New N-4 piperazinyl derivatives of norfloxacin: design, synthesis, and correlation of calculated physicochemical parameters with antibacterial activity

Article abstract of DOI:10.3906/kim-1706-16

A group of N?4 piperazinyl derivatives of norfloxacin was synthesized and identified by different spectroscopic techniques. The N?4 piperazinyl substituent in target compounds 2a–2k, 3a–3c, and 4a and 4b was designed to have different electronic, steric,

Full text of DOI:10.3906/kim-1706-16

Turk J Chem
Turkish Journal of Chemistry
(2018) 42: 1072 – 1085
© TÜBİTAK
http://journals.tubitak.gov.tr/chem/
doi:10.3906/kim-1706-16
Research Article
New N -4 piperazinyl derivatives of norfloxacin: design, synthesis, and correlation
of calculated physicochemical parameters with antibacterial activity
1
1
Gamal El-Din ABUO-RAHMA ,, Samar ABBAS ,, Mai SHOMAN1,∗,,
2
3
Ebtihal SAMIR ,, Rehab ABDEL-BAKY ,
1
Department of Medicinal Chemistry, Faculty of Pharmacy, Minia University, Minia, Egypt
2
Physical Chemistry, Department of Analytical Chemistry, Faculty of Pharmacy, Deraya University, New Minia,
Minia, Egypt
3
Department of Microbiology, Faculty of Pharmacy, Minia University, Minia, Egypt
Received: 08.06.2017
•
Accepted/Published Online: 15.04.2018
•
Final Version: 03.08.2018
Abstract: A group of N−4 piperazinyl derivatives of norfloxacin was synthesized and identified by different spectroscopic
techniques. The N−4 piperazinyl substituent in target compounds 2a–2k, 3a–3c, and 4a and 4b was designed to
have different electronic, steric, and physicochemical properties. The antibacterial activity of the newly synthesized
compounds was evaluated against Pseudomonas aeruginosa, Escherichia coli, Klebsiella pneumoniae, and Staphylococcus
aureus strains using norfloxacin as a reference. Results showed that most of the tested compounds had higher activity
against E. coli and K. pneumoniae than norfloxacin, whereas only five derivatives were more active against P. aeruginosa.
On the other hand, all derivatives were less active than norfloxacin against S. aureus. The biological activity of the target
compounds, expressed in log MIC, is correlated with lipophilicity, polarizability, and topology parameters. Results showed
that none of the calculated parameters could determine the biological activity. Consequently, the total volume of the
molecule, bulkiness at C-7, electronic factors, and lipophilicity are important factors that should be considered in the
design of new fluoroquinolones.
Key words: Norfloxacin, fluoroquinolones, antibacterial, quantitative structure activity relationship, lipophilicity,
polarizability, thermodynamic parameters
1. Introduction
1
Fluoroquinolones represent an important class of synthetic antibiotics during the last decades. They possess
a broad antibacterial effect against a wide variety of gram-positive and gram-negative microorganisms. Flu-
oroquinolones act by targeting two isozymes: DNA-gyrase and topoisomerase IV. The two enzymes, bound
2
3
to fluoroquinolones, are transformed into toxic enzymes that target bacterial chromosomes. The 1,4-dihydro-
3-carboxylic-4-one skeleton is considered an essential pharmacophore for binding fluoroquinolones with DNA-
gyrase. Meanwhile, it is believed that the 6-fluoro and 7-piperazinyl groups are responsible for the broad-
2
spectrum and anti-Pseudomonas activities of fluoroquinolones. The physicochemical parameters of fluoro-
quinolones are believed to be essential for the cell permeability of these antibiotics and, consequently, their
4
antibacterial activity. Chemical modifications at C-7 of the fluoroquinolone skeleton enable the control of
pharmacokinetic properties and thus might cause a change in the cell permeability, potency, and spectrum of
∗Correspondence: shoman mai@yahoo.com
1072
This work is licensed under a Creative Commons Attribution 4.0 International License.

ABUO-RAHMA et al./Turk J Chem
2
activity. Although fluoroquinolones are broad-spectrum antibiotics, bacterial resistance to them has made the
search for newer antibiotics a continuous challenge.
Moreover, a recent quantitative structure activity relationship (QSAR) study used quantum chemistry
and chemometric methods to prove the importance of molecular properties for the anti-P. aeruginosa activity
5
for selected fluoroquinolones. In addition, QSAR studies showed a linear correlation between the antibacte-
rial activity of benzene sulfonamide fluoroquinolone derivatives with electronic parameters along with steric
6
parameters. On the other hand, hydrophobic properties showed a minor role in the activity. Additionally, a
moderate correlation was found among 15 developed quinolones between the minimum inhibitory concentra-
tions (MICs) and hydrophobicity (r = 0.61). On the other hand, analysis of QSAR among 40 fluoroquinolones
7
revealed that the MIC increment ratio was significantly correlated to the bulkiness of the C-7 substituent. The
thermodynamic parameters for the association of some quinolones with double-strand and single-strand DNA
showed that the change in Gibbs free energy is negative, indicating that the complex formation is spontaneous.
This observation suggests that the association of the quinolone with double-strand as well as single-strand DNA
is an energetically favorable (exothermic) process, whereas the entropy change is unfavorable. The effect of the
substituents at the quinolone ring is less prominent in the single-strand DNA case compared to double-strand
8
DNA due to the steric effect. Meanwhile, another study showed the importance of electronic and acid-base
properties of quinolones for biological activity. Deprotonation of the piperazinyl–NH group greatly affects the
9
charge distribution in the studied compounds.
Collectively, optimizing the overall molecular configurations enhances the number of intracellular targets
10
for antimicrobial action and impedes the efficiency of efflux proteins that diminish intracellular penetration.
Based on the above findings and in continuation of our research to explore the effect of N-4 piperazinyl
11
substitution on antibacterial activity, a number of different N-4 piperazinyl substituents are designed. The
target substituents have variable physicochemical, volume, and electronic properties. Moreover, this research
aims at investigating the correlation between biological activity expressed in log MIC with different lipophilicity,
topological, electronic, and thermal parameters theoretically calculated.
2. Results and discussion
2.1. Synthesis of norfloxacin analogs
Synthesis of the target compounds is outlined in the Scheme. Alkylated norfloxacin derivatives 2a–2k were
prepared by refluxing equimolar ratios of norfloxacin 1 and the respective alkyl halide in acetonitrile using tri-
ethylamine as a base (Scheme). Acylated intermediates of PABA (p-aminobenzoic acid, Figure A), adamantine-
1-amine (Figure 1B), 4-p-tolylthiazol-2-amine (Figure C), and 5-(3,4,5-trimethoxyphenyl)-1,3,4-thiadiazol-2-
amine (Figure D) were prepared through the reaction of the respective amine with bromoacetyl bromide or
chloroacetyl chloride in dichloromethane using potassium carbonate as a base.
Quaternary ammonium derivatives of norfloxacin 3a–3c were prepared using a procedure reported for a
12
similar reaction by heating norfloxacin 1 in acetonitrile with five equivalents of the respective alkyl halide.
1
H NMR spectra of compounds 3a–3c showed a downfield shift of the piperazine protons to δ 3.0–4.0 ppm.
Similarly, acylated norfloxacin derivatives 4a and 4b were synthesized by heating equimolar ratios of 1
with the respective acyl halide in acetonitrile using triethylamine as a base.
All the synthesized compounds were confirmed using NMR spectroscopy; the definite pattern of nor-
floxacin (two doublets and a singlet at δ 7.2–8.9 ppm) supported the success of the coupling processes at the
1073

