Search for factors affecting antibacterial activity and toxicity of 1,2,4-triazole-ciprofloxacin hybrids

Article abstract of DOI:10.1016/j.ejmech.2015.04.058

A series of 1,2,4-triazole-based compounds was designed as potential antibacterial agents using molecular hybridization approach. The target compounds (23-44) were synthesized by Mannich reaction of 1,2,4-triazole-3-thione derivatives with ciprofloxacin (

Full text of DOI:10.1016/j.ejmech.2015.04.058

Accepted Manuscript
Search for factors affecting antibacterial activity and toxicity of 1,2,4-triazole-
ciprofloxacin hybrids
Tomasz Plech, Barbara Kaproń, Agata Paneth, Urszula Kosikowska, Anna Malm,
Aleksandra Strzelczyk, Paweł Stączek, Łukasz Świątek, Barbara Rajtar, Małgorzata
Polz-Dacewicz
PII:
S0223-5234(15)30022-2
10.1016/j.ejmech.2015.04.058
EJMECH 7873
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 13 February 2015
Revised Date: 12 April 2015
Accepted Date: 27 April 2015
Please cite this article as: T. Plech, B. Kaproń, A. Paneth, U. Kosikowska, A. Malm, A. Strzelczyk, P.
Stączek, Ł. Świątek, B. Rajtar, M. Polz-Dacewicz, Search for factors affecting antibacterial activity and
toxicity of 1,2,4-triazole-ciprofloxacin hybrids, European Journal of Medicinal Chemistry (2015), doi:
10.1016/j.ejmech.2015.04.058.
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ACCEPTED MANUSCRIPT
Search for factors affecting antibacterial activity and toxicity of 1,2,4-triazole-
ciprofloxacin hybrids
Tomasz Plech,* Barbara Kaproń, Agata Paneth, Urszula Kosikowska, Anna Malm,
Aleksandra Strzelczyk, Paweł Stączek, Łukasz Świątek, Barbara Rajtar, Małgorzata Polz-
Dacewicz
•
•
primary effect on antibacterial activity
CPX
the presence of a phenyl ring is essential for strong
antibacterial effect
N
N
•
•
EDGs on a phenyl ring are more favourable than EWGs
R1
S
N
negative correlation between the alkyl substituent length
and antibacterial activity
R2
•
•
secondary effect on antibacterial activity
the presence of a phenyl ring is not indispensable
for strong antibacterial effect
•
•
negative correlation between the alkyl substituent length
and antibacterial activity
the presence of methylene linker (between triazole and phenyl rings)
increases toxicity against human cells

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Search for factors affecting antibacterial activity and toxicity
of 1,2,4-triazole-ciprofloxacin hybrids
Tomasz Plech,1* Barbara Kaproń,1 Agata Paneth,1 Urszula Kosikowska,2 Anna Malm,2
Aleksandra Strzelczyk,3 Paweł Stączek,3 Łukasz Świątek,4 Barbara Rajtar,4
Małgorzata Polz-Dacewicz4
1Department of Organic Chemistry, Faculty of Pharmacy, Medical University,
Chodzki 4A, 20-093 Lublin, Poland
2Department of Pharmaceutical Microbiology, Faculty of Pharmacy, Medical University,
Chodzki 1, 20-093 Lublin, Poland
3Department of Genetics of Microorganisms, University of Lodz,
Banacha 12/16, 90-237 Lodz, Poland
4Department of Virology, Faculty of Medicine, Medical University,
Chodźki 1, 20-093 Lublin, Poland
*Correspondence to Tomasz Plech, Department of Organic Chemistry, Faculty of Pharmacy,
Medical University, Chodzki 4A, 20-093 Lublin, Poland
Phone: +48 081 532 05 19. Fax: +48 081 532 45 46.
Email: tomasz.plech@umlub.pl
Keywords:
1,2,4-triazole-3-thiones; fluoroquinolones; gyrase DNA; topoisomerase IV; MTT assay

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Abstract
A series of 1,2,4-triazole-based compounds was designed as potential antibacterial agents
using molecular hybridization approach. The target compounds (23-44) were synthesized by
Mannich reaction of 1,2,4-triazole-3-thione derivatives with ciprofloxacin (CPX) and
formaldehyde. Their potent antibacterial effect on Gram-positive bacteria was accompanied
by similarly strong activity against Gram-negative strains. The toxicity of the CPX-triazole
hybrids for bacterial cells was even up to 18930 times higher than the toxicity for human
cells. The results of enzymatic studies showed that the antibacterial activity of the CPX-
triazole hybrids is not dependent solely on the degree of their affinity to DNA gyrase and
topoisomerase IV.
1. Introduction
Bacterial type II topoisomerases (DNA gyrase and topoisomerase IV) are essential in the
process of replication, transcription, repair and recombination of genetic material [1]. The
action of topoisomerases consists in introducing changes to the spatial structure of DNA by
catenation and decatenation of duplex DNA rings, relaxation of supercoiled DNA, and in the
case of DNA gyrase – introduction of negative supercoils into DNA in an energy-dependent
reaction [2]. Until today, fluoroquinolones (FQs) have been the most effective antibacterials,
the molecular target of which is DNA gyrase (gyrDNA) and topoisomerase IV (topoIV) [3].
In the case of Gram-negative bacteria, a primary target of fluoroquinolones is gyrDNA
whereas topoIV inhibition is merely an additional element that contributes to the total
antibacterial action of FQs. DNA gyrase catalyses ATP-dependent reaction of introducing
negative supercoils in the circular DNA of bacteria, which allows the double strand to be
uncoiled during the replication process. In Gram-positive bacteria, however, the primary
molecular target of FQs is topoIV. The main function of topoIV appears to be decatenation of
the daughter circular chromosomes [4]. The aim of the research that we have conducted so far
has been to design potential antibacterial drugs which inhibit bacterial topoisomerases [5, 6].
Our research revealed that by joining ciprofloxacin (CPX) with different 1,2,4-triazole
derivatives, the antibacterial effect of CPX may be enhanced [6]. The rationale for combining
these two molecules was clarified in our previous paper [6]. The utilization of the molecular
hybridization strategy for obtaining novel CPX derivatives (hybrids) was also described by
other authors [7, 8]. It was obvious that the differences in the activity of CPX-triazole hybrids

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resulted from the structure of substituents connected to the 1,2,4-triazole ring. The most
favourable antibacterial effect was obtained when the aforementioned five-membered
heterocyclic ring was connected with a hydroxyphenyl fragment. By exploring this subject,
we have attempted to trace the way in which further modification of the structure of CPX-
triazole hybrids affects the antibacterial activity. Our aim was also to check whether there is
an interrelation between the structure of the synthesized compounds and their toxicity for
human cells. Finally, the enzymatic studies presented now are an attempt to gain a better
understanding of the molecular mechanisms responsible for antibacterial activity of the CPX-
triazole hybrids.
2. Results and discussion
2.1. Chemistry
4,5-Disubstituted-1,2,4-triazole-3-thiones (1-22) were obtained by dehydrocyclization of the
respective thiosemicarbazides (described in Ref. [5]) in alkaline medium. Their chemical
1
13
structures were confirmed on the basis of H-NMR and C-NMR spectra, and the results of
elemental analysis. The CPX-triazole hybrids (23-44) were synthesized according to the
method described previously [6], and presented in Scheme 1.
Scheme 1
2.2. Antibacterial activity
The microbiological tests conducted on the reference strains of Gram-positive and Gram-
negative bacteria revealed that the newly obtained CPX-triazole hybrids were, in large
majority, much more potent than CPX itself. In the group of the Gram-positive bacteria tested,
the Staphylococcus aureus strain is of the greatest epidemiological relevance. Asymptomatic
carrier state of these bacteria is quite common – even in 50% of the population, but the risk of
developing symptoms mainly concerns persons with impaired immune system [9]. The
greatest mortality rate is noted in individuals infected with methicillin-resistant S. aureus
(MRSA). Treating MRSA infections is difficult due to the fact that these strains are resistant
to β-lactam antibiotics, and additionally over 90% of MRSA strains exhibit cross-resistance
with macrolides and fluoroquinolones (including CPX) [10]. In the group of the derivatives

