Synthesis of novel 1,2-diarylpyrazolidin-3-one–based compounds and their evaluation as broad spectrum antibacterial agents

Article abstract of DOI:10.1016/j.bioorg.2020.103759

There is a continuous need to develop new antibacterial agents with non-traditional mechanisms to combat the nonstop emerging resistance to most of the antibiotics used in clinical settings. We identified novel pyrazolidinone derivatives as antibacterial hits in an in-house library screening and synthesized several derivatives in order to improve the potency and increase the polarity of the discovered hit compounds. The oxime derivative 24 exhibited promising antibacterial activity against E. coli TolC, B. subtilis and S. aureus with MIC values of 4, 10 and 20 μg/mL, respectively. The new lead compound 24 was found to exhibit a weak dual inhibitory activity against both the E. coli MurA and MurB enzymes with IC₅₀ values of 88.1 and 79.5 μM, respectively, which could partially explain its antibacterial effect. A comparison with the previously reported, structurally related pyrazolidinediones suggested that the oxime functionality at position 4 enhanced the activity against MurA and recovered the activity against the MurB enzyme. Compound 24 can serve as a lead for further development of novel and safe antibiotics with potential broad spectrum activity.

Full text of DOI:10.1016/j.bioorg.2020.103759

Journal Pre-proofs
Synthesis of novel 1,2-diarylpyrazolidin-3-one–based compounds and their
evaluation as broad spectrum antibacterial agents
Salma A. Mokbel, Reem K. Fathalla, Lina Y. El-Sharkawy, Ashraf H. Abadi,
Matthias Engel, Mohammad Abdel-Halim
PII:
S0045-2068(20)30366-7
DOI:
https://doi.org/10.1016/j.bioorg.2020.103759
YBIOO 103759
Reference:
To appear in:
Bioorganic Chemistry
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Accepted Date:
17 February 2020
13 March 2020
Please cite this article as: S.A. Mokbel, R.K. Fathalla, L.Y. El-Sharkawy, A.H. Abadi, M. Engel, M. Abdel-
Halim, Synthesis of novel 1,2-diarylpyrazolidin-3-one–based compounds and their evaluation as broad spectrum
antibacterial agents, Bioorganic Chemistry (2020), doi: https://doi.org/10.1016/j.bioorg.2020.103759
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Synthesis of novel 1,2-diarylpyrazolidin-3-one–
based compounds and their evaluation as broad
spectrum antibacterial agents
AUTHOR NAMES
Salma A. Mokbel1, Reem K. Fathalla2, Lina Y. El-Sharkawy1,3, Ashraf H. Abadi1,
Matthias Engel2* and Mohammad Abdel-Halim1*
AUTHOR ADDRESS
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy and Biotechnology, German
University in Cairo, Cairo 11835, Egypt.
2Pharmaceutical and Medicinal Chemistry, Saarland University, Campus C2.3, D-66123
Saarbrücken, Germany
3Division of Pharmacy & Optometry, School of Health Sciences, Faculty of Biology, Medicine
and Health, University of Manchester, M13 9PT, UK
*Correspondence:
Dr. Matthias Engel. Phone: +49 681 302 70312. Fax: +49 681 302 70308. E-mail:
ma.engel@mx.uni-saarland.de.
Dr. Mohammad Abdel-Halim: Phone +201000006222. Email: mohammad.abdel-
halim@guc.edu.eg

Abstract
There is a continuous need to develop new antibacterial agents with non-traditional mechanisms
to combat the nonstop emerging resistance to most of the antibiotics used in clinical settings. We
identified novel pyrazolidinone derivatives as antibacterial hits in an in-house library screening
and synthesized several derivatives in order to improve the potency and increase the polarity of
the discovered hit compounds. The oxime derivative 24 exhibited promising antibacterial activity
against E. coli TolC, B. subtilis and S. aureus with MIC values of 4, 10 and 20 µg/mL, respectively.
The new lead compound 24 was found to exhibit a weak dual inhibitory activity against both the
E. coli MurA and MurB enzymes with IC50 values of 88.1 and 79.5 µM, respectively, which could
partially explain its antibacterial effect. A comparison with the previously reported, structurally
related pyrazolidinediones suggested that the oxime functionality at position 4 enhanced the
activity against MurA and recovered the activity against the MurB enzyme. Compound 24 can
serve as a lead for further development of novel and safe antibiotics with potential broad spectrum
activity.
1. Introduction
The extensive appearance of resistant bacterial strains represents an alarming threat to
public health. This resistance has been exacerbated by the decrease in industrial efforts to discover
and develop new antibacterial agents during the last decades.[1] In light of this situation, efforts
toward the discovery of new antibacterial agents with novel mechanisms of action need to be
strengthened which represents one of the greatest challenges in drug discovery. The inhibition of
bacterial peptidoglycan synthesis is an established strategy for the development of new
antibacterial agents.[2-4] To date, the focus was on the late steps of peptidoglycan biosynthesis
that are catalyzed by transpeptidases and led to important inhibitors, such as the -lactam
antibiotics.[5-7] Only in the last decade, drug discovery efforts increasingly turned towards the
cytoplasmic steps of the peptidoglycan biosynthesis, which are catalyzed by the Mur enzymes
MurA-F.[2, 3] Starting with MurA, the cytoplasmic Mur enzymes synthesize the final
peptidoglycan biosynthesis precursor uridine 5'-diphosphate (UDP)-N-acetylmuramyl-
pentapeptide.[8, 9]

In previous studies, several pyrazolidine-3,5-diones were reported as inhibitors of bacterial cell
wall biosynthesis through inhibition of the MurB enzyme (UDP-N-acetylenolpyruvyl glucosamine
reductase), [10-12] with some derivatives showing activity also against MurA and MurC enzymes
(Figure 1). The 4-alkyl and 4,4-dialkyl pyrazolidinediones were described as weak inhibitors of S.
aureus and E. coli MurB, and were shown to have modest antibacterial activity against Gram-
positive bacteria, particularly against penicillin-resistant Streptococcus pneumonia (PRSP),
compound A in Figure 1 is an example.[11] A second series of related pyrazolidinediones with
4-benzoyl and 4-arylamido substituents was reported to inhibit E. coli MurB in the low micromolar
range, and also showed moderate inhibitory activity against MurA and MurC (cpds. B and C,
Figure 1).[10, 12]
F3CO
O
O
HN
O
O
O
O
O
O
N N
N
B
N
N
N
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
C
A
MurB E. coli IC50 = 4.8 µM
MIC (µg/mL)
MIC (µg/mL) S. aureus GC 1131 = 16
MurB E. coli IC50 = 50 µM
MurB E. coli IC50 = 5.4 µM
MIC (µg/mL)
MIC (µg/mL) S. aureus GC 1131 = 12.7
MIC (µg/mL)
E. coli imp > 128
E. coli imp = 200
E. coli imp = 25.4
MIC (µg/mL) S. aureus GC 1131 = 100
Figure 1. Representative examples of the previously reported pyrazolidine-3,5-diones
The latter group of compounds was shown to inhibit biosynthesis of peptidoglycan, as evaluated
by measuring the amount of accumulated soluble peptidoglycan after incubating the compounds
with Streptococcus epidermidis. [10] However, the inhibitors suffered from the following
drawbacks: i) activity against Gram-positive bacteria but much less against Gram-negative ones
ii) loss of activity in the presence of 4% bovine serum albumin (BSA), indicative of a strong BSA
binding.[10-12] It can be assumed that the strong BSA binding was caused by the high lipophilicity
of the structures, which required three aryl systems for biological activity. The replacement of the
aryl system in position 4 by branched alkyl chains had previously resulted in almost inactive

compounds against Gram-negative bacteria (compound A, Figure 1). Thus, the pyrazolidinedione
scaffold gave promising MurB inhibitors with a good activity against Gram-positive bacteria in
vitro, but was not suitable for in vivo applications. In the present study, novel, structurally related
pyrazolidin-3-one derivatives with anti-bacterial activity were developed from initial hits
identified in a screening of an in–house library.
2. Results and Discussion
2.1. Compound design
Inspired by the reported pyrazolidine-3,5-dione series, we decided to screen an in-house library of
compounds with some structural similarities, but one major difference, which is the absence of the
second carbonyl group at position 5 of the pyrazolidine ring (compounds D-I, Figure 2). Since this
also disrupted the coplanarity of the bis-aryl-pyrazolidine-3,5-dione core, it seemed interesting to
investigate the effect of the changed shape on target inhibition and anti-bacterial activity. In spite
of their limited size, the screened small library of compounds offered some diversity of functional
group variations. In particular, it allowed us to evaluate the potential influence of the substituent
at position 4 of the pyrazolidinone ring.
Compounds D-I were screened for their ability to inhibit the growth of several bacterial
species including S. aureus (strain Newman), B. subtilis (ATCC 6633) and E. coli (both E. coli
BL-21 and a TolC mutant in which the AcrAB-TolC multidrug efflux pump gene is deleted). The
results are shown in Table 1. Interestingly, our preliminary screening results showed that a plain
methyl group (compound D) rather than the bulky and lipophilic benzoyl (compound E) or
pivaloyl (compound H) moieties in position 4 gave the highest overall potency. This was a
significant progress compared to the pyrazolidinedione scaffold that required such substituents to
achieve a significant activity.[10-12] Compound D even seemed to be active against a wider
bacterial spectrum when compared to the most relative compounds in the previously reported 4-
alkylpyrazolidinedione series,[11] as it showed improved activity against the Gram-negative E.
coli TolC. Although its activity was reduced against the E. coli BL-21 (non-efflux pump deficient),
the 3-fold drop of activity (Table1) is less pronounced than found with some antibacterial drugs in
the market: e.g., a 60-fold drop in activity was reported for the macrolide antibiotics erythromycin