ABUO-RAHMA et al./Turk J Chem
O
N
O
O
N
COOH
F
F
COOH
RX ( 5 equivalents)
F
COOH
N
RCOCl
CH3CN, TEA
N
N
N
N
R
X
N
HN
R
R
3a-c
1
4a,b
3a, R = -CH2CH3
X = I
3b, R = -CH2CH2C l
3c, R = CH2-CH=CH2
X = Cl
X = Br
R
X
CH3CN, TEA
O
F
COOH
N
N
N
R
2a-k
Scheme. Synthesis of the target compounds 2a–2k, 3a–3c, and 4a and 4b.
Figure. Structure of acylated intermediates used in the synthesis of the designed compounds. A- Acylated derivative
of PABA (p-aminobenzoic acid); B- adamantine-1-amine; C- 4-p-tolylthiazol-2-amine; D- 5-(3,4,5-trimethoxyphenyl)-
1,3,4-thiadiazol-2-amine.
norfloxacin nucleus. Aliphatic protons appeared as expected. For example, compound 2d showed characteristic
allylic protons at δ 5.13, 5.19, and 5.8 ppm. A singlet at δ 3.7 ppm accounting for the spacer CH2 at 2j and
the pattern at δ 1.6–2.9 ppm of the adamantane motif for compound 2f also confirm the success of the coupling
processes, and so on. Elemental analyses also confirmed the formation of the designed compounds.
2.2. Antibacterial activity
Antibacterial activity of the prepared compounds 2a–2k, 3a–3c, and 4a and 4b in addition to the parent
norfloxacin was determined against four standard strains of S. aureus (ATCC 6538), K. pneumoniae (ATCC
10031), P. aeruginosa (ATCC 10145), and E. coli (ATCC 8739). Results are expressed as minimum concentra-
tion required to inhibit bacterial growth (MIC, µg/mL) and are listed in Table 1. Results were judged according
to Clinical and Laboratory Standards Institute (CLSI) guidelines as follows; if the MIC of the tested compound
is ≤ 4µg/mL, the microorganism is considered sensitive; at ≥16 the microorganism is resistant. If the MIC is
around 8 µg/mL, the compound is regarded as intermediately active (Table 2).
Results showed that compounds 2a, 2h, 2j, and 4b had comparable or higher activity than the reference
norfloxacin. They had MICs of 1.10–4.70 µg/mL against gram-positive bacteria while compounds 2a, 2b, 2d,
2g, 2k, 3a, and 4a showed the best activity against E. coli (MIC values were 1.1–2.52 µg/mL vs. 1.85–1.95
1074

ABUO-RAHMA et al./Turk J Chem
Table 1. Different substitutions and minimum inhibitory concentrations (MICs) (µg/mL) of the tested compounds
2a–2k, 3a–3c, 4a, 4b, and norfloxacin 1 against S. aureus, E. coli, K. pneumoniae, and P. aeruginosa.
O
F
COOH
N
N
N
R
Comp.
No.
R
Minimum inhibitory concentration (MIC, µg/mL)
Staph. aureus E. coli Klebsiella
ATCC 6538* ATCC pneumonia
Ps. aeruginosa
ATCC 10145
8739
1.95
ATCC 10031
2.48
1
H
1.85
27.69
2a
2b
2c
2d
2e
2f
Ethyl
1.40
1.10
1.41
27.63
1.40
4.10
157.8
3.437
3.61
6.17
Isopropyl
CH2COOH
13.19
120.22
149.06
208.70
304.40
5.30
5.24
349.86
193.13
52.80
CH2 CH
CH2
2.33
H
C
30.57
115.44
COOCH2CH3
CH3
NHCOCH2
233.32
H
N
2g
20.27
1.84
1.90
15.50
CH2
H3CH2COOC
O
O
C
2h
2i
4.70
NA
82.99
1.69
22.60
1.87
4.22
NA
CH2
O
H
2 C
CH3
HN
O
2j
1.88
15.24
2.39
20.09
1.88
498.09
96.82
S
H2C
HN
N
OCH3
OCH3
OCH3
2k
38.48
N
N
HN
S
H2C
O
3a
3b
3c
4a
88.90
2.50
1.20
21.7
2.52
1.10
151.8
19.02
295.70
NA
B is-(ethyl)-
113.50
166.88
55.92
93.5
Bis-(2-chloroethyl)-
378.05
12.97
Bis-(allyl)-
O
BrH2C
C
O
4b
1.70
7.33'
57.90
NA
F
C
NA: Not actve.
1075

