acinetobacter baumannii
CC50
Comp
(µM)a
11
14
3.12
50
-
17
21R
-
22P
-
24
-
We have also prepared both pure enantiomers of the racemic
compound 20 (42 and 43). They were both active against the
same strains and showed almost similar MIC values than 20
(Table 2).
12
20
21
22
25
28
36
37
38
-
-
112.4
73.2
-
-
6.25
25
6.25
12.5
12.5
6.25
1.56
-
6.25
-
As it was one of the of the broadest spectrum derivatives,
including the two most colistin-resistant strains (Ab 21R, Ab
22P), compound 41 was chosen to develop bactericidal studies
against them by time kill curves. We tested it at different
conditions: 1xMIC (6.25 µM), 2xMIC (12.5 µM), and 4xMIC
(25 µM) for each strain. We found that compound 41 showed
bactericidal effect at 4xMIC after 4 hours against Ab21R.
Furthermore, it reached a reduction close to 3-log10 after 4 hours
at 2xMIC, compared to the initial inoculum, against Ab21R
(Figure 2). No bactericidal activity was observed at any condition
against Ab22P strain (Figure 3).
-
-
148.1
200.0
-
50
12.5
50
3.12
-
-
12.5
25
25
-
-
-
-
3.12 142.2
25 82.0
6.25 129.7
3.12
3.12
3.12
-
6.25
3.12
-
3.12
-
3.12
-
3.12
-
91.8
-
3.12
104.3

For compounds exhibiting good antimicrobial activity it is
important to study if they can selectively kill microorganisms
without being significantly toxic to host cells. All active
compounds were tested towards A549 cell line at several
concentrations (Supporting Information) and their CC50 values
were also calculated (Supplementary Information, Table S13).
Selectivity index (SI) of selected compounds (12, 28, 36, 37, 40
and 41) was determined (Supplementary Information, Table
S13). The results showed that these compounds had relatively
high safety index, with values in the range of 8.69-52.56, being
most of them > 20, suggesting high selective toxicity towards
bacterial cells over host ones.24-26
Microbial infections are always associated with the triggering
of inflammatory processes, necessary for the body to fight the
cause of the damage and stimulate the healing process.27
However, if the inflammatory process becomes chronic it can
lead to severe tissue damage that can be associated with a loss of
the functionality of the affected tissue, providing greater harm
than benefit.28 In recent years, numerous in vitro and in vivo
studies highlighted the unexplored anti-inflammatory potential of
molecules having a piperazine moiety.13,29-32 This action seems to
be due to the ability of these molecules to act, in macrophages, as
antagonists of histamine receptors ,29,33,34 involved in the
modulation of different inflammatory mediators, such as NO,
pro-inflammatory cytokines and TNF-α.35,36 Furthermore, several
studies have highlighted the remarkable versatility of piperazine-
scaffold, whose anti-inflammatory activity often coexists with the
anti-bacterial one.37-39
Figure 2. Bactericidal activity of compound 41 against A.
baumannii 21R.
This scenario prompted us strongly to evaluate the anti-
inflammatory potential of the piperazine-derived thioureas
subject of this study, in order to reveal their possible double
effect that would find a useful use in the clinical practice. The
anti-inflammatory potential was assessed by monitoring the
ability of different compounds to inhibit, in the macrophage line
RAW 264.7, the production of nitric oxide (NO), one of the main
mediators of phlogistic processes.40
Figure 3. Bactericidal activity of compound 41 against A.
baumannii 22P.
Considering the drawbacks of COL and other potential active
antibiotics in monotherapy, combination therapy has been raised
as an interesting strategy to overcome these limitations. The
synergistic activity of 41 in combination with standard antibiotic
COL against Ab 21R and Ab 22P was also investigated.23 The
outcomes were determined by calculation of Fractional Inhibitory
Concentration (FIC) Index values and presented in Table 3.
Compound 41 and COL displayed FICI values of 0.5 and 0.37
against Ab 21R and Ab 22P, which indicated a synergistic
interaction. It was noted that the compound markedly reduced the
MIC value of COL by fourfold against both strains.