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tested, the compounds with 16-fold greater activity than CPX (i.e., 38, 41) with respect to S.
aureus ATCC25923 were identified. Moreover, complete inhibition of the growth of the
second methicillin-susceptible S. aureus strain (i.e. S. aureus ATCC6538) by compounds 30,
34 and 38 occurred at about 8-fold lower concentrations when compared to CPX (Table 1).
Surprisingly, the tested MRSA strain turned out to be more sensitive to the synthesized CPX-
triazole hybrids than the MSSA strains. Merely 3 of 22 derivatives obtained exhibited activity
that was comparable to or lower than vancomycin – a drug used for treatment of MRSA
infections. The remaining compounds demonstrated more potent action than the
aforementioned drug and, what must be emphasised, the minimal inhibitory concentrations
(MICs) for compounds 26 and 38 were about 15 times lower than in the case of vancomycin
(i.e., 0.046 ꢀM vs. 0.68 ꢀM). Furthermore, it must be noted that all CPX-triazole hybrids
were characterised by bactericidal effects (MBC/MIC≤4) towards both MSSA and MRSA
strains. Moreover, the fact that Bacillus cereus ATCC10876 was found to be highly sensitive
to the synthesized compounds is also important from the microbiological point of view. It is
crucial, as Bacillus cereus is phylogenetically related to Bacillus anthracis (i.e. an etiologic
agent of anthrax) [11]. Due to this similarity, the screening tests on new candidates for anti-
anthrax drugs are conducted using B. cereus strain since it is assumed that compounds which
inhibit its growth will also be effective against B. anthracis. Thus, taking into account the
MIC values, one can assume that the CPX-triazole hybrids could also be effective in the
treatment of anthrax.
Tables 1 & 2
As shown in Table 2, the potent antibacterial effect on Gram-positive bacteria exerted by the
obtained CPX-triazole hybrids is also accompanied by similarly strong activity against Gram-
negative bacteria. The Gram-negative strains tested included, among others, Escherichia coli
ATCC25922, Klebsiella pneumoniae ATCC13883 and Pseudomonas aeruginosa ATCC9027.
Epidemiological reports concerning hospital-acquired infections indicate that, next to the
MRSA, these strains are the most common causes of such infections [12]. Due to the fact that
the obtained CPX-triazole hybrids demonstrated more potent antibacterial effects on the three
bacterial strains mentioned above as compared with CPX itself, these compounds might
become efficacious treatment option in the management of hospital-acquired infections. In the

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case of P. aeruginosa, which is responsible for approximately 10% of hospital-acquired
infections, an alarming trend is observed consisting in the selection of strains resistant to
antibiotics and chemotherapeutics, including fluoroquinolones [13]. It is assumed that this
phenomenon results from a capacity of P. aeruginosa to produce membrane proteins, called
efflux pumps, responsible for removing drugs from the inside of bacterial cells. In the group
of the obtained derivatives (23-44), all compounds, with only one exception, were
characterised by more potent activity than CPX. In the cases of 4-methoxybenzyl (30) and 4-
methoxyphenyl (43) derivatives, an approximately 31-fold increase in antibacterial activity
was observed when compared to CPX (MICs equalled 0.023 ꢀM for 30 and 43 vs. 0.72 ꢀM
for CPX).
2.3. Toxicity evaluation
In the process of searching for new antibacterial drugs, it is important to differentiate between
compounds toxic only to the bacterial cells from compounds that act non-selectively and are
toxic both for bacterial and human cells. This is even more important in the cases of such
compounds, the antibacterial effects of which result from inhibition of bacterial
topoisomerases. There is a risk that bacterial topoisomerase inhibitors will also affect human
topoisomerases leading to dysfunction of human cells. With the use of the MTT assay, it was
investigated how the newly-obtained CPX-triazole hybrids affect the viability of human HEK-
293 cells. For the purposes of the assay, we selected structurally diversified derivatives,
including compounds 30, 38 and 43 that were considered to be the most promising due to
their potent antibacterial activity. The toxicity of the derivatives tested (i.e., 24, 30, 31, 34, 37,
38, 41, 43) was expressed in the form of concentrations that decrease the viability of cells by a
half (EC50). The EC50/MIC ratio was used as a parameter representing the selectivity of the
CPX-triazole hybrids towards bacterial cells [14]. The bigger the difference between EC50 and
MIC values, the lesser the risk of toxic effects. EC50 values for the tested compounds ranged
from 62.89 ± 0.43 ꢀM to 139.55 ± 17.77 ꢀM, and were considerably higher than doses needed
to obtain the antibacterial effect (Table 3). The toxicity of the CPX-triazole hybrids for
bacterial cells was even up to 18930 times higher than the toxicity for human HEK-293 cells.
The most selective action of the tested compounds was observed for E. coli ATCC25922
strain.
Table 3

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2.4. Enzymatic studies
Considerable increase in antibacterial activity of the obtained CPX-triazole hybrids may result
from the fact that:
1) these derivatives inhibit bacterial topoisomerases in a more potent way than CPX itself;
2) CPX-triazole hybrids permeate inside the bacterial cells more easily than CPX;
3) CPX-triazole hybrids are not substrates for bacterial efflux pumps, thus, they are not
removed from the bacterial cells;
4) there is an additional, so far unknown mechanism of antibacterial activity of CPX-triazole
hybrids that is not associated with topoisomerases inhibition.
In order to verify the first hypothesis, the affinity of the selected compounds towards bacterial
type II topoisomerases (i.e., DNA gyrase and topoisomerase IV) was analysed. For testing, the
derivatives described in our earlier article [6] were selected (Table 4).
Table 4
The results obtained demonstrated that the antibacterial activity of the CPX-triazole hybrids is
not dependent solely on the degree of their affinity to DNA gyrase and topoisomerase IV.
Even though all three derivatives (A-C) inhibited DNA gyrase in a much weaker way than
CPX itself, the compounds A and B were characterised by potent antibacterial activity (Figure
1). Moreover, the influence of other factors on the total antibacterial effect is confirmed by the
fact that derivatives A and C differed considerably in their activity despite their nearly
identical affinity to DNA gyrase (IC50 values for compounds A and C were 6.05 ꢀM and 6.28
ꢀM, respectively). The same example demonstrates to what degree the structure of the
substituent in the C-5 position of 1,2,4-triazole-3-thione ring affects the activity of such
compounds. The affinity of the derivatives tested was comparable to (in the cases of
compounds A and B, containing hydroxyphenyl fragment) or approximately twofold lower (in
the case of compound C, containing alkyl chain at the same position of the 1,2,4-triazole ring)
than the affinity of CPX. However, there was no correlation between the affinity of the CPX-
triazole hybrids towards topoisomerase IV and their antibacterial activity. Again, this also
confirms that inhibition of bacterial type II topoisomerases in not the sole factor responsible
for the antibacterial activity of the compounds discussed. Therefore, the first hypothesis has to

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be refuted as untrue. Verification of the remaining hypotheses will require further
microbiological studies during which the influence of the bacterial outer membranes
permeability and efflux pumps on the antibacterial effect will be tested. Such studies,
conducted with the use of so-called permeabilizers and efflux pumps inhibitors, have already
been initiated and the results will be presented in due course.
Figure 1
2.5. Structure-activity observations
As can be seen from Scheme 1, compounds 23-44 differ in the position of the hydroxyl group
in the phenyl ring and the structure of the substituent in the position 4 of the 1,2,4-triazole-3-
thione core. The results of the MTT assay (Table 3) clearly show that the presence of the
-CH2- linker between 1,2,4-triazole ring and aryl substituent negatively affects toxicity of the
obtained CPX-triazole hybrids. EC50 values for the compounds in which aryl substituent was
connected directly to the1,2,4-triazole ring ranged from 105.34 ± 2.75 ꢀM (for compound 43)
to 139.55 ± 17.77 ꢀM (for compound 31). The toxicity of the respective benzyl derivatives
was approximately twice as high. The data presented in Table 3 also shows that substitution
pattern of the hydroxyphenyl moiety has no major effect on the toxicity profile of the
compounds tested. Similarly, the change of hydroxyl group position affected antibacterial
activity to a slight degree only. However, its presence alone seems to be crucial. It may be
therefore concluded that it is not the position of the hydroxyl group that determines a potent
antibacterial activity, but rather the particular properties of this group (e.g., the potential of
forming hydrogen bonds, strong electrondonating character) and its influence on the
electronic properties of the molecule that is the source of antibacterial properties.
Nevertheless, having interpreted these results in connection with those presented previously
[6], a number of structure – activity correlations were observed, as shown in Scheme 2.
Scheme 2
3. Conclusions
To sum up, thanks to combining 1,2,4-triazole-3-thione derivatives with the molecule of
ciprofloxacin, new compounds endowed with potent antibacterial activity against Gram-

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positive and Gram-negative strains were obtained. More importantly, it was observed that
their antibacterial action was particularly potent in relation to strains that cause life-
threatening infections. The concentrations that effectively inhibit bacterial growth were even
several thousand times greater than respective toxic concentrations, which confirms that these
derivatives may be treated as the lead compounds for novel chemotherapeutics. The
enzymatic assay carried out for the selected compounds suggests that considerable increase in
antibacterial activity of the obtained CPX-triazole hybrids does not result from an increase in
their affinity to bacterial type II topoisomerases (DNA gyrase and topoisomerase IV).
4. Experimental
4.1. Chemistry
4.1.1. General comments
All reagents and solvents were purchased from Alfa Aesar (Ward Hill, USA) and Merck Co.
(Darmstadt, Germany). Melting points were determined by using Fisher-Johns apparatus
1
(Fisher Scientific, Schwerte, Germany) and are uncorrected. The H-NMR and 13C-NMR
spectra (in DMSO-d6) were recorded on a Bruker Avance spectrometer (Bruker BioSpin
GmbH, Rheinstetten, Germany) using TMS as an internal standard. FT-IR spectra were
recorded using ATR Platinum Diamond A 225 device. Elemental analyses were performed on
an AMZ 851 CHX analyser (PG, Gdańsk, Poland) and the results were within ± 0.4% of the
theoretical value.
4.1.2. General procedure for the synthesis of 1,2,4-triazole-3-thione derivatives (1-22)
Respective thiosemicarbazide derivatives, described in Ref. [5], were dissolved in 2% NaOH
and refluxed for 2h. After cooling, the mixture was neutralized with 3M HCl. The precipitate
formed was filtered and washed with distilled water. The compounds were crystallized from
EtOH. In the cases of already known compounds, information about their properties may be
retrieved in the Chemical Abstract Service database (CAS numbers are given below).
5-(2-Hydroxyphenyl)-4-phenyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (1)
Yield: 78%. CAS: 81518-26-5

