Design, synthesis and molecular modeling studies on novel moxifloxacin derivatives as potential antibacterial and antituberculosis agents

Article abstract of DOI:10.1016/j.bioorg.2019.102965

Twenty-one novel alkyl/acyl/sulfonyl substituted fluoroquinolone derivatives were designed, synthesized and evaluated for their anti-tuberculosis and antibacterial activity. The targeted compounds were synthesized by the introduction of alkyl, acyl or sulfonyl moieties to the basic secondary amine moiety of moxifloxacin. Structures of the compounds were enlightened by FT-IR, ¹H NMR, ¹³C NMR and HRMS data besides elemental analysis. Compounds were initially tested in vitro for their anti-mycobacterial activity against Mycobacterium tuberculosis H37Rv using microplate alamar blue assay. Minimal inhibitory concentration (MIC) values of all compounds were found between > 25.00–0.39 μg/mL while compounds 1, 2 and 13 revealed an outstanding activity against M. tuberculosis H37Rv with MIC values of 0.39 μg/mL. Activities of compounds 1–21 against to a number of Gram-positive and Gram-negative bacteria and fast growing mycobacterium strain were also investigated by agar well diffusion and microdilution methods. According to antimicrobial activity results, compound 13 was found the most potent derivative with a IC₅₀ value of <1.23 μg/mL against Staphylococcus aureus and clinical strain of methicillin-resistant clinical strain of S. aureus.

Full text of DOI:10.1016/j.bioorg.2019.102965

Accepted Manuscript
Design, synthesis and molecular modeling studies on novel moxifloxacin de-
rivatives as potential antibacterial and antituberculosis agents
Aslı Türe, Necla Kulabaş, Serap İpek Dingiş, Kaan Birgül, Arif Bozdeveci,
Şengül Alpay Karaoğlu, Vagolu Siva Krishna, Dharmarajan Sriram, İlkay Kü
ç ükgüzel
PII:
S0045-2068(18)31299-9
https://doi.org/10.1016/j.bioorg.2019.102965
102965
DOI:
Article Number:
Reference:
YBIOO 102965
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
10 November 2018
27 April 2019
29 April 2019
Please cite this article as: A. Türe, N. Kulabaş, S. İpek Dingiş, K. Birgül, A. Bozdeveci, S. Alpay Karaoğlu, V. Siva
Krishna, D. Sriram, I. Kü ç ükgüzel, Design, synthesis and molecular modeling studies on novel moxifloxacin
derivatives as potential antibacterial and antituberculosis agents, Bioorganic Chemistry (2019), doi: https://doi.org/
10.1016/j.bioorg.2019.102965
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Design, synthesis and molecular modeling studies on novel moxifloxacin
¤
derivatives as potential antibacterial and antituberculosis agents
Aslı Türe a, Necla Kulabaş a, Serap İpek Dingiş a, Kaan Birgül a §, Arif Bozdeveci b,
Şengül Alpay Karaoğlu b, Vagolu Siva Krishna c, Dharmarajan Sriram c, İlkay Küçükgüzel a *
a
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Marmara University, Haydarpaşa 34668 İstanbul,
Turkey
b
c
Department of Biology, Recep Tayyip Erdoğan University, 53100 Rize, Turkey
Medicinal Chemistry Research Laboratory, Pharmacy group, Birla Institute of Technology and Science, 333031
Pilani, India
Abstract: Twenty-one novel alkyl/acyl/sulfonyl substituted fluoroquinolone derivatives were designed,
synthesized and evaluated for their anti-tuberculosis and antibacterial activity. The targeted compounds
were synthesized by the introduction of alkyl, acyl or sulfonyl moieties to the basic secondary amine moiety
of moxifloxacin. Structures of the compounds were enlightened by FT-IR, 1H-NMR, 13C-NMR and HRMS
data besides elemental analysis. Compounds were initially tested in vitro for their anti-mycobacterial
activity against Mycobacterium tuberculosis H37Rv using microplate alamar blue assay. Minimal
inhibitory concentration (MIC) values of all compounds were found between > 25.00 - 0.39 µg/mL while
compounds 1, 2 and 13 revealed an outstanding activity against M. tuberculosis H37Rv with MIC values
of 0.39 µg/mL. Activities of compounds 1-21 against to a number of Gram-positive and Gram-negative
bacteria and fast growing mycobacterium strain were also investigated by agar well diffusion and
microdilution methods. According to antimicrobial activity results, compound 13 was found the most potent
derivative with a IC50 value of <1.23 μg/mL against Staphylococcus aureus and clinical strain of
methicillin-resistant clinical strain of S. aureus.
Keywords: Fluoroquinolones; moxifloxacin; antibacterial activity; anti-tuberculosis activity; DNA
supercoiling assay; molecular docking; resistant strains.
* Corresponding author. E-mail address: ikucukguzel@marmara.edu.tr; Tel: +90 216 349 12 16; Fax: +90
216 345 29 52; ORCID no: 0000-0002-7188-1859.
§
Present address: Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Altınbaş University,
Bakırköy 34145 İstanbul, Turkey.
¤
This research was presented at the XXIV European Federation for Medicinal Chemistry International
Symposium on Medicinal Chemistry (Manchester, UK) on Aug 28 - Sep 1, 2016 and at the 5th International
Bahçeşehir University (BAU) Drug Design Congress (İstanbul, Turkey) on October 19-21, 2017.
Abbreviations: FQ, fluoroquinolone; TB, tuberculosis; WHO, World Health Organization; MDR/RR TB,
multi-drug resistant / rifampicin resistant tuberculosis; MABA, microplate alamar blue assay.
1. Introduction
Bacterial infections and antimicrobial agent resistance are seen as a public health problem
worldwide [1]. Fluoroquinolones (FQs) are broad-spectrum antibacterial agents that are used for
the treatment of various bacterial infections such as urinary tract infections, sexually transmitted
diseases, respiratory tract infections and etc. over the past three decades [2-5]. They are also
1

recommended as second line anti-tuberculosis agents by World Health Organization (WHO) [6].
Structures of certain FQs in clinical use are presented in Figure 1.
Tuberculosis (TB), caused by pathogen Mycobacterium tuberculosis, is an emerging global health
threat and the ninth leading cause of death worldwide. According to the report by WHO published
in 2017, TB is responsible for approximately 1.3 million deaths annually among HIV-negative
people and additional 374,000 deaths among HIV-positive people [7]. The standard treatment
regimen for TB includes a combination of rifampin, isoniazid, pyrazinamide and ethambutol.
Bedaquiline and delemanid have been approved by the authorities for the treatment of multidrug
resistant / rifampicin resistant tuberculosis (MDR/RR TB) [8]. MDR/RR TB is a growing problem
and affects thousands of people’s lives. Nearly 600,000 people were diagnosed with and 240,000
people died of MDR/RR TB in 2016 [7]. FQs show successful profile for drug-susceptible TB
treatment and drug-resistant TB treatment. Especially moxifloxacin and gatifloxacin, which are
fourth generation FQs, have shown promising anti-mycobacterial activity in clinical studies [9]. It
is important to note that phase IV clinical trials on moxifloxacin for TB treatment are currently
being conducted [10].
Figure 1. Chemical structures of the FQ derivatives.
FQs are known as DNA topoisomerase inhibitors. DNA topoisomerases can be divided into two
main types as topo I and topo II. Topoisomerase II enzymes are also classified as DNA gyrase and
topo IV. Especially topo II enzymes are attractive targets for the development of antibacterial
agents. FQs inhibit bacterial DNA gyrase enzymes which are closely related to topo IV [11, 12].
Although several crystal structures of DNA topoisomerases obtained from different
microorganisms were reported previously, the X-ray structure of M. tuberculosis DNA gyrase, co-
crystallized with DNA and several ligands (moxifloxacin, gatifloxacin, levofloxacin,
ciprofloxacin) has lately been published in 2016 by Blower et al (Figure 2) [13]. This breakthrough
development has opened the way to the discovery of new anti-tuberculosis agents which can
specifically inhibit M. tuberculosis DNA gyrase [14].
2