and clarithromycin between E. coli TolC and wild type strains.[13] This suggested that the hit
compound D is a weaker substrate of the AcrAB-TolC pump and sufficiently active compounds
can be obtained after optimization of the potency.
O
O
O
O
N
N
N
N
N
N
Cl
Cl
Cl
Cl
Cl
Cl
D
E
F
O
O
O
O
O
O
N
N
N
N
N
Cl
N
Cl
N N
Cl
Cl
Cl
Cl
Cl
Cl
G
H
J
I
Figure 2. The small library of pyrazolidinone derivatives (compounds D-I) and the reference
compound J
A comparison of compound D with its 4-unsubstituted precursor I proved that the 4-methyl
substituent enhanced the activity against E. coli TolC (MIC50: 2.6 vs. 4.9 µg/mL respectively).
Nevertheless, compound I still inhibited the bacterial growth (MIC=20 µg/mL), whereas the
unsubstituted pyrazolidinedione J (compound 17 in ref [10]) - prepared for comparison - was much
less active against E. coli TolC (MIC> 50 µg/mL in our assays). This result demonstrated that the
new scaffold might be superior for the development of new antibacterial drugs especially against
Gram-negative bacteria, because the core structure I already showed significant activity compared
to J. Although the pyrazolidinedione derivative J showed higher potency against Gram-positive
bacteria when compared to compound I (in accordance with the data reported in ref [10]), this was
overcome already by the plain methyl substitution in D. Thus, the 1,2-diarylpyrazolidin-3-one core
was identified as a new anti-bacterial scaffold with potential broad spectrum activity. On the other
hand, it was noted that introduction of the methyl group markedly decreased the solubility of D in

the growth medium compared with I, precluding the use of higher concentration to determine the
MIC for compound D. An enlarged aryl system as in compound F (Table 1) aggravated the
solubility issue, so that its potency to inhibit bacterial growth could not be precisely evaluated.
Table 1. MIC50 values of the new pyrazolidinone inhibitors (µg/mL)*
Compound no.
D
E
F
G
H
I
J
S. aureus
B. subtilis
3.6
4.4
2.6
7.8
>25
>25
>25
>25
n. db
n. d
n. d
n. d
>25
>25
7.3
>25
>25
>25
8.5
4.9
>25
4.6
6.8
>25
>25
>25
E. coli TolC
E. coli BL-21
>25
>25
aMIC50 were used for initial comparison to overcome solubility problems of some compounds; bn.d.: not determined
because of poor solubility in the assay medium. * Experiments were made at least two times and standard deviation
was less than 20 % (most cases: < 15 %).
Hence, one of the major goals of this work is to synthesize derivatives of D and I with higher
polarity along with improved potency. This was planned through the variation of both the 1,2-
diaryl substituents and the 4-substituent at the pyrazolidine ring. Additionally, we wanted to test
whether the MurA/MurB enzymes were still the cellular targets of the novel compounds, and
finally, we aimed at evaluating the cytotoxicity against human cells.
2.2. Chemistry
The synthesis of the desired 3-pyrazolidinones (compounds 1-8) was accomplished via a three-
step sequence as shown in Scheme 1. The procedure was started by oxidative coupling of the
aniline derivative using manganese IV oxide under reflux in toluene for 8 hours and a water trap
(Dean-Stark apparatus) was used to separate water released from the reaction. The coloured
diazene was then reduced to the respective hydrazine using Zn/NH4Cl in acetone at room
temperature (Scheme 1).

The cyclization reaction was performed through reacting the diaryl hydrazine derivatives with 3-
chloropropionyl chloride in the presence of potassium carbonate as a base at room temperature.
The cyclization was completed after 72 hours. Under the same reaction conditions, the use of
1,2-bis(2-chlorophenyl)hydrazine (compound b2) did not give rise to a new product, which can be
attributed to the steric hindrance caused by the bulky ortho-chloro substituents. In the latter case,
harsher reaction conditions were used to get the respective pyrazolidinone, where the diaryl
hydrazine was deprotonated by sodium hydride in DMF, followed by the addition of 3-
chloropropionyl chloride and heating at 60 °C for 3 hours, to obtain the desired product in a poor
yield (15%) (Scheme 1).
The bis-(bromophenyl) derivatives, compounds 5 and 6, were subjected to Suzuki coupling in
order to replace the bromine atoms with phenyl, 3-thienyl, or 2-furyl rings (Scheme 2). The
coupling was achieved by the reaction of compounds 5 or 6 with the respective aryl boronic acid
derivative in the presence of Cs2CO3 as a base, tetrakis(triphenylphosphine) palladium as catalyst
and dioxane/water as solvent; the same reaction conditions did not give successful coupling with
pyridine and pyrazole boronic acids, however.
Substitution of the pyrazolidinone derivative (3) at position 4 was initiated by the deprotonation
of this acidic methylene by using lithium diisoprpyl amide (LDA) at -78 °C for 1 hour, followed
by trapping the anion by adding the suitable electrophiles like methyl iodide to give compound 13
(Scheme 3). Carboxylation of position 4 was similarly achieved by treating the anion with carbon
dioxide (dry ice) to give the 4-carboxy derivative 14, basically as described in ref[14]. It is worth
mentioning that lithium bis(trimethylsilyl)amide (LiHMDS) could also do the required
deprotonation successfully under the same conditions. Several amide derivatives were prepared by
reacting the carboxylic acid derivative 14 with different amines using HBTU as coupling agent in
the presence of TEA as shown in Scheme 3. Synthesis of the oxime (compound 24) was
accomplished by treatment of 14 with sodium nitrite and HCl as in ref [14] (Scheme 3). Reduction
of the oxime group in 24 was achieved using Zn dust in acetic acid to yield the respective primary
amine 25 (Scheme 3). It is worth mentioning that trials to prepare the ester derivatives of 14 in the
presence of an acid catalyst did not give the desired product but mainly returned the decarboxylated
precursor 3. This can be explained by the decarboxylation mechanism of β-ketoacids under acidic
conditions.[15]

Scheme 1
R
O
R
iii
ii
HN
NH
N
i
N
N
H2N
N
R
R
R
b1-b8
a1-a8
R
R
1-8
Reagents and conditions i) MnO2, toluene, reflux. ii) Zn, NH4Cl, acetone, RT. iii) acetone, K2CO3 followed by 3-
chloropropionyl chloride, RT (or DMF, NaH, followed by 3-chloropropionyl chloride, heat at 60 °C for 3h to get
compound 2) .
Cpd #
R
Cpd #
R
Cpd #
R
a1, b1, 1
a2, b2, 2
a3, b3, 3
H
a4, b4, 4
a5, b5, 5
a6, b6, 6
4-fluoro
3-bromo
4-bromo
a7, b7, 7
a8, b8, 8
4-methyl
3,4-dimethyl
2-chloro
3-chloro
Scheme 2
O
O
i
N
N
N
N
Br
Ar
Br
Ar
5 or 6
9-12
Reagents and conditions i) Pd(PPh3)4, Cs2CO3, dioxane: water (4:1), reflux overnight.
Cpd#
9
Attachment
Ar
Cpd#
11
Attachment
Ar
O
para
meta
10
para
12
para
S
O

Scheme 3
O
O
N
ii
N
i
N N
13
Cl
Cl
Cl
Cl
3
O
R
HO
O
O
O
N
iii
N
N
N
Cl
Cl
Cl
Cl
14
iv
15-23
OH
N
NH2
O
v
O
N
N
N N
Cl
Cl
Cl
Cl
25
24
Reagents and conditions i) 1.5 equiv of LDA, 1.5 equiv of MeI, THF, −78 °C then room temperature for 20 h. ii)
14 equiv of LDA, excess CO2, THF, −78 °C then room temperature for 1 h. iii) HBTU, TEA, amine derivative,
DCM, room temperature, 5h. iv) NaNO2, HCl, Et2O/H2O. v) Zn dust, acetic acid, NH4Cl, heat at 50 °C, overnight.
Cpd#
15
R
Cpd#
R
Cpd#
21
R
N
18
N
N
N
O
16
17
19
20
22
23
O
HN
N
N
H
N
HN
N
O

2.3. Biological Evaluation
Bacterial Growth Inhibition. The synthesized compounds were tested against Gram-positive and
negative bacteria for growth inhibition, using serial dilutions of each compound with a maximum
concentration of 25 µg/mL to minimize solubility problems during the assay. The results are listed
in Table 2. For the more potent compounds, the assay was repeated with an adapted concentration
range to allow a more precise evaluation of the MIC values. However, some of the compounds
possessed a poor solubility, leading to precipitation above a certain concentration threshold; for
some of these compounds, a strong initial inhibition was observed at low concentrations, allowing
determining a MIC50 value, while a plateau was reached at the higher concentrations. Therefore,
we included in Table 1 the MIC50 values to allow a comparison of potency between all compounds,
and the MIC where it could be determined.