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4-Benzyl-5-(2-hydroxyphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (2)
Yield: 83%. CAS: 312319-66-7
4-(4-Fluorophenyl)-5-(2-hydroxyphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (3)
1
Yield: 81%, m.p. 246-248˚C, H-NMR (250 MHz): 6.76-6.92 (m, 2H, Ar-H), 7.22-7.42 (m,
6H, Ar-H), 10.06 (s, 1H, OH), 14.14 (s, 1H, NH). 13C-NMR (75 MHz): 111.66, 113.92,
114.21, 114.28, 117.61, 128.72, 128.86, 129.24, 129.31, 130.17, 130.92, 148.42, 154.18,
158.32, 162.22, 166.18. Anal. calc. for C14H10FN3OS (287.31): C 58.53, H 3.51, N 14.63.
Found: C 58.69, H 3.20, N 14.56.
4-(4-Fluorobenzyl)-5-(2-hydroxyphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (4)
Yield: 85%, m.p. 210-211˚C, 1H-NMR (250 MHz): 5.20 (s, 2H, -CH2-), 6.80-7.16 (m, 7H, Ar-
H), 7.38-7.47 (m, 1H, Ar-H), 10.55 (s, 1H, OH), 14.05 (s, 1H, NH). 13C-NMR (75 MHz):
44.50, 111.81, 113.52, 113.86, 114.60, 117.94, 127.86, 127.99, 129.91, 130.56, 131.23,
148.59, 154.19, 158.07, 161.94, 165.54. Anal. calc. for C15H12FN3OS (301.34): C 59.79, H
4.01, N 13.94. Found: C 59.65, H 3.86, N 14.08.
4-(4-Chlorophenyl)-5-(2-hydroxyphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (5)
Yield: 87%. CAS: 26131-65-7
4-(4-Chlorobenzyl)-5-(2-hydroxyphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (6)
Yield: 77%, m.p. 218-219˚C, 1H-NMR (250 MHz): 5.20 (s, 2H, -CH2-), 6.84-6.93 (m, 1H, Ar-
H), 6.98 (dd, 2H, Ar-H, J = 1.9 Hz, 6.5 Hz), 7.03-7.17 (m, 2H, Ar-H), 7.30 (dd, 2H, Ar-H, J =
13
1.9 Hz, 6.5 Hz), 7.40-7.47 (m, 1H, Ar-H), 10.56 (s, 1H, OH), 14.18 (s, 1H, NH). C-NMR
(75 MHz): 44.56, 111.67, 114.60, 117.94, 126.90, 127.63, 129.95, 130.68, 131.21, 133.33,
148.64, 154.17, 165.57. Anal. calc. for C15H12ClN3OS (317.79): C 56.69, H 3.81, N 13.22.
Found: C 56.49, H 3.95, N 13.12.
5-(2-Hydroxyphenyl)-4-(4-methoxyphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (7)
Yield: 80%, m.p. 288-290˚C, 1H-NMR (250 MHz): 3.76 (s, 3H, OCH3), 6.77-6.90 (m, 2H, Ar-
H), 6.95 (dd, 2H, Ar-H, J = 2.2 Hz, 6.8 Hz), 7.22 (dd, 2H, Ar-H, J = 2.2 Hz, 6.8 Hz), 7.26-
7.38 (m, 2H, Ar-H), 10.04 (s, 1H, OH), 14.06 (s, 1H, NH). 13C-NMR (75 MHz): 53.90,
111.94, 112.34, 114.20, 114.30, 117.51, 125.58, 127.79, 130.14, 130.74, 148.56, 154.34,

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154.50, 157.69, 166.32, 166.48. Anal. calc. for C15H13N3O2S (299.35): C 60.18, H 4.38, N
14.04. Found: C 60.10, H 4.21, N 13.86.
5-(2-Hydroxyphenyl)-4-(4-methoxybenzyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (8)
1
Yield: 75%, m.p. 186-187˚C, H-NMR (250 MHz): 3.70 (s, 3H, OCH3), 5.16 (s, 2H, -CH2-),
6.76 (dd, 2H, Ar-H, J = 2.0 Hz, 6.7 Hz), 6.86-6.95 (m, 3H, Ar-H), 7.05-7.15 (m, 2H, Ar-H),
13
7.41-7.50 (m, 1H, Ar-H), 10.55 (s, 1H, OH), 14.01 (s, 1H, NH). C-NMR (75 MHz): 44.64,
53.64, 112.01, 112.22, 114.61, 117.98, 126.40, 127.38, 129.98, 131.14, 148.64, 154.10,
157.20, 165.45. Anal. calc. for C16H15N3O2S (313.37): C 61.32, H 4.82, N 13.41. Found: C
61.20, H 5.05, N 13.26.
5-(3-Hydroxyphenyl)-4-phenyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (9)
Yield: 85%. CAS: 26028-80-8
4-Benzyl-5-(3-hydroxyphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (10)
Yield: 81%, m.p. 161-163˚C, 1H-NMR (250 MHz): 5.39 (s, 2H, -CH2-), 6.91-7.42 (m, 9H, Ar-
H), 9.98 (s, 1H, OH), 14.18 (s, 1H, NH). 13C-NMR (75 MHz): 45.40, 113.72, 116.38, 117.46,
125.14, 125.61, 126.13, 127.18, 128.73, 134.36, 150.12, 156.04, 156.18, 166.44, 166.60.
Anal. calc. for C15H13N3OS (283.35): C 63.58, H 4.62, N 14.83. Found: C 63.51, H 4.50, N
14.67.
4-(4-Fluorophenyl)-5-(3-hydroxyphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (11)
Yield: 74%. CAS: 727982-83-4
4-(4-Fluorobenzyl)-5-(3-hydroxyphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (12)
Yield: 81%, m.p. 161-163˚C, 1H-NMR (250 MHz): 5.28 (s, 2H, -CH2-), 6.84-6.93 (m, 3H, Ar-
H), 7.02-7.14 (m, 4H, Ar-H), 7.21-7.28 (m, 1H, Ar-H), 9.89 (s, 1H, OH), 14.10 (s, 1H, NH).
13C-NMR (75 MHz): 44.75, 113.70, 113.84, 114.19, 116.40, 117.54, 125.55, 127.33, 127.46,
128.78, 130.54, 150.01, 156.03, 156.18, 158.11, 162.00, 166.41. Anal. calc. for C15H12FN3OS
(301.34): C 59.79, H 4.01, N 13.94. Found: C 59.60, H 3.80, N 14.16.
4-(4-Chlorophenyl)-5-(3-hydroxyphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (13)
Yield: 84%, m.p. 220-221˚C, 1H-NMR (250 MHz): 6.71-6.90 (m, 3H, Ar-H), 7.20 (t, 1H, Ar-
H, J = 8.0 Hz), 7.44 (dd, 2H, Ar-H, J = 2.0 Hz, 6.6 Hz), 7.62 (dd, 2H, Ar-H, J = 2.0 Hz, 6.6