An initial screening was performed by treating macrophages
with all the different molecules at a concentration of 50 μM
(Supplementary Information, Table S14). Surprisingly, the
compounds that showed, in this first phase, an inhibitory capacity
on the production of NO were all characterized by possessing
two trifluoromethyl substituents (12, 20, 28, 36, 37, 38, 39, 40,
41), which previously turned out to be the best antimicrobial
agents. Trifluoromethyl group, indeed, is well known moiety in
pharmaceutical chemistry, with peculiar chemical and
physiological stability and able to enhance the anti-inflammatory
potential of bioactive compounds.41,42 Among our results, an
exception is represented by the compounds 21, 23, 30, 33,
differently substituted on the phenyl ring, characterized by a
weak or absent antibacterial activity against A. baumannii, but
which responded positively to the anti-inflammatory screening.
Subsequently, the active compounds were tested at lower
concentrations (1 and 10 μM), in order to highlight reductions in
NO production at concentrations that are closest to the previously
identified MICs (Figure 4A). Highest reduction of NO
production (percentage of inhibition ≥ 80%) was observed with
2-phenylpiperazine derivatives 37 (3,3-dimethylbutanoyl
derivative), 39 (tert-butoxycarbonyl derivative) and 40 (2-
cyclohexylacetyl derivative), while the inhibition observed with
their methyl analogues (20, 12 and 28 respectively) was
significantly lower (highest value observed 40%). The phenyl
substituent on piperazine ring seems to improve the anti-
inflammatory profile.
Table 3. Determination of FIC values and interaction effects of
compound 41 and COL against two colistin-resistant A.
baumannii clinical strains.
MIC (µM)
FICa
Strain
Agent
Alone Combination
FIC
FIC
Individual
Index
Comp.
6.25
0.78
0.12
Ab
41
0.5 (S)
21R
COL
1178
12.5
443
0.38
0.12
Comp.
1.56
Ab 22P
41
0.37 (S)
COL
3545
886
0.25
aThe FICI was interpreted as follow: synergism=FICI≤0.5; indifferent> 0.5 to
≤4; antagonism=FICI > 4. The experiments were performed in triplicate.

In order to assess if the observed reduction of the NO
production could be by macrophages death, the effect on the cell
viability of our compounds was also evaluated (Supporting
information). In Figure 4B it is shown these results expressed as
percentage of cell viability versus control. Compounds 39 and 40
were the only ones with cytotoxic effects on the macrophage line
under examination.
Development Regional Fund A way to achieve Europe,
Operative program Intelligent Growth 2014-2020. ). T.C.C. is
supported by the V Plan Propio of the University of Seville with
a postdoctoral contract as research personnel in training. JSC is a
researcher belonging to the program “Nicolás Monardes” (C-
0059-2018), Servicio Andaluz de Salud, Junta de Andalucía,
Spain. MEPI is a researcher belonging to the program “Nicolás
Monardes” (C1-0038-2019), Servicio Andaluz de Salud, Junta de
Andalucía, Spain. S.M, F.A, L.F and A.R.C. thank Dipartimento
di Farmacia
e Scienze della Salute e della Nutrizione-
Dipartimento di Eccellenza MIUR L 232/2016". Authors also
thank CITIUS (Centro de Investigación, Tecnología e Innovación
de la Universidad de Sevilla) for its important contribution in
recording NMR and Mass spectra.
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Figure 4. Anti-inflammatory activity of Piperazine-derived
thioureas. Nitrites production (A) and cell viability (B)
assessments after treatment of LPS-stimulated RAW 264.7 cell
line with different concentration of compounds for 24 hours.
5.
6.
In summary, we have evaluated the antibacterial activity of a
collection of 39 piperazine-derived thioureas. All active
compounds showed growth inhibition against 46% of the tested
colistin-resistant A. baumannii clinical strains. The SAR analysis
7.
8.
has indicated that those compounds with
a
3,5-bis-
9.
trifluoromethyl phenyl ring at the thiourea function (12, 28, 36,
37, 40 and 41) were the most effective. Their potent activity, no
cytotoxicity, anti-inflammatory activity and easy synthetic
accessibility allow us to consider them as potentially strong
candidates for development of new anti-Acinetobacter agents.