Modification of already known FQ derivatives is a commonly used approach in drug discovery.
Several reports highlight that increasing the lipophilicity of FQs can improve their activity [15-
17]. In the literature, there are miscellaneous examples of the synthesis and activity evaluation of
modified FQ derivatives [18-40]. Moreover, benzenesulfonyl-substituted FQs have recently been
reported as a new class of FQs [41]. In the light of the literature and our recent report [42] that
reveals the efficacy of modified FQ analogues, some novel FQ derivatives were designed and
synthesized. Possible mode of action of the synthesized compounds was studied through enzymatic
studies and docking calculations, as well.
2. Results and Discussion
2.1. Chemistry
Novel moxifloxacin-derived compounds were designed and synthesized in this study. The
synthetic strategy of the targeted compounds is described in the Scheme 1. To obtain compounds
1-7, moxifloxacin was reacted with several substituted benzyl chlorides in tetrahydrofuran
containing sodium hydroxide. For the synthesis of compounds 8-14, moxifloxacin was reacted
with the corresponding benzoyl chlorides in dimetylformamide in the presence of anhydrous
K2CO3. Compounds 15-21 were synthesized by the reaction of aryl sulfonyl chlorides with
moxifloxacin in acetonitrile containing triethylamine.
Scheme 1. Synthesis of the targeted derivatives. i. (Non) Substituted benzyl chloride, 5N NaOH, THF. ii.
(Non) Substituted benzoyl chloride, K2CO3, DMF. iii. (Non) Substituted benzenesulfonyl chloride, TEA,
ACN.
Purity of the synthesized compounds was checked by using TLC and HPLC. The structures of the
1
13
final compounds were confirmed by FTIR and H-NMR, C-NMR besides elemental analysis.
Molecular weights and empirical formulae of compounds 1-21 were also determined by high
resolution mass spectrometry (HRMS). According to ESI-HRMS findings, calculated and
measured m/z values were found within satisfactory range.
3

Well documented spectral data is presented in Supplementary Material.
2.2. Biological studies
2.2.1. Anti-mycobacterial activity studies
MIC calculation of compounds 1-21 against M. tuberculosis were conducted with microplate
alamar blue assay (MABA). Determination of cytotoxicity in RAW 264.7 cell culture and also M.
tuberculosis DNA gyrase supercoiling assay for the determination of anti-tuberculosis activity
were performed. Results are presented in Table 1.
According to the MABA results, introduction of alkyl moiety to moxifloxacin resulted in better
anti-mycobacterial activity compared to the introduction of acyl or sulfonyl moieties. Compounds
1-7 possessed of MIC values at 0.39 - 3.13 µg/mL, compounds 8-14 possessed of MIC values at
0.39 - 25.00 µg/mL and compounds 15-21 possessed of MIC values at 25.00 - >25.00 µg/mL. It
is obvious that introduction of sulfonyl group to secondary amine’s nitrogen does not enhance anti-
tuberculosis activity. Compounds with acyl group substitution show moderate anti-tuberculosis
activity.
Cytotoxicity of the compounds possessing MIC values less than 25.00 µg/mL against M.
tuberculosis was evaluated at 25.00 µg/mL concentration. Percentage viability of RAW 264.7 cells
which were exposed to 25.00 µg/mL concentrations of compounds were found between 85.20-
60.40%. Cytotoxicity results are presented in the Table 1. Percentage viability of RAW 264.7 cells
which were exposed to 25.00 µg/mL moxifloxacin was found 73.20%. Compared to moxifloxacin,
compounds 1, 2, 4-6, 9, 12, 14 were found as favorably less toxic to RAW 264.7 cell lines.
Related to the mycobacterial activity studies, compounds 1-3, 5-6 and 13 possessing MIC values
under 2.00 µg/mL were selected for M. tuberculosis supercoiling assay since they exhibited better
anti-tuberculosis activity. DNA gyrase supercoiling assay was also applied to the reference drug
moxifloxacin. Based on the supercoiling assay, IC50 values of compounds 1-3, 5-6 and 13 were
found between 4.60 - 12.52 µM whereas IC50 value of moxifloxacin was found 4.20 µM.
Compounds 1, 2 and 13 were identified as the most active moxifloxacin derivatives among the
synthesized compounds, by their MIC value of 0.39 µg/mL against M. tuberculosis. Of these
selected derivatives, IC50 value of compound 1 was 4.60±0.12 µM on M. tuberculosis DNA
supercoiling assay while viability percentage was found 74.24% on RAW 264.7 cells exposed to
this compound. Further to the inhibition value which was found less than 30.00%, it can be asserted
that compound 1 showed acceptable toxicity to RAW 264.7 cells. Besides, as seen in Table 1, it is
observed that compound 1 is less toxic than moxifloxacin and supercoiling assay result of
compound 1 reveals an IC50 value comparable to moxifloxacin (4.20±0.23 µM). In addition,
compounds 2 and 13 which possess the same MIC value with compound 1, reveal IC50 values of
6.10±0.32 and 8.69±0.56 µM on supercoiling assay, respectively.
4

Table 1. Anti-tuberculosis activity results of the compounds.
IC50 (µM)
MIC
(µg/mL)
MIC
(µM)
Cytotoxicity*
(viability%)
Compound
L
-R
GScore
Log P**
Supercoiling
assay
4.60±0.12
6.10±0.32
9.12±0.41
ND
6.12±0.18
12.52±0.68
ND
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
-CH2-
-CH2-
-CH2-
-CH2-
-CH2-
-CH2-
-CH2-
-CO-
-CO-
-CO-
-CO-
-CO-
-CO-
-CO-
-SO2-
-SO2-
-SO2-
-SO2-
-SO2-
-SO2-
-SO2-
-
H
Me
F
OMe
Cl
NO2
Br
H
Me
F
OMe
Cl
NO2
Br
H
0.39
0.39
1.56
3.13
1.56
0.79
0.77
3.06
6.00
2.97
74.24
79.24
64.60
81.05
81.04
75.44
72.17
ND
80.20
60.40
72.40
77.17
66.39
85.20
ND
ND
ND
ND
ND
ND
ND
73.30
-12.939
-12.847
-13.814
-13.177
-13.470
-11.874
-13.993
-12.465
-10.673
-11.703
-11.883
-11.666
-12.509
-12.000
-11.259
-8.979
3.42
3.88
3.47
3.33
4.01
3.15
4.19
1.93
2.39
2.15
2.45
2.69
1.89
2.87
2.92
3.38
3.24
3.44
3.78
3.12
3.96
1.60
1.56
3.13
2.91
5.49
25.00
12.50
12.50
12.50
6.25
49.45
24.06
23.88
23.34
11.57
0.71
21.39
> 46.16
> 44.99
44.68
> 43.73
> 43.40
> 42.62
40.29
0.44
ND
ND
ND
ND
ND
0.39
8.69±0.56
ND
12.50
> 25.00
> 25.00
25.00
> 25.00
> 25.00
> 25.00
25.00
0.18
ND
ND
ND
ND
ND
ND
ND
Me
F
OMe
Cl
NO2
Br
17
18
19
20
-12.830
-12.256
-10.942
-5.055
-12.964
-13.569
21
Moxifloxacin
-
4.20±0.23
*
Cytotoxicity of the compounds was tested at RAW 264.7 cell line at 25.0 µg/mL. Results were presented as viability% of RAW
264.7 cells.
** Calculations were performed using Molinspiration online property calculation toolkit (http://www.molinspiration.com).
In this study, Mycobacterium smegmatis which is a rapidly growing mycobacterium was
considered as a useful surrogate model for tuberculosis research. Compounds 1-21 were tested
against M. smegmatis with agar dilution and microdilution assays. It was noteworthy that, the
compounds which were found to be active against M. smegmatis, also possessed good inhibitory
properties on M. tuberculosis (Table 2 and Table 3).
Log P values of compounds were calculated theoretically and presented in Table 1. It should be
stated that no correlation was determined between Log P values and anti-tuberculosis activity of
the target compounds.
2.2.2. Antimicrobial activity studies
5