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Hz), 9.84 (s, 1H, OH), 14.18 (s, 1H, NH). 13C-NMR (75 MHz): 113.70, 116.07, 117.62,
125.27, 128.02, 128.42, 129.24, 132.10, 132.65, 149.07, 155.74, 166.97. Anal. calc. for
C14H10ClN3OS (303.77): C 55.35, H 3.32, N 13.83. Found: C 55.23, H 3.12, N 13.98.
4-(4-Chlorobenzyl)-5-(3-hydroxyphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (14)
Yield: 80%, m.p. 160-162˚C, 1H-NMR (250 MHz): 5.34 (s, 2H, -CH2-), 6.91-7.00 (m, 3H, Ar-
H), 7.06-7.14 (m, 2H, Ar-H), 7.27-7.35 (m, 1H, Ar-H), 7.40 (dd, 2H, Ar-H, J = 2.0 Hz, 6.6
Hz), 9.94 (s, 1H, OH), 14.17 (s, 1H, NH). 13C-NMR (75 MHz): 44.86, 113.67, 116.48,
117.53, 125.48, 127.09, 127.19, 128.81, 130.73, 133.36, 150.00, 156.04, 166.38. Anal. calc.
for C15H12ClN3OS (317.79): C 56.69, H 3.81, N 13.22. Found: C 56.78, H 3.62, N 13.04.
5-(3-Hydroxyphenyl)-4-(4-methoxyphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (15)
Yield: 73%, m.p. 238-240˚C, 1H-NMR (250 MHz): 3.85 (s, 3H, OCH3), 6.71-6.90 (m, 3H, Ar-
H), 7.10 (dd, 2H, Ar-H, J = 2.1 Hz, 6.9 Hz), 7.18 (t, 1H, Ar-H, J = 7.7 Hz), 7.30 (dd, 2H, Ar-
13
H, J = 2.1 Hz, 6.9 Hz), 9.84 (s, 1H, OH), 14.11 (s, 1H, NH). C-NMR (75 MHz): 54.04,
113.09, 113.61, 115.89, 117.51, 125.56, 125.74, 128.31, 128.45, 149.29, 155.67, 158.15,
167.29. Anal. calc. for C15H13N3O2S (299.35): C 60.18, H 4.38, N 14.04. Found: C 60.23, H
4.50, N 14.20.
5-(3-Hydroxyphenyl)-4-(4-methoxybenzyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (16)
1
Yield: 78%, m.p. 188-190˚C, H-NMR (250 MHz): 3.74 (s, 3H, OCH3), 5.31 (s, 2H, -CH2-),
6.82-7.04 (m, 7H, Ar-H), 7.28-7.36 (m, 1H, Ar-H), 9.94 (s, 1H, OH), 14.08 (s, 1H, NH). 13C-
NMR (75 MHz): 44.84, 53.66, 112.53, 113.76, 116.37, 117.60, 125.70, 126.30, 126.72,
128.75, 150.01, 156.04, 157.18, 166.32, 166.48. Anal. calc. for C16H15N3O2S (313.37): C
61.32, H 4.82, N 13.41. Found: C 61.10, H 4.73, N 13.23.
5-(4-Hydroxyphenyl)-4-phenyl-2,4-dihydro-3H-1,2,4-triazole-3-thione (17)
Yield: 82%. CAS: 26028-88-6
4-Benzyl-5-(4-hydroxyphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (18)
Yield: 84%. CAS: 174573-91-2
4-(4-Fluorophenyl)-5-(4-hydroxyphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (19)
Yield: 75%. CAS: 451502-00-4

ACCEPTED MANUSCRIPT
4-(4-Chlorophenyl)-5-(4-hydroxyphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (20)
Yield: 77%. CAS: 54918-96-6
5-(4-Hydroxyphenyl)-4-(4-methoxyphenyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (21)
Yield: 83%. CAS: 69626-14-8
5-(4-Hydroxyphenyl)-4-(4-methoxybenzyl)-2,4-dihydro-3H-1,2,4-triazole-3-thione (22)
1
Yield: 86%, m.p. 178-180˚C, H-NMR (250 MHz): 3.66 (s, 3H, OCH3), 5.24 (s, 2H, -CH2-),
6.75-6.84 (m, 4H, Ar-H), 6.94 (m, 2H, Ar-H), 7.33 (dd, 2H, Ar-H, J = 1.9 Hz, 6.7 Hz), 10.11
13
(s, 1H, OH), 13.95 (s, 1H, NH). C-NMR (75 MHz): 44.72, 53.66, 112.53, 114.20, 115.18,
126.42, 126.72, 128.67, 150.23, 157.17, 157.92, 166.07. Anal. calc. for C16H15N3O2S
(313.37): C 61.32, H 4.82, N 13.41. Found: C 61.19, H 4.69, N 13.30.
4.1.3. General procedure for the synthesis of 1,2,4-triazole-ciprofloxacin hybrids (23-44)
1 mmol of the respective 1,2,4-triazole derivative (1-22) was dissolved (with heating) in 40
ml of anhydrous ethanol and then equimolar amount of ciprofloxacin and formaldehyde
solution were added. The obtained suspension was stirred at room temperature for 12 hours.
The precipitate formed was filtered off, dried, and crystallized from ethanol to give
compounds 23-44.
1-Cyclopropyl-6-fluoro-7-[4-{[3-(2-hydroxyphenyl)-4-phenyl-5-thioxo-4,5-dihydro-1H-
1,2,4-triazol-1-yl]methyl}piperazin-1-yl]-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (23)
Yield: 69%, m.p. 241-243˚C, 1H-NMR (250 MHz): 1.13-1.17 (m, 2H, cyclopropyl), 1.28-1.32
(m, 2H, cyclopropyl), 2.88 (bs, 4H, piperazine), 3.30 (bs, 4H, piperazine), 3.74-3.80 (m, 1H,
cyclopropyl), 4.67 (s, 2H, CH2), 7.04-8.10 (m, 11H, Ar-H), 8.65 (s, 1H, Ar-H), 10.35 (s, 1H,
13
OH), 14.94 (s, 1H, COOH). C-NMR (75 MHz): 7.68, 36.21, 46.11, 50.47, 61.07, 106.32,
106.80, 110.72, 111.33, 114.20, 116.89, 117.84, 126.14, 126.87, 127.14, 129.45, 130.11,
132.02, 139.20, 144.17, 147.20, 148.31, 152.42, 154.67, 164.92, 166.20, 176.19. Elemental
analysis for C32H29FN6O4S (612.67). Calculated: C 62.73, H 4.77, N 13.72. Found: C 62.85,
H 4.62, N 13.87.
1-Cyclopropyl-6-fluoro-7-[4-{[4-benzyl-3-(2-hydroxyphenyl)-5-thioxo-4,5-dihydro-1H-1,2,4-
triazol-1-yl]methyl}piperazin-1-yl]-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (24)

ACCEPTED MANUSCRIPT
Yield: 74%, m.p. 204-205˚C, 1H-NMR (250 MHz): 1.20-1.23 (m, 2H, cyclopropyl), 1.27-1.30
(m, 2H, cyclopropyl), 2.70 (s, 4H, piperazine), 3.27 (bs, 4H, piperazine), 3.94-3.97 (m, 1H,
cyclopropyl), 4.65 (s, 2H, CH2), 5.14 (s, 2H, CH2), 7.06-8.01 (m, 11H, Ar-H), 8.65 (s, 1H, Ar-
H), 10.50 (s, 1H, OH), 14.88 (s, 1H, COOH). 13C-NMR (75 MHz): 8.14, 37.81, 45.23, 46.18,
51.73, 62.40, 106.38, 107.17, 111.06, 112.18, 113.52, 116.25, 117.62, 125.14, 125.69, 126.90,
127.09, 128.52, 134.17, 139.66, 145.60, 147.82, 148.71, 152.92, 156.04, 164.71, 166.43,
178.50. Elemental analysis for C33H31FN6O4S (626.70). Calculated: C 63.24, H 4.99, N 13.41.
Found: C 63.11, H 4.82, N 13.47.
1-Cyclopropyl-6-fluoro-7-[4-{[4-(4-fluorophenyl)-3-(2-hydroxyphenyl)-5-thioxo-4,5-
dihydro-1H-1,2,4-triazol-1-yl]methyl}piperazin-1-yl]-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid (25)
Yield: 70%, m.p. 233-235˚C, 1H-NMR (250 MHz): 1.09-1.11 (m, 2H, cyclopropyl), 1.15-1.18
(m, 2H, cyclopropyl), 2.78 (bs, 4H, piperazine), 3.26 (bs, 4H, piperazine), 3.77-3.88 (m, 1H,
cyclopropyl), 4.58 (s, 2H, CH2), 7.11-7.98 (m, 10H, Ar-H), 8.67 (s, 1H, Ar-H), 10.10 (s, 1H,
OH), 15.06 (s, 1H, COOH). IR (ATR): 3405 (O-H, stretch), 3121, 2946, 2867 (C-H, stretch),
1724 (C=O, stretch), 1573 (C=N, stretch), 1426 (C-O, stretch), 1303 (C=S, stretch), 1289 (O-
H, bend), 1048 (C-F, stretch). Elemental analysis for C32H28F2N6O4S (630.66). Calculated: C
60.94, H 4.48, N 13.33. Found: C 60.83, H 4.42, N 13.37.
1-Cyclopropyl-6-fluoro-7-[4-{[4-(4-fluorobenzyl)-3-(2-hydroxyphenyl)-5-thioxo-4,5-
dihydro-1H-1,2,4-triazol-1-yl]methyl}piperazin-1-yl]-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid (26)
Yield: 65%, m.p. 160-161˚C, 1H-NMR (250 MHz): 1.20-1.23 (m, 2H, cyclopropyl), 1.28-1.31
(m, 2H, cyclopropyl), 2.70 (bs, 4H, piperazine), 3.42 (bs, 4H, piperazine), 3.72-3.76 (m, 1H,
cyclopropyl), 4.70 (s, 2H, CH2), 5.23 (s, 2H, CH2), 7.18-7.85 (m, 10H, Ar-H), 8.69 (s, 1H, Ar-
H), 10.53 (s, 1H, OH), 14.98 (s, 1H, COOH). IR (ATR): 3446 (O-H, stretch), 3101, 2946,
2782 (C-H, stretch), 1714 (C=O, stretch), 1580 (C=N, stretch), 1406 (C-O, stretch), 1294
(C=S, stretch), 1256 (O-H, bend), 1021 (C-F, stretch). Elemental analysis for C33H30F2N6O4S
(644.69). Calculated: C 61.48, H 4.69, N 13.04. Found: C 61.57, H 4.62, N 13.17.