The optimization process will be focused on improving their
potency as both antibacterial and anti-inflammatory agents. They
potentially could be used as adjuvants in combination therapy in
order to restore the efficacy of current employed antibiotics in the
clinical setting.
10. Nural, Y.; Gemili, M.; Ulger, M.; Sari, H.; c, De Coen, L. M.;
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Res. 2016, 9, 261-269.
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Dat. Collect. 2018, 15/16, 197-206.
14. Al-Wahaibi, L. H.; Hassan, H. M.; Abo-Kamar, A. M.; Ghabbour,
H. A.; El-Emam, A. A. Molecules 2017, 22, 710.
15. Patila, M.; Poyilb, A.N.; Joshic, S.D.: Patild, S.A.; Patila, S.A.;
Bugarine, A. Bioorganic Chemistry 2019, 92, 103217
Declaration of Competing Interest
16. Mazzotta, S.; Marrugal Lorenzo, J. A.; Vega-Holm, M.; Serna-
Gallego, A.; Álvarez-Vidal, J.; Berastegui-Cabrera, J.; Pérez del
Palacio, J.; Díaz, C.; Aiello, F.; Pachón, J.; Iglesias-Guerra, F.;
Vega-Pérez, J. M.; Sánchez-Céspedes, J. Eur. J. Med. Chem.
2020, 185, 111840.
17. Vega-Pérez, J. M.; Periñán, I.; Argandoña, M.; Vega-Holm, M.;
Palo-Nieto, C.; Burgos-Morón, E.; López-Lázaro, M.; Vargas, C.;
Nieto, J. J.; Iglesias-Guerra, F. Eur. J. Med. Chem. 2012, 58, 591-
612.
18. Synthesis of the thiourea derivatives (39-43). To a solution of the
monoacyl derivative (0.75 mmol) in dry dichloromethane (10 mL)
was added the corresponding isothiocyanate (0.9 mmol). The
reaction mixture was stirred at room temperature until TLC
showed that all the starting material had reacted, than was
evaporated to dryness. Compounds were purified by flash
chromatography on silica gel using the appropriate eluent.
19. Spectra data of the representative compound 41. 1H NMR (500
MHz, DMSO-d6)  9.81 (s, 1H), 8.11 (s, 2H), 7.77 (s, 1H), 3.97 (t,
J = 5.1 Hz, 4H), 3.48 (t, J = 5.1 Hz, 4H), 1.44 (s, 9H). 13C NMR
(125 MHz, DMSO-d6)  180.8, 153.8, 143.1, 130.1, 129.8, 129.6,
129.3, 129.2, 124.4, 124.3, 122.2, 116.6, 79.2, 47.8, 42.8, 28.0.
HRMS (m/z): calcd. for C18H21F6N3O2SNa 480.1151 M+Na+;
found 480.1143. Anal. calcd for C18H21F6N3O2S: C, 47.26; H,
4.63; N, 9,19. Found: C, 46.95; H, 4.77; N, 9.17.
The authors declare no competing financial interest or
personal relationships that could have appeared to influence the
work reported in this paper. S.M, TCC, M.V.H, J.S.C., J.P.,
J.M.V.P., F.I.G., M. V.H. and M.E.P.I. are co-inventors of the
European patent EP16382073.1 (Name of the Invention:
Piperazine derivatives as antiviral agents with increased
therapeutic activity; year of application: 2016).
Acknowledgments
This work has been supported by Ministerio de Economía y
Competitividad, Plan Estatal 2013-2016 Retos-Proyectos I+D+i
(CTQ2016-78580-C2-2-R). MVH also thanks Ministerio de
Economía y Competitividad, Plan Estatal 2013-2016 Excelencia
I+D+i (CTQ2016-78703-P). This work has been also supported
by Plan Nacional de I+D+i 2013-2016 and Instituto de Salud
Carlos III, Subdirección General de Redes y Centros de
Investigación Cooperativa, Ministerio de Economía, Industria y
Competitividad, Spanish Network for Research in Infectious
Diseases (REIPI RD16/0016/0009)—co-financed by European
20. Valencia, R.; Arroyo, L. A.; Conde, M.; Aldana, J. M.; Torres, M.
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Tale, R. H.; Kamble, V. M. (2012). Bioorganic & medicinal
chemistry letters 2012, 22(20), 6385-6390.