ABUO-RAHMA et al./Turk J Chem
Table 2. MIC of norfloxacin against the tested organisms according to CLSI.
MIC (µg/mL)
Microorganism
S
I
R
S. aureus
E. coli
≤ 4
8
8
8
8
≥ 16
≥ 16
≥ 16
≥ 16
≤ 4
K. pneumoniae ≤ 4
P. aeruginosa
≤ 4
|S= Sensitive, I = intermediate, R= resistant.
µg/mL for norfloxacin). The same compounds appeared to be less active against the rest of the gram-negative
bacteria; they showed MICs of 2.50 to >100 µg/mL against different gram-negative bacteria.
On the other hand, introducing a bulky N 4-adamantane carbamoyl group in compound 2f did not show
any significant activity against all the tested microorganisms (MICs = 115–304 µg/mL, Table 1). Meanwhile,
compound 3b having a bis-(2-chloroethyl) group and 3a with a bis-ethyl group showed moderate activity against
the gram-positive S. aureus (MIC = 88.90 µg/mL) but higher activity against E. coli and K. pneumoniae
(MIC = 2.50 and 1.10 µg/mL, respectively, Table 1). Consequently, the N -isopropyl compounds 2b and 2g
showed moderate activity against S. aureus, while compounds 2a, 2b, 2d, 2g, 2i, 3b, and 4b showed better
activity than the reference norfloxacin against E. coli with MIC values that ranged between 1.00 and 1.84
µg/mL. In addition, compounds 2e and 3a showed good activity, but less so than norfloxacin. Compounds 2b,
2d, 2g, 2i, 2k, and 3a showed comparable activity to norfloxacin against Klebsiella pneumoniae, while 2a, 2b,
2g, and 2h showed better activity against P. aeruginosa.
Overall, most compounds showed considerable activity against S. aureus and E. coli and lower activity
against K. pneumoniae but low or no activity against P. aeruginosa.
2.3. Calculation of lipophilicity, polarizability, and topology parameters
Calculation of lipophilicity, topological, electronic, and thermal parameters of the target compounds and
norfloxacin were carried out using the ChemBio 3D Ultra 2014 program. Calculated parameters include the
Balaban index as a topological parameter. Diameter, electronic energy, molecular mass, and Wiener index were
calculated as steric parameters. Some electronic parameters were also calculated including dipole moment and
N-4 piperazinyl charge density. Moreover, the heat of formation, molar refractivity, Gibbs free energy, and
log p, as thermodynamic parameters, were measured. It is expected that these aforementioned parameters
have an effect on the permeability of fluoroquinolones to bacterial cells. They may also affect binding with the
target site, DNA-gyrase or its isozyme topoisomerase IV. The Wiener index is a measure of the compactness
13
of molecules. It was also reported to be connected with the molecular Van der Waals areas. The Wiener
index provides a rough measure of the molecular surface, though it cannot be considered as either a measure
14
of molecular volume or of the volume-to-surface ratio. Moreover, some reports showed the importance of the
9
steric effect and Gibbs free energy or heat of formation on the binding of quinolones with DNA. Additionally,
partitioning of quinolones in 1-octanol/water was found to be an entropy-driving process with absorbing heat
and water solvating drug molecules more easily than 1-octanol. The enthalpy changes for quinolone molecules
15
partitioning in 1-octanol/water are all positive, and the degrees of the system decrease. As several reports
focused on the importance of these parameters for the activity of fluoroquinolones, lipophilicity, polarizability,
and topology parameters for the prepared compounds are calculated and outlined in Table 3.
1076

ABUO-RAHMA et al./Turk J Chem
1077

ABUO-RAHMA et al./Turk J Chem
Correlations were established using Pearson’s correlation coefficient (r) in bivariate linear correlations (P
< 0.05). All statistical analyses were performed using SPSS 17 (SPSS Inc., Chicago, IL, USA). The Pearson
correlation is +1 in the case of a perfect direct (increasing) linear relationship (correlation), –1 in the case of a
16
perfect decreasing (inverse) linear relationship (anticorrelation), and some value in the open interval (–1, 1)
in all other cases, indicating the degree of linear dependence between the variables. As it approaches zero, there
is a decreased relationship (closer to uncorrelated). The closer the coefficient is to either –1 or 1, the stronger
the correlation is between the variables. If the variables are independent, Pearson’s correlation coefficient is 0,
but the converse is not true because the correlation coefficient detects only linear dependencies between two
variables.
Correlation results between the antibacterial activity expressed in log MIC and the calculated parameters
of the tested compounds and the reference norfloxacin are outlined in Table 4. It was clear that the N−4
substituent has an important impact on log P, N -4 charge density, and steric energy among other parameters.
The N -4 charge ranged from 0.7 with a bis alkyl substitution to –0.12 with large bulky adamantyl group.
Additionally, the N -4 charge had an impact on antibacterial activity, especially against gram-positive bacteria
(correlation coefficient = 0.34), while changing the N -4 charge did not cause any significant change in anti-E.
coli activity (correlation coefficient = 0.01, Table 4)
Table 4. Correlation coefficient between log MIC of the tested strains and the calculated parameters of the target
compounds and the reference norfloxacin.
Correlation cooffiecient
Klebsiella
Staph. aureus E. coli
pneumoniae
P. aeruginosa
ATCC 6538
0.211600
–0.001138
0.14130
ATCC 8739 ATCC 10031 ATCC 10145
Molecular mass
Balaban index
Wiener index
0.205800
0.081990
0.19880
0.19910
0.27010
0.10250
–0.040480
0.13680
0.01965
0.04281
–0.131400
–0.244200
–0.19520
–0.19260
0.15590–
0.01530
0.132300
–0.05435
0.20450
0.280200
0.106100
0.15530
0.19470
0.20250
0.17250
–0.004659
0.06598
0.05620
0.09526
Topological diameter bond(s) 0.01722
Molar refractivity, cm−1/mol 0.13680
Log P
–0.23160
–0.319500
–0.08832
0.34210
Heat of formation [kJ/mol]
Gibbs free energy [kJ/mol]
N4-charge
Steric energy [kcal/mol]
–0.28180
–0.21380
Moreover, molecular mass, diameter, and steric energy also had an impact on the antibacterial activity of
the tested norfloxacin derivatives. Increasing molecular mass had a small negative impact on the activity against
K. pneumoniae (correlation coefficient = –0.13) and a higher positive impact on the other microorganisms
17
(correlation coefficient = 0.21–0.28). These data are consistent with the published data in the literature.
The diameter of the molecules had a weak impact on the anti-E. coli and P. aeruginosa activity (correlation
coefficient = 0.19) and increasing diameter negatively affected activity against K. pneumoniae (correlation
coefficient = –0.19).
1078