ACCEPTED MANUSCRIPT
1-Cyclopropyl-6-fluoro-7-[4-{[4-(4-chlorophenyl)-3-(2-hydroxyphenyl)-5-thioxo-4,5-
dihydro-1H-1,2,4-triazol-1-yl]methyl}piperazin-1-yl]-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid (27)
Yield: 75%, m.p. 235-237˚C, 1H-NMR (250 MHz): 1.12-1.14 (m, 2H, cyclopropyl), 1.20-1.23
(m, 2H, cyclopropyl), 2.86 (bs, 4H, piperazine), 3.32 (bs, 4H, piperazine), 3.80-3.85 (m, 1H,
cyclopropyl), 4.48 (s, 2H, CH2), 6.94 (dd, 2H, Ar-H, J = 1.8 Hz, 6.7 Hz), 7.11-7.75 (m, 8H,
Ar-H), 8.68 (s, 1H, Ar-H), 10.41 (s, 1H, OH), 15.09 (s, 1H, COOH). IR (ATR): 3482 (O-H,
stretch), 3062, 2861 (C-H, stretch), 1738 (C=O, stretch), 1618 (C=N, stretch), 1426 (C-O,
stretch), 1321 (C=S, stretch), 1273 (O-H, bend), 1012 (C-F, stretch). Elemental analysis for
C32H28ClFN6O4S (647.12). Calculated: C 59.39, H 4.36, N 12.99. Found: C 59.46, H 4.50, N
13.10.
1-Cyclopropyl-6-fluoro-7-[4-{[4-(4-chlorobenzyl)-3-(2-hydroxyphenyl)-5-thioxo-4,5-
dihydro-1H-1,2,4-triazol-1-yl]methyl}piperazin-1-yl]-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid (28)
Yield: 69%, m.p. 208-210˚C, 1H-NMR (250 MHz): 1.15-1.21 (m, 2H, cyclopropyl), 1.30-1.34
(m, 2H, cyclopropyl), 2.86 (bs, 4H, piperazine), 3.35 (bs, 4H, piperazine), 3.86-3.95 (m, 1H,
cyclopropyl), 4.62 (s, 2H, CH2), 5.22 (s, 2H, CH2), 6.94-7.75 (m, 10H, Ar-H), 8.67 (s, 1H, Ar-
H), 10.50 (s, 1H, OH), 14.98 (s, 1H, COOH). IR (ATR): 3452 (O-H, stretch), 3185, 2971,
2732 (C-H, stretch), 1726 (C=O, stretch), 1582 (C=N, stretch), 1439 (C-O, stretch), 1323
(C=S, stretch), 1281 (O-H, bend), 1054 (C-F, stretch). Elemental analysis for
C33H30ClFN6O4S (661.14). Calculated: C 59.95, H 4.57, N 12.71. Found: C 60.13, H 4.68, N
12.90.
1-Cyclopropyl-6-fluoro-7-[4-{[3-(2-hydroxyphenyl)-4-(4-methoxyphenyl)-5-thioxo-4,5-
dihydro-1H-1,2,4-triazol-1-yl]methyl}piperazin-1-yl]-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid (29)
Yield: 74%, m.p. 246-247˚C, 1H-NMR (250 MHz): 1.12-1.15 (m, 2H, cyclopropyl), 1.21-1.24
(m, 2H, cyclopropyl), 2.94 (s, 4H, piperazine), 3.37 (s, 4H, piperazine), 3.61 (s, 3H, OCH3),
3.82-3.86 (m, 1H, cyclopropyl), 4.60 (s, 2H, CH2), 6.99 (dd, 2H, Ar-H, J = 2.0 Hz, 6.7 Hz),
7.10-7.85 (m, 8H, Ar-H), 8.66 (s, 1H, Ar-H), 10.16 (s, 1H, OH), 14.86 (s, 1H, COOH). IR

ACCEPTED MANUSCRIPT
(ATR): 3427 (O-H, stretch), 3162, 2861, 2782 (C-H, stretch), 1753 (C=O, stretch), 1604
(C=N, stretch), 1430 (C-O, stretch), 1274 (C=S, stretch), 1250 (O-H, bend), 1052 (C-F,
stretch). Elemental analysis for C33H31FN6O5S (642.70). Calculated: C 61.67, H 4.86, N
13.08. Found: C 61.55, H 4.72, N 13.19.
1-Cyclopropyl-6-fluoro-7-[4-{[3-(2-hydroxyphenyl)-4-(4-methoxybenzyl)-5-thioxo-4,5-
dihydro-1H-1,2,4-triazol-1-yl]methyl}piperazin-1-yl]-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid (30)
Yield: 66%, m.p. 166-167˚C, 1H-NMR (250 MHz): 1.20-1.24 (m, 2H, cyclopropyl), 1.32-1.35
(m, 2H, cyclopropyl), 2.92 (bs, 4H, piperazine), 3.30 (bs, 4H, piperazine), 3.61 (s, 3H, OCH3),
3.86-3.93 (m, 1H, cyclopropyl), 4.54 (s, 2H, CH2), 5.20 (s, 2H, CH2), 6.90 (dd, 2H, Ar-H, J =
2.1 Hz, 6.7 Hz), 6.94-7.75 (m, 8H, Ar-H), 8.67 (s, 1H, Ar-H), 10.50 (s, 1H, OH), 14.94 (s, 1H,
COOH). IR (ATR): 3405 (O-H, stretch), 3167, 3052, 2861 (C-H, stretch), 1719 (C=O,
stretch), 1582 (C=N, stretch), 1440 (C-O, stretch), 1307 (C=S, stretch), 1289 (O-H, bend),
1042 (C-F, stretch). Elemental analysis for C34H33FN6O5S (656.73). Calculated: C 62.18, H
5.06, N 12.80. Found: C 62.10, H 5.17, N 12.94.
1-Cyclopropyl-6-fluoro-7-[4-{[3-(3-hydroxyphenyl)-4-phenyl-5-thioxo-4,5-dihydro-1H-
1,2,4-triazol-1-yl]methyl}piperazin-1-yl]-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (31)
Yield: 78%, m.p. 232-233˚C, 1H-NMR (250 MHz): 1.13-1.16 (m, 2H, cyclopropyl), 1.27-1.30
(m, 2H, cyclopropyl), 2.94 (bs, 4H, piperazine), 3.35 (bs, 4H, piperazine), 3.76-3.81 (m, 1H,
cyclopropyl), 4.65 (s, 2H, CH2), 7.10-7.86 (m, 11H, Ar-H), 8.69 (s, 1H, Ar-H), 10.09 (s, 1H,
13
OH), 14.99 (s, 1H, COOH). C-NMR (75 MHz): 7.50, 35.00, 45.19, 51.21, 59.27, 105.78,
106.26, 111.02, 111.64, 113.86, 116.74, 117.64, 125.54, 126.04, 127.32, 128.64, 129.13,
131.64, 138.22, 144.32, 147.20, 148.80, 151.62, 154.64, 164.74, 165.87, 178.37. Elemental
analysis for C32H29FN6O4S (612.67). Calculated: C 62.73, H 4.77, N 13.72. Found: C 62.80,
H 4.65, N 13.77.
1-Cyclopropyl-6-fluoro-7-[4-{[4-benzyl-3-(3-hydroxyphenyl)-5-thioxo-4,5-dihydro-1H-1,2,4-
triazol-1-yl]methyl}piperazin-1-yl]-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (32)
Yield: 62%, m.p. 244-246˚C, 1H-NMR (250 MHz): 1.15-1.22 (m, 2H, cyclopropyl), 1.27-1.31