Table 2. Biological activity of the synthesized compounds against bacteria*
OH
O
O
R
R
N
O
O
O
N
N
N
N
N N
N
N
Cl
Cl
Cl
Cl
Cl
Cl
R
R
1-12
14-23
24
13 or 25
E. coli tolC
MIC50
µg/mL
E. coli tolC
MIC
µg/mL
B. Subitilis
MIC50
µg/mL
B. Subitilis
MIC
µg/mL
S. aureus
MIC
µg/mL
Cpd #
R
1
2
-H
>25
13
>25
43
>25
>25
>25
>25
>25
>25
2-chloro
3
4
5
6
7
8
3-chloro
4-fluoro
3-bromo
4-bromo
4-methyl
2.7
>25
2.8
2.2
6.2
3.8
10
>25
NR
NR
24
4.9
>25
4.2
7.7
18.2
NR
>25
NR
NR
>25
>25
>25
>25
>25
>25
25
3,4-dimethyl
14
5.5
25
9
>25
>25
>25
>25
>25
>25
>25
>25
para
10
>25
>25
S
para
meta
para
11
14.5
16.5
25
25
16
12
>25
>25
>25
>25
O
12
O
13
14
-CH3
-OH
1.8
19.5
NR
>25
3.8
>25
NR
>25
>25
>25
15
23
>25
>25
>25
>25
N
N

16
17
>25
>25
>25
>25
>25
>25
O
N
>25
>25
>25
>25
H
N
O
18
19
N
O
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
N
20
21
N
>25
4.6
>25
>25
>25
>25
>25
>25
>25
>25
>25
>25
N
22
23
>25
>25
HN
>25
HN
24
25
-
1.8
8
4
25
4
15
10
44
20
>25
-NH2
NR: Not reached due to solubility problems. * Experiments were made at least two times and
standard deviation was less than 20 % (most cases: < 15 %).

2.3.1. Activity against E. coli TolC
First round of structural optimization. Since our main focus was to take advantage of the new
scaffold’s improved activity against the Gram-negative E. coli TolC, we based our rounds of
optimisation and SAR analysis on the assay results obtained from this phenotypic screen. The first
round of structural optimization for the discovered hits involved the synthesis of several 4-
unsubstituted pyrazolidinone derivatives to determine the optimum substitution type and pattern
at the 1,2-diaryl system. To this end, several analogues of compound I (MIC = 20 µg/mL) were
prepared, starting with compound 1, in which the halogen substituents were removed. This almost
led to abolishing the antibacterial activity against E. coli TolC, indicating that a halogen or at least
hydrophobic substitution is essential for the antibacterial efficacy. Shifting the chloro substituents
to the ortho positions greatly reduced the antibacterial activity as well (compound 2).
Contrastingly, the shift of the chloro substituents from the para (compound I) to the meta positions
gave compound 3, showing a two-fold increase in antibacterial potency (MIC = 10 µg/mL).
Compound 4, bearing fluorine which is smaller and less lipophilic than chlorine, almost lost the
activity against E. coli TolC. On the other hand, compounds 5 and 6, endowed with the bulkier
and more lipophilic bromine, at the meta or para position of the aromatic rings, respectively,
showed somewhat lower MIC50 values compared to that of the respective chloro congeners
(compounds 2 and I). However, the poor water solubility did not permit to determine a MIC value
for 5 and 6. Replacing the p-chloro substitution in compound I with the lipophilic, electron-
donating methyl kept the antibacterial activity with almost similar potency (compound 7 compared
with compound I). A slight increase of the antibacterial activity was observed when compound 7
was substituted with an additional methyl group at the meta-positions of the 1,2-bisphenyl system
(compound 8, MIC = 14 µg/mL).
As the bulky bromo substitution in compounds 5 and 6 retained the antibacterial activity, it was
conceivable that aryl systems having steric requirements comparable to bromine might also be
tolerated and lead to an improved potency and/or water solubility. While the replacement of
bromine at position 4 with phenyl or the bioisosteric thiophene rings had a negative impact on the
biological activity (compare 9 and 10 with compound 6), the more polar furan ring system in
compounds 11 and 12 recovered some of the antibacterial potency but with about 3-6 fold increase
in the MIC50 values compared to their bromo precursors. The furan derivatives exhibited almost

full inhibition of bacterial growth at 25 µg/mL, which is most probably attributed to their improved
solubility in the assay medium compared to their respective bromo analogues. However, the bi-
aryl systems did not appear promising with respect to increasing the potency. In summary, the 3-
chloro-substituted analogue 3 showed the best balance between potency (MIC = 10 µg/mL) and
solubility and was chosen for further optimization
Second round of optimization. Keeping bis-(m-chlorophenyl) rings of compound 3 constant,
different substituents were tested at position 4 of the pyrazolidine ring. Like in hit compound D,
the 4-methyl substituent showed some improvement in the antibacterial potency as indicated by
the reduction in the MIC50 (compound 13), however, solubility remained a limiting factor for
further development of other 4-alkyl derivatives. Hence, we decided to move towards developing
new compounds with more polar substituents at position 4. This was primarily achieved by the
carboxylation of position 4 to give compound 14. Unfortunately, the carboxyl function led to a
tenfold decrease in potency (MIC50) compared with the methylated analogue 13. In an attempt to
regain the potency, several amide derivatives were synthesized, including the polar alicyclic
amides 15 and 16, which were virtually inactive. The methoxy, alkyl, cycloalkyl and allyl amide
series (17-23), also did not yield active congeners except the cyclopropyl amide derivative 22,
however, MIC values were not reached within the tested concentration range.
In a different trial to boost the activity of compound 3 with a more polar analogue, the oxime
derivative 24 was synthesized. Compound 24 showed an MIC value of 4 µg/mL, representing the
most potent antibacterial agent against E. coli TolC among the present series.
Since incorporation of a primary amine was reported to increase accumulation of compounds
inside E. coli,[16] and was also assumed to significantly increase the polarity similar to the active
oxime derivative 24, we finally synthesized compound 25. Although the primary amine showed a
better activity than the carboxylic acid in compound 14, adding this amino deteriorated the MIC
by 2.5-fold compared to the unsubstituted compound 3.
2.3.2. Activity against E. coli with wild type efflux pump
Compounds 3 and 24, the most potent compounds against E. coli TolC, were also tested against
the non-efflux pump deficient E. coli BL-21 strain. The activity of the compounds was found to

be greatly reduced, as only a weak inhibition was observed at 25 µg/mL, indicating that the
compounds’ activity is aborted by efflux transport. This adds an extra goal in the future
optimization of this class of compounds which is developing new structures that are not substrates
for efflux pumps which represents one of the major problems in antibiotics' development.[17, 18]
2.3.3. Activity against Gram-positive bacteria.
All new compounds were tested against B. subtilis and S. aureus. Altogether, the activity pattern
of the compounds against B. subtilis was very similar to that obtained against E. coli TolC,
although the MIC values tended to be somewhat increased. It is possible that the compounds hit
the same conserved target in both the Gram-positive and Gram-negative bacteria, so that they have
the potential to be developed to broad spectrum antibiotics. The oxime derivative 24 showed the
highest potency against B. subtilis with a MIC value of 10 µg/mL. Additionally, 24 was also the
most potent compound against S. aureus with MIC value of 20 µg/mL. Interestingly, methyl
substitution on both the 1,2-diphenyl rings enhanced the antibacterial activity against S. aureus as
can be seen from the MIC values of compounds 7 and 8 (MIC = 25 µg/mL for both compounds).
2.3.4. Investigation of MurA and MurB inhibition as a possible mechanism of action.
As the newly presented series possess a structural similarity to the previously reported
pyrazolidinedione type MurB inhibitors,[10-12] we screened both compounds 3 and 24 for their
ability to inhibit recombinant MurA and MurB E. coli enzymes at 100 µM concentration.
Fosfomycin and compound J from ref [10] were used as positive controls for the MurA and MurB
assay, respectively (Table 3). Compound 3 showed a weak effect against both MurA and MurB at
100 µM (30.7 µg/mL), indicating that its antibacterial effect is likely due to other mechanisms. Its
dicarbonyl analogue J had more potent MurB inhibition, indicating that the additional carbonyl (at
position 5) in compound J enhanced the MurB inhibition. On the other hand, the oxime derivative
24 showed a dual inhibition of both MurA and MurB enzymes (IC50´s were 88.1 µM and 79.5 µM
against MurA and MurB, respectively), which can be directly attributed to the oxime group at
position 4 that might recover the potency against MurB and boost the activity against MurA.
Nevertheless, the weak inhibitory potency of compound 24 against MurA and MurB enzymes can
only partially explain the antibacterial activity against E.coli TolC and B. Subitilis (MIC = 4 µg/mL
and 10 µg/mL respectively). It is likely that 24 is affecting bacterial growth through additional,
unknown mechanisms.