ABUO-RAHMA et al./Turk J Chem
Additionally, a negative to weak correlation was shown between the Balaban index, the Wiener index,
and the tested activities. Log P had a moderate to weak correlation with the antibacterial activity in most
of the tested strains, which means that hydrophobicity of the molecule is not the only factor affecting the
entrance of the molecule into the bacterial cell. The highest impact for hydrophobicity was observed against S.
aureus strains, where higher log P values decreased the observed antibacterial activity (correlation coefficient =
–0.23, Table 4). A previous correlation study reported that passive diffusion is not the defining mechanism for
18
fluoroquinolone entry to bacterial cells minimizing the role of lipophilicity as observed in the current study.
Similarly, heat of formation and Gibbs energy showed weak correlations with the activity against S. aureus,
whereas they showed weak or no impact on the activity against different gram-negative strains. Change in Gibbs
free energy is usually negative, indicating that the complex formation between a compound and target enzyme
is spontaneous. The association is driven by favorable ∆H (negative enthalpies) and therefore is exothermic.
Gibbs free energy did not show a significant correlation with the antibacterial activity. Additionally, the increase
in steric energy moderately decreased both anti-Staphylococcus and anti-Klebsiella activities, while no similar
effect was observed on the rest of the tested strains.
Moreover, data in Table 5 show that there is a highly significant correlation between molar mass and
Weiner index (r = 0.965) and a strong negative correlation between steric energy and N-4 charge (r = –0.948).
It is obvious that the increase of N -4 charge density has a deleterious effect on log P (r = –0.940). As expected,
there was a medium correlation between Gibbs energy and the heat of formation (r = 0.314). It was obvious
that there was a weak to moderate effect of the topological parameters on log P that may affect the permeability
of quinolones. On the other hand, there was no significant correlation between log P and the Weiner index. In
summary, there was no calculated factor that could determine the biological activity. However, the total volume
of the molecule, bulkiness at C-7, electronic factors, and lipophilicity can determine the activity. It is obvious
that when designing newer fluoroquinolones, the total volume of the molecule, bulkiness at C-7, electronic
factors, and lipophilicity factors should be considered. Abuo-Rahma et al. previously studied correlations of
other fluoroquinolone properties with antimicrobial activity and their results supported the fact that no single
parameter is most effective in determining the activity of fluoroquinolones where biological activity is a factor
12
of a group of lipophilicity, molecular mass, and electronic factors. Similar results were found in the literature
as the antibacterial activity of fluoroquinolones depended on certain electronic effects and steric effects of bulky
groups positioned on the piperazine ring where electronic properties of OCH3 and Cl groups significantly
enhanced the antibacterial activity of a series of sulfonamide-substituted fluoroquinolones while the CH3 steric
19
effect decreased the activity.
2.4. Conclusion
Fifteen norfloxacin derivatives containing different substituents at the N 4-piperazinyl moiety have been syn-
thesized. These derivatives are designed to have different lipophilicity, polarizability, and topology properties.
Some of the prepared compounds showed reasonable antibacterial activity parallel to or greater than the ref-
erence norfloxacin. Isopropyl derivative 2b and p−aminobenzoic acid derivative 2g showed broad-spectrum
antibacterial activity against all the tested strains. Generally, test compounds have higher activity against
S. aureus and E. coli among the examined microorganisms. Correlation between antibacterial activities, ex-
pressed in log MIC, and lipophilicity, polarizability, and topology parameters showed that none of the studied
parameters could exclusively affect the antibacterial activity. Indeed, the total volume of the molecules, bulk-
iness, electronic factors, and lipophilicity can determine the antibacterial activity. For example, though some
1079

ABUO-RAHMA et al./Turk J Chem
Table 5. Correlation coefficient between all tested parameters of the new compounds and the reference norfloxacin.
Molecular mass
0.974323
0.965624
0.969158
0.909384
0.936761
0.103195
–0.58673
0.45710
–0.27782
0.527731
Balaban !ndex
W!ener !ndex
0.97951
0.92457
0.97777
0.98582
0.28444
–0.42964
–0.60270
0.64539
0.54360
–0.47708
–0.31828
0.682676
0.51235
0.073290
Topolog!cal
d!ameter
bond(s)
0.96683
0.44737
0.22063
–0.25877
0.78560
–0.64054
0.80082
Molar
refract!v!ty,
–0.46759
0.742911
0.67662
0.79667
0.313555
–0.46845
–0.94043
–0.56107
0.63129
0.89379
0.366455
cm–1/mol
Log P
Heat of
format!on
[kJ/mol]
G!bbs free
–0.95347
0.922978
–0.94803
energy [kJ/mol]
N4-charge
parameters like the charge on N−4 affect activity against S. aureus, others such as log P showed almost no
effect on activity on K pneumoniae. Thus, all these parameters should be considered in the design of new
fluoroquinolones.
3. Experimental
3.1. Materials
All reagents and solvents were of commercially available reagent grade quality and were used without further
purification. The IR spectra were recorded on a Nicolet iS5 FT-IR spectrometer (Fisher Scientific, USA).
1
13
The H NMR and
C NMR spectra were recorded on a Bruker Avance III (USA) at 400 MHz or 600
MHz. Chemical shifts are reported in δ ppm using TMS as an internal standard and coupling constants
(J) are expressed in Hz. Abbreviations indicating multiplicity were used as follows: s = singlet, bs = broad
singlet, d = doublet, t = triplet, q = quartet, and m = multiplet. Melting points were measured on a Stuart
SMP10 melting point apparatus (Germany). Elemental analyses were carried out at the Regional Center
1080

