Design, synthesis, antimicrobial, and DNA gyrase inhibitory properties of fluoroquinolone–dichloroacetic acid hybrids

Article abstract of DOI:10.1111/cbdd.13638

A series of new fluoroquinolone conjugates 8a–g and 9a–f were synthesized via benzotriazole-mediated synthetic approach with good yield and purity. Some of the synthesized analogs exhibited significant antibacterial properties against Escherichia coli and

Full text of DOI:10.1111/cbdd.13638

MRS. ISRAA ABDALLA MOUSTAFA SLIEM (Orcid ID : 0000-0001-9708-4017)
DR SIVA S PANDA (Orcid ID : 0000-0003-3668-104X)
DR RIHAM GEORGE (Orcid ID : 0000-0002-2493-1315)
DR RAJEEV SAKHUJA (Orcid ID : 0000-0002-9284-9137)
Article type : Research Article
Design, synthesis, antimicrobial and DNA gyrase inhibitory properties of
fluoroquinolone-dichloroacetic acid hybrids
Israa A. Seliem a,b, Siva S. Panda a,*, Adel S. Girgis c, Yosra I. Nagy d, Riham F. George e, Walid
Fayad f, Nehmedo G Fawzy c, Tarek S. Ibrahim b,g, Amany M. M. Al-Mahmoudy b, Rajeev
Sakhuja h, Zakaria K. M. Abdel-samii b
a Department of Chemistry & Physics, Augusta University, Augusta, GA 30912, USA.
b
Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Zagazig University,
Zagazig, 44519, Egypt.
c Department of Pesticide Chemistry, National Research Centre, Dokki, Giza 12622, Egypt.
d
Microbiology and Immunology Department, Faculty of Pharmacy, Cairo University, Cairo
11562, Egypt.
e
Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, Cairo 11562,
Egypt.
f
Drug Bioassay-Cell Culture Laboratory, Pharmacognosy Department, National Research
Centre, Dokki, Giza, 12622. Egypt.
This article has been accepted for publication and undergone full peer review but has not been through the
copyediting, typesetting, pagination and proofreading process, which may lead to differences between this
version and the Version of Record. Please cite this article as doi: 10.1111/CBDD.13638
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g
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, King Abdulaziz University,
Jeddah, 21589, Saudi Arabia.
h Department of Chemistry, Birla Institute of Technology and Science, Pilani 333031, Rajasthan,
India.
* Corresponding author. E-mail: sspanda12@gmail.com, sipanda@augusta.edu
Tel: +1-706-667-4022; fax: +1-706-667-4519
Running head: fluoroquinolone-dichloroacetic acid hybrid conjugates
Keywords: Fluoroquinolone, Dichloroacetic acid, Antibacterial, Hybrid conjugates, DNA gyrase.
Abstract
A series of new fluoroquinolone conjugates 8a–g and 9a–f were synthesized via
benzotriazole-mediated synthetic approach with good yield and purity. Some of the synthesized
analogs exhibited significant antibacterial properties against E. coli and S. aureus with potency
higher than that of the parent drugs through in vitro standard bio-assay procedure (conjugates 8c
and 8d reveal antimicrobial properties with potency 1.9, 61.9, 20.7 and 2.4, 37.1, 8.3 folds relative
to the parent antibiotic 6 against E. coli, S. aureus and E. faecalis, respectively). The observed
experimental data supported by enzymatic DNA gyrase inhibitory property. Developed BMLR-
QSAR model validates the observed experimental data and recognizes the parameters responsible
for the enhanced antibacterial properties.
Introduction
Bacterial infections caused by both Gram-positive and Gram-negative pathogens are
responsible for the majority of hospital-acquired infections, which affects the global healthcare
systems (Peleg & Hooper, 2010). According to the latest World Health Organization (WHO)
report, around 10 million deaths every year (over 15% of all deaths) are due to infectious disease
(Balaban et al., 2016). In addition, the drug resistance organisms keep on increasing at an alarming
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rate associated with considerable mortality (Sprenger & Fukuda, 2016). This forces an immense
need to develop potential antimicrobial agents active against both drug sensitive and drug-resistant
bacterial infections (Brown & Wright, 2016; Silver, 2011).
Quinolone and fluoroquinolones are among different class of antibiotics used for the
treatment of bacterial infections (Fig. 1). The pharmacological importance of fluoroquinolones is
continually attracting medicinal chemists’ to use as a scaffold for the development of potent
antibacterial agents. Fluoroquinolones can show adverse effects in the central nervous system, skin
and gastrointestinal tract (Sarro & Sarro, 2001). Over the past few years, different positions of the
fluoroquinolones were modified to improve the potency and overcome the associated drug
resistance (Towle et al., 2018; Bartzatt et al., 2013; Allaka et al., 2016; Zhang et al., 2014; Rajulu
et al., 2014).
Dichloroacetate (DCA) is known for inhibition of pyruvate dehydrogenase kinase (PDK),
which is a key step that leads to reestablishment of the mitochondrial oxidative phosphorylation
pathway (Sutendra & Michelakis, 2013). Although, the preliminary study of DCA shows the
slowdown growth of certain tumors the mechanism is not well developed. Several antitumor
agents show improved efficacy when taken in combination with DCA (Florio et al., 2018). As far
as we are aware, only one article reported the dichloroacetic acid derivatives for antibacterial
properties (Casini et al., 1966).