Table 3: E. coli MurA and MurB Inhibition for compounds 3 and 24*
OH
N
O
O
O
O
N
N
N
N
N
N
Cl
Cl
Cl
Cl
Cl
Cl
J
3
24
MurA
MurB
Cpd #
% inhibition at
100 µM
28.3
IC50 (µM)
% inhibition at
100 µM
26.9
IC50 (µM)
3
24
J
ND
88.1
ND
ND
79.5
122
NT
54.7
13.2
76
58.9
39.7
NT
Fosfomycin
22.3
*Values are mean values of at least two experiments; standard deviation <10%; NT: not tested.
2.3.5. Cytotoxicity assay against HepG2 cells
To evaluate the suitability of the scaffold as potential antibiotics, we evaluated the cytotoxicity of
the lead compound 24 against the HepG2 human liver cancer cell line using the MTT assay.
Compound 24 showed only low toxicity towards HepG2 cells (LD50 > 50 µg/mL, 16 % growth
inhibition at 50 µg/mL). This indicates that this new lead compound is not particularly cytotoxic
against mammalian cells, leaving a sufficient therapeutic window for further optimization.
3. Conclusion
In the present study, a new antibacterial lead compound, 24, was developed based on an initial
screening hit. The still low molecular weight of 336 g/mol leaves room for further optimizations
while it already displayed good broad spectrum potency. Importantly, 24 showed promising
activity against Gram-negative E. coli TolC, and the related hit compound D even against the non-
efflux pump defective E. coli BL-21, suggesting that the new 1,2-diarylpyrazolidin-3-one scaffold
can be optimized to a new antibiotic agent against Gram-negative bacteria, for which there is a
very high medical need.[19] While the targets of the new inhibitors could only be partially
identified, they were sufficiently selective for the bacterial cells.

4. EXPERIMENTAL SECTION
4.1. Chemistry
All reagents and solvents were obtained from commercial suppliers and were used as received. 1H
13
NMR and C NMR spectra were measured using A Bruker DRX 500 spectrometer, for some
compounds Varian Mercury VX 400 spectrometer was used. The chemical shifts are referenced to
the residual protonated solvent signals. All the tested compounds had at least 95% purity as
obtained by means of HPLC-MS. Mass spectra (HPLC-ESI-MS) were obtained using a TSQ
quantum (Thermo Electron Corporation) instrument with a triple quadrupole mass detector
(Thermo Finnigan) and an ESI source. All samples were inserted using an autosampler (Surveyor,
Thermo Finnigan) by an injection volume of 10 μL. The MS detection was determined using a
source CID of 10 V and carried out at a spray voltage of 4.2 kV, a nitrogen sheath gas pressure of
4.0 X 105 Pa, a capillary temperature of 400 oC , a capillary voltage of 35 V and an auxiliary gas
pressure of 1.0 X 105 Pa. Reversed phase C18 NUCLEODUR 100-3 (125 X 3 mm) column
(Macherey-Nagel) was used to separate analytes. The mobile phase consisted of water containing
0.1% TFA (A) and acetonitrile containing 0.1% TFA (B). HPLC-Method: flow rate 0.4 mL/min.
The percentage of B started at an initial of 5%, was increased up to 100% during 16 min, kept at
100% for 2 min, and flushed back to 5% in 2 min. Melting points were measured by a Mettler FP1
melting point apparatus and are uncorrected.
4.1.1. General synthetic procedures and experimental details
4.1.1.1. General procedure for synthesis of the diazene derivatives (a1-a8). To a suspension of the
aniline derivative (100 mmol) in toluene (350 mL), manganese (IV) oxide (500 mmol, 43.45 g)
was added while stirring. The mixture was refluxed with water removal using (Dean-Stark
apparatus) for 8 hours. The mixture was left to cool then filtered through a pad of silica gel. The
filter cake was washed with several portions of toluene and methylene chloride. The collected

wash and the filtrate were evaporated under reduced pressure to afford the diazene derivative as
an orange to red solid. The product was used in the next step with no need for further purification.
4.1.1.1.1. 1,2-Bisphenyldiazene (a1). Synthesized according to the general procedure for diazene
synthesis using aniline; mp 68-69 °C; MS (ESI): m/z = 183 (M+H) +.
4.1.1.1.2. 1,2-Bis(2-chlorophenyl)diazene (a2). Synthesized according to the general procedure
for diazene synthesis using 2-chloroaniline; mp 135-137 °C; MS (ESI): m/z = 251 (M+H) +.
4.1.1.1.3. 1,2-Bis(3-chlorophenyl)diazene (a3). Synthesized according to the general procedure
for diazene synthesis using 3-chloroaniline; mp 101-103 °C; MS (ESI): m/z = 251 (M+H) +.
4.1.1.1.4. 1,2-Bis(4-fluorophenyl)diazene (a4). Synthesized according to the general procedure for
diazene synthesis using 4-fluoroaniline; mp 101-102 °C; MS (ESI): m/z = 219 (M+H) +.
4.1.1.1.5. 1,2-Bis(3-bromophenyl)diazene (a5). Synthesized according to the general procedure
for diazene synthesis using 3-bromoaniline; mp 150-151 °C; MS (ESI): m/z = 340.8 (M+H) +.
4.1.1.1.6. 1,2-Bis(4-bromophenyl)diazene (a6). Synthesized according to the general procedure
for diazene synthesis using 4-bromoaniline; mp 203-204 °C; MS (ESI): m/z = 340.8 (M+H) +.
4.1.1.1.7. 1,2-Bis-p-tolyldiazene (a7). Synthesized according to the general procedure for diazene
synthesis using p-toluidine; mp 143-145 °C; MS (ESI): m/z = 211 (M+H) +.
4.1.1.1.8. 1,2-Bis(3,4-dimethylphenyl)diazene (a8). Synthesized according to the general
procedure for diazene synthesis using 3,4-dimethylaniline; mp 210-211 °C; MS (ESI): m/z = 239
(M+H) +.
4.1.1.2. General procedure for synthesis of the hydrazine compounds (b1-b8). To a suspension of
the diazene derivative (22.5 mmol) in acetone (100 mL) saturated aqueous ammonium chloride
(30 mL) and zinc dust (172.3 mmol, 11.3 g) were added. The suspension was stirred at room
temperature for 5 h and filtered through a pad of silica gel. The silica cake was washed with acetone
till obtaining a clear filtrate. The filtrate was collected, and the solvent was evaporated under
reduced pressure till reaching half of the initial volume. The residue was diluted with ice/water

(150 mL) to form a precipitate. The precipitate was filtered off, dried and stored under inert
atmosphere. The product was used in the next step without further purification.
4.1.1.2.1. 1,2-Bisphenylhydrazine (b1). Synthesized according to the general procedure for
hydrazine synthesis using 1,2-bisphenyldiazene (a1); m.p 131-132°C; MS (ESI): m/z = 185 (M+H)
+
.
4.1.1.2.2. 1,2-Bis(2-chlorophenyl)hydrazine (b2). Synthesized according to the general procedure
for hydrazine synthesis using 1,2-bis(2-chlorophenyl)diazene (a2); m.p 87-88 °C; MS (ESI): m/z
= 253 (M+H) +.
4.1.1.2.3. 1,2-Bis(3-chlorophenyl)hydrazine (b3). Synthesized according to the general procedure
for hydrazine synthesis using 1,2-bis(3-chlorophenyl)diazene (a3); m.p 97-99 °C; MS (ESI): m/z
= 253 (M+H) +.
4.1.1.2.4. 1,2-Bis(4-fluorophenyl)hydrazine (b4). Synthesized according to the general procedure
for hydrazine synthesis using 1,2-bis(4-fluorophenyl)diazene (a4); m.p 94-95 °C; MS (ESI): m/z
= 221 (M+H) +.
4.1.1.2.5. 1,2-Bis(3-bromophenyl)hydrazine (b5). Synthesized according to the general procedure
for hydrazine synthesis using 1,2-bis(3-bromophenyl)diazene (a5); m.p 160-161 °C; MS (ESI):
m/z = 340.8 (M+H) +.
4.1.1.2.6. 1,2-Bis(4-bromophenyl)hydrazine (b6). Synthesized according to the general procedure
for hydrazine synthesis using 1,2-bis(4-bromophenyl)diazene (a6); m.p 123-125 °C; ;MS (ESI):
m/z = 340.8 (M+H) +.
4.1.1.2.7. 1,2-Bis-p-tolylhydrazine (b7). Synthesized according to the general procedure for
hydrazine synthesis using 1,2-bis-p-tolyldiazene (a7); m.p 39-40 °C; MS (ESI): m/z = 213 (M+H)
+
.
4.1.1.2.8. 1,2-Bis(3,4-dimethylphenyl)hydrazine (b8). Synthesized according to the general
procedure for hydrazine synthesis using 1,2-bis(3,4-dimethylphenyl)diazene (a8); m.p 125- 126
°C; MS (ESI): m/z = 241 (M+H) +.