Antimicrobial activity of compounds 1-21 was first investigated by agar well diffusion method
against to Gram-positive / Gram-negative bacteria, a mycobacterial strain and two yeast-like fungi
(Table 2). MIC values of the compounds were investigated after the determination of the
quantitative values of the compounds' antibacterial activity (Table 3).
Compounds 1-7, in which alkyl moieties were substituted to moxifloxacin, showed significant
antibacterial activity against both Gram-positive and Gram-negative species. Compounds bearing
aroyl (8-14) or arylsulfonyl (15-21) moieties showed significant antibacterial activity against
Gram-positive bacteria. It can be asserted that substitution of arylalkyl moiety to moxifloxacin
results in broad-spectrum antibacterial activity, while substitution of either aroyl or arylsulfonyl
moieties provides selectivity towards Gram-positive bacteria.
Among the synthesized FQ derivatives, compound 13 was found to be the most potent one in
accordance with antibacterial activity results. It was shown to be active against both Gram-positive
and Gram-negative strains without possessing selectivity. In addition, compound 13 showed a MIC
value of <1.23 μg/mL against Staphylococcus aureus and MRSA. Antibacterial activity of
compound 13 against S. aureus was shown to be superior in comparison with the reference
compounds.
Synthesized compounds did not demonstrate substantive activity against P. aeruginosa.
Compound 4 revealed the best anti-pseudomonal activity with the MIC value of 85.16 μg/mL. It
was also determined that compounds 1-21 did not have inhibitory activity against yeast-like fungi
(Table 2). It should be mentioned that the designed compounds did not show significant
antibacterial activity against Lactobacillus acidophilus. L. acidophilus already presents in human
microbiota and it has an important role in fermentation, especially in the dairy industry [43]. Less
antibacterial activity of the compounds against L. acidophilus could be evaluated as a favorable
effect, as this species is not a pathogenic strain.
Synthesized compounds were tested against Bacillus cereus strain, which is a spore-producing
bacterium and one of the food-borne infectious agents. MIC values against B. cereus were found
between <1.23 and 18.00 μg/mL.
6

Table 2. Screening for antimicrobial activity of the compounds using agar well diffusion method.
Microorganisms and Inhibition Zone Values (mm)
Mycobacterial
strain
Compounds
Gram-Negative Bacteria
Gram-Positive Bacteria
Yeast-Like Fungi
Ec.
Yp.
Kp.
Pa.
Sa.
MRSA
Ef.
Lm.
La.
Bc.
Ms.
Ca.
Sc.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
22
22
22
22
20
22
20
15
12
12
12
12
26
12
15
16
18
12
15
18
20
32
35
35
33
16
12
20
20
18
18
17
10
6
9
6
9
21
6
12
6
16
-
14
8
15
30
32
35
30
15
15
20
21
18
20
18
6
6
8
6
6
20
6
6
6
15
6
9
-
8
17
9
6
-
-
-
-
-
-
12
-
8
-
8
-
8
25
24
28
28
28
30
24
32
28
30
28
25
30
25
24
20
24
21
20
20
26
28
30
35
35
22
19
24
25
22
24
20
24
20
24
22
20
20
18
20
16
21
18
18
18
22
30
30
30
35
19
16
22
25
19
22
19
24
20
22
22
18
22
18
20
15
19
15
14
15
18
30
30
32
35
20
16
20
18
17
18
16
20
20
16
20
15
15
16
14
12
14
14
10
15
16
25
27
30
30
12
13
12
15
-
16
6
8
10
-
-
6
15
-
-
-
-
-
-
-
-
8
18
17
19
20
18
19
16
22
20
20
21
18
22
17
18
15
16
15
15
15
16
28
28
30
30
25
35
35
35
30
35
35
25
25
25
20
25
30
20
14
12
20
10
15
10
35
>40
>40
>40
>40
7
6
-
-
-
6
-
-
7
-
7
-
-
-
-
-
-
-
-
-
-
-
6
7
-
10
6
-
-
-
-
-
-
-
6
-
6
-
6
-
6
-
-
-
18
19
20
21
14
6
-
18
30
32
35
30
12
35
38
40
35
-
-
-
-
NFX
CFX
CFX.HCl
MFX.HCl
22
26
41
-
-
NFX, Norfloxacin; CFX, Ciprofloxacin; CFX.HCl, Ciprofloxacin hydrochloride; MFX.HCl, Moxifloxacin hydrochloride; Ec., Escherichia coli ATCC 25922; Yp.,
Yersinia pseudotuberculosis ATCC 911; Kp., Klebsiella pneumoniae ATCC13883, Pa., Pseudomonas aeruginosa ATCC 43288; Sa. Staphylococcus aureus ATCC
25923; MRSA, Methicillin-resistant clinical strain of Staphylococcus aureus; Ef., Enterococcus faecalis ATCC 29212, Lm., Listeria monocytogenes ATCC43251;
La., Lactobacillus acidophilus RSKK 06029; Bc., Bacillus cereus Roma 709; Ms., Mycobacterium smegmatis ATCC607; Ca., Candida albicans ATCC 60193
and Sc., Saccharomyces cerevisiae RSKK 251, (-): no activity.
7

Table 3. Screening for antimicrobial activity of the compounds using microdilution method.
Microorganisms and Minimal Inhibitory Concentration (µg/mL)
Mycobacter
ial strain
Yeast-Like Fungi
Compounds
Gram-Negative Bacteria
Gram-Positive Bacteria
Ec.
Yp.
Kp.
Pa.
Sa.
MRSA
Ef.
Lm.
La.
Bc.
Ms.
Ca.
Sc.
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
2.71
2.64
2.69
2.66
9.57
43.36
84.38
10.74
5.32
38.28
19.92
41.41
153.13
331.25
325.00
337.50
165.63
9.86
318.75
165.63
321.88
81.25
-
84.38
328.13
39.84
0.60
43.36
42.19
10.74
5.32
38.28
19.92
41.41
306.25
331.25
325.00
337.50
331.25
9.86
346.88
<2.71 <2.71
<2.64 2.64
2.71
2.64
<2.69
<2.66
2.39
<2.49
<2.59
4.79
<2.59
<2.54
<2.64
<2.59
1.23
<2.49
<2.59
2.51
<2.54
2.42
10.55
2.56
2.49
2.71 138.75 18.00
2.64 67.50 2.64
2.69 137.50 2.69
5.32 170.31 <2.66
25.00
2.64
2.69
2.66
4.79
2.49
2.59
9.57
10.35
10.16
10.55
5.18
1.23
4.98
20.70
20.12
20.31
9.67
42.19
20.51
4.98
346.88
337.50
-
-
-
343.75
85.16
306.25
318.75
<2.69 <2.69
<2.66 <2.66
<2.39 <2.39
<2.49 <2.49
<2.59 <2.59
-
-
-
343.75
340.63
2.39
2.49
-
2.39
2.49
-
4.98
79.69
318.75
127.50
331.25
10.35
38.28
82.81
81.25
89.38
82.81
1.23
79.69
82.81
80.47
40.63
154.69
84.38
82.03
19.92
<0.60
<0.60
<0.60
<0.60
-
-
-
-
-
-
2.59 263.00 2.59
4.79 245.00 4.79
<2.59 132.00 <2.59
-
-
4.79
4.79
-
-
-
-
-
-
-
<2.59 <2.59
10.16 <2.54
<2.64 <2.64
<2.59 <2.59
<1.23 <1.23
<2.49 <2.49
<2.59 <2.59
<2.51 2.516
<2.54 <2.54
<2.42 <2.42
<2.64 <2.64
331.25
2.54
<2,64
-
-
2.54
<2.64
-
337.50
<2.59 256.00 <2.59
1.23 157.81 <1.23
-
-
-
-
-
-
-
-
-
-
-
-
-
-
315.63
-
331.25
-
325.00
-
337.50
-
159.38
5.27
1.31
1.26
5.08
318.75
265
2.49
5.18
5.03
2.54
2.42
21.09
5.56
2.49
-
-
-
-
2.49
2.59
2.51
2.54
2.42
5.27
2.56
2.49
331.25
321.88
81.25
309.38
84.38
328.13
19.92
1.32
0.60
<0.60
<0.60
-
325.00
18
19
20
21
-
-
-
-
-
337.50
<2.56
2.56
-
<2.49 <2.49
318.75
NFX
CFX
CFX.HCl
MFX.HCl
1.32
1.31
2.51
1.27
0.60
1.31
1.26
2.64
2.61
1.26
<0.60
2.64 675.00 2.64
5.27
-
-
-
-
0.60
<0.60
<0.60
1.31
1.26
83.59
80.47
1.31
0.60
<0.60
<0.60
<0.60
<0.60
<0.60 40.63 <0.60
NFX, Norfloxacin; CFX, Ciprofloxacin; CFX.HCl, Ciprofloxacin hydrochloride; MFX.HCl, Moxifloxacin hydrochloride; Ec., Escherichia coli ATCC 25922; Yp.,
Yersinia pseudotuberculosis ATCC 911; Kp., Klebsiella pneumoniae ATCC13883, Pa., Pseudomonas aeruginosa ATCC 43288; Sa. Staphylococcus aureus ATCC
25923; MRSA, Methicillin-resistant clinical strain of Staphylococcus aureus; Ef., Enterococcus faecalis ATCC 29212, Lm., Listeria monocytogenes ATCC43251;
La., Lactobacillus acidophilus RSKK 06029; Bc., Bacillus cereus Roma 709; Ms., Mycobacterium smegmatis ATCC607; (-): no activity.
8