ACCEPTED MANUSCRIPT
(m, 2H, cyclopropyl), 2.84-2.93 (m, 4H, piperazine), 3.45-3.52 (m, 4H, piperazine), 3.88-
3.93 (m, 1H, cyclopropyl), 4.52 (s, 2H, CH2), 5.22 (s, 2H, CH2), 7.04-7.64 (m, 11H, Ar-H),
8.69 (s, 1H, Ar-H), 10.02 (s, 1H, OH), 14.93 (s, 1H, COOH). 13C-NMR (75 MHz): 7.59,
35.87, 44.65, 46.16, 52.34, 60.64, 105.97, 106.27, 110.34, 111.68, 113.22, 115.26, 117.62,
124.34, 126.60, 126.90, 128.06, 128.68, 135.17, 138.16, 144.61, 148.71, 147.34, 151.14,
155.64, 165.64, 166.46, 179.61. Elemental analysis for C33H31FN6O4S (626.70). Calculated: C
63.24, H 4.99, N 13.41. Found: C 63.18, H 4.81, N 13.33.
1-Cyclopropyl-6-fluoro-7-[4-{[4-(4-fluorophenyl)-3-(3-hydroxyphenyl)-5-thioxo-4,5-
dihydro-1H-1,2,4-triazol-1-yl]methyl}piperazin-1-yl]-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid (33)
Yield: 59%, m.p. 247-249˚C, 1H-NMR (250 MHz): 1.13-1.18 (m, 2H, cyclopropyl), 1.21-1.25
(m, 2H, cyclopropyl), 2.85 (bs, 4H, piperazine), 3.35 (bs, 4H, piperazine), 3.82-3.88 (m, 1H,
cyclopropyl), 4.42 (s, 2H, CH2), 6.92-7.68 (m, 10H, Ar-H), 8.66 (s, 1H, Ar-H), 10.34 (s, 1H,
OH), 14.87 (s, 1H, COOH). IR (ATR): 3479 (O-H, stretch), 3042, 2804 (C-H, stretch), 1723
(C=O, stretch), 1621 (C=N, stretch), 1445 (C-O, stretch), 1327 (C=S, stretch), 1286 (O-H,
bend), 1027 (C-F, stretch). Elemental analysis for C32H28F2N6O4S (630.66). Calculated: C
60.94, H 4.48, N 13.33. Found: C 60.80, H 4.35, N 13.27.
1-Cyclopropyl-6-fluoro-7-[4-{[4-(4-fluorobenzyl)-3-(3-hydroxyphenyl)-5-thioxo-4,5-
dihydro-1H-1,2,4-triazol-1-yl]methyl}piperazin-1-yl]-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid (34)
Yield: 66%, m.p. 238-240˚C, 1H-NMR (250 MHz): 1.20-1.23 (m, 2H, cyclopropyl), 1.27-1.31
(m, 2H, cyclopropyl), 2.86 (bs, 4H, piperazine), 3.31 (bs, 4H, piperazine), 3.74-3.80 (m, 1H,
cyclopropyl), 4.67 (s, 2H, CH2), 5.25 (s, 2H, CH2), 6.90-7.54 (m, 10H, Ar-H), 8.64 (s, 1H, Ar-
H), 9.92 (s, 1H, OH), 14.98 (s, 1H, COOH). IR (ATR): 3505 (O-H, stretch), 2963, 2861 (C-H,
stretch), 1703 (C=O, stretch), 1591 (C=N, stretch), 1417 (C-O, stretch), 1329 (C=S, stretch),
1270 (O-H, bend), 1011 (C-F, stretch). Elemental analysis for C33H30F2N6O4S (644.69).
Calculated: C 61.48, H 4.69, N 13.04. Found: C 61.50, H 4.56, N 13.07.

ACCEPTED MANUSCRIPT
1-Cyclopropyl-6-fluoro-7-[4-{[4-(4-chlorophenyl)-3-(3-hydroxyphenyl)-5-thioxo-4,5-
dihydro-1H-1,2,4-triazol-1-yl]methyl}piperazin-1-yl]-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid (35)
Yield: 75%, m.p. 250-252˚C, 1H-NMR (250 MHz): 1.20-1.23 (m, 2H, cyclopropyl), 1.27-1.30
(m, 2H, cyclopropyl), 2.90 (bs, 4H, piperazine), 3.35 (bs, 4H, piperazine), 3.80-3.85 (m, 1H,
cyclopropyl), 4.65 (s, 2H, CH2), 6.82-7.53 (m, 8H, Ar-H), 7.60 (dd, 2H, Ar-H, J = 2.0 Hz, 6.5
Hz), 8.67 (s, 1H, Ar-H), 9.81 (s, 1H, OH), 14.89 (s, 1H, COOH). IR (ATR): 3440 (O-H,
stretch), 3103, 2943, 2767 (C-H, stretch), 1736 (C=O, stretch), 1595 (C=N, stretch), 1428 (C-
O, stretch), 1319 (C=S, stretch), 1260 (O-H, bend), 1032 (C-F, stretch). Elemental analysis for
C32H28ClFN6O4S (647.12). Calculated: C 59.39, H 4.36, N 12.99. Found: C 59.52, H 4.43, N
13.11.
1-Cyclopropyl-6-fluoro-7-[4-{[4-(4-chlorobenzyl)-3-(3-hydroxyphenyl)-5-thioxo-4,5-
dihydro-1H-1,2,4-triazol-1-yl]methyl}piperazin-1-yl]-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid (36)
Yield: 68%, m.p. 250-251˚C, 1H-NMR (250 MHz): 1.15-1.19 (m, 2H, cyclopropyl), 1.27-1.32
(m, 2H, cyclopropyl), 2.92-3.30 (m, 8H, piperazine), 3.85-3.92 (m, 1H, cyclopropyl), 4.62 (s,
2H, CH2), 5.30 (s, 2H, CH2), 6.94-7.61 (m, 10H, Ar-H), 8.67 (s, 1H, Ar-H), 10.05 (s, 1H,
OH), 14.82 (s, 1H, COOH). IR (ATR): 3457 (O-H, stretch), 3067, 2974, 2861 (C-H, stretch),
1735 (C=O, stretch), 1605 (C=N, stretch), 1418 (C-O, stretch), 1315 (C=S, stretch), 1267 (O-
H, bend), 1017 (C-F, stretch). Elemental analysis for C33H30ClFN6O4S (661.14). Calculated: C
59.95, H 4.57, N 12.71. Found: C 59.90, H 4.58, N 12.82.
1-Cyclopropyl-6-fluoro-7-[4-{[3-(3-hydroxyphenyl)-4-(4-methoxyphenyl)-5-thioxo-4,5-
dihydro-1H-1,2,4-triazol-1-yl]methyl}piperazin-1-yl]-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid (37)
Yield: 76%, m.p. 224-226˚C, 1H-NMR (250 MHz): 1.23-1.27 (m, 2H, cyclopropyl), 1.30-1.34
(m, 2H, cyclopropyl), 2.94 (bs, 4H, piperazine), 3.37 (bs, 4H, piperazine), 3.80 (s, 3H, OCH3),
3.82-3.86 (m, 1H, cyclopropyl), 4.60 (s, 2H, CH2), 6.92-7.65 (m, 10H, Ar-H), 8.65 (s, 1H, Ar-
H), 9.85 (s, 1H, OH), 14.92 (s, 1H, COOH). IR (ATR): 3483 (O-H, stretch), 3052, 2939, 2842
(C-H, stretch), 1737 (C=O, stretch), 1597 (C=N, stretch), 1429 (C-O, stretch), 1328 (C=S,

ACCEPTED MANUSCRIPT
stretch), 1257 (O-H, bend), 1022 (C-F, stretch). Elemental analysis for C33H31FN6O5S
(642.70). Calculated: C 61.67, H 4.86, N 13.08. Found: C 61.60, H 4.93, N 13.05.
1-Cyclopropyl-6-fluoro-7-[4-{[3-(3-hydroxyphenyl)-4-(4-methoxybenzyl)-5-thioxo-4,5-
dihydro-1H-1,2,4-triazol-1-yl]methyl}piperazin-1-yl]-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid (38)
Yield: 73%, m.p. 233-235˚C, 1H-NMR (250 MHz): 1.18-1.23 (m, 2H, cyclopropyl), 1.29-1.34
(m, 2H, cyclopropyl), 2.90 (bs, 4H, piperazine), 3.35 (bs, 4H, piperazine), 3.61 (s, 3H, OCH3),
3.86-3.93 (m, 1H, cyclopropyl), 4.54 (s, 2H, CH2), 5.20 (s, 2H, CH2), 6.90 (dd, 2H, Ar-H, J =
2.1 Hz, 6.7 Hz), 6.94-7.75 (m, 8H, Ar-H), 8.67 (s, 1H, Ar-H), 10.50 (s, 1H, OH), 14.94 (s, 1H,
COOH). IR (ATR): 3403 (O-H, stretch), 3067, 2885 (C-H, stretch), 1707 (C=O, stretch), 1582
(C=N, stretch), 1440 (C-O, stretch), 1327 (C=S, stretch), 1295 (O-H, bend), 1010 (C-F,
stretch). Elemental analysis for C34H33FN6O5S (656.73). Calculated: C 62.18, H 5.06, N
12.80. Found: C 62.13, H 5.00, N 12.92.
1-Cyclopropyl-6-fluoro-7-[4-{[3-(4-hydroxyphenyl)-4-phenyl-5-thioxo-4,5-dihydro-1H-
1,2,4-triazol-1-yl]methyl}piperazin-1-yl]-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (39)
Yield: 65%, m.p. 254-255˚C, 1H-NMR (250 MHz): 1.13-1.16 (m, 2H, cyclopropyl), 1.26-1.30
(m, 2H, cyclopropyl), 2.90 (bs, 4H, piperazine), 3.33 (bs, 4H, piperazine), 3.80-3.85 (m, 1H,
cyclopropyl), 4.63 (s, 2H, CH2), 7.06-7.66 (m, 11H, Ar-H), 8.67 (s, 1H, Ar-H), 10.13 (s, 1H,
13
OH), 14.84 (s, 1H, COOH). C-NMR (75 MHz): 8.21, 34.32, 45.19, 52.25, 58.47, 106.78,
107.72, 111.02, 112.63, 114.81, 115.82, 117.95, 124.84, 125.94, 127.52, 128.21, 129.93,
130.27, 137.44, 145.17, 147.20, 149.06, 152.62, 154.64, 165.83, 166.22, 176.54. Elemental
analysis for C32H29FN6O4S (612.67). Calculated: C 62.73, H 4.77, N 13.72. Found: C 62.85,
H 4.67, N 13.67.
1-Cyclopropyl-6-fluoro-7-[4-{[4-benzyl-3-(4-hydroxyphenyl)-5-thioxo-4,5-dihydro-1H-1,2,4-
triazol-1-yl]methyl}piperazin-1-yl]-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (40)
Yield: 71%, m.p. 258-259˚C, 1H-NMR (250 MHz): 1.22-1.27 (m, 2H, cyclopropyl), 1.30-1.34
(m, 2H, cyclopropyl), 2.74 (bs, 4H, piperazine), 3.30 (bs, 4H, piperazine), 3.76-3.82 (m, 1H,
cyclopropyl), 4.54 (s, 2H, CH2), 5.20 (s, 2H, CH2), 6.91-7.69 (m, 11H, Ar-H), 8.63 (s, 1H, Ar-