4.1.1.3. General procedures for synthesis of the pyrazolidinone derivatives (1–8).
Procedure A. The hydrazine derivative (2 mmol) was dissolved in 30 mL acetone and K2CO3 (2.5
equivalent, 5 mmol, 0.8 g) was added. The mixture was stirred under ice cooling for 15 minutes.
3-Chloropropionyl chloride (1.5 equivalent, 5 mmol, 0.69 g) was added. The mixture was stirred
for 3 days at room temperature. Afterwards, acetone was evaporated under reduced pressure till
half of the initial volume was reached. The mixture was poured into an ice/water mixture (100 mL)
to form a precipitate. The product was collected after vacuum filtration and was purified by silica
gel column chromatography.
Procedure B. The hydrazine derivative (2 mmol) was dissolved in 20 mL DMF then NaH (4 mmol,
0.096 g) was added under ice cooling. The mixture was stirred for 15 min then 3-chloropropionyl
chloride (1.5 equivalent, 5 mmol, 0.69 g) was added. After that, the resulting solution was heated
at 60 oC under argon atmosphere for 3h. The dark solution was poured on ice/water mixture and
the obtained solid was collected using vacuum filtration and was purified by silica gel column
chromatography.
4.1.1.3.1. 1,2-Diphenylpyrazolidin-3-one (1). Synthesized according to method A for
pyrazolidinone synthesis using 1,2-bisphenylhydrazine (b1); dark brown solid; yield: 56%; mp
1
126.3-129 °C; H NMR (400 MHz, CDCl3) δ 7.82 – 7.77 (m, 2H), 7.34 – 7.27 (m, 4H), 7.12 –
13
7.01 (m, 2H), 7.01 – 6.96 (m, 2H), 4.02 (t, J = 7.3 Hz, 2H), 2.73 (t, J = 7.3 Hz, 2H); C NMR
(101 MHz, CDCl3) δ 172.29, 150.18, 138.62, 129.70, 129.33, 124.88, 124.14, 118.92, 118.85,
55.41, 30.17; MS (ESI): m/z = 239.01 (M+H) +
.
4.1.1.3.2. 1,2-Bis(2-chlorophenyl)pyrazolidin-3-one (2). Synthesized according to method B for
pyrazolidinone synthesis using 1,2-bis(2-chlorophenyl)hydrazine (b2); white solid; yield: 15%;
1
mp 72-74 °C; H NMR (500 MHz, CDCl3) δ 7.57 – 7.49 (m, 2H), 7.45 – 7.39 (m, 1H), 7.37 (dt,
J = 5.7, 2.5 Hz, 1H), 7.24 – 7.20 (m, 2H), 7.20 – 7.16 (m, 1H), 7.08 – 7.01 (m, 1H), 4.01 (dd, J =
7.7, 6.5 Hz, 2H), 2.81 (t, J = 7.7 Hz, 2H); MS (ESI): m/z = 307.06 (M+H) +
.
4.1.1.3.3. 1,2-Bis(3-chlorophenyl)pyrazolidin-3-one (3). Synthesized according to method A for
pyrazolidinone synthesis using 1,2-bis(3-chlorophenyl)hydrazine (b3); yellow solid; yield: 83%;
1
mp 84-86 °C; H NMR (500 MHz, CDCl3) δ 7.87 (t, J = 2.1 Hz, 1H), 7.72 – 7.67 (m, 1H), 7.31 –

7.27 (m, 1H), 7.27 – 7.21 (m, 1H), 7.14 – 7.06 (m, 2H), 7.03 – 6.99 (m, 1H), 6.89 – 6.83 (m, 1H),
4.05 (t, J = 7.3 Hz, 2H), 2.77 (t, J = 7.3 Hz, 2H); 13C NMR (126 MHz, CDCl3) δ 171.16, 147.32,
136.79, 132.74, 132.52, 130.29, 129.66, 125.15, 124.39, 120.80, 118.77, 116.24, 115.35, 49.59,
31.32; MS (ESI): m/z = 307.03 (M+H) +
.
4.1.1.3.4. 1,2-Bis(4-fluorophenyl)pyrazolidin-3-one (4). Synthesized according to the method A
for pyrazolidinone synthesis using 1,2-bis(4-fluorophenyl)hydrazine (b4); beige solid; yield:
1
75%; mp 114-116 °C; H NMR (500 MHz, CDCl3) δ 7.88 – 7.56 (m, 2H), 7.04 – 6.86 (m, 6H),
3.93 (t, J = 7.4 Hz, 2H), 2.71 (t, J = 7.4 Hz, 2H); 13C NMR (126 MHz, CDCl3) δ 171.37, 159.38
(d, J = 243.2 Hz), 159.36 (d, J = 244.4 Hz), 145.47 (d, J = 2.5 Hz), 133.94 (d, J = 2.7 Hz), 120.15
(d, J = 7.9 Hz), 120.04 (d, J = 8.1 Hz), 116.04 (d, J = 22.8 Hz), 115.57 (d, J = 22.5 Hz), 55.07,
31.14; MS (ESI): m/z = 275.02 (M+H) +
.
4.1.1.3.5. 1,2-Bis(3-bromophenyl)pyrazolidin-3-one (5). Synthesized according to the method A
for pyrazolidinone synthesis using 1,2-bis(3-bromophenyl)hydrazine (b5); brown solid; yield:
1
81%; mp 97-99 °C; H NMR (500 MHz, CDCl3) δ 8.09 (t, J = 2.0 Hz, 1H), 7.83 – 7.76 (m, 1H),
7.33 (ddd, J = 8.0, 1.9, 1.1 Hz, 1H), 7.31 – 7.27 (m, 2H), 7.23 (td, J = 7.9, 6.0 Hz, 2H), 6.99 – 6.93
(m, 1H), 4.11 (t, J = 7.3 Hz, 2H), 2.83 (t, J = 7.3 Hz, 2H); 13C NMR (126 MHz, CDCl3) δ 171.62,
150.77, 138.90, 130.53, 130.22, 127.54, 126.93, 123.12, 122.64, 121.44, 120.84, 116.64, 116.63,
54.87, 31.33.; MS (ESI): m/z = 394.8 (M+H)+
.
4.1.1.3.6. 1,2-Bis(4-bromophenyl)pyrazolidin-3-one (6). Synthesized according to the method A
for pyrazolidinone synthesis using 1,2-bis(4-bromophenyl)hydrazine (b6); light brown solid;
1
yield: 75%; mp 180-181 °C; H NMR (500 MHz, CDCl3) δ 7.69 – 7.63 (m, 2H), 7.43 – 7.35 (m,
4H), 6.85 – 6.79 (m, 2H), 3.98 (t, J = 7.3 Hz, 2H), 2.72 (t, J = 7.3 Hz, 2H); 13C NMR (126 MHz,
CDCl3) δ 171.66, 148.58, 137.02, 132.46, 132.10, 120.21, 119.95, 117.54, 116.88, 54.96, 31.55;
MS (ESI): m/z = 394.8 (M+H) +
.
4.1.1.3.7. 1,2-Bis-p-tolylpyrazolidin-3-one (7). Synthesized according to method A for
pyrazolidinone synthesis using 1,2-bis-p-tolylhydrazine (b7); dark brown solid; yield: 42%; mp
1
108-110 °C; H NMR (500 MHz, CDCl3) δ 7.69 – 7.68 (m, 1H), 7.68 – 7.66 (m, 1H), 7.12 – 7.10
(m, 1H), 7.08 (tdd, J = 1.8, 0.8 Hz, 2H), 7.07 – 7.04 (m, 1H), 6.90 – 6.88 (m, 1H), 6.88 – 6.86 (m,
13
1H), 3.95 (t, J = 7.3 Hz, 2H), 2.71 (t, J = 7.3 Hz, 2H), 2.28 (d, J = 3.1 Hz, 6H); C NMR (126

MHz, CDCl3) δ 171.70, 147.48, 135.84, 134.08, 133.42, 129.90, 129.49, 118.70, 118.58, 55.10,
31.66, 20.97, 20.74; MS (ESI): m/z = 267.04 (M+H) +
.
4.1.1.3.8. 1,2-Bis(3,4-dimethylphenyl)pyrazolidin-3-one (8). Synthesized according to method A
for pyrazolidinone synthesis using 1,2-bis(3,4-dimethylphenyl)hydrazine (b8); brown solid; yield:
1
35%; mp 86-87 °C; H NMR (500 MHz, DMSO-d6) δ 7.54 (d, J = 2.0 Hz, 1H), 7.39 (dd, J = 8.3,
2.2 Hz, 1H), 7.05 (t, J = 6.9 Hz, 1H), 6.99 (t, J = 8.3 Hz, 1H), 6.82 (t, J = 3.9 Hz, 1H), 6.67 – 6.59
(m, 1H), 3.92 (t, J = 7.3 Hz, 2H), 2.61 (t, J = 7.3 Hz, 2H), 2.14 (m, 12H); 13C NMR (126 MHz,
DMSO-d6) δ 13C NMR (101 MHz, DMSO-d6) δ 171.91, 148.24, 137.52, 136.96, 136.56, 132.42,
131.69, 130.34, 130.14, 120.20, 119.46, 116.12, 115.83, 54.90, 31.39, 20.21, 20.18, 19.21, 19.01;
MS (ESI): m/z = 295.13 (M+H) +
.
4.1.1.4. General procedure for Suzuki coupling (9-12). The aryl boronic acid derivative (1.5 mmol)
was added to a mixture of the bis-bromophenyl derivative (0.5 mmol, 0.2 g), caesium carbonate
(4 equivalent, 0.65 g, 2 mmol) and tetrakis (triphenylphosphine) palladium (0.05 equivalent, 0.025
mmol, 0.02 g) in 30 mL of dioxane/water (4 : 1). The mixture was refluxed overnight at 100 oC.
The solvent was evaporated under reduced pressure, then brine was added and the product was
extracted with EtOAc (3×40 mL). The combined organic extracts were dried with Mg2SO4,
evaporated to dryness under reduced pressure and purified by silica gel column chromatography.
4.1.1.4.1. 1,2-Bis([1,1'-biphenyl]-4-yl)pyrazolidin-3-one (9). Synthesized according to the method
outlined above using phenylboronic acid and 1,2-bis(4-bromophenyl)pyrazolidin-3-one (6) ; beige
1
solid; yield: 30%; mp 155-157 °C; H NMR (500 MHz, CDCl3) δ 7.77 (d, J = 8.7 Hz, 2H), 7.46
– 7.33 (m, 8H), 7.26 (t, J = 7.6 Hz, 4H), 7.20 – 7.12 (m, 2H), 6.93 (d, J = 8.6 Hz, 2H), 3.91 (t, J =
13
7.3 Hz, 2H), 2.63 (t, J = 7.2 Hz, 2H); C NMR (126 MHz, CDCl3) δ 172.19, 149.28, 140.74,
140.60, 137.74, 137.57, 137.10, 129.15, 129.12, 128.31, 127.91, 127.48, 127.20, 127.15, 119.15,
119.04, 115.73, 55.33, 32.06; MS (ESI): m/z = 391.32 (M+H) +.
4.1.1.4.2. 1,2-Bis(4-(thiophen-3-yl)phenyl)pyrazolidin-3-one (10). Synthesized according to the
method outlined above using 3-thiophene boronic acid and 1,2-bis(4-bromophenyl)pyrazolidin-3-
one (6); light brown solid; yield: 35%; mp 176-177 °C; 1H NMR (500 MHz, CDCl3) δ 7.88 – 7.83
(m, 2H), 7.57 – 7.53 (m, 2H), 7.53 – 7.49 (m, 2H), 7.39 (dd, J = 2.8, 1.4 Hz, 1H), 7.37 – 7.32 (m,
4H), 7.32 – 7.30 (m, 1H), 7.04 – 7.00 (m, 2H), 4.05 (t, J = 7.3 Hz, 2H), 2.77 (t, J = 7.3 Hz, 2H);