ABUO-RAHMA et al./Turk J Chem
of Mycology and Biotechnology, Al-Azhar University, Cairo. LC/MS/MS was carried out using an Agilent
UPLC/LC/MS/MS 1260 Infinity II (USA) with 6420 Triple Quad LC/MS detector at the Faculty of Pharmacy,
Minia University. Norfloxacin was purchased from Medical Union Pharmaceutical (MUP) Abu-Sultan, Ismailia,
20
21
1
22
12
Egypt. Compounds 2a, 2b, 2d, 2h, and 3a were prepared as reported.
3.1.1. N-((3s,5s,7s)-Adamantan-1-yl)-2-bromoacetamide23
B
1
H NMR (400 MHz, DMSO-d6) 1.59–1.60 (6H, m, adamantane 3CH 2), 1.89–1.90 (3H, m, 3 CH (CH2)),
1.96–1.98 (6H, m, adamantane 3NCH 2), 4.02 (1H, s, COCH 2), 7.23 (1H, s, NH).
3.1.2. 2-Chloro-N-(4-(p-tolyl)thiazol-2-yl)acetamide C24
1
Pale yellow crystals, mp: 162–164 ◦ C (reported: 165–167 ◦ C); H NMR (400 MHz, CDCl3) δ 2.43 (3H, s,
CH 3), 4.43 (2H, s, COCH 2), 7.29–7.30 (3H, m, thiazole C5 & 2 Ar H), 7.78 (2H, d, J = 7.50, Ar H), 10.90
(1H, s, NH).
3.1.3. 2-Bromo-N-(5-(3,4,5-trimethoxyphenyl)-1,3,4-thiadiazol-2-yl) acetamide D
1
Yellow powder; 3.10 g, 84.00% yield; mp: 274–276 ◦ C; H NMR (400 MHz, DMSO-d6) δ 3.74 (3H, s, OCH 3),
3.89 (6H, s, OCH 3), 4.22 (2H, s, -COCH 2), 7.21 (2H, s, Ar-H), 12.29 (1H, s, -NH).
3.1.4. 7-(4-Allylpiperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid 2d
1
Pale yellow powder; yield = 65%, mp = 237–238 ◦ C, H NMR (600 MHz, DMSO-d6) δ1.35 (3H, t,J = 6.9
Hz, NCH2 CH 3), 2.95–3.05 (4H, m, piperazinyl-H), 2.99 (2H, d, J = 6.2 Hz, NCH 2 CH), 3.34–3.36 (4H, m,
piperazinyl-4H), 4.54 (2H, q, J = 6.9 Hz, CH 2 CH3), 5.13 (H, d, Jcis = 10.3 Hz, N -CH2 -CH=CH 2), 5.19
(H, d, Jtrans = 17.2 Hz, NCH 2 CH), 5.79–5.82 (1H, m, N-CH2 -CH=CH2), 7.14 (1H, d, J = 6.8 Hz, H−8),
13
7.88 (1H, d, J = 13 Hz, H -5), 8.91 (1H, s, H -2), 15.32 (1H, brs, COOH); C NMR (150 MHz, DMSO-d6) δ
14.32, 49.08, 49.50, 52.21, 60.67, 106.10, 107.44, 111.57, 118.10, 120.48, 137.09, 144.60, 148.50, 166.32, 176.40.
Anal. Calcd. for C19 H22 FN3 O3 (359.16): C, 63.50; H, 6.17; N, 11.69. Found: C, 63.64; H, 6.21; N, 11.84.
3.1.5. 7-(4-(1-Ethoxy-1-oxopropan-2-yl)piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-
3-carboxylic acid 2e
1
Pale yellow powder; yield = 74%, mp = 209–211 ◦ C, H NMR (600 MHz, DMSO-d6) δ1.17 (3H, d, J = 7.00
Hz, CH3 CH-), 1.18 (3H, t, J = 6.8 Hz, -OCH2 CH 3), 1.37 (3H, t,J = 6.9 Hz, NCH2 CH 3), 2.72 (1H, d,J =
7.0 Hz, CHCH3), 3.35–3.95 (8 H, m, piperazinyl-8H), 4.08 (2H, q, J = 6.8 Hz, OCH 2 CH3), 4.55 (2H, q, J =
6.9 Hz, -N-CH 2 CH3), 7.14 (1H, d, J = 6.9 Hz, H−8), 7.88 (1H, d, J = 14 Hz, H -5), 8.91 (1H, s, H -2);
13
C NMR (150 MHz, DMSO-d6) δ 14.46, 14.57, 40.28, 48.57, 49.50, 50.07, 60.06, 61.52, 106.10, 107.31, 111.30,
119.48, 137.47, 146.05, 148.82, 157.50, 166.44, 172.36, 175.50. Anal. Calcd. for C21 H26 FN3 O5 (419.19): C,
60.13; H, 6.25; N, 10.01. Found: C, 60.41; H, 6.39 1; N, 10.43.
1081