B
A
C
Figure 2. A. Crystal structure of M. tuberculosis DNA gyrase in complex with moxifloxacin (PDB ID: 5BTA). B. Moxifloxacin in the binding site
of M. tuberculosis DNA gyrase. C. Interactions between moxifloxacin and M. tuberculosis DNA gyrase.
9

2.3. In silico docking studies
FQs are known to inhibit bacterial topoisomerase II enzymes [11,12]. The X-ray structure of M.
tuberculosis DNA gyrase co-crystallized with DNA and moxifloxacin was published in 2016 [13].
To gain more molecular insight into the activity of the compounds, docking calculations were
carried out. Docking studies with M. tuberculosis DNA gyrase in addition to S. aureus DNA gyrase
were conducted to estimate the possible inhibitory activities of the designed compounds. Ligands
were prepared as (1S,6S)-configured stereoisomers of 7-[2,8-diazabicyclo[4.3.0]nonan-8-yl
moiety since 1S,6S configuration is present in the 3D structure of the starting compound
moxifloxacin. DNA, Mg2+ and conserved water molecules were kept during the calculations.
GScores which were obtained from the docking studies of the compounds 1-21 and moxifloxacin
with M. tuberculosis DNA gyrase and S. aureus DNA gyrase by using Schrödinger Glide-SP
Flexible Docking are presented in Supplementary Material.
As a preliminary SAR finding, it can be said that compounds 1-7, which showed better anti-
tuberculosis activity compared to other series 8-21, demonstrated higher GScores. There was no
similar correlation with S. aureus, as most of the synthesized compounds were active against this
strain regardless of their GScores.
According to MIC values and supercoiling assay results, compound 1 was demonstrated as the
most active compound against M. tuberculosis. Figure 3 shows the model conformation of
compound 1 in the binding site of M. tuberculosis DNA gyrase.
Figure 3. Compound 1 in M. tuberculosis DNA gyrase binding site. Amino acids which can be mutated or
which interact with the ligand are shown in the figure. Hydrogen bonds are depicted as blue dashes.
Attractive charge and salt bridge interactions are shown as orange dashes. Metal acceptor interactions are
shown as gray dashes.
10

Owing to the docking studies, carboxylate group of compound 1 and Arg128 make salt bridge
interaction. Carboxylate group also makes attractive charge interaction with Mg2+. As a metal
acceptor, oxygen atom of quinolone ring interacts with Mg2+ as well. Hydrogen bonds between
the two of the conserved water molecules and compound 1 were detected. Van der Waals contacts
between Ser90 and compound 1 contribute to the binding. Conventional H bond, alkyl, pi-pi
stacking and halogen interactions between compound 1 and DNA helix were observed
additionally.
Figure 4. Compound 13 in S. aureus DNA gyrase binding site. Amino acids which can be mutated or which
interact with the ligand are presented. Hydrogen bonds are depicted as blue dashes. Attractive charge and
salt bridge interactions are shown as orange dashes. Metal acceptor interactions are shown as gray dashes.
Asn499Lys, Thr500Asn and Glu501Val mutations are found in quinolone-resistance determining
region [13]. In addition to docking studies with wild-type M. tuberculosis DNA Gyrase; whether
compounds make significant interaction with any of these amino acid residues, docking was
11

performed with mutated M. tuberculosis DNA Gyrase as well. Mutant form of M. tuberculosis
DNA Gyrase was prepared by using MOE (Chemical Computing Group) rotamer explorer.
However, no special interaction between mutated amino acid residues and compounds was
observed through the docking studies of synthesized compounds into mutated proteins.
Compound 13 was shown as the most active compound against S. aureus. Figure 4 represents
possible binding conformation of compound 13 in S. aureus DNA gyrase binding site. Arg122
interacts with carboxylate group of compound 13. Carboxylate group also interacts with Mg2+.
Oxygen atom of quinolone ring makes a metal acceptor interaction with Mg+2 ion as well. Oxygen
atom of quinolone ring and carboxylate group make hydrogen bond interactions with conserved
water molecules. Van der Waals contacts were observed between Lys417, Asn476 and Glu477.
Carbon-hydrogen bond interaction was detected between methoxy group of compound 13 and
Gly459. Contribution of alkyl, pi-alkyl and van der Waals contacts were observed additionally.
3. Conclusions
Novel FQ derivatives were designed as potential anti-tuberculosis and antibacterial agents.
Targeted compounds were synthesized by the introduction of arylalkyl, aroyl or arylsulfonyl
moieties to basic secondary amine group of moxifloxacin.
Anti-tuberculosis activity of the designed compounds 1-21 was evaluated in vitro. MABA method
was used to determine the MIC values of the synthesized compounds against M. tuberculosis
H37Rv. MIC values of all compounds were found between > 25 and 0.39 µg/mL. Compounds 1,
2 and 13 expressed an outstanding activity against M. tuberculosis H37Rv with the MIC value of
0.39 µg/mL. Cytotoxicity of the compounds with the MIC values which were found less than 25.00
µg/mL against M. tuberculosis was tested. Viability% of RAW 264.7 cell lines were found
between 85.20-60.40% whereas some of the designed compounds were less toxic than
moxifloxacin to RAW 264.7 cells. DNA gyrase supercoiling assay was applied to compounds 1-
3, 5-6 and 13 as well as reference drug moxifloxacin and IC50 values of these selected compounds
were found between 4.60-12.52 µM.
Antibacterial activity of the designed compounds against Gram-positive, Gram-negative and
acidoresistant bacterial strains and yeast-like fungi was evaluated with zone value measurement
and microdilution assays. Compounds 1-7 were found to be active against both Gram-positive and
Gram-negative strains. Compounds 8-21 were found to be selectively active against Gram-positive
bacteria. Remarkably, compound 13 showed a MIC value of <1.23 μg/mL against S. aureus and
MRSA. Thus, this compound can be considered as a useful antibacterial agent against antibiotic
resistant S. aureus. Noteworthy, compounds which were active against M. smegmatis also showed
good activities towards M. tuberculosis.
4. Experimental Protocols
12