13C NMR (126 MHz, CDCl3) δ 171.58, 148.49, 141.50, 141.32, 136.94, 131.91, 131.53, 127.18,
126.76, 126.19, 126.11, 126.02, 125.98, 119.76, 119.64, 118.71, 118.54, 54.76, 31.58; MS (ESI):
m/z = 403.33 (M+H) +.
4.1.1.4.3. 1,2-Bis(3-(furan-2-yl)phenyl)pyrazolidin-3-one (11). Synthesized according to the
method outlined above using 2-furan boronic acid and 1,2-bis(3-bromophenyl)pyrazolidin-3-one
1
(5); dark brown solid; yield: 18%; mp 121-123 °C; H NMR (500 MHz, CDCl3) δ 7.66 – 7.60 (m,
2H), 7.58 – 7.50 (m, 1H), 7.48 – 7.40 (m, 2H), 7.09 – 6.98 (m, 3H), 6.94 – 6.90 (m, 2H), 6.87 (t,
J = 2.0 Hz, 1H), 6.77 (ddd, J = 8.3, 2.5, 0.8 Hz, 1H), 6.62 (ddd, J = 8.1, 2.2, 1.0 Hz, 1H), 6.16 (s,
13
1H), 3.81 – 3.70 (m, 2H), 2.69 (s, 2H); C NMR (126 MHz, CDCl3) δ 176.09, 149.91, 148.50,
132.24, 132.22, 132.16, 132.08, 131.94, 131.11, 130.78, 130.65, 128.65, 128.55, 123.56, 123.41,
122.96, 122.03, 115.70, 115.26, 111.35, 111.15, 46.99, 31.24.; MS (ESI): m/z = 371.22 (M+H) +.
4.1.1.4.4. 1,2-Bis(4-(furan-2-yl)phenyl)pyrazolidin-3-one (12). Synthesized according to the
method outlined above using 2-furan boronic acid and 1,2-bis(4-bromophenyl)pyrazolidin-3-one
1
(6); brown solid; yield: 15%; mp 158-160 °C; H NMR (500 MHz, CDCl3) δ 7.88 – 7.83 (m, 2H),
7.57 – 7.49 (m, 4H), 7.40 – 7.28 (m, 6H), 7.04 – 7.00 (m, 2H), 4.05 (t, J = 7.3 Hz, 2H), 2.77 (t, J
= 7.3 Hz, 2H); 13C NMR (126 MHz, CDCl3) δ 171.67, 148.58, 141.59, 141.41, 137.03, 132.00,
131.62, 127.28, 126.85, 126.28, 126.20, 126.11, 126.07, 119.85, 119.74, 118.80, 118.63, 54.85,
31.67.; MS (ESI): m/z = 371.26 (M+H) +
.
4.1.1.5. 1,2-Bis(3-chlorophenyl)-4-methylpyrazolidin-3-one (13). To a three-necked flask, lithium
diisopropylamide (LDA) (0.6 mL) (2M in THF/n-heptane/ethylbenzene) was added and mixed
with THF (10 mL) under argon atmosphere. The solution was stirred for 20 min at 0 oC, and further
cooled to -78 oC. A solution of compound 3 (1 mmol, 0.306 g) dissolved in THF (10 mL) was
added to the mixture via a syringe dropwise. After stirring for 1 h at -78oC, methyl iodide (2 mmol,
0.12 mL) was added. The mixture was left to stir for additional 30 min at -78 oC, then left to attain
room temperature and left to stir overnight. Afterwards, the solvent was evaporated to dryness
under reduced pressure and the residue was partitioned between brine and EtOAc. The EtOAc
layer was separated and the aqueous layer was washed with 50 mL EtOAc for two times. The
combined organic layers were dried over anhydrous MgSO4, evaporated under vacuum and the
residue was purified using silica gel column chromatography to give the title compound as a

yellow solid; yield: 52%; mp 141-142; 1H NMR (500 MHz, CDCl3) δ 7.83 (dd, J = 3.1, 1.1 Hz,
1H), 7.68 – 7.65 (m, 1H), 7.25 – 7.22 (m, 1H), 7.21 – 7.17 (m, 1H), 7.08 (ddd, J = 8.0, 2.0, 1.0 Hz,
1H), 7.02 (tdd, J = 3.8, 1.9, 0.9 Hz, 1H), 6.97 – 6.95 (m, 1H), 6.81 (ddd, J = 8.2, 2.3, 0.9 Hz, 1H),
4.07 (dd, J = 11.5, 7.5 Hz, 1H), 3.71 (t, J = 11.5 Hz, 1H), 2.95 (dp, J = 11.5, 7.1 Hz, 1H), 1.21 (d,
J = 7.0 Hz, 3H); 13C NMR (126 MHz, CDCl3) δ 174.21, 150.85, 139.01, 135.10, 134.68, 130.25,
129.96, 124.52, 123.74, 118.34, 118.00, 116.08, 115.98, 62.11, 36.06, 12.63; MS (ESI): m/z =
321.10 M+H) +
.
4.1.1.6. 1,2-Bis(3-chlorophenyl)-3-oxopyrazolidine-4-carboxylic acid (14). To a three-necked
flask, lithium diisopropylamide (LDA) (35 mL) (2 M in THF/n-heptane/ethylbenzene) was added
and mixed with THF (10 mL) under argon atmosphere. The solution was stirred for 20 minutes at
0 oC, and further cooled to -78 oC. A solution of compound 3 (5 mmol, 1.53 g) dissolved in THF
(10 mL) was added via a syringe dropwise. After stirring for 1 h at -78 oC, crushed dry ice was
added until excess solid dry ice was observed, and left till no effervescence remains in the flask
(about 1 h). The mixture was evaporated to dryness then brine was added. The medium was
acidified using 2M HCl. The separated yellow residue was extracted with EtOAc. The aqueous
layer was washed with 50 mL EtOAc for two times. The combined organic layers were dried over
anhydrous MgSO4, evaporated under vacuum and the residue was purified using silica gel column
chromatography, using (CH2Cl2, 1% HCOOH) as mobile phase to give the title compound as a
1
yellow solid; yield: 89%; mp 160-162 °C; H NMR (500 MHz, DMSO-d6) δ 13.15 (s, 1H), 7.79
(t, J = 2.1 Hz, 1H), 7.57 (ddd, J = 8.3, 2.1, 1.0 Hz, 1H), 7.37 (dt, J = 35.9, 8.1 Hz, 2H), 7.24 (ddd,
J = 8.0, 2.1, 1.0 Hz, 1H), 7.14 – 7.05 (m, 2H), 6.92 (ddd, J = 8.2, 2.3, 1.0 Hz, 1H), 4.34 (qd, J =
13
12.0, 7.8 Hz, 2H), 3.94 (t, J = 7.7 Hz, 1H); C NMR (126 MHz, DMSO- d6) δ 168.68, 168.14,
150.90, 138.95, 133.86, 133.66, 131.15, 130.96, 124.90, 123.43, 117.98, 117.88, 116.92, 116.53,
57.20, 48.62; MS (ESI): m/z = 351.12 (M+H) +
.
4.1.1.7. General procedure for amide synthesis (15- 23). To a solution of compound 14 (1.2 mmol,
0.4 g) in 20 mL CH2Cl2, HBTU (1.3 mmol, 0.5 g), triethylamine (0.4 mL) and the respective
amine (4 equivalent, 4.8 mmol) were added sequentially. The mixture was stirred for 5 h at room