ABUO-RAHMA et al./Turk J Chem
3.1.6. 7-(4-(2-((3s,5s,7s)-Adamantan-1-ylamino)-2-oxoethyl)piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-
1,4-dihydroquinoline-3-carboxylic acid 2f
1
Pale yellow powder; yield = 62%, mp >300 ◦ C; H NMR (400 MHz, DMSO-d6) δ1.40 (3H, t, J = 6.8
NCH2 CH 3), 1.60–1.65 (6H, m, adamantane 3CH 2), 1.93–2.06 (9H, m, adamantane 3NCH 2 and 3 CH(CH2)),
2.63–3.65 (4H, m, piperazinyl-H), 3.32–3.34 (6H, m, piperazinyl-4H and COCH2 N), 4.58 (2H, q, J = 6.8 Hz,
NCH 2 CH3), 7.11 (1H, s, NH), 7.17 (1H, d, J = 7.2 Hz, H−8), 7.89 (1H, d, J = 13Hz, H -5), 8.95 (1H, s,
H -2), 15.10 (1H, brs, COOH). Anal. Calcd. for C28 H35 FN4 O4 (510.26): Calcd. C, 65.86; H, 6.91; N, 10.97.
Found: C, 65.98; H, 6.98; N, 11.09.
3.1.7. 7-(4-(2-((4-(Ethoxycarbonyl)phenyl)amino)-2-oxoethyl)piperazin-1-yl)-1-ethyl-6-fluoro-4-
oxo-1,4-dihydroquinoline-3-carboxylic acid 2g
1
Pale yellow powder; yield = 66%, mp = 270–273 ◦ C, H NMR (600 MHz, DMSO-d6) δ1.26 (3H, t, J = 6.8
OCH2 CH 3), 1.36 (3H, t,J = 7.6 Hz, NCH2 CH 3), 2.73–2.85 (4H, m, piperazinyl-H), 3.36 (2H, s, -COCH 2 NH),
3.47–3.49 (4H, m, piperazinyl-4H), 4.24 (2H, q, J = 6.8 Hz, OCH 2 CH3), 4.58 (2H, q, J = 7.6 Hz, NCH 2 CH3),
7.22 (1H, d, J = 6.9 Hz, H−8), 7.75 (2H, d, J = 6.8 Hz, Ar−H), 7.85 (2H, d, J = 6.8 Hz, Ar−H), 7.89
13
(1H, d, J = 12Hz, H -5), 8.93 (1H, s, H -2), 10.10 (1H, brs, NH), 15.10 (1H, brs, COOH); C NMR (150
MHz, DMSO-d6) δ 14.20, 14.44, 42.72, 46.59, 49.13, 52.26, 60.48, 106.80, 107.14, 107.28, 111.60, 118.88, 130.25,
137.21, 137.31, 144.00, 148.87, 152.70, 153.10, 158.80, 165.44, 166.14, 176.31. Anal. Calcd. for C27 H29 FN4 O6
(524.21): C, 61.82; H, 5.57; N, 10.68. Found: C, 61.94; H, 5.54; N, 10.84.
3.1.8. 7-(4-((p-Tolylcarbamoyl)methyl)piperazin-1-yl)-1-ethyl-6-fluoro-1,4-dihydro-4-oxoquinoline-
3-carboxylic acid 2i
1
Pale yellow powder; yield = 72%, mp = 294–296 ◦ C, H NMR (400 MHz, DMSO-d6) 1.43 (3H, t,J = 7.2 Hz,
NCH2 CH 3), 2.26 (3H, s, p-tolyl-CH 3), 3.16–3.89 (8H, m, piperazinyl-H), 4.33 (2H, s, -CH2 -CO), 4.63 (2H,
q, J = 7.6 Hz, NCH 2 CH3), 7.15 (2H, d, J = 8.00 Hz, Ar−H), 7.24 (1H, d, J = 6.9 Hz, H−8), 7.54 (2H, d, J
13
= 8.00 Hz, Ar−H), 7.94 (1H, d, J = 12.8 Hz, H -5), 8.94 (1H, s, H -2), 10.98(1H, brs, NH); C NMR (100
MHz, DMSO-d6) δ 14.98, 20.94, 42.39, 46.81, 49.63, 51.78, 57.03, 106.97, 107.66, 111.79, 112.02, 119.97, 120.40,
129.76, 133.71, 135.93, 137.59, 144.32, 144.42, 149.15, 151.89, 154.37, 162.91, 166.50, 176.61. Anal. Calcd. for
C25 H27 FN4 O4 (466.2): LC/MS/MS data: Calculated (466.2), Found: 466.2
3.1.9. 1-Ethyl-6-fluoro-4-oxo-7-(4-(2-oxo-2-((4-(p-tolyl)thiazol-2-yl)amino)ethyl)piperazin-1-yl)-1,4-
dihydroquinoline-3-carboxylic acid 2j
1
Pale yellow powder; yield = 49%, mp = 294–296 ◦ C, H NMR (600 MHz, DMSO-d6) δ1.38 (3H, t,J = 6.8
Hz, NCH2 CH 3), 2.29 (3H, s, CH 3), 2.45–2.46 (4H, m, piperazinyl-H), 3.51–3.55 (6H, m, piperazinyl-4H and
COCH 2), 4.58 (2H, q, J = 6.8 Hz, CH 2 CH3), 7.21 (3H, d, J = 8 Hz, H−8 and 2 ArH), 7.61 (1H, s, ArH),
13
7.75 (2H, d, J = 8 Hz, ArH), 7.93 (1H, d, J = 12.4 Hz, H -5), 8.94 (1H, s, H -2), 15.32 (1H, brs, COOH);
C
NMR (150 MHz, DMSO-d6) δ 14.79, 21.12, 39.34, 40.34, 49.47, 100.49, 107.58, 111.35, 114.5, 122.50, 126.07,
129.77, 131.90, 135.10, 137.74, 146.50, 149.10, 151.20, 152.50, 159.80, 163.40, 166.53, 176.10. Anal. Calcd. for
C28 H28 FN5 O4 S (549.18): C, 61.19; H, 5.13; N, 12.74. Found: C, 61.04; H, 5.23; N, 12.90.
1082