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H), 10.22 (s, 1H, OH), 15.11 (s, 1H, COOH). 13C-NMR (75 MHz): 7.66, 36.89, 45.34, 46.59,
50.14, 61.10, 106.16, 107.22, 110.34, 111.28, 114.72, 115.66, 117.12, 125.58, 126.07, 126.91,
127.82, 129.18, 135.27, 137.02, 145.28, 147.93, 148.34, 152.54, 156.14, 164.21, 165.71,
177.82. Elemental analysis for C33H31FN6O4S (626.70). Calculated: C 63.24, H 4.99, N 13.41.
Found: C 63.20, H 4.85, N 13.49.
1-Cyclopropyl-6-fluoro-7-[4-{[4-(4-fluorophenyl)-3-(4-hydroxyphenyl)-5-thioxo-4,5-
dihydro-1H-1,2,4-triazol-1-yl]methyl}piperazin-1-yl]-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid (41)
Yield: 71%, m.p. 256-257˚C, 1H-NMR (250 MHz): 1.24-1.28 (m, 2H, cyclopropyl), 1.33-1.39
(m, 2H, cyclopropyl), 2.72 (bs, 4H, piperazine), 3.31 (bs, 4H, piperazine), 3.82-3.86 (m, 1H,
cyclopropyl), 4.52 (s, 2H, CH2), 7.07-7.70 (m, 10H, Ar-H), 8.69 (s, 1H, Ar-H), 9.76 (s, 1H,
OH), 14.94 (s, 1H, COOH). IR (ATR): 3502 (O-H, stretch), 3021, 2917 (C-H, stretch), 1727
(C=O, stretch), 1593 (C=N, stretch), 1448 (C-O, stretch), 1336 (C=S, stretch), 1275 (O-H,
bend), 1048 (C-F, stretch). Elemental analysis for C32H28F2N6O4S (630.66). Calculated: C
60.94, H 4.48, N 13.33. Found: C 60.83, H 4.28, N 13.49.
1-Cyclopropyl-6-fluoro-7-[4-{[4-(4-chlorophenyl)-3-(4-hydroxyphenyl)-5-thioxo-4,5-
dihydro-1H-1,2,4-triazol-1-yl]methyl}piperazin-1-yl]-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid (42)
Yield: 67%, m.p. 222-224˚C, 1H-NMR (250 MHz): 1.21-1.24 (m, 2H, cyclopropyl), 1.28-1.32
(m, 2H, cyclopropyl), 2.91 (bs, 4H, piperazine), 3.32 (bs, 4H, piperazine), 3.82-3.86 (m, 1H,
cyclopropyl), 4.55 (s, 2H, CH2), 6.92 (dd, 2H, Ar-H, J = 1.9 Hz, 6.4 Hz), 7.10-7.48 (m, 6H,
Ar-H), 7.62 (dd, 2H, Ar-H, J = 2.0 Hz, 6.5 Hz), 8.68 (s, 1H, Ar-H), 9.92 (s, 1H, OH), 14.73 (s,
1H, COOH). IR (ATR): 3432 (O-H, stretch), 3121, 2921 (C-H, stretch), 1717 (C=O, stretch),
1565 (C=N, stretch), 1436 (C-O, stretch), 1317 (C=S, stretch), 1274 (O-H, bend), 1020 (C-F,
stretch). Elemental analysis for C32H28ClFN6O4S (647.12). Calculated: C 59.39, H 4.36, N
12.99. Found: C 59.45, H 4.46, N 13.06.

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1-Cyclopropyl-6-fluoro-7-[4-{[3-(4-hydroxyphenyl)-4-(4-methoxyphenyl)-5-thioxo-4,5-
dihydro-1H-1,2,4-triazol-1-yl]methyl}piperazin-1-yl]-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid (43)
Yield: 74%, m.p. 224-226˚C, 1H-NMR (250 MHz): 1.24-1.27 (m, 2H, cyclopropyl), 1.31-1.34
(m, 2H, cyclopropyl), 2.94 (bs, 4H, piperazine), 3.34 (bs, 4H, piperazine), 3.56 (s, 3H, OCH3),
3.84-3.87 (m, 1H, cyclopropyl), 4.61 (s, 2H, CH2), 6.97 (dd, 2H, Ar-H, J = 2.1 Hz, 6.4 Hz),
7.13-7.56 (m, 6H, Ar-H), 7.66 (dd, 2H, Ar-H, J = 2.0 Hz, 6.5 Hz) 8.69 (s, 1H, Ar-H), 10.13 (s,
1H, OH), 14.96 (s, 1H, COOH). IR (ATR): 3409 (O-H, stretch), 3117, 2917, 2783 (C-H,
stretch), 1725 (C=O, stretch), 1604 (C=N, stretch), 1439 (C-O, stretch), 1321 (C=S, stretch),
1268 (O-H, bend), 1011 (C-F, stretch). Elemental analysis for C33H31FN6O5S (642.70).
Calculated: C 61.67, H 4.86, N 13.08. Found: C 61.56, H 4.98, N 13.15.
1-Cyclopropyl-6-fluoro-7-[4-{[3-(4-hydroxyphenyl)-4-(4-methoxybenzyl)-5-thioxo-4,5-
dihydro-1H-1,2,4-triazol-1-yl]methyl}piperazin-1-yl]-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid (44)
Yield: 76%, m.p. 249-251˚C, 1H-NMR (250 MHz): 1.19-1.23 (m, 2H, cyclopropyl), 1.26-1.30
(m, 2H, cyclopropyl), 2.93 (s, 4H, piperazine), 3.34 (s, 4H, piperazine), 3.67 (s, 3H, OCH3),
3.89-3.92 (m, 1H, cyclopropyl), 4.58 (s, 2H, CH2), 5.22 (s, 2H, CH2), 6.97 (dd, 2H, Ar-H, J =
2.1 Hz, 6.7 Hz), 7.10-7.69 (m, 8H, Ar-H), 8.67 (s, 1H, Ar-H), 10.21 (s, 1H, OH), 14.87 (s, 1H,
COOH). IR (ATR): 3452 (O-H, stretch), 3060, 2849 (C-H, stretch), 1707 (C=O, stretch), 1582
(C=N, stretch), 1435 (C-O, stretch), 1330 (C=S, stretch), 1267 (O-H, bend), 1028 (C-F,
stretch). Elemental analysis for C34H33FN6O5S (656.73). Calculated: C 62.18, H 5.06, N
12.80. Found: C 62.29, H 5.21, N 12.68.
4.2. Antimicrobial activity evaluation
The antimicrobial activity of the compounds was tested on the Gram-positive strains
(Staphylococcus aureus ATCC 25923, Staphylococcus aureus ATCC 6538, Staphylococcus
aureus Microbank 14001, Staphylococcus epidermidis ATCC 12228, Bacillus subtilis ATCC
6633, Bacillus cereus ATCC 10876, Micrococcus luteus ATCC 10240), and on the Gram-
negative strains (Escherichia coli ATCC 25922, Klebsiella pneumoniae ATCC 13883,
Proteus mirabilis ATCC 12453, Pseudomonas aeruginosa ATCC 9027). Ciprofloxacin and