4.1. Chemistry
Apart from moxifloxacin.HCl (CAS number: 186826-86-8), all starting materials, reagents and
solvents were of high-grade commercial products purchased from Aldrich or Merck. Purity
determination of the compounds were performed by TLC on Merck silica gel 60 F254 plates. For
the purification of benzoyl substituted FQs 8-14, silica gel column chromatography was utilized
using dichloromethane : methanol (90:10, v/v) as eluent and Merck silica gel 60 F254 (0.063-0.200
mm) as adsorbent. Melting points (°C, uncorrected) were determined using
Schmelzpunktbestimmer SMP II basic model melting point apparatus. Elemental analysis was
performed by using LECO CHNS-932 instrument and the results were found to be in consistent
with the assigned structures. Infrared spectra were recorded on a Shimadzu FTIR 8400S and data
are expressed in wavenumbers, ν (cm-1). 1H- and 13C-NMR (decoupled) spectra were recorded on
Brüker AVANCE-DPX 400 at 300 MHz, the chemical shifts were expressed in δ (ppm) downfield
from tetramethylsilane (TMS) using DMSO-d6 or CDCl3 as solvent. High resolution mass spectra
were acquired by using Jeol JMS700 instrument.
4.1.1. General procedure for the synthesis of compounds 1-7
Moxifloxacin.HCl (1.50 mmol) was stirred in tetrahydrofuran (30 mL) in the presence of aq. 5N
NaOH (0.6 mL). Appropriate benzyl chloride (2.25 mmol) was added to the medium gradually
afterwards. Reaction was maintained at 50 °C for 24 h. When moxifloxacin spot disappeared from
TLC sheet, reaction medium was treated with ice-cold water. Solution was neutralized with 10%
acetic acid. The precipitate was then filtered and washed with water. Crude product was
recrystallized from EtOH or acetone.
4.1.1.1. 6-Fluoro-8-methoxy-4-oxo-1-cyclopropyl-7-(2-benzyl-2,8-diazabicyclo[4.3.0]nonan-8-
yl)-1,4-dihydroquinoline-3-carboxylic acid 1
(CAS Number: 2254156-04-0) M.p. 150 °C. Elemental analysis calculated for C28H30FN3O4; C
68.42, H 6.15, N 8.55; found C 68.12, H 5.67, N 8.51. IR (cm-1): 1732, 1622 (C=O). 1H-NMR 
ppm (300 MHz, CDCl3): 15.11 (s, 1H, COOH), 8.80 (s, 1H, C2-H), 7.93 (m, 1H, Ar-H'), 7.83 (d,
1H, C5-H, J = 13.8 Hz), 7.62-7.29 (m, 3H, Ar-H'), 7.32-3-7.28 (m, 1H), 3.59 (s, 3H, OCH3),
4.08-0.82 (aliphatic C-H). HRMS (ESI+) m/z calculated for C28H30FN3O4 [M+Na]+, 514.2118,
found 514.2124.
4.1.1.2. 6-Fluoro-8-methoxy-4-oxo-1-cyclopropyl-7-(2-(4-methylphenyl)methyl-2,8-
diazabicyclo[4.3.0]nonan-8-yl)-1,4-dihydroquinoline-3-carboxylic acid 2
M.p. 138 °C. Elemental analysis calculated for C29H32FN3O4; C 68.89, H 6.38, N 8.31; found C
69.23, H 6.07, N 8.06. IR (cm-1): 1722, 1620 (C=O). 1H-NMR  ppm (300 MHz, CDCl3): 15.14
(s, 1H, COOH), 8.77 (s, 1H, C2-H), 7.80 (d, 1H, C5-H, J = 14.1 Hz), 7.14 (d, 2H, Ar-H2'6', J = 7.8
Hz), 7.05 (d, 2H, Ar-H3'5', J = 7.8 Hz), 3.59 (s, 3H, OCH3), 4.03-0.87 (aliphatic C-H). 13C-NMR
δ ppm (75 MHz, CDCl3): 176.69 (quinolone C4=O), 167.19 (-COOH), 153.68 (C6, J = 249.8 Hz),
149.44, 140.52, 140.42, 137.63 (quinolone C7, J = 11.3 Hz), 136.50, 135.93, 134.48, 128.88,
13

128.40, 117.88, 107.48, 107.55, 107.86, 62.20, 60.86, 59.67, 53.76, 53.27, 50.39, 40.48, 37.62,
23.78, 22.35, 21.03 (CH3), 9.86, 9.09. HRMS (ESI+) m/z calculated for C29H31FN3O4 [M+Na]+,
528.2280, found 528.2275.
4.1.1.3. 6-Fluoro-8-methoxy-4-oxo-1-cyclopropyl-7-(2-(4-fluorophenyl)methyl-2,8-
diazabicyclo[4.3.0]nonan-8-yl)-1,4-dihydroquinoline-3-carboxylic acid 3
M.p. 169 °C. Elemental analysis calculated for C28H29F2N3O4; C 66.00, H 5.74, N 8.25; found C
65.81, H 5.64, N 8.12. IR (cm-1): 1716, 1624 (C=O). 1H-NMR  ppm (300 MHz, CDCl3): 15.11
(s, 1H, COOH), 8.77 (s, 1H, C2-H), 7.80 (d, 1H, C5-H, J = 14.1 Hz), 7.24-7.20 (m, 2H, Ar-H2'6'),
6.97-6.91 (m, 2H, Ar-H3'5'), 3.59 (s, 3H, OCH3), 4.00-0.88 (aliphatic C-H). HRMS (ESI+) m/z
calculated for C28H29F2N3O4 [M+H]+, 510.2199, found 510.2199.
4.1.1.4. 6-Fluoro-8-methoxy-4-oxo-1-cyclopropyl-7-(2-(4-methoxyphenyl)methyl-2,8-
diazabicyclo[4.3.0]nonan-8-yl)-1,4-dihydroquinoline-3-carboxylic acid 4
M.p. 164 °C. Elemental analysis calculated for C29H32FN3O5; C 66.78, H 6.18, N 8.06; found C
66.38, H 6.06, N 8.00. IR (cm-1): 1620 (C=O). 1H-NMR  ppm (300 MHz, CDCl3): 15.13 (s, 1H,
COOH), 8.77 (s, 1H, C2-H), 7.80 (d, 1H, C5-H, J = 13.8 Hz), 7.16 (d, 2H, Ar-H2'6', J = 8.7 Hz),
6.78 (d, 2H, Ar-H3'5', J = 8.7 Hz), 3.75 (s, 3H, Ar-OCH3), 3.59 (s, 3H, OCH3), 4.03-0.91 (aliphatic
C-H). 13C-NMR δ ppm (75 MHz, CDCl3): 176.71, 167.19 (-COOH), 158.60 (phenyl C4), 153.69
(quinolone C6, J = 249.0 Hz), 149.45, 140.51, 140.42, 137.63 (quinolone C7, J = 10.5 Hz), 134.49,
130.92, 129.57, 117.90, 113.57, 107.87, 107.55, 107.48, 62.12, 60.86, 59.34, 55.20, 53.83, 53.15,
50.21, 40.49, 37.58, 23.82, 22.38, 9.11, 9.83. HRMS (ESI+) m/z calculated for C29H32FN3O5
[M+H]+, 522.2399, found 522.2400.
4.1.1.5. 6-Fluoro-8-methoxy-4-oxo-1-cyclopropyl-7-(2-(4-chlorophenyl)methyl-2,8-
diazabicyclo[4.3.0]nonan-8-yl)-1,4-dihydroquinoline-3-carboxylic acid 5
M.p. 119 °C. Elemental analysis calculated for C28H29ClFN3O4; C 63.94, H 5.56, N 7.99; found C
63.18, H 5.62, N 7.90. IR (cm-1): 1726, 1622 (C=O). 1H-NMR  ppm (300 MHz, CDCl3): 15.11
(s, 1H, COOH), 8.77 (s, 1H, C2-H), 7.82 (d, 1H, C5-H, J = 14.1 Hz), 7.26-7.20 (m, 4H, Ar-H'),
3.57 (s, 3H, OCH3), 4.00-0.87 (aliphatic C-H). HRMS (ESI+) m/z calculated for C28H29ClFN3O4
[M+H]+, 526.1903, found 526.1903.
4.1.1.6. 6-Fluoro-8-methoxy-4-oxo-1-cyclopropyl-7-(2-(4-nitrophenyl)methyl-2,8-
diazabicyclo[4.3.0]nonan-8-yl)-1,4-dihydroquinoline-3-carboxylic acid 6
M.p. 115 °C. Elemental analysis calculated for C28H29FN4O4; C 62.68, H 5.45, N 10.44; found C
1
62.41, H 5.35, N 10.16. IR (cm-1): 1726, 1620 (C=O). H-NMR  ppm (300 MHz, DMSO-d6):
15.00 (s, 1H, COOH), 8.76 (s, 1H, C2-H), 8.25-7.54 (m, 5H, Ar-H', C5-H), 3.62 (s, 3H, OCH3),
4.19-1.00 (aliphatic C-H). HRMS (ESI+) m/z calculated for C28H29FN4O6 [M+Na]+, 559.1968,
found 559.1977.
4.1.1.7. 6-Fluoro-8-methoxy-4-oxo-1-cyclopropyl-7-(2-(4-bromophenyl)methyl-2,8-
diazabicyclo[4.3.0]nonan-8-yl)-1,4-dihydroquinoline-3-carboxylic acid 7
14