temperature. The solvent was evaporated under reduced pressure then purification was done using
silica gel column chromatography.
4.1.1.7.1. 1,2-Bis(3-chlorophenyl)-4-(4-methylpiperazine-1-carbonyl)pyrazolidin-3-one (15).
Synthesized according to the general procedure for amide synthesis using N-methylpiperazine;
1
white solid; yield: 76%; mp 159-161 °C; H NMR (400 MHz, DMSO-d6) δ 7.77 (t, J = 2.0 Hz,
1H), 7.54 (ddd, J = 8.3, 2.0, 1.0 Hz, 1H), 7.41 (t, J = 8.2 Hz, 1H), 7.31 (t, J = 8.0 Hz, 1H), 7.22
(ddd, J = 8.0, 2.0, 1.0 Hz, 1H), 7.12 – 6.99 (m, 2H), 6.96 – 6.88 (m, 1H), 4.63 (dd, J = 9.7, 7.8 Hz,
1H), 4.56 – 4.37 (m, 1H), 4.21 (dd, J = 11.7, 7.8 Hz, 1H), 3.75 – 3.46 (m, 3H), 3.46 – 3.23 (m,
3H), 2.40 (s, J = 16.1 Hz, 2H), 2.21 (s, 3H); 13C NMR (101 MHz, DMSO-d6) δ 168.88, 164.05,
150.91, 138.84, 133.58, 133.47, 130.97, 130.71, 124.63, 122.98, 117.70, 117.54, 116.57, 116.45,
56.92, 54.80, 54.11, 44.55, 41.57; MS (ESI): m/z = 433.13 (M+H) +
.
4.1.1.7.2.
1,2-Bis(3-chlorophenyl)-4-(morpholine-4-carbonyl)pyrazolidin-3-one
(16).
Synthesized according to the general procedure for amide synthesis using morpholine; white solid;
1
yield: 77%; mp 93-95 °C; H NMR (500 MHz, DMSO-d6 ) δ 7.78 (t, J = 2.0 Hz, 1H), 7.54 (ddd,
J = 8.4, 2.1, 0.9 Hz, 1H), 7.41 (t, J = 8.2 Hz, 1H), 7.31 (t, J = 8.1 Hz, 1H), 7.23 (ddd, J = 8.0, 2.1,
0.9 Hz, 1H), 7.13 – 7.02 (m, 2H), 6.93 (ddd, J = 8.3, 2.3, 0.8 Hz, 1H), 4.64 (dd, J = 10.0, 7.8 Hz,
1H), 4.46 (dd, J = 11.7, 10.2 Hz, 1H), 4.21 (dd, J = 11.8, 7.7 Hz, 1H), 3.74 – 3.46 (m, 7H), 3.39 –
3.34 (m, 1H); 13C NMR (126 MHz, DMSO-d6) δ 168.84, 164.18, 150.91, 138.81, 133.61, 133.48,
131.00, 130.75, 124.68, 123.04, 117.72, 117.57, 116.60, 116.49, 66.44, 56.86, 46.07, 44.42; MS
(ESI): m/z =420.4 (M+H)+
.
4.1.1.7.3.
1,2-Bis(3-chlorophenyl)-N-methoxy-3-oxopyrazolidine-4-carboxamide
(17).
Synthesized according to the general procedure for amide synthesis using methoxyamine
hydrochloride ; white creamy semisolid; yield: 25.1%; 1H NMR (400 MHz, DMSO- d6) δ 11.35
(s, 1H), 7.79 (t, J = 2.0 Hz, 1H), 7.56 (ddd, J = 8.3, 2.0, 0.9 Hz, 1H), 7.42 (t, J = 8.2 Hz, 1H), 7.33
(t, J = 8.1 Hz, 1H), 7.24 (ddd, J = 8.0, 2.0, 0.9 Hz, 1H), 7.12 (dd, J = 7.6, 1.5 Hz, 1H), 7.07 (t, J =
2.1 Hz, 1H), 6.91 (dd, J = 7.9, 1.9 Hz, 1H), 4.29 (dd, J = 14.3, 6.6 Hz, 2H), 3.68 (dd, J = 9.7, 8.2
Hz, 1H), 3.58 (s, 3H); 13C NMR (101 MHz, DMSO-d6) δ 169.22, 163.13, 151.39, 139.24, 134.32,

134.05, 131.59, 131.47, 125.33, 124.07, 118.50, 118.12, 117.14, 117.00, 63.98, 57.25, 46.55; MS
(ESI): m/z = 380 (M+H) +
.
4.1.1.7.4. 1,2-Bis(3-chlorophenyl)-N-methoxy-N-methyl-3-oxopyrazolidine-4-carboxamide (18).
Synthesized according to the general procedure for amide synthesis using N-methoxy-N-
methylamine hydrochloride; white solid; yield: 53%; mp 112-114; 1H NMR (400 MHz, DMSO-
d6) δ 7.78 (t, J = 2.0 Hz, 1H), 7.55 (ddd, J = 8.3, 2.0, 0.9 Hz, 1H), 7.42 (t, J = 8.2 Hz, 1H), 7.31 (t,
J = 8.0 Hz, 1H), 7.24 (ddd, J = 8.0, 2.0, 0.9 Hz, 1H), 7.09 (dt, J = 4.2, 1.5 Hz, 2H), 6.94 – 6.87 (m,
13
1H), 4.52 (t, J = 8.8 Hz, 1H), 4.32 (dd , J = 11.4 Hz, 2H), 3.73 (s, 3H), 3.14 (s, 3H); C NMR
(101 MHz, DMSO- d6) δ 169.14, 167.11, 151.31, 139.23, 134.18, 133.99, 131.50, 131.29, 125.27,
123.69, 118.35, 118.17, 117.18, 116.80, 62.08, 57.34, 45.10, 32.22; MS (ESI): m/z = 394.10
(M+H) +
.
4.1.1.7.5.
1,2-Bis(3-chlorophenyl)-N,N-dimethyl-3-oxopyrazolidine-4-carboxamide
(19).
Synthesized according to the general procedure for amide synthesis using dimethylamine; light
1
yellow powder; yield: 39.7%; mp 152-154 °C; H NMR (400 MHz, DMSO- d6) δ 7.77 (t, J = 2.0
Hz, 1H), 7.54 (ddd, J = 8.3, 2.0, 1.0 Hz, 1H), 7.46 – 7.37 (m, 1H), 7.31 (dd, J = 10.5, 5.6 Hz, 1H),
7.22 (ddd, J = 7.9, 2.0, 1.0 Hz, 1H), 7.07 (ddd, J = 5.4, 3.1, 1.3 Hz, 2H), 6.91 (ddd, J = 8.3, 2.2,
0.8 Hz, 1H), 4.59 (dd, J = 9.7, 7.9 Hz, 1H), 4.41 (dd, J = 11.5, 9.9 Hz, 1H), 4.22 (dd, J = 11.6, 7.9
Hz, 1H), 3.10 (s, 3H), 2.86 (s, 3H); 13C NMR (100 MHz, DMSO- d6) δ 168.71, 165.39, 150.70,
138.54, 133.34, 133.18, 130.68, 130.45, 124.36, 122.71, 117.45, 117.31, 116.32, 116.09, 56.72,
44.55, 36.81, 35.10; MS (ESI): m/z = 378.15 (M+H) +
.
4.1.1.7.6.
1,2-Bis(3-chlorophenyl)-N,N-diethyl-3-oxopyrazolidine-4-carboxamide
(20).
Synthesized according to the general procedure for amide synthesis using diethylamine; beige
1
solid; yield: 43%; mp 117-119 °C; H NMR (400 MHz, DMSO- d6) δ 7.78 (t, J = 2.0 Hz, 1H),
7.54 (ddd, J = 8.3, 2.1, 1.0 Hz, 1H), 7.42 (dd, J = 10.8, 5.5 Hz, 1H), 7.31 (t, J = 8.1 Hz, 1H), 7.23
(ddd, J = 7.9, 2.1, 1.0 Hz, 1H), 7.11 – 7.02 (m, 2H), 6.91 (ddd, J = 8.2, 2.3, 0.8 Hz, 1H), 4.42 (dd,
J = 18.9, 8.3 Hz, 2H), 4.29 (dd, J = 10.3, 6.9 Hz, 1H), 3.60 – 3.49 (m, 1H), 3.45 – 3.34 (m, 1H),
3.33 – 3.16 (m, 2H), 1.12 (t, J = 7.1 Hz, 3H), 0.99 (t, J = 7.0 Hz, 3H); 13C NMR (101 MHz,
DMSO- d6) δ 169.69, 165.66, 151.48, 139.38, 134.06, 133.97, 131.46, 131.18, 125.11, 123.38,