Insert fig. 1
The present study describes synthesis of quinolone conjugates with amino acids to improve
the antibacterial property and increase the cell-permeability (Faidallah et al., 2018; Panda et al.,
2016; Panda et al., 2015; Tiwari et al., 2014; Panda et al., 2014; Ibrahim et al., 2014). The present
work focuses on ciprofloxacin and norfloxacin, second-generation fluoroquinolone that inhibits
DNA gyrase and topoisomerase IV. For the first time herein, we report the hybrid conjugates of
DCA and fluoroquinolone, which may overcome the resistance while maintaining or improving
the antibacterial property as well as diminishing associated adverse effects.
E. coli, P. aeruginosa, S. aureus and E. faecalis were selected for the synthesized hybrid
conjugate screening because of their physiological relevance and close relation to pathogenic
strains which cause several diseases in humans.
Experimental Section
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Chemistry
Melting points were determined on a capillary point apparatus equipped with a digital
thermometer. NMR spectra were recorded in (DMSO-d6) on Bruker NMR spectrometers operating
1
at 500 MHz for H (with TMS as an internal standard) and 125 MHz for 13C. All microwave
assisted reactions were carried out with a single mode cavity Discover Microwave Synthesizer
(CEM Corporation, NC). The reaction mixtures were transferred into a 10 mL glass pressure
microwave tube equipped with a magnetic stirrer bar. The tube was closed with a silicon septum
and the reaction mixture was subjected to microwave irradiation (Discover mode; run time: 60 s;
Power Max-cooling mode).
General procedure for the preparation of N-(Boc-aminoacyl)-benzotriazoles 10a–g (Panda et
al., 2014).
Compounds 10a–g were synthesized by irradiating an equimolar amount of Boc protected
amino acid with 1-(methylsulfonyl)-1H-benzo[d][1,2,3]triazole (BtSO2Me) in the presence of 2.0
eq. of triethylamine for 2 min run time and 60 min hold time at 70 °C and 50 W irradiation power.
Completion of the reaction was monitored by TLC. After completion of the reaction, the mixture
was quenched with water. The product obtained extracted with ethyl acetate and then washed with
a saturated solution of sodium carbonate and water to afford compound 10a–g.
General procedure for the synthesis of Boc-protected amino acid-fluoroquinolone conjugates
11a–g and 12a–f
A dried heavy-walled Pyrex tube containing a small stir bar was charged with N-(Boc-
aminoacyl)benzotriazoles (1.0 eq.) and fluoroquinolone (1.0 eq.) dissolved in DMF along with
triethylamine (2.0 eq.). The reaction mixture was exposed to microwave irradiation (20 W) at 50
°C for 60 min. Each mixture was allowed to cool through an inbuilt system until the temperature
fell below 30 °C (ca. 10 min). Each reaction mixture was quenched with ice cold water and the
solid obtained was filtered and washed with 4N HCl and water to give the desired compound.
General procedure for the synthesis of hybrid conjugates 8a-g and 9a-f
Boc protected amino acid-fluoroquinolone conjugate was stirred in HCl gas saturated
dioxane for 2 h. Dioxane was evaporated under reduced pressure and the residue was treated with
diethyl ether. The resultant solid was treated without further purification with benzotriazole
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derivatives of DCA 3 in the presence of triethylamine (2.0 eq.) in acetonitrile-water mixture (3.5
mL + 1.5 mL) and stirred at room temperature for 4-6 h. Acetonitrile was removed under vacuum
and the residue quenched in ice cold water. The precipitate obtained was washed with 4N HCl,
water and then dried over vacuum to obtain the desired hybrid conjugates 8a-g and 9a-f.
(S)1-cyclopropyl-7-(4-((2,2-dichloroacetyl)glycyl)piperazin-1-yl)-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (DCA-L-Gly-Cip, 8a).
White microcrystals, m.p 241–243 °C, yield 61%. IR: νmax/cm-1 3300, 3010, 2923, 1702, 1247,
1
1114, 804; H NMR (DMSO-d6) δ: 15.17 (s, 1H), 8.72–8.67 (m, 2H), 7.92 (d, J = 12.4 Hz, 1H),
7.58 (s, 1H), 6.70 (s, 1H), 4.20–4.14 (m, 2H), 4.10–4.06 (m, 1H), 3.89–3.54 (m, 8H), 1.35–1.30
13
(m, 2H), 1.22–1.17 (m, 2H) . C NMR (DMSO-d6) δ: 176.3, 166.1, 165.9, 163.7, 152.0, 148.0,
144.8, 139.2, 118.9, 111.1, 111.0, 106.7, 66.5, 49.3, 49.1, 43.7, 42.3, 41.2, 35.9, 7.6. HRMS m/z
for C21H21Cl2FN4O5 [M+Na]+ Calcd. 521.0765. Found: 521.077
(S)1-cyclopropyl-7-(4-((2,2-dichloroacetyl)-L-alanyl)piperazin-1-yl)-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (DCA-L-Ala-Cip, 8b).