M.p. 118 °C. Elemental analysis calculated for C28H29BrFN3O4; C 58.95, H 5.12, N 7.37; found C
59.04, H 5.35, N 6.87. IR (cm-1): 1726, 1620 (C=O). 1H-NMR  ppm (300 MHz, DMSO-d6): 15.22
(s, 1H, COOH), 8.77 (s, 1H, C2-H), 7.81 (d, 1H, C5-H, J = 14.7 Hz), 7.33 (d, 2H, Ar-H2'6', J = 8.4
Hz), 7.13 (d, 2H, Ar-H3'5', J = 8.4 Hz), 3.58 (s, 3H, OCH3), 4.13-0.85 (aliphatic C-H). HRMS
(ESI+) m/z calculated for C28H29BrN3O4 [M+Na]+, 592.1231, found 592.1231.
4.1.2. General procedure for the synthesis of compounds 8-14
To a solution of moxifloxacin.HCl (1.50 mmol) in DMF (30 mL), K2CO3 (3.00 mmol) was added.
The mixture was allowed to stir at 60 °C for 2 h. Related benzoyl chloride (2.25 mmol) was then
added to the medium dropwise. When the reaction was complete, the mixture was poured into ice-
water. The precipitate was filtered and the resultant raw material was purified by column
chromatography using DCM : MeOH (90:10, v/v) as mobile phase.
4.1.2.1. 6-Fluoro-8-methoxy-4-oxo-1-cyclopropyl-7-(2-(phenyl)carbonyl-2,8-
diazabicyclo[4.3.0]nonan-8-yl)-1,4-dihydroquinoline-3-carboxylic acid 8
M.p. 162 °C. Elemental analysis calculated for C28H28FN3O5.3/2 H2O; C 63.15, H 5.87, N 7.89;
found C 63.63, H 5.55, N 7.85. IR (cm-1): 3536 (O-H); 1732, 1618 (C=O). 1H-NMR  ppm (300
MHz, DMSO-d6): 15.14 (s, 1H, COOH), 8.66 (s, 1H, C2-H), 7.66 (d, 1H, C5-H, J = 13.8 Hz),
7.73-7.45 (m, 5H, Ar-H'), 3.58 (s, 3H, OCH3), 4.16-0.87 (aliphatic C-H). HRMS (ESI+) m/z
calculated for C28H28FN3O5 [M+Na]+, 528.1911, found 528.1920.
4.1.2.2. 6-Fluoro-8-methoxy-4-oxo-1-cyclopropyl-7-(2-(4-methylphenyl)carbonyl-2,8-
diazabicyclo[4.3.0]nonan-8-yl)-1,4-dihydroquinoline-3-carboxylic acid 9
M.p. 148 °C, Elemental analysis calculated for C29H30FN3O5; C 67.04, H 5.82, N 8.09; found C
66.63, H 6.04, N 8.11. IR (cm-1): 1724, 1616 (C=O). 1H-NMR  ppm (300 MHz, CDCl3): 14.97
(s, 1H, COOH), 8.79 (s, 1H, C2-H), 7.81 (d, 1H, C5-H, J = 14.1 Hz), 7.37-7.22 (m, 4H, Ar-H'),
3.59 (s, 3H, OCH3), 2.39 (s, 3H, Ar-CH3), 4.05-0.84 (aliphatic C-H). 13C-NMR δ ppm (75 MHz,
CDCl3): 176.75 (quinolone C4=O), 171.95 (amide C=O), 166.94 (-COOH), 153.69, (quinolone
C6, J = 249.0 Hz), 149.73, 141.15, 140.06, 140.02, 137.10 (quinolone C7, J = 10.5 Hz), 134.39,
133.11, 129.22, 126.85, 118.90, 108.19, 107.87, 107.70, 61.22, 56.56, 56.45, 48.56, 40.41, 35.80,
25.59, 24.58, 21.40, 10.54, 8.53. HRMS (ESI+) m/z calculated for C28H30FN3O5 [M+Na]+,
542.2067, found 542.2072.
4.1.2.3. 6-Fluoro-8-methoxy-4-oxo-1-cyclopropyl-7-(2-(4-fluorophenyl)carbonyl-2,8-
diazabicyclo[4.3.0]nonan-8-yl)-1,4-dihydroquinoline-3-carboxylic acid 10
M.p. 115 °C. Elemental analysis calculated for C28H27F2N3O5.1/3 H2O; C 63.51, H 5.27, N 7.94;
found C 63.05, H 5.38, N 7.90. IR (cm-1): 1716, 1620 (C=O). 1H-NMR  ppm (300 MHz, CDCl3):
14.96 (s, 1H, COOH), 8.80 (s, 1H, C2-H), 7.83 (d, 1H, C5-H, J = 13.8 Hz), 7.48-7.10 (m, 4H, Ar-
H'), 3.60 (s, 3H, OCH3), 4.10-0.86 (aliphatic C-H). HRMS (ESI+) m/z calculated for
C28H27F2N3O5 [M+Na]+, 546.1816, found 546.1822.
15