118.21, 118.05, 117.06, 116.82, 57.71, 45.45, 42.10, 14.32, 13.23; MS (ESI): m/z = 406.1 (M+H)
+
.
4.1.1.7.7. 1,2-Bis(3-chlorophenyl)-4-(piperidine-1-carbonyl)pyrazolidin-3-one (21). Synthesized
according to the general procedure for amide synthesis using piperidine; beige solid; yield: 34%;
1
mp 163-165 °C; H NMR (400 MHz, DMSO- d6) δ 7.77 (t, J = 2.0 Hz, 1H), 7.54 (ddd, J = 8.3,
1.9, 0.7 Hz, 1H), 7.41 (t, J = 8.2 Hz, 1H), 7.30 (t, J = 8.1 Hz, 1H), 7.22 (ddd, J = 8.0, 2.0, 0.8 Hz,
1H), 7.09 – 7.02 (m, 2H), 6.94 – 6.88 (m, 1H), 4.61 (dd, J = 9.6, 7.9 Hz, 1H), 4.44 (dd, J = 11.5,
9.9 Hz, 1H), 4.21 (dd, J = 11.7, 7.8 Hz, 1H), 3.63 – 3.42 (m, 3H), 3.39 – 3.30 (m, 1H), 1.67 – 1.46
(m, 4H), 1.46 – 1.35 (m, 2H); 13C NMR (101 MHz, DMSO- d6) δ 169.50, 164.22, 151.40, 139.33,
134.01, 133.91, 131.41, 131.14, 125.03, 123.36, 118.12, 117.96, 116.99, 116.85, 57.48, 46.91,
45.03, 43.11, 26.62, 25.75, 24.38; MS (ESI): m/z = 418.04 (M+H) +
,
4.1.1.7.8.
1,2-Bis(3-chlorophenyl)-N-cyclopropyl-3-oxopyrazolidine-4-carboxamide
(22).
Synthesized according to the general procedure for amide synthesis using cyclopropylamine;
1
brown solid; yield: 63%; mp 138-140 °C; H NMR (500 MHz, DMSO- d6) δ 8.24 (d, J = 4.2 Hz,
1H), 7.79 (t, J = 2.0 Hz, 1H), 7.55 (ddd, J = 8.4, 2.1, 0.9 Hz, 1H), 7.44 – 7.38 (m, 1H), 7.35 – 7.29
(m, 1H), 7.23 (ddd, J = 8.0, 2.1, 0.9 Hz, 1H), 7.13 – 7.08 (m, 1H), 7.05 (t, J = 2.1 Hz, 1H), 6.90
(ddd, J = 8.3, 2.3, 0.8 Hz, 1H), 4.24 (dd, J = 19.2, 9.3 Hz, 2H), 3.78 (dd, J = 9.9, 8.1 Hz, 1H), 2.68
– 2.58 (m, 1H), 0.69 – 0.54 (m, 2H), 0.43 – 0.27 (m, 2H); 13C NMR (126 MHz, DMSO- d6) δ
169.31, 166.64, 150.97, 138.80, 133.76, 133.49, 131.01, 130.90, 124.61, 123.43, 117.86, 117.43,
116.45, 116.44, 57.13, 48.16, 22.49, 5.79, 5.69; MS (ESI): m/z = 390.08 (M+H) +
.
4.1.1.7.9. N-Allyl-1,2-bis(3-chlorophenyl)-3-oxopyrazolidine-4-carboxamide (23). Synthesized
according to the general procedure for amide synthesis using allylamine; grey solid; yield: 42.9%;
1
mp 93-95 °C; H NMR (400 MHz, DMSO- d6) δ 8.33 (t, J = 5.7 Hz, 1H), 7.80 (t, J = 2.0 Hz, 1H),
7.57 (ddd, J = 8.3, 2.1, 1.0 Hz, 1H), 7.45 – 7.29 (m, 2H), 7.23 (ddd, J = 8.0, 2.1, 1.0 Hz, 1H), 7.14
– 7.04 (m, 2H), 6.91 (ddd, J = 8.2, 2.3, 0.8 Hz, 1H), 5.77 (ddt, J = 17.2, 10.1, 4.9 Hz, 1H), 5.21
(dd, J = 17.2, 1.8 Hz, 1H), 5.06 (dd, J = 10.3, 1.7 Hz, 1H), 4.28 (dd, J = 9.1, 3.8 Hz, 2H), 3.96 (t,
J = 9.1 Hz, 1H), 3.80 – 3.65 (m, 2H); 13C NMR (101 MHz, DMSO- d6) δ 169.38, 165.45, 150.98,

138.81, 134.48, 133.78, 133.50, 131.02, 130.92, 124.64, 123.45, 117.89, 117.46, 116.50, 116.44,
115.20, 57.10, 48.22, 41.03; MS (ESI): m/z = 390.00 (M+H) +
.
4.1.1.8. 1,2-Bis(3-chlorophenyl)-4-(hydroxyimino)pyrazolidin-3-one (24). To a stirred solution of
compound 14 in 5:1 Et2O/H2O (10 mL) cooled at 0 °C, NaNO2 (400 mg, 5.8 mmol) was added
followed by conc. HCl (3 mmol, 0.25 mL) cautiously divided on four portions for 3 h. After stirring
for 2.5 hours at room temperature, saturated aqueous ammonium chloride was added (15 mL). The
mixture was stirred for 1 more hour. The mixture was extracted with EtOAc (4 times). Organic
layers were collected, dried over MgSO4 and the solvent was evaporated under reduced pressure.
Then the resulting residue was purified by silica gel column chromatography to afford the title
compound as a yellow oil; yield: 19%; 1H NMR (500 MHz, DMSO- d6) δ 12.83 (s, 1H), 7.88 (t,
J = 2.0 Hz, 1H), 7.63 (ddd, J = 8.4, 2.1, 0.9 Hz, 1H), 7.46 – 7.40 (m, 1H), 7.34 – 7.28 (m, 1H),
7.28 – 7.24 (m, 1H), 7.12 (dtt, J = 2.9, 2.0, 1.0 Hz, 2H), 6.95 (ddd, J = 8.3, 2.2, 1.0 Hz, 1H), 4.68
(s, 2H); 13C NMR (126 MHz, DMSO- d6) δ 160.27, 151.93, 148.32, 139.15, 133.90, 133.40,
131.07, 130.90, 125.19, 123.99, 118.46, 118.12, 117.07, 116.76, 66.34; MS (ESI): m/z = 336.05
(M+H) +
.
4.1.1.9. 4-Amino-1,2-bis(3-chlorophenyl)pyrazolidin-3-one (25). To a stirred solution of oxime
24 (0.167 g, 0.5 mmol) in acetic acid (20 ml) at room temperature was added zinc dust (1.29 g,
19.8 mmol) and NH4Cl (0.56 g, 10.5 mmol). After stirring at 50 °C overnight, the mixture was
filtered over a pad of silica gel and the filtrate was concentrated under reduced pressure. To the
resulting residue, saturated aq. NaHCO3 was added and the mixture was extracted with methylene
chloride. The combined organic layers were dried over anhydrous Na2SO4. The solvent was
removed under reduced pressure then resulting residue was purified by silica gel chromatography
to afford the title compound as while solid; yield: 42%; mp 180-182; 1H NMR (500 MHz, DMSO-
d6) δ 7.80 (t, J = 2.0 Hz, 1H), 7.54 (ddd, J = 8.4, 2.1, 0.9 Hz, 1H), 7.41 (t, J = 8.2 Hz, 1H), 7.30 (t,
J = 8.1 Hz, 1H), 7.24 – 7.14 (m, 1H), 7.13 – 7.03 (m, 2H), 6.90 (ddd, J = 8.3, 2.2, 0.8 Hz, 1H),
4.21 (dd, J = 11.2, 7.1 Hz, 1H), 3.82 (dd, J = 11.4, 7.1 Hz, 1H), 3.72 (t, J = 11.3 Hz, 1H), 2.05 (s,

2H); 13C NMR (126 MHz, DMSO-d6) δ 174.58, 150.99, 139.36, 133.78, 133.40, 130.90, 130.85,
124.14, 122.88, 117.40, 117.03, 116.04, 115.93, 61.46, 51.64. MS (ESI): m/z = 322.02 (M+H) +
.
4.2. Biology
4.2.1 Minimal Inhibitory Concentration (MIC) determination
MIC values for E. coli TolC, S. aureus (strain Newman), B. subtilis (ATCC 6633) and E. coli BL-
21 were determined for all compounds with a maximal DMSO concentration of 1% as previously
described in ref [20]. Final compound concentrations prepared from serial dilutions ranged from
1 to 25 µg/mL and were adapted for each compound depending on their antibacterial activity and
the observation of compound precipitation in the growth medium. The ODs were determined after
addition of the compounds and again after incubation for 16 h at 37 °C and 50 rpm in 96 well
plates using a POLARstar Omega Microplate Reader (BMG LABTECH). Given MIC values are
means of two independent determinations. They are defined as the lowest concentration of
compounds that reduced OD600 by ≥ 95 % and were read off the inhibition curves. Experiments
were made at least two times and standard deviation was less than 20 % (most cases: < 15 %). LB
broth was used for E.coli and B. subtilis, and Müller Hinton medium was used for S. aureus.
4.2.2. Determination of cytotoxicity
HepG2 cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS), 100
units/mL penicillin, and 100 μg/mL streptomycin. Cells were cultured in a 96-well plate at a
density of 3x104 cell/well, and then incubated in 5% CO2 at 37oC for 12 hours. Media was replaced
with a fresh one containing variable concentrations of the tested compounds dissolved in DMSO
and left for 36 hours in 5% CO2 at 37oC. To access the cytotoxicity a MTT assay was performed.
The media was replaced with a fresh one containing MTT (5mg/mL) and incubated for 3 hours.
The precipitate was dissolved in DMSO and measured at 570 nm using a POLARstar Omega
Microplate Reader (BMG LABTECH). 0.1% DMSO was used as a control and the samples were
assayed in triplicates in 4 different cultures. To determine the LD50 probit analysis was used.