White microcrystals, m.p 253–255 °C, yield 73%. IR: νmax/cm-1 3371, 3010, 2865, 1696, 1245,
1
1113, 805; H NMR (DMSO-d6) δ: 13.84 (br s, 1H), 8.92 (s, 1H), 8.65 (s, 1H), 7.91 (d, J = 12.9
Hz, 1H), 7.58 (s, 1H), 6.57 (s, 1H), 4.89-4.80 (m, 1H), 4.18 (s, 1H), 3.81–3.61 (m, 8H), 1.38–1.19
13
(m, 7H). C NMR (DMSO-d6) δ: 176.3, 169.5, 165.9, 162.7, 153.9, 151.9, 148.1, 139.1, 118.9,
111.1, 110.9, 106.8, 66.5, 49.6, 49.2, 45.4, 44.5, 41.3, 35.9, 17.6, 7.6. HRMS m/z for
C22H23Cl2FN4O5 [M+H]+ Calcd. 512.1025. Found: 513.1033
(R),(S)1-cyclopropyl-7-(4-((2,2-dichloroacetyl)alanyl)piperazin-1-yl)-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (DCA-DL-Ala-Cip, 8c)
White microcrystals, m.p 216–218 °C, yield 57%. IR: νmax/cm-1 3308, 3009, 2865, 1721, 1241,
1111, 804; 1H NMR (DMSO-d6) δ: 15.16 (s, 1H), 8.92 (s, 1H), 8.65 (s, 1H), 7.91 (d, J = 12.7 Hz,
1H), 7.58 (s, 1H), 6.57 (s, 1H), 4.91–4.79 (m, 1H), 4.39–4.32(m, 1H), 3.81–3.62 (m, 8H), 1.32–
1.19 (m, 7H). 13C NMR (DMSO-d6) δ: 176.3, 169.5, 165.9, 162.7, 152.0, 148.1, 145.0, 139.1,
119.1, 111.1, 111.0, 106.7, 66.5, 50.2, 49.6, 49.2, 45.4, 44.4, 36.0, 17.6, 7.6. HRMS m/z for
C22H23Cl2FN4O5 [M+H]+ Calcd. 512.1025. Found: 513.1099
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(R)1-cyclopropyl-7-(4-((2,2-dichloroacetyl)-D-alanyl)piperazin-1-yl)-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (DCA-D-Ala-Cip, 8d).
White microcrystals, m.p 204–206 °C, yield 77%. IR: νmax/cm-1 3300, 3050, 2865, 1716, 1240,
1109, 803; 1H NMR (DMSO-d6) δ: 15.16 (s, 1H), 8.66 (s, 1H), 8.12 (s, 1H), 7.92 (d, J = 11.6 Hz,
1H), 7.58 (s, 1H), 6.58 (s, 1H), 4.88–4.80 (m, 1H), 4.53–4.41 (m, 1H), 3.82–3.55 (m, 8H), 1.37–
1.19 (m, 7H). 13C NMR (DMSO-d6) δ: 176.4, 169.5, 165.9, 161.0, 152.0, 148.1, 145.0, 139.1,
119.0, 111.1, 111.0, 107.0, 66.5, 50.2, 49.1, 45.4, 44.4, 41.3, 36.0, 17.6, 7.6. HRMS m/z for
C22H23Cl2FN4O5 [M+H]+ Calcd. 512.1030. Found: 513.1103
(S)7-(4-((2,2-dichloroacetyl)isoleucyl)piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (DCA-L-ILe-Cip, 8e).
White microcrystals, m.p 212‒214 °C, yield 83%. IR: νmax/cm-1 3200, 3000, 2968, 1712, 1256,
1
1112, 804; H NMR (DMSO-d6) δ: 15.16 (s, 1H), 8.89–8.88 (m, 1H), 8.65 (s, 1H), 7.91 (d, J =
12.5 Hz, 1H), 7.58 (s, 1H), 6.61 (s, 1H), 4.85–4.72 (m, 2H), 3.82–3.61 (m, 8H), 1.46–0.85 (m,
13H). 13C NMR (DMSO-d6) δ: 176.3, 168.8, 165.9, 163.4, 151.9, 148.0, 144.7, 139.1, 118.9,
111.1, 110.9, 106.8, 66.4, 53.0, 49.7, 49.3, 45.5, 44.0, 36.7, 35.9, 23.7, 15.5, 11.0, 7.6. HRMS m/z
for C25H29Cl2FN4O5 [M+Na]+ Calcd. 577.1391. Found: 577.1373
(S)1-cyclopropyl-7-(4-((2,2-dichloroacetyl)valyl)piperazin-1-yl)-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (DCA-L-Val-Cip, 8f).
White microcrystals, m.p 241‒243 °C, yield 74%. IR: νmax/cm-1 3280, 3050, 2931, 1709, 1239,
1114, 804; 1H NMR (DMSO-d6) δ: 15.18 (s, 1H), 8.83 (d, J = 9.4 Hz, 1H), 8.68 (s, 1H), 7.94 (d, J
= 13.1 Hz, 1H), 7.59 (d, J = 8.1 Hz, 1H), 6.63 (s, 1H), 4.74–4.69 (m, 1H), 3.82–3.61 (m, 7H),
2.10–2.03 (m, 1H), 1.33–1.19 (m, 6H), 0.92–0.87 (m, 6H). 13C NMR (DMSO-d6) δ: 176.3, 168.7,
165.8, 163.5, 151.9, 148.0, 144.8, 139.1, 118.9, 111.1, 110.9, 106.7, 66.4, 53.8, 49.7, 49.3, 45.3,
45.0, 35.9, 30.5, 19.3, 17.4, 7.6. HRMS m/z for C24H27Cl2FN4O5 [M+H]+ Calcd. 540.1341. Found:
541.1413
(S)1-cyclopropyl-7-(4-((2,2-dichloroacetyl)phenylalanyl)piperazin-1-yl)-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (DCA-L-Phe-Cip, 8g).