21. The activity screening studies were performed as follows: All
compounds were screened at 50 µM in 96-well plate to identify
which produced inhibition of growth against 13 colistin-resistant
A. baumannii clinical strains. For each strain, approximately 1x105
CFU/mL bacteria in Müller Hinton Broth (MHB) were dispensed
into the plates containing 50µM of each piperazine derivate. Plates
were incubated at 37º C with humidity for 18-24 hours. The
compounds were dissolved in DMSO, which was used as negative
control. As a positive control, 1x105 CFU/mL bacteria in broth,
without compound was incubated in the same conditions. Growth
was indicated by turbidity, a single large dot, or a filamentous
network of growth. The experiments were performed in triplicate
and were read independently by two observers.
40. Inhibition of nitric oxide (NO) production in lipopolysaccharide
(LPS)-stimulated RAW 264.7 cells. Briefly, RAW 264.7 cells
were seeded at a density of 2 × 105 cells/well in 24 well plates and
incubated overnight in complete medium. Media were aspirated
and replaced with fresh DMEM, then, cells were simultaneously
treated for 24 h with LPS (1 µg/mL) and different concentrations
of extracts or isolated compounds. DMSO was used as a vehicle
control. After treatment end, the presence of nitrite (a stable
oxidized product of NO) was measured in cell culture media by
using the Griess assay, and results are expressed as percentage of
nitrites production versus control (untreated LPS-stimulated cells).
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22. Minimal inhibitory concentration (MIC) was determined by the
broth microdilution method (cation-adjusted Mueller-Hinton
broth; Becton Dickinson, Cockeysville, Md.), in accordance with
the CLSI guidelines (Clinical and Laboratory Standards Institute,
2012).The compounds for which inhibition of the bacterial growth
was observed in the screening studies were tested (6clinical
strains). A range of piperazine derivates concentrations from
50µM to 0.195µM was tested. MICs were read as the lowest
concentrations of piperazine derivate which inhibited visible
growth after incubation for 24 h. The experiments were performed
in triplicate and were read independently by two observers.
23. Xu, J.; Pachón-Ibañez, M. E.; Cebrero-Cangueiro, T.; Chen, H.;
Sánchez-Céspedes, J.; Zhou, J. Bioorg. Med. Chem. Lett. 2019,
29, 1399-1402.
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A.; Ferrario, A.; Gomer, C.; Aiello, F.; Adams, J. D. Heliyon
2019, 5, e01366.
Supplementary Material
Full
description
of
biological
assays,
chemical
characterization and copies of NMR spectra of piperazine
derivatives 39-43, growth Inhibition screening data, MIC values
and NO production screening for compounds 5-43; Selectivity
Index for the six selected compounds (12, 28, 36, 37, 40 and 41)
can be found in the Supplementary Material.
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Declaration of interests
26. Ong, Z. Y.; Gao, S. J.; Yang, Y. Y. Adv. Funct. Mater. 2013, 23,
3682–3692.
27. Carullo, G.; Cappello, A. R.; Frattaruolo, L.; Badolato, M.;
Arme
☐X The authors declare that they have no known
ntano
As corresponding author I sign this declaration in the name of all authors of this manuscript
Margarita Vega Holm
,
B.;
Aiell
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Futur
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Med.
Chem
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2017,
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relationships that could have appeared to influence
the work reported in this paper.
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☐The authors declare the following financial
interests/personal relationships which may be
considered as potential competing interests:
32. Ma, Y.; Zheng, X.; Gao, H.; Wan, C.; Rao, G.; & Mao, Z.
Molecules 2016, 21(12), 1684.
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Highlights for: Exploration of piperazine-
derived thioureas as antibacterial and anti-
inflammatory agents. In vitro evaluation against
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Comp.
8
Structure
R1
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
CH3
Ph
R3
H
R4
F
R5
H
clinical isolates of colistin-resistant Acinetobacter
baumannii.