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vancomycin were used as control antibacterial agents. Microbial suspensions with an optical
density of 0.5 McFarland standard 150 x 106 CFU/mL (CFU – colony forming units) were
prepared in sterile 0.85 % NaCl. All stock solutions of the tested compounds were dissolved
in dimethyl sulfoxide (DMSO). The medium with DMSO in the final concentration and
without the tested compounds served as a control - no microbial growth inhibition was
observed. Preliminary antibacterial in vitro potency of the tested compounds was screened
using an agar dilution method on the basis of the growth inhibition on a Mueller-Hinton agar
to which the tested compounds in concentration of 1000 µg/mL were added. In the next assay
in vitro antibacterial activity of the compounds with inhibitory effect was determined by a
broth microdilution method. The 96-well microplates were used; 198 ꢀL of Mueller–Hinton
broth with a series of two-fold dilutions of the tested compound in the range of the final
concentrations from 0.24 to 1000 ꢀg/mL was inoculated with 2 ꢀL of microbial suspension.
After incubation (at 37ᵒC for 18 h), spectrophotometric measurements of optical density
(OD600) of the bacterial cultures with the tested compounds were performed in order to
determine MIC. OD600 of bacterial cultures in the medium without the tested compounds was
used as a control. The activity was expressed as the minimal concentration of the compound
that inhibits the visible growth of the bacteria (MIC, minimal inhibitory concentration). The
MBC (minimal bactericidal concentration), defined as the lowest concentration of each
compound that resulted in >99.9% reduction in CFU of the initial inoculum, was also
assessed.
4.3. Cytotoxicity assay
HEK-293 (human embryonic kidney) cells were obtained from the American Type Culture
Collection (ATCC CRL-1573) and were grown in Eagle’s Minimal Essential Medium (MEM;
Sigma) supplemented with 10% foetal bovine serum (FBS; Sigma). 100 U/mL of penicillin
and 100 µg/mL of streptomycin were added to the media. The cell cultures were incubated at
37˚C in a humidified atmosphere with 5% CO2. The investigated compounds were dissolved
in dimethyl sulfoxide (50 mg/mL) and then diluted in cell culture media supplemented with
2% FBS. HEK-293 cells were placed into 96-well plastic plates (Nunc, Roskilde, Denmark) at
a cell density of 3 x 105 cells per well. After 24 h of incubation at 37˚C, the media were
removed and cells treated with the derivatives, diluted in media at final concentrations of 2-
500 ꢀg/mL. Cell cultures were incubated at 37˚C for 48 h. The cytotoxicity was estimated
using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) that is cleaved
into a coloured formazan product by metabolically active cells, according to the assay

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described by Takenouchi and Munekata [15]. The quantity of the formazan product was
measured in an automatic plate reader. From the obtained results the EC50 (concentration of
the substance which inhibits cells growth in 50% in proportion to the growth of control cells)
values were calculated. The results were given as mean ± SD of three independent
experiments.
4.4. Enzymatic assays
The inhibitory activity of DNA gyrase and topoisomerase IV from E. coli was evaluated using
gyrase supercoling assay kit and topoisomerase IV decatenation kit (both kits obtained from
Inspiralis). Briefly, supercoiled pBR322 plasmid DNA (0.5ꢀg) was incubated with 1 U
gyrase, in the dedicated supercoiling assay buffer supplied by the manufacturer, in the
presence of varying concentrations of the test compounds. Reactions were carried out at 37 °C
for 1 h and then terminated by the addition of equal volume of 2x STOP Buffer (40%
sucrose,100 mM Tris Cl pH 7.5, 1 mM EDTA, 0.5 mg/ml bromophenol blue) and
chloroform/isoamyl alcohol. Samples were vortexed, centrifuged and run through a 15 cm 1%
agarose gel in TAE buffer (40 mM Tris-acetate, 2 mM EDTA) for 3 h at 50 V. Gels were
stained with ethidium bromide and visualized under UV light. The decatenation assay was
performed using E. coli topoisomerase IV decatenation kit (Inspiralis). Interlinked kDNA
substrate (0.5 ꢀg) was incubated with 1 U topoisomerase IV (Inspiralis), in the dedicated
decatenation assay buffer supplied by the manufacturer, in the presence of varying
concentrations of the test compounds. Reactions were carried out at 37°C for 1 h and then
terminated by the addition of equal volume of 2x STOP Buffer (40% sucrose, 100 mM Tris-
Cl pH 7.5, 1 mM EDTA, 0.5 mg/ml bromophenol blue) and chloroform/isoamyl alcohol.
Samples were vortexed, centrifuged and run through a 15 cm 1% agarose gel in TAE buffer
for 1.5 h at 80 V. Gels were stained with ethidium bromide and visualized under UV light.
Concentrations of inhibitor that prevented 50% of the kinetoplast DNA from being converted
into decatenated minicircles (IC50 values) were determined by plotting the results obtained
from the densytometric analyses of the gel images using Quantity One software (BioRad).
Acknowledgements
This research was supported by the Ministry of Science and Higher Education under Iuventus
Plus grant No. IP2014 037473.

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Dr. Tomasz Plech is a recipient of the Fellowship for Young Researchers with Outstanding
Scientific Achievements from the Medical University of Lublin (Lublin, Poland).
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Table 1. Antibacterial activity of compounds 23-44 against Gram-positive strains
Minimal inhibitory concentrations (µM)
R1
R2
MSSA-1*
MSSA-2**
MRSA***
S. epidermidis
ATCC 12228
0.098
B. subtilis
B. cereus
M. luteus
ATCC 6638 ATCC 10876 ATCC 10240
2-OH
2-OH
2-OH
2-OH
2-OH
2-OH
2-OH
2-OH
3-OH
3-OH
3-OH
3-OH
3-OH
3-OH
3-OH
3-OH
4-OH
4-OH
4-OH
C6H5-
C6H5-CH2-
0.80
0.38
1.55
0.76
0.76
0.74
0.76
0.36
0.80
0.38
0.76
0.37
0.76
1.48
0.74
0.18
1.60
3.11
0.19
0.39
0.19
0.38
0.19
0.18
0.18
0.37
0.091
0.39
0.19
0.38
0.093
0.37
1.48
0.37
0.091
0.39
1.56
0.19
0.19
0.38
0.19
0.046
0.093
0.36
0.18
0.091
0.39
0.19
0.38
0.37
0.37
1.48
0.093
0.046
0.39
1.56
0.19
0.39
0.048
0.19
0.098
0.19
1.59
1.56
3.09
0.76
1.51
1.48
1.52
1.49
1.60
1.56
1.55
0.76
1.51
2.95
1.52
1.49
1.60
6.24
1.55
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
0.38
0.38
4-F-C6H4-
0.19
4-F-C6H4-CH2-
4-Cl-C6H4-
0.046
0.18
0.011
0.093
0.045
0.093
0.011
0.098
0.048
0.095
0.023
0.093
0.18
0.093
0.18
4-Cl-C6H4-CH2-
4-OCH3-C6H4-
4-OCH3-C6H4-CH2-
C6H5-
0.091
0.093
0.046
0.19
0.091
0.37
0.091
0.19
C6H5-CH2-
0.096
0.19
0.096
0.19
4-F-C6H4-
4-F-C6H4-CH2-
4-Cl-C6H4-
0.19
0.093
0.18
0.093
0.18
4-Cl-C6H4-CH2-
4-OCH3-C6H4-
4-OCH3-C6H4-CH2-
C6H5-
0.18
0.19
0.37
0.19
0.091
0.098
0.19
0.091
0.024
0.78
0.18
0.098
0.19
C6H5-CH2-
4-F-C6H4-
0.095
0.048
0.095

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4-OH
4-OH
4-OH
4-Cl-C6H4-
4-OCH3-C6H4-
0.76
0.76
2.97
2.96
-
0.37
0.19
0.75
0.72
-
0.093
0.093
0.75
-
0.18
0.37
1.49
1.48
-
0.046
0.023
0.18
0.09
-
0.18
0.19
0.091
0.36
-
1.51
1.52
5.95
5.88
-
42
43
4-OCH3-C6H4-CH2-
44
CPX
0.68
VCN
„*” – S. aureus ATCC 25923, „**” – S. aureus ATCC 6538, „***” – S. aureus MICROBANK 14001; CPX – ciprofloxacin; VCN – vancomycin .

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Table 2. Antibacterial activity of compounds 23-44 against Gram-negative strains
Minimal inhibitory concentrations (µM)
Compounds
E. coli
ATCC 25922
0.012
K. pneumoniae
ATCC 13883
0.049
P. mirabilis
ATCC 12453
0.024
P. aeruginosa
ATCC 9027
0.39
23
0.006
0.011
0.011
0.011
0.023
0.011
0.011
0.012
0.006
0.012
0.006
0.012
0.023
0.006
0.011
0.024
0.048
0.024
0.011
0.012
0.046
0.024
0.096
0.048
0.093
0.023
0.023
0.047
0.023
0.049
0.048
0.048
0.046
0.046
0.18
0.024
0.024
0.023
0.011
0.046
0.047
0.023
0.024
0.012
0.024
0.006
0.024
0.023
0.047
0.023
0.049
0.024
0.048
0.012
0.023
0.091
0.045
0.096
0.095
0.046
0.37
0.36
0.76
0.023
0.19
0.19
0.38
0.093
0.37
0.36
0.19
0.18
0.20
0.38
0.38
0.18
0.023
0.36
0.72
24
25
26
27
28
29
30
31
32
33
34
35
36
0.093
0.091
0.049
0.19
37
38
39
40
0.19
41
0.093
0.093
0.091
0.36
42
43
44
Ciprofloxacin

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Table 3. Toxic effects of the selected triazole-CPX hybrids towards human HEK-293 cells
Compounds
EC50 ± SD (µM)
69.09 ± 10.15
78.72 ± 9.18
24
30
31
34
37
38
41
43
139.55 ± 17.77
69.49 ± 5.04
113.58 ± 2.43
62.89 ± 0.43
106.24 ± 16.60
105.34 ± 2.75