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White microcrystals, m.p 153‒155 °C, yield 67%. IR: νmax/cm-1 3220, 3008, 2980, 1719, 1256,
1111, 805; 1H NMR (DMSO-d6) δ: 15.18 (br s, 1H), 9.11 (s, 1H), 8.65 (s, 1H), 8.42 (s, 1H), 7.88
(d, J = 13.2 Hz, 1H), 7.48–7.21 (m, 5H), 6.55 (s, 1H), 5.1–4.99 (m, 1H), 4.77–4.70 (m, 1H), 3.81–
2.91 (m, 10H), 1.33–1.19 (m, 4H). 13C NMR (DMSO-d6) δ: 176.3, 168.4, 165.9, 163.0, 151.9,
148.1, 144.7, 139.1, 129.9, 129.6, 128.7, 128.2, 126.7, 118.9, 111.1, 110.9, 106.7, 66.3, 50.3, 49.1,
45.4, 44.7, 41.2, 37.5, 35.9, 8.5, 7.6. HRMS m/z for C28H27Cl2FN4O5 [M+H]+ Calcd. 588.1338.
Found: 589.1408
(S)7-(4-((2,2-dichloroacetyl)glycyl)piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid (DCA-Gly-Nor, 9a).
White microcrystals, m.p 242–244 °C, yield 62%. IR: νmax/cm-1 3383, 3052, 2923, 1720, 1244,
1111, 808; 1H NMR (DMSO-d6) δ: 15.32 (s, 1H), 8.97 (s, 1H), 8.72 (s, 1H), 7.94 (d, J = 12.8 Hz,
1H), 7.20 (s, 1H), 6.69 (s, 1H), 4.63–4.57 (m, 2H), 4.18–4.13 (m, 2H), 3.69–3.65 (m, 4H), 3.38–
3.33 (m, 4H), 1.45–1.39 (m, 3H). 13C NMR (DMSO-d6) δ: 176.2, 166.1, 163.8, 163.7, 151.8,
148.6, 145.2, 137.1, 119.9, 111.3, 106.8, 106.2, 66.5, 49.5, 49.4, 49.2, 49.1, 43.8, 41.2, 14.4.
HRMS m/z for C20H21Cl2FN4O5 [M+H]+ Calcd. 486.0867. Found: 487.0947
(S)7-(4-((2,2-dichloroacetyl)-L-alanyl)piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (DCA-L-Ala-Nor, 9b).
White microcrystals, m.p 256–258 °C, yield 85%. IR: νmax/cm-1 3301, 3050, 2927, 1706, 1239,
1
1111, 804; H NMR (DMSO-d6) δ: 15.32 (s, 1H), 8.97–8.91 (m, 2H), 7.94 (d, J = 11.9 Hz, 1H),
7.21 (s, 1H), 6.57 (s, 1H), 4.88–4.80 (m, 1H), 4.64–4.55 (m, 2H), 3.74–3.67 (m, 8H), 1.42–1.40
13
(m, 3H), 1.27–1.23 (m, 3H). C NMR (DMSO-d6) δ: 176.2, 169.5, 166.1, 162.7, 153.8, 151.9,
148.6, 145.1, 137.1, 111.4, 111.2, 107.1, 106.3, 66.4, 49.7, 49.3, 49.1, 45.4, 44.6, 17.7, 14.4.
HRMS m/z for C21H23Cl2FN4O5 [M+H]+ Calcd. 523.0922. Found: 523.0917
(R)7-(4-((2,2-dichloroacetyl)-D-alanyl)piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (DCA-D-Ala-Nor, 9c).
White microcrystals, m.p 237–239 °C, yield 78%. IR: νmax/cm-1 3308, 3050, 2930, 1708, 1241,
1
1111, 804; H NMR (DMSO-d6) δ: 15.30 (br s, 1H), 8.95–8.91 (m, 2H), 7.93 (d, J = 15.5 Hz,
1H), 7.21 (s, 1H), 6.57 (s, 1H), 4.63–4.54 (m, 2H), 4.88–4.80 (m, 1H), 3.74-3.48 (m, 8H), 1.44–
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1.39 (m, 3H), 1.26 (d, J = 5.0 Hz, 3H). 13C NMR (DMSO-d6) δ: 176.2, 169.5, 166.1, 162.8, 151.8,
148.6, 145.1, 137.1, 119.6, 111.3, 107.1, 106.3, 66.5, 49.7, 49.1, 45.4, 44.6, 42.4, 41.3, 17.7, 14.4.
HRMS m/z for C21H23Cl2FN4O5 [M+H]+ Calcd. 500.1024. Found: 501.11
(S)7-(4-((2,2-dichloroacetyl)isoleucyl)piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (DCA-l-ILe-Nor, 9d)
White microcrystals, m.p 219‒221 °C, yield 75%. IR: νmax/cm-1 3269, 3050, 2980, 1687, 1241,
1117, 805; 1H NMR (DMSO-d6) δ: 15.30 (br s, 1H), 8.94-8.89 (m, 2H), 7.90 (d, J = 10 Hz, 1H),
7.44 (s, 1H), 6.63 (s, 1H), 4.72–4.59 (m, 3H), 3.82–3.36 (m, 8H), 1.84 (s, 1H), 1.58–1.30 (m, 3H),
1.20–0.86 (m, 8H). 13C NMR (DMSO-d6) δ: 176.1, 168.8, 166.1, 163.4, 151.8, 148.5, 145.0,
137.1, 119.5, 111.3, 107.1, 106.2, 66.4, 53.0, 52.9, 49.4, 49.1, 45.4, 45.0, 36.8, 23.7, 15.5, 14.4,
11.0. HRMS m/z for C24H29Cl2FN4O5 [M+H]+ Calcd. 542.1482. Found: 543.1562
(S)7-(4-((2, 2-dichloroacetyl)valyl)piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4 dihydroquinoline-3-
carboxylic acid (DCA-L-Val-Nor 9e).