ABUO-RAHMA et al./Turk J Chem
3.1.10. 7-(4-((5-(3,4,5-Trimethoxyphenyl)-1,3,4-thiadiazol-2-ylcarbamoyl)methyl) piperazin-1-yl)-
1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid 2k
1
Pale yellow powder; yield = 74%, mp = 278–280 ◦ C, H NMR (400 MHz, DMSO-d6) 1.42 (3H, t,J = 7.2 Hz,
NCH2 CH 3), 2.80 (4H, m, piperazinyl-H), 3.55 (4H, m, piperazinyl-H), 3.73 (2H, s, -CH2 -CO), 3.84 (3H, s,
-OCH3), 3.88 (6H, s, 2-OCH3), 4.59 (2H, q, J = 7.6 Hz, NCH 2 CH3), 7.20 (2H, s, 2Ar H), 7.18 (2H, d, J =
13
7.2 Hz, H-8), 7.94 (1H, d, J = 12.8 Hz, H -5), 8.94 (1H, s, H -2), 15.15 (1H, brs, -COOH); C NMR (100
MHz, DMSO-d6) δ 14.81, 49.54, 49.96, 52.48, 56.59, 60.22, 60.64, 104.72, 106.34, 107.54, 111.53, 111.76, 119.97,
126.03, 137.67, 139.94, 142.00, 145.97, 148.94, 153.87, 154.02, 158.47, 162.33, 166.59, 169.19, 176.64. Anal.
Calcd. for C29 H31 FN6 O7S (626.20): LC/MS/MS data: Calculated: (626.2), Found: (626.2).
3.1.11. 4-(3-Carboxy-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinolin-7-yl)-1,1-diethylpiperazin-1-ium
iodide 3a
1
Pale yellow powder; yield = 68%, mp >300 ◦ C, H NMR (600 MHz, DMSO-d6) δ1.22 (6H, t,J = 6.8 Hz,
NCH2 CH 3), 1.38 (3H, t,J = 6.8 Hz, NCH2 CH 3), 3.20–3.32 (4H, m, piperazinyl-H), 3.48–3.85 (4H, m,
piperazinyl-H), 4.57 (2H, t,J = 6.8 Hz, NCH 2 CH3), 4.00 (4H, q,J = 6.8 Hz, NCH 2 CH3), 4.57 (2 H, q,J
= 6.8 Hz, NCH 2 CH3), 7.20 (1H, d, J = 7 Hz, H−8), 7.90 (1H, d, J = 12 Hz, H -5), 8.91 (1H, s, H -2);
13
C NMR (150 MHz, DMSO-d6) δ 9.10, 14.45, 42.75, 46.56, 49.17, 50.22, 50.89, 106.65, 107.23, 111.55, 120.01,
137.16, 148.78, 151.77, 153.76, 166.11, 176.26. Anal. Calcd. for C20 H27 FIN3 O3 (503.11): C, 47.72; H, 5.41;
N, 8.35. Found: C, 47.89; H, 5.49; N, 8.51.
3.1.12. 4-(3-Carboxy-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinolin-7-yl)-1,1-bis(2-chloroethyl)pipe-
razin-1-ium chloride 3b
1
Pale yellow powder; yield = 44%, mp = 237–239 ◦ C, H NMR (600 MHz, DMSO-d6) δ1.37 (3H, t,J = 7.5 Hz,
NCH2 CH 3), 2.58 (4H, t, J = 6.8 Hz, ClCH2 CH2 N-), 3.26–3.46 (4H, m, piperazinyl-H), 3.46–3.51 (4H, m,
piperazinyl-4H), 4.07 (4H, q, J = 7.0 Hz, CH2 CH 2 Cl), 4.57 (4H, t,J = 6.8 Hz, -NCH 2 CH3), 7.22 (1H, d, J
13
= 6.8 Hz, H−8), 7.93 (1H, d, J = 13 Hz, H -5), 8.94 (1H, s, H -2); C NMR (150 MHz, DMSO-d6) δ 14.61,
42.75, 46.66, 46.71, 49.29, 51.75, 60.12, 69.24, 106.10, 107.44, 111.57, 120.28, 137.39, 144.63, 149.04, 152.40,
166.32, 176.48. Anal. Calcd. for C20 H25 Cl3 FN3 O3 (479.09): C, 49.96; H, 5.24; N, 8.74. Found: C, 50.03; H,
5.22; N, 8.89.
3.1.13. 1,1-Diallyl-4-(3-carboxy-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinolin-7-yl)piperazin-1-ium
bromide 3c
1
Pale yellow powder; yield = 63%, mp = 229–230 ◦ C, H NMR (400 MHz, DMSO-d6) δ1.43 (3H, t,J = 8
Hz, NCH2 CH 3), 2.52–2.59 (4H, m, piperazinyl-H), 3.05 (4H, d, J = 6.2 Hz, 2NCH 2 CH), 3.32–3.34 (4H, m,
piperazinyl-H), 4.60 (2H, q, J = 8 Hz, CH 2 CH3), 5.18 (2H, d, Jcis = 4 Hz, 2N-CH2 -CH=CH 2), 5.24 (2H,
d, Jtrans = 12 Hz, 2NCH 2 CH), 5.85–5.87 (2H, m, 2N-CH2 -CH=CH2), 7.19 (1H, d, J = 8 Hz, H−8), 7.92
(1H, d, J = 12 Hz, H -5), 8.96 (1H, s, H -2), 15.37 (1H, brs, COOH). Anal. Calcd. for C22 H27 BrFN3 O3
(479.12): C, 55.01; H, 5.67; N, 8.75. Found: C, 55.17; H, 5.71; N, 8.89.
1083