A
A
A
A
9
H
CF3
OCH3
CH3
H
H
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39a
40a
41a
42a
43a
H
H
S. Mazzotta, T.Cebrero-Cangueiro, L. Frattaruolo,
M. Vega-Holm*, M. Carretero-Ledesma, J.
Sánchez-Céspedes, A.R. Cappello, F. Aiello, J.
Pachón, J.M. Vega-Pérez*, F. Iglesias-Guerra, M.E.
Pachón-Ibáñez.
H
H
CF3
H
CF3
H
A
B
NO2
Cl
H
H
B
B
B
B
B
B
B
C
C
C
C
C
C
C
C
D
D
D
D
D
D
D
D
B
D
A
C
A
B
B
H
CN
F
H
E-mail: mvegaholm@us.es
E-mail: vega@us.es
H
H
H
CF3
OCH3
CH3
H
H
 A set of 39 piperazine-derived thioureas
was evaluated against colistin-resistant
Acinetobacter baumannii clinical strains.
 Six derivatives inhibited bacterial growth of
46% of the A. baumannii strains at low
micromolar concentrations.
H
H
H
H
CF3
H
CF3
H
NO2
Cl
H
H
 Bactericidal activity (time kill curve) was
tested for compound 41 against two colistin-
resistant A. baumannii strains.
H
CN
F
H
H
H
H
CF3
OCH3
CH3
H
H
 Compound 41 was also tested for
synergistic combination with colistin
against the same two clinical strains.
H
H
H
H
CF3
H
CF3
H
 Their potential anti-inflammatory activity
was assessed by their ability to inhibit the
production of nitric oxide
NO2
Cl
H
H
H
CN
F
H
H
H
Table 1. 4-Acyl-1-phenylaminothiocarbonyl-2-substituted-
piperazine derivatives 5-43.
H
CF3
OCH3
CH3
H
H
H
H
O
O
R1
R1
N
O
N
H
H
H
H
R5
R4
N
N
R5
R4
N
N
S
S
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
CF3
B
A
R3
R5
R3
R5
H
R4
R3
R4
R3
Ph
H
S
S
N
N
N
N
Ph
H
H
H
N
N
R1
R1
Ph
H
O
O
C
D
H
H
(R)-CH3
(S)-CH3
H
Comp.
Structure
A
R1
R3
R4
R5
H
H
H
H
5
6
7
CH3
CH3
CH3
H
H
H
NO2
Cl
aNew compounds.
A
A
CN

Table 2. MIC values (µM) of the selected piperazine-derived
thioureas and COL against colistin-resistant A. baumannii
clinical strains and CC50 values (M).
MIC (µM) Acinetobacter baumannii
CC50
Comp
(µM)a
11
14
3.12
50
-
17
21R
-
22P
-
24
-
12
20
-
-
112.4
73.2
-
-
6.25
25
6.25
12.5
12.5
6.25
1.56
-
6.25
-
21
-
-
148.1
200.0
22
-
50
12.5
50
3.12
-
-
12.5
25
25
25
-
-
3.12 142.2
25 82.0
6.25 129.7
28
-
6.25
3.12
-
-
3.12
-
3.12
3.12
3.12
-
36
37
3.12
-
3.12
91.8
104.3
127
38
-
3.12
6.25
-
-
-
39
-
12.5
-
-
-
40
3.12
12.5
-
3.12
6.25
6.25
6.25
1772
6.25
12.5
12.5
6.25
3545
3.12 124.6
108.6
6.25 162.1
41
6.25
-
6.25
-
-
42
43
-
-
-
12.5
7
73.5
-
COL
14
56
> 111
a Cytotoxic concentration 50%.
Table 3. Determination of FIC values and interaction effects
of compound 41 and COL against two colistin-resistant A.
baumannii clinical strains.
MIC (µM)
FICa
Strain
Agent
Alone Combination
FIC
FIC
Individual
Index
Comp.
6.25
0.78
0.12
Ab
41
0.5 (S)
21R
COL
1178
12.5
443
0.38
0.12
Comp.
1.56
Ab 22P
41
0.37 (S)
COL
3545
886
0.25
aThe FICI was interpreted as follow: synergism=FICI≤0.5; indifferent> 0.5 to
≤4; antagonism=FICI > 4. The experiments were performed in triplicate