White microcrystals, m.p 240‒242 °C, yield 76%. IR: νmax/cm-1 3280, 3050, 2931, 1688, 1234,
1
1112, 806.; H NMR (DMSO-d6) δ: 15.30 (brs, 1H), 8.96 (s, 1H), 8.82 (d, J = 7.1 Hz, 1H), 7.94
(d, J = 12.3 Hz, 1H), 7.21 (s, 1H), 6.64 (s, 1H), 4.75–4.69 (m, 1H), 4.63-4.55 (m, 2H), 3.81–3.72
(m, 8H), 2.10–2.01 (m, 1H), 1.44–1.39 (m, 3H), 0.91–0.88 (m, 6H). 13C NMR (DMSO-d6) δ:
176.2, 168.7, 166.1, 163.4, 151.8, 148.6, 145.1, 137.1, 119.6, 111.3, 107.1, 106.3, 66.4, 54.0, 53.8,
49.8, 49.4, 49.1, 45.0, 30.6, 19.3, 17.4, 14.4. HRMS m/z for C23H27Cl2FN4O5 [M+H]+ Calcd.
528.1341. Found: 529.1416
(S)7-(4-((2,2-dichloroacetyl)phenylalanyl)piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-
dihydroquinoline-3-carboxylic acid (DCA-L-Phe-Nor, 9f).
White microcrystals, m.p 114‒116 °C, yield 64%. IR: νmax/cm-1 3220, 3029, 2980, 1697, 1240,
1113, 807; 1H NMR (DMSO-d6) δ: 15.30 (br s, 1H), 9.10 (s, 1H), 8.95 (s, 1H), 7.90 (d, J = 11 Hz,
1H), 7.43–7.04 (m, 6H), 6.55 (s, 1H), 5.04–4.98 (m, 1H), 4.57–4.54 (m, 2H), 3.68–2.73 (m, 10H),
1.47–1.32 (m, 3H). 13C NMR (DMSO-d6) δ: 176.2, 168.4, 166.1, 163.0, 151.8, 148.6, 145.0,
137.1, 136.5, 129.5, 129.3, 128.4, 128.2, 126.7, 111.3, 111.2, 107.1, 106.1, 66.3, 50.3, 49.3, 49.1,
45.4, 44.7, 41.3, 37.5, 14.4. HRMS m/z for C27H27Cl2FN4O5 [M+H]+ Calcd. 576.1335. Found:
577.141
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1-Cyclopropyl-7-(4-(2,2-dichloroacetyl)piperazin-1-yl)-6-fluoro-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid (DCA-Cip, 15).
Yellow microcrystals, m.p 250–252 °C, yield 88%. IR: νmax/cm-1 3017, 2865, 1720, 1245, 1097,
1
804; H NMR (DMSO-d6) δ: 15.17 (s, 1H), 8.66 (s, 1H), 7.93 (d, J = 12.4 Hz, 1H), 7.33 (s, 1H),
6.41 (s, 1H), 3.81–3.54 (m, 9H), 1.31–1.19 (m, 4H). 13C NMR (DMSO-d6) δ: 176.4, 165.9, 161.9,
151.9, 148.2, 148.1, 139.1, 119.0, 111.2, 106.8, 99.5, 65.8, 49.2, 48.9, 42.6, 42.3, 35.9, 7.6. HRMS
m/z for C19H18Cl2FN3O4 [M+H]+ Calcd. 441.0658. Found: 441.0689
7-(4-(2,2-dichloroacetyl)piperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic
acid (DCA-Nor, 16).
Yellow microcrystals, m.p 220–222°C, yield 63%. IR: νmax/cm-1 3017, 2931, 1713, 1240, 1110,
804; 1H NMR (DMSO-d6) δ: 15.29 (br s, 1H), 8.94 (s, 1H), 7.90 (d, J = 12.4 Hz, 1H), 7.33 (s, 1H),
13
6.40 (s, 1H), 4.63–4.52 (m, 2H), 3.78–3.54 (m, 8H), 1.45–1.33 (m, 3H). C NMR (DMSO-d6) δ:
176.1, 166.1, 161.9, 151.8, 148.6, 144.9, 137.1, 119.6, 111.3, 107.1, 106.4, 65.8, 49.3, 49.1, 45.3,
42.6, 42.4, 14.4. HRMS m/z for C18H18Cl2FN3O4 [M+H]+ Calcd. 429.0658. Found: 429.0678
Biological studies
Antimicrobial properties
Methodology of the antimicrobial activity determination is mentioned in the supplementary
material.
Antiproliferative properties
Methodology of the antiproliferative properties of the synthesized compounds against
RPE1 cell line is mentioned in the supplementary material.
E-coli DNA gyrase inhibitory properties
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Methodology of the E. coli DNA gyrase inhibitory properties of the tested compounds is
mentioned in the supplementary material.
2D-QSAR studies
Methodology of the 2D-QSAR studies is mentioned in the supplementary material
Results and discussion
Chemistry
N-Acylbenzotriazoles are efficient reagents for acylation reactions (Panda et al., 2014).