ABUO-RAHMA et al./Turk J Chem
3.1.14. 7-(4-(2-Bromoacetyl)piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carbox-
ylic acid25 4a
1
White powder; yield: 0.370 g (84%); mp: 247–249 ◦ C; H NMR (400 MHz, DMSO-d6): 1.45 (3H, t, J = 7.6
Hz, NCH2 CH 3), 3.38–3.42 (4H, m, piperazinyl-H), 3.70–3.74 (4H, m, piperazinyl-H), 4.20 (2H, s, BrCH 2),
4.59 (2H, q, J = 7.6 Hz, NCH 2 CH3), 7.22 (1H, d, J H-F = 7.6 Hz, H 8), 7.96 (1H, d, J H-F = 13.6 Hz, H 5),
13
8.94 (1H, s, H 2), 15.22 (1H, s, COOH); C NMR (100 MHz, DMSO-d6): 14.76, 28.29, 41.61, 46.11, 49.56,
106.86, 107.58, 111.73 (d, J = 23 Hz), 120.06 (d, J = 8 Hz), 137.62, 145.56 (d, J = 11 Hz), 149.14, 153.29 (d,
J = 247 Hz), 165.49, 166.57, and 176.67; Anal. Calcd for C18 H19 BrFN3 O4 : C, 49.11; H, 4.35; N, 9.54. Found:
C, 49.34; H, 4.31; N, 9.78.
3.2. Antibacterial activity
3.2.1. Bacterial strains
Standard strains of S. aureus (ATCC 6538), K. pneumoniae (ATCC 10031), P. aeruginosa (ATCC 10145), and
E. coli (ATCC 8739) were obtained from the Microbiological Resource Centre (MIRCIN), Agriculture Faculty,
Ain Shams University.
3.2.2. Determination of minimum inhibitory concentrations26
6
Microorganisms (0.5 mL) of 1 × 10 CFU/mL (0.5 McFarland turbidity) were plated in sterile petri dishes and
then 20 mL of sterile, molten, and cooled (45 ◦ C) Muller Hinton agar medium was added to all petri dishes.
The plates then were slowly rotated to ensure the uniform distribution of the microorganisms, allowing the
solidification on a flat surface. After solidification, four equidistant and circular wells of 10 mm in diameter
were carefully punched using a sterile cork bore.
Twofold serial dilutions were performed on the tested compounds and the reference norfloxacin. Equal
volumes of the tested compounds and the reference were well applied separately to each one in the three
replicates using a micropipette. All plates were incubated overnight at 37 ◦ C and then collected. Zones of
inhibition that developed were measured. The average of the zones of inhibition was calculated. The MIC was
calculated by plotting the natural logarithm of the concentration of extract against the square of the zones of
inhibition. A regression line was drawn through the points. The antilogarithm of the intercept on the logarithm
of the concentration axis gave the MIC value.
3.3. Statistical analysis
Correlations were established using Pearson’s correlation coefficient (r) in bivariate linear correlations (P <
0.05). All statistical analyses were performed using SPSS 17 (SPSS Inc., Chicago, IL, USA).
References
1. Koga, H.; Itoh, A.; Murayama, S.; Suzue, S.; Irikura, T. J. Med. Chem. 1980, 23, 1358-1363.
2. Foroumadi, A.; Emami, S.; Mehni, M.; Moshafi, M. H.; Shafiee, A. Bioorg. Med. Chem. Lett. 2005, 15, 4536-4539.
3. Drlica, K.; Zhao, X. Curr. Opin. Antiinfect. Investig. Drugs 1999, 1, 435-442.
4. Domagala, J. M.; Hanna, L. D.; Heifetz, C. L.; Hutt, M. P.; Mich, T. F.; Sanchez, J. P.; Solomon, M. J. Med.
Chem. 1986, 29, 394-404.
1084

ABUO-RAHMA et al./Turk J Chem
5. Li, X.; Zhang, R.; Yang; X. Front. Chem. China 2010, 5, 80-87.
6. Nieto, M. J.; Alovero, F. L.; Manzo, R. H.; Mazzieri, M. R. Eur. J. Med. Chem. 2005, 40, 361-369.
7. Takenouchi, T.; Tabata, F.; Iwata, Y.; Hanzawa, H.; Sugawara, M.; Ohaya, S. Antimicrob. Agents Chemother.
1996, 40, 1835-1842.
8. Lee, B. H.; Ga-Kyeung, G. Y.; Jang, J.; Lee, D. J.; Noh, S. G.; Cho, T. Bull. Korean Chem. Soc. 2009, 30,
1031-1034.
9. Shabaan, A. K. E.; Hassan, A. E.; Saadullah; G. A. Chin. Sci. Bull. 2012, 57, 1665-1671.
10. Peterson, L. R. Clin. Infect. Dis. Off. Publ. Infect. Dis. Soc. Am. 2001, 33 (Suppl. 3), S180-186.
11. Abdel-Aziz, M.; Park, S. E.; Abuo-Rahma, G. E. D. A. A.; Sayed, M. A.; Kwon, Y. Eur. J. Med. Chem. 2013,
69, 427-438.
12. Abuo-Rahma, G. E. D. A. A.; Sarhan, H. A.; Gad, G. F. M. Bioorg. Med. Chem. 2009, 17, 3879-3886.
13. Wiener, H. J. Am. Chem. Soc. 1947, 69, 17-20.
14. Gutmanan, I.; Körtvélyesia, T. Z. Naturforsch. 1995, 50a, 669.
15. Zhang, C.; Wang, Y. Chem. Res. Chin. Univ. 2010, 26, 636-639.
16. Dowdy, S.; Wearden, S. Statistics for Research; Wiley: New York, NY, USA, 1983.
17. Piddock, L. J.; Jin, Y. F.; Griggs; D. J. J. Antimicrob. Chemother. 2001, 47, 261-270.
18. Breznica-Selmani, P.; Mladenovska, K.; Dräger, G.; Mikhova, B.; Panovski, N.; Kaftandzieva, A.; Kavrakovski, Z.;
Hoxha, A.; Sheqerxhiu, N.; Pavlova, M. J. et al. Maced. J. Chem. Chemical Eng. 2016, 35, 179-197.
19. Abdel-Aziz, A. A. M; Asiri, Y. A; Al-Agamy, M. H. M. Eur. J. Med. Chem. 2011, 46, 5487-5497.
20. Emami, S.; Shafiee, A.; Foroumadi, A. Mini Rev. Med. Chem. 2006, 6, 375-386.
21. Bagchi, M. C.; Mills, D.; Basak, S. C. J. Mol. Model. 2007, 13, 111-120.
22. Patel, M. M.; Patel, L. J. Scientific World Journal 2014, 2014, 897187.
23. Lengyel, I.; M. J. Aaronson. Can. J. Spectrosc. 1974, 19, 95-99.
24. Pravst, I.; Zupan, M.; Stavber, S. Tetrahedron 2008, 64, 5191-5199.
25. Aziz, H.; Abbasa, S. H.; Moustafa, G. I.; Abuo-Rahmaa, G. E. D. A. A. Malaysian Journal of Medical Research
2017, 27, 56-58.
26. Cockerill, F. Performance Standards for Antimicrobial Susceptibility Testing: Twenty-First Informational Supple-
ment. Clinical and Laboratory Standards Institute: Wayne, PA, USA, 2011.
1085