4.1.2.4. 6-Fluoro-8-methoxy-4-oxo-1-cyclopropyl-7-(2-(4-methoxyphenyl)carbonyl-2,8-
diazabicyclo[4.3.0]nonan-8-yl)-1,4-dihydroquinoline-3-carboxylic acid 11
M.p. 187 °C. Elemental analysis calculated for C29H32FN3O5; C 65.04, H 5.65, N 7.85; found C
1
64.68, H 5.37, N 7.85. IR (cm-1): 3473 (O-H); 1724, 1614 (C=O). H-NMR  ppm (300 MHz,
CDCl3): 14.98 (s, 1H, COOH), 8.79 (s, 1H, C2-H), 7.81 (d, 1H, C5-H, J = 14.1 Hz), 7.41 (d, 2H,
Ar-H3'5', J = 8.4 Hz), 6.93 (d, 2H, Ar-H2'6', J = 8.7 Hz), 3.84 (s, 3H, Ar-OCH3), 3.59 (s, 3H,
OCH3), 4.09-0.78 (aliphatic C-H). 13C-NMR δ ppm (75 MHz, CDCl3): 176.77 (quinolone C4=O),
171.73 (amide C=O), 166.94 (-COOH), 160.88 (phenyl C4), 153.71, (quinolone C6, J = 249.8
Hz), 149.73, 141.17, 141.08, 137.10 (quinolone C7, J = 11.3 Hz), 137.02, 134.40, 128.86, 128.11,
119.96, 113.87, 108.24, 107.92, 107.75, 61.22, 56.57, 56.47, 55.38, 48.54, 40.39, 35.82, 25.62,
24.62, 10.53, 8.53. HRMS (ESI+) m/z calculated for C29H31FN3O6 [M+Na]+, 536.2191, found
536.2199.
4.1.2.5. 6-Fluoro-8-methoxy-4-oxo-1-cyclopropyl-7-(2-(4-chlorophenyl)carbonyl-2,8-
diazabicyclo[4.3.0]nonan-8-yl)-1,4-dihydroquinoline-3-carboxylic acid 12
M.p. 170 °C. Elemental analysis calculated for C28H27ClFN3O5.1/2H2O; C 61.26, H 5.14, N 7.65;
found C 61.07, H 5.05, N 7.50. IR (cm-1): 3482 (O-H); 1732, 1614 (C=O). 1H-NMR  ppm (300
MHz, CDCl3): 14.95 (s, 1H, COOH), 8.79 (s, 1H, C2-H), 7.81 (d, 1H, C5-H, J = 14.1 Hz), 7.44-
7.35 (m, 4H, ArH'), 3.59 (s, 3H, OCH3), 4.08-0.78 (aliphatic C-H). HRMS (ESI+) m/z calculated
for C28H28ClFN3O5 [M+Na]+, 562.1521, found 562.1517.
4.1.2.6. 6-Fluoro-8-methoxy-4-oxo-1-cyclopropyl-7-(2-(4-nitrophenyl)carbonyl-2,8-
diazabicyclo[4.3.0]nonan-8-yl)-1,4-dihydroquinoline-3-carboxylic acid 13
M.p. 138-142 °C. Elemental analysis calculated for C28H27FN4O5; C 61.09, H 4.94, N 10.18; found
C 61.18, H 5.46, N 10.12. IR (cm-1): 3587 (OH), 1723, 1618 (C=O). 1H-NMR  ppm (300 MHz,
CDCl3): 15.04 (s, 1H, COOH), 8.77 (s, 1H, C2-H), 8.12 (d, 2H, Ar-H3'5', J = 8.7 Hz), 7.80 (d, 1H,
C5-H, J = 14.1 Hz), 7.46 (d, 2H, Ar-H2'6', J = 9.0 Hz), 3.60 (s, 3H, OCH3), 4.04-0.86 (aliphatic
C-H). HRMS (ESI+) m/z calculated for C28H27FN4O7 [M-CH3+H]+, 537.1780, found 537.2154.
4.1.2.7. 6-Fluoro-8-methoxy-4-oxo-1-cyclopropyl-7-(2-(4-bromophenyl)carbonyl-2,8-
diazabicyclo[4.3.0]nonan-8-yl)-1,4-dihydroquinoline-3-carboxylic acid 14
M.p. 160-162 °C. Elemental analysis calculated for C28H27FBrN3O5.H2O; C 55.82, H 4.85, N 6.97;
found C 56.09, H 4.44, N 6.98. IR (cm-1): 1726, 1620 (C=O). 1H-NMR  ppm (300 MHz, CDCl3):
15.13 (s, 1H, COOH), 8.76 (s, 1H, C2-H), 7.79 (d, 1H, C5-H, J = 14.1 Hz), 7.24-7.18 (m, 4H, Ar-
H'), 3.59 (s, 3H, OCH3), 4.03-0.86 (aliphatic C-H). HRMS (ESI+) m/z calculated for
C28H27F2N3O5 [M+Na]+, 584.1191, found 584.1205.
4.1.3. General procedure for the synthesis of compounds 15-21
To moxifloxacin.HCl (1.50 mmol) and triethlyamine (2.25 mmol) solution in acetonitrile (50 mL),
related arylsulfonyl chloride (2.25 mmol) was added. The reaction medium was maintained at
16

room temperature for 4 h. At the end of reaction, ACN was evaporated and the resultant precipitate
was washed with water. Final crude product was recrystallized from EtOH.
4.1.3.1. 6-Fluoro-8-methoxy-4-oxo-1-cyclopropyl-7-(2-phenylsulfonyl-2,8-
diazabicyclo[4.3.0]nonan-8-yl)-1,4-dihydroquinoline-3-carboxylic acid 15
M.p. 165-168 °C. Elemental analysis calculated for C27H28FN3O6S.H2O; C 57.95, H 5.40, N 7.51,
S 5.73; found C 57.70, H 5.55, N 7.45, S 5.76. IR (cm-1): 1722, 1616 (C=O). 1H-NMR  ppm (300
MHz, CDCl3): 8.80 (s, 1H, C2-H), 7.87-7.33 (m, 6H, Ar-H', C5-H), 3.52 (s, 3H, OCH3), 4.67-0.78
(aliphatic C-H). HRMS (ESI+) m/z calculated for C27H28FN3O6S [M+Na]+, 564.1575, found
564.1581.
4.1.3.2. 6-Fluoro-8-methoxy-4-oxo-1-cyclopropyl-7-(2-(4-methylphenyl)sulfonyl-2,8-
diazabicyclo[4.3.0]nonan-8-yl)-1,4-dihydroquinoline-3-carboxylic acid 16
(CAS number: 1253700-48-9) M.p. 209 °C. Elemental analysis calculated for C27H28FN3O6S; C
60.53, H 5.44, N 7.56, S 5.77; found C 60.49, H 5.52, N 7.75, S 5.72. IR (cm-1): 1737, 1622 (C=O);
1155 (S=O). 1H-NMR  ppm (300 MHz, DMSO-d6): 15.14 (s, 1H, COOH), 8.64 (s, 1H, C2-H),
7.76 (d, 2H, Ar-H2'6', J = 8.1 Hz), 7.65 (d, 1H, C5-H, J = 13.8 Hz), 7.43 (d, 2H, Ar- Ar-H3'5', J =
8.1 Hz), 3.49 (s, 3H, Ar-OCH3), 2,39 (s, 3H, Ar-CH3). 13C-NMR δ ppm (75 MHz, CDCl3): 176.74
(quinolone C4=O), 166.89 (-COOH), 153.70 (quinolone C6, J = 249.0 Hz), 149.75, 143.31,
141.23, 141.14, 136.98, 136.89 (quinolone C7, J = 11.3 Hz), 134.30, 129.83, 127.08, 119.02,
108.15, 107.83, 107.73, 61.20 (-OCH3), 55.75, 53.97, 47.95, 40.93, 40.34, 35.55, 24.68, 23.66,
21.50 (-CH3), 10.47, 8.57. HRMS (ESI+) m/z calculated for C28H30FN3O6S [M+H]+, 556.1912,
found 556.1936 [44].
4.1.3.3. 6-Fluoro-8-methoxy-4-oxo-1-cyclopropyl-7-(2-(4-fluorophenyl)sulfonyl-2,8-
diazabicyclo[4.3.0]nonan-8-yl)-1,4-dihydroquinoline-3-carboxylic acid 17
M.p. 163 °C. Elemental analysis calculated for C27H27F2N3O6S; C 57.95, H 4.86, N 7.51, S 5.73;
found C 57.22, H 4.92, N 7.67, S 5.50. IR (cm-1): 1726, 1620 (C=O); 1153 (S=O). 1H-NMR  ppm
(300 MHz, CDCl3): 14.92 (s, 1H, COOH), 8.78 (s, 1H, C2-H), 7.90-7.85 (m, 2H, Ar-H2'6'), 7.78
(d, 1H, C5-H, J = 13.8 Hz), 7.25-7.19 (m, 2H, Ar-H3'5'), 3.54 (s, 3H, OCH3), 4.65-0.78 (aliphatic
C-H). HRMS (ESI+) m/z calculated for C27H28F2N3O6S [M+H]+, 560.1661, found 560.1661.
4.1.3.4. 6-Fluoro-8-methoxy-4-oxo-1-cyclopropyl-7-(2-(4-methoxyphenyl)sulfonyl-2,8-
diazabicyclo[4.3.0]nonan-8-yl)-1,4-dihydroquinoline-3-carboxylic acid 18
M.p. 206 °C. Elemental analysis calculated for C28H30FN3O7S.H2O; C 57.04, H 5.47, N 7.13, S
5.44; found C 56.96, H 5.28, N 7.13, S 5.24. IR (cm-1): 1726, 1620 (C=O); 1147 (S=O). 1H-NMR
 ppm (300 MHz, CDCl3): 14.94 (s, 1H, COOH), 8.78 (s, 1H, C2-H), 7.80-7.76 (m, 3H, Ar-H2'6',
C5-H), 6.99 (d, 2H, Ar-H3'5', J = 9.0 Hz), 3.90 (s, 3H, Ar-OCH3), 3.53 (s, 3H, OCH3), 4.63-0.79
(alifatik C-H). 13C-NMR δ ppm (75 MHz, CDCl3): 176.73 (quinolone C4=O), 166.89 (-COOH),
163.00 (phenyl C4), 153.67 (quinolone C6, J = 249.8 Hz) 149.75, 141.21, 141.12, 136.93
(quinolone C7, J = 10.5 Hz), 134.31, 131.41, 129.17, 118.98, 114.36, 108.13, 107.81, 107.60,
17