The carboxyl group of dichloroacetic acid 1 was activated by a modified reported procedure. The
synthesis of benzotriazolide of dichloroacetic acid 3 was reported by katritzky et al (Katritzky et
al., 2003). However, we were unable to get the product by following the described procedure.
After several trials, we observed that the compound is not stable in basic conditions. So we
modified the method from basic workup to acidic workup. The advantage of benzotriazole
chemistry is the accessibility to use either acid or base to remove the excess benzotriazole from the
reaction mixture.
DCA hybrid conjugates with antibiotics and amino acid as linker were prepared by using
two different routes. In route I, the benzotriazolide of dichloroacetic acid 3 was treated with amino
acids 4 in the presence of triethylamine in the acetonitrile-water mixture. We were unable to
activate the DCA-amino acid conjugate by using benzotriazole chemistry. To prepare our target
hybrid conjugates we explored different coupling reagents like HOBt, EDC and DCC. Every time
we confirmed the formation of the product 8 by NMR but in the impure form (Scheme 1). We also
failed to purify the hybrid conjugates by column chromatography. We switched to an alternative
synthetic route.
Insert Scheme 1
In route II, we utilized Boc protected amino acid–fluoroquinolone conjugates 11a–g and
12a–f prepared by our previously reported method (Panda et al., 2014). Boc group deprotection
with dioxane/HCl mixture at 20 °C for 2 h gave the unprotected amino acid–fluoroquinolone
conjugates, which were further used for the next step without characterization. The target hybrid
conjugates 8a–g and 9a–f were prepared by coupling unprotected amino acid–fluoroquinolone
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conjugates with DCA–benzotriazolide 3 in the presence of triethylamine at 20 °C for 4-6 h in good
yields (31–77%) (Scheme 2).
Insert Scheme 2
To better understand the importance of amino acid in these hybrid conjugates, we have
also synthesized DCA-fluoroquinolone conjugates without any linker. The DCA-benzotriazole 3
was treated with the ciprofloxacin 6 and norfloxacin 7 under microwave irradiation in the presence
of TEA to obtain the conjugates in good yields (Scheme 3)
Insert Scheme 3
Biological studies
Antimicrobial properties
Antimicrobial properties of the synthesized hybrid conjugates 8a‒g, 9a‒f, 15 and 16 were
determined by the standard techniques and compared with the parent fluoroquinolone antibiotics 6
and 7 (Clinical & Laboratory Standards Institute, 2012). The antimicrobial properties of the tested
conjugates and their standard precursors (6, 7) against Gram-negative (E. coli, P. aeruginosa) and
Gram-positive (S. aureus, E. faecalis) bacteria were determined in MIC (μM) values (Table 1).
From the observed data, it has been noticed that the parent antibiotics show antimicrobial
properties with higher potency against the tested Gram-negative bacteria relative to the Gram-
positive strains. Considering that ciprofloxacin 6 is of higher efficacy/potency than norfloxacin 7
(MIC = 0.377, 6.263; 5.030, 12.526, 12.072, 83.506; 24.144, 100.207 μM for 6 and 7 against E.
coli, P. aeruginosa, S. aureus, and E. faecalis, respectively). The synthesized hybrid conjugates
8c and 8d reveal enhanced antimicrobial properties against three of the tested microorganisms
with potency 1.9, 61.9, 20.7 and 2.4, 37.1, 8.3 folds relative to the parent precursor antibiotic 6
against E. coli, S. aureus and E. faecalis, respectively. Compound 15 also reveals remarkable
antimicrobial properties with potency 5.5, 1.1, 42.7 and 9.2 folds relative to the antibiotic 6 against
E. coli, P. aeruginosa, S. aureus, and E. faecalis, respectively. It has also been noticed that the D-
amino acid bearing conjugates are of higher antimicrobial properties than the L-analogues as
exhibited in pairs 8b/8d and 9b/9c. We also observed phenylalanine containing conjugates are less
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active compared to others. For better understanding, the SAR (structure-activity relationships),
computational studies were considered.
Antiproliferative properties
Antiproliferative properties of the synthesized conjugates (8a‒g, 9a‒f, 15 and 16) were
determined by the standard MTT technique utilizing RPE1 (normal human immortalized retinal
epithelial) cell line (Ismail et al., 2016).Table S1 (Supplementary material) reveals that all the
synthesized conjugates reveal safe profile (antiproliferation < 20%) against the tested normal
human cell line utilized at 100 μM (% of cell proliferation = 98.2‒80.0). This observation may
support the safe application of the synthesized compounds.
E-coli DNA gyrase inhibitory properties
DNA replication, transcription, and other cellular transactions usually initiated by
uncoiling and unwinding DNA helices. The DNA helices are super-coiled inside the cell.
Topoisomerase enzymes are responsible for the DNA topological steps (Badshah & Ullah, 2018).
Two main types of topoisomerases are well known. Type-I catalyzes single DNA strand break
while type-II (gyrase) capable for break two DNA strands at single circular and twisted around
each other i.e. removing the links in the DNA replication forks (Tiz et al., 2019; Ponnusamy et al.,
2018; Gençer et al., 2017). DNA gyrase is a famous drug discovery target for developing
antibacterial active agents due to the negative DNA super-coiling effect capable to block DNA
replication and eventually bacterial cell death (Tomašič et al., 2017; Zhang et al., 2016).
Fluoroquinolones are well-known DNA gyrase inhibitors in many bacterial species (Towle et al.,
2018; Carta et al., 2019).
The conjugates synthesized revealing potent antimicrobial properties against E. coli (8c, 8d
and 15) were subjected for DNA gyrase supercoiling bioassay and the data compared with that of
their parent antibiotic, ciprofloxacin (CIP). The study can validate the observed antimicrobial
properties and also reveal the selectivity towards the targeted enzyme (mode of action). From the
results obtained (Table 2, Fig. 2) it can be concluded that CIP seems the most effective/selective
agent towards the targeted enzyme relative to the other tested conjugates (IC50 = 2.27 μM).
Conjugate 8c reveals higher potency/selectivity (about 3 folds) than 8d (IC50 = 3.25, 9.80 μM for
8c and 8d, respectively). Also, compound 15 reveals promising efficacy towards the targeted
enzyme with lower potency/selectivity than the parent antibiotic CIP (IC50 = 3.55 μM, 64%
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relative efficacy to CIP). We believe there must be an additional mechanism responsible for the
activity of the conjugates and we assume DCA must be playing an important role in inhibiting
pyruvate dehydrogenase kinase. Based on a report from 2012 by Birkenstock et al., pyruvate
dehydrogenase is an important target for antibiotics (Birkenstock et al., 2012).
Insert Fig. 2
2D-QSAR studies
The conjugates synthesized with variable antimicrobial properties against E. coli, S. aureus and E.
faecalis were subjected for 2D-QSAR studies to better understand the factors affecting/controlling
the biological properties. The QSAR models (CODESSA-Pro software) (Srour et al., 2018), as
well as an explanation of their descriptors, are mentioned in the supplementary material (Tables
S2‒S10, Figs. S1‒S3). The estimated antimicrobial properties due to the QSAR models are close
to the experimental values. Additionally, the statistical parameters (including cross-validation
leave one-out and many-out “up to 20% of the training set” correlation coefficients) of the QSAR
models support their goodness for utilization in predication of effective hits (R2 = 0.924, 0.958,
0.962; R2cvOO = 0.870, 0.923, 0.928; R2cvMO = 0.886, 0.928, 0.936 for E. coli, S. aureus and E.
faecalis, respectively).
Conclusions
A set of dichloroacetic acid-fluoroquinolone conjugates 8a–g, 9a–f, 15 and 16 were
synthesized in good yields using benzotriazole chemistry. The synthesized compounds were bio-
assayed against Gram positive and Gram negative pathogens following the in vitro standard
procedure. Some of the synthesized hybrids exhibited antibacterial properties with higher potency
than the starting precursor, fluoroquinolones (used as standard reference). Conjugates 8c and 8d
revealed antimicrobial properties against with potency 1.9, 61.9, 20.7 and 2.4, 37.1, 8.3 folds
relative to the parent antibiotic 6 against E. coli, S. aureus and E. faecalis, respectively. Also,
compound 15 revealed promising antimicrobial properties with potency 5.5, 1.1, 42.7 and 9.2
folds relative to 6 against E. coli, P. aeruginosa, S. aureus, and E. faecalis, respectively. The
DNA gyrase enzymatic study supports the experimental data. We believe there is more than one
mode of mechanisms are involved in the antibacterial property of the conjugates. 2D-QSAR is
explored in the present study for validating the observed data and determining the most important
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structural parameters responsible for the properties. A robust BMLR-QSAR model developed for
the antibacterial activities using CODESSA-Pro software.
Acknowledgments
We thank the Egyptian Cultural and Educational Bureau Scholarship. Authors also thank the
Center for Undergraduate Research and Scholarship (CURS) and Translational Research Program
(TRP) at Augusta University for financial support.
Conflict of interest
The authors state no conflict interest.
Data Availability Statement
All data generated or analyzed during this study are included in this published article and its
Supplementary Information Files.
Figure Legends
Fig. 1. Examples of different class of antibiotics.
Scheme 1. Route I for the synthesis of hybrid conjugates 8a–g and 9a–f.
Scheme 2. Route II for the synthesis of hybrid conjugates.
Scheme 3. Synthesis of DCA-ciprofloxacin and DCA-norfloxacin conjugates.
Fig. 2. Preview depicting the E. coli DNA gyrase supercoiling assay of CIP, conjugates 8c, 8d and
15 at different concentrations (0.1, 1, 10, and 100 μM).
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R
O
O
N
H
N
OH
OH
S
O
O
O
H
H
H2N
S N
O
Cl
N
O
H
OH
Cl
O
Sulfonamides
Chloramphenicol
B-Lactams
O
O
O
N
O
F
O
N
F
F
OH
OH
O
N
O
H
N
N
N
N
HN
HN
O
Oxazolidinones
Norfloxacin
Ciprofloxacin
Fluoroquinolones
Fig. 1. Examples of different class of antibiotics.
R
OH
H2N
O
O
O
R
N
4
THF, SOCl2
3 h, 20 o
O
Cl
Cl
Cl
O
OH
N
+
OH
N
N
N
H
5
N
C
CH3CN, H2O
TEA, 3 h, 20 o
Cl
Cl
Cl
O
H
N
C
2
3
1
O
F
OH
coupling
reagent
N
N
R1
HN
6 =
7 =
O
O
F
OH
O
R
N
N
R1
Cl
N
N
H
8a-g =
9a-f =
Cl
O
impure product
Scheme 1. Route I for the synthesis of hybrid conjugates 8a–g and 9a–f.