61.19, 55.78, 55.63, 53.93, 47.91, 40.89, 40.35, 35.63, 24.23, 23.69, 10.45, 8.55. HRMS (ESI+)
m/z calculated for C28H30FN3O7S [M+Na]+, 594.1686, found 594.1685.
4.1.3.5. 6-Fluoro-8-methoxy-4-oxo-1-cyclopropyl-7-(2-(4-chlorophenyl)sulfonyl-2,8-
diazabicyclo[4.3.0]nonan-8-yl)-1,4-dihydroquinoline-3-carboxylic acid 19
M.p. 206 °C. Elemental analysis calculated for C27H28FN3O6S; C 56.30, H 4.72, N 7.29, S 5.57;
found C 56.08, H 4.72, N 7.35, S 5.35. IR (cm-1): 1726, 1620 (C=O); 1147 (S=O). 1H-NMR  ppm
(300 MHz, CDCl3): 14.92 (s, 1H, COOH), 8.78 (s, 1H, C2-H), 7.81-7.77 (m, 3H, Ar-H2'6', C5-H),
7.53 (d, 2H, Ar-H3’H5’, J = 9.0 Hz), 3.53 (s, 3H, OCH3), 4.65-0.79 (alifatik C-H). HRMS (ESI+)
m/z calculated for C27H27ClFN3O6 [M+Na]+, 576.1365, found 576.1378.
4.1.3.6. 6-Fluoro-8-methoxy-4-oxo-1-cyclopropyl-7-(2-(4-nitrophenyl)sulfonyl-2,8-
diazabicyclo[4.3.0]nonan-8-yl)-1,4-dihydroquinoline-3-carboxylic acid 20
M.p. 199 °C. Elemental analysis calculated for C27H27FN4O8S; C 55.28, H 4.64, N 9.55, S 5.47;
found C 54.53, H 4.56, N 9.58, S 5.44. IR (cm-1): 1726, 1622 (C=O); 1151 (S=O). 1H-NMR  ppm
(300 MHz, CDCl3): 14.86 (s, 1H, COOH), 8.77 (s, 1H, C2-H), 8.39 (d, 2H, Ar-H3'5', J = 9.0 Hz),
8.06 (d, 2H, Ar-H2'6', J = 9.3 Hz), 7.76 (d, 1H, C5-H, J = 13.8 Hz), 3.56 (s, 3H, OCH3), 4.69-0.79
(aliphatic C-H). HRMS (ESI+) m/z calculated for C27H27FN4O8S [M+Na]+, 609.1431, found
609.1430.
4.1.3.7. 6-Fluoro-8-methoxy-4-oxo-1-cyclopropyl-7-(2-(4-bromophenyl)sulfonyl-2,8-
diazabicyclo[4.3.0]nonan-8-yl)-1,4-dihydroquinoline-3-carboxylic acid 21
M.p. 164 °C. Elemental analysis calculated for C27H27BrFN3O6S.H2O; C 52.26, H 4.39, N 6.77, S
5.17; found C 52.37, H 4.31, N 6.99, S 4.88. IR (cm-1): 1726, 1620 (C=O). 1H-NMR  ppm (300
MHz, CDCl3): 14.92 (s, 1H, COOH), 8.78 (s, 1H, C2-H), 7.80 (d, 1H, C5-H, J = 13.8 Hz), 7.74-
7.66 (m, 4H, Ar-H'), 3.53 (s, 3H, OCH3), 4.64-0.78 (aliphatic C-H). HRMS (ESI+) m/z calculated
for C27H27BrFN3O6S [M+Na]+, 642.0686, found 642.0690.
4.2. Biological Studies
4.2.1. Anti-mycobacterial activity
Anti-tuberculosis activity of the designed compounds 1-21 was evaluated in vitro. MABA method
was used to determine the MIC values of the synthesized compounds against M. tuberculosis
H37Rv [45, 46]. Results are represented in the Table 1. Method is explained in Supplementary
Material.
4.2.2. Cytotoxicity
Compounds revealing MIC values less than 25.00 µg/mL were further examined for toxicity in a
RAW 264.7 cell line at a concentration of 25.00 µg/mL [47]. Method is commented in
Supplementary Material.
4.2.3. DNA supercoiling assay
18

DNA supercoiling [48] assay was performed using MTB DNA supercoiling assay kits (Inspiralis
Ltd., Norwich, UK). Method is presented in Supplementary Material.
4.2.4. Antimicrobial activity
Detailed information can be found in Supplementary Material.
4.2.4.1. Zone inhibition assay
Simple susceptibility screening test using agar well diffusion was used to determine the
antibacterial activity [49]. Antimicrobial activity was evaluated by the measurement of the zone
inhibition of the tested organism.
4.2.4.2. Minimum inhibitory concentration assay
Dilution susceptibility testing method [50] was used to determine the minimal concentration of the
compound needed to inhibit or kill the microorganism.
4.3. Molecular modeling studies
Molecular modeling studies with M. tuberculosis DNA gyrase and S. aureus DNA gyrase were
carried out to simulate the potential inhibition profile of the synthesized compounds. Protein
structures of M. tuberculosis and S. aureus DNA gyrase enzymes were obtained from protein data
bank with 5BTA and 5CDQ PDB codes, respectively. During molecular modeling studies,
prepared ligand structures were docked into protein binding site at pH: 7.0±1.0 using Schrödinger
Glide-SP Flexible Docking procedure. Carboxylic acid and tertiary amine moieties were generated
as negatively and positively charged functions, respectively. 7-[2,8-Diazabicyclo[4.3.0]nonan-8-
yl moieties of each compound were specially preserved as (1S,6S)-configured stereoisomers.
Docking results were evaluated according to ligand efficiency results alongside docking scores.
Furthermore; N499K, T500N, E501V mutated proteins of M. tuberculosis DNA gyrase were
prepared using MOE (Chemical Computing Group) rotamer explorer. Docking calculations were
run for mutant proteins. However, no special interaction between mutated amino acid residues and
compounds was observed after docking of synthesized compounds into mutated proteins.
Figures were prepared by using PyMOL software.
Acknowledgments
The authors are grateful to Dr. Jürgen Gross from the Institute of Organic Chemistry, University
of Heidelberg, for his generous help on obtaining high resolution mass spectra of the compounds.
We would like to thank Prof. Dr. habil. Wolfgang Sippl from Institute of Pharmacy, Faculty of
Natural Sciences, Martin Luther University, for giving us the opportunity of working in his
molecular modeling research laboratory. Moxifloxacin hydrochloride was generously donated by
Atabay Pharmaceuticals and Fine Chemicals Inc.
19

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