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O
O
O
O
F
N
R
N
N
F
OH
OH
DMF, TEA
Boc
N
H
R
N
N
+
N
N
R1
R1
11a-g =
12a-f =
O
MW, 70 oC, 1 h
Boc
N
HN
N
10a-g
H
O
6 =
7 =
dioxane-HCl(g)
20 oC, 2 h
O
O
O
Cl
O
O
N
F
F
OH
Cl
N
N
OH
3
O
R
N
N
R1
R
N
N
R1
Cl
N
N
N
CH3CN, TEA
20 oC, 4-6 h
H3N
Cl
H
Cl
O
O
13a-g =
14a-f =
8a-g =
9a-f =
Scheme 2. Route II for the synthesis of hybrid conjugates.
O
O
O
O
F
OH
F
O
OH
DMF, TEA
Cl
Cl
N
N
R1
+
N
N
N
N
R1
MW, 70 oC, 1 h
N
Cl
N
Cl
HN
15 =
16 =
3
O
6 =
7 =
Scheme 3. Synthesis of DCA-ciprofloxacin and DCA-norfloxacin conjugates.
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Fig. 2. Preview depicting the E. coli DNA gyrase supercoiling assay of CIP, conjugates 8c, 8d and
15 at different concentrations (0.1, 1, 10, and 100 μM).
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Table 1. Antimicrobial properties of the tested compounds (8a‒g, 9a‒f, 15, 16) and standard references (6, 7).
Minimum inhibitory concentration (MIC), μg/mL (μM)
Entry Compd.
E. coli
P. aeruginosa
S. aureus
E. faecalis
O
O
N
1
2
3
0.125 (0.377)
1.667 (5.030)
4.000 (12.072)
8.000 (24.144)
F
OH
N
HN
CIP, 6
O
N
O
2.000 (6.263)
0.333 (0.668)
4.000 (12.526)
9.333 (18.692)
26.667 (83.506)
32.000 (100.207)
F
OH
N
HN
NOR, 7
O
N
O
0.750 (1.502)
2.000 (4.005)
F
OH
O
N
Cl
N
N
H
Cl
O
DCA-Gly-Cip, 8a
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O
N
O
4
0.667 (1.299)
21.333 (41.557)
0.250 (0.487)
1.667 (3.247)
F
OH
O
N
Cl
N
N
H
Cl
O
DCA-L-Ala-Cip, 8b
O
N
O
5
6
0.100 (0.195)
5.333 (10.389)
0.100 (0.195)
0.600 (1.169)
F
OH
O
N
Cl
N
N
H
Cl
O
DCA-DL-Ala-Cip, 8c
O
O
0.082 (0.159)
4.000 (7.792)
0.167 (0.325)
1.500 (2.922)
F
OH
O
N
N
Cl
N
N
H
Cl
O
DCA-D-Ala-Cip, 8d
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O
N
O
7
8
0.333 (0.600)
0.333 (0.616)
10.667 (19.204)
17.067 (31.523)
0.267 (0.480)
0.333 (0.616)
3.733 (6.722)
0.800 (1.478)
F
OH
O
N
Cl
N
N
H
Cl
O
DCA-L-ILe-Cip, 8e
O
O
F
OH
O
N
N
Cl
N
N
H
Cl
O
DCA-L-Val-Cip, 8f
O
O
9
0.800 (1.357)
25.600 (43.430)
1.067 (1.810)
4.267 (7.238)
F
OH
O
N
N
Cl
N
N
H
Cl
O
DCA-L-Phe-Cip, 8g
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O
N
O
10
11
12
1.667 (3.420)
4.667 (9.308)
1.000 (1.995)
64.000 (131.333)
128.000 (255.316)
13.333 (26.595)
16.000 (32.833)
6.667 (13.298)
1.000 (1.995)
53.333 (109.444)
21.333 (42.553)
6.867 (13.697)
F
OH
O
O
O
N
Cl
Cl
Cl
N
N
H
Cl
Cl
Cl
O
O
O
DCA-Gly-Nor, 9a
O
O
F
OH
N
N
N
N
H
DCA-L-Ala-Nor, 9b
O
O
F
OH
N
N
N
N
H
DCA-D-Ala-Nor, 9c
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O
N
O
13
14
6.400 (11.777)
5.333 (10.074)
>51.3 (>94.402)
>51.3 (>96.904)
2.133 (3.926)
2.133 (4.030)
25.600 (47.109)
21.350 (40.329)
F
OH
O
N
Cl
N
N
H
Cl
O
DCA-L-ILe-Nor, 9d
O
O
F
OH
O
N
N
Cl
N
N
H
Cl
O
DCA-L-Val-Nor, 9e
O
O
15
5.333 (9.236)
>51.3 (>88.842)
2.667 (4.618)
>51.3 (>88.842)
F
OH
O
N
N
Cl
N
N
H
Cl
O
DCA-L-Phe-Nor, 9f
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O
N
O
16
17
0.030 (0.068)
0.250 (0.581)
2.000 (4.522)
0.125 (0.283)
1.000 (2.324)
1.167 (2.638)
F
OH
Cl
N
N
Cl
O
DCA-Cip, 15
O
N
O
9.333 (21.692)
5.333 (12.396)
F
OH
Cl
N
N
Cl
O
DCA-Nor, 16
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Table 2. Inhibitory properties of E. coli DNA gyrase supercoiling for the tested compounds.
Entry
Compd.
6 (CIP)
8c
IC50 (μM) ± SD
2.27 ± 0.16
3.25 ± 0.11
9.80 ± 0.42
3.55 ± 0.17
1
2
3
4
8d
15
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