Design, synthesis and antimicrobial evaluation of novel glycosylated-fluoroquinolones derivatives

Article abstract of DOI:10.1016/j.ejmech.2020.112513

Herein we report the design, synthesis and biological evaluation of structurally modified ciprofloxacin, norfloxacin and moxifloxacin standard drugs, featuring amide functional groups at C-3 of the fluoroquinolone scaffold. In vitro antimicrobial testing against various Gram-positive bacteria, Gram-negative bacteria and fungi revealed potential antibacterial and antifungal activity. Hybrid compounds 9 (MIC 0.2668 ± 0.0001 mM), 10 (MIC 0.1358 ± 00025 mM) and 13 (MIC 0.0898 ± 0.0014 mM) had potential antimicrobial activity against a fluoroquinolone-resistant Escherichia coli clinical isolate, compared to ciprofloxacin (MIC 0.5098 ± 0.0024 mM) and norfloxacin (MIC 0.2937 ± 0.0021 mM) standard drugs. Interestingly, compound 10 also exerted potential antifungal activity against Candida albicans (MIC 0.0056 ± 0.0014 mM) and Penicillium chrysogenum (MIC 0.0453 ± 0.0156 mM). Novel derivatives and standard fluoroquinolone drugs exhibited near-identical cytotoxicity levels against L6 muscle cell-line, when measured using the MTT assay.

Full text of DOI:10.1016/j.ejmech.2020.112513

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Design, Synthesis and Antimicrobial Evaluation of Novel Glycosylated-
fluoroquinolones Derivatives
Aya A.M. Mohammed, Ghadeer A.R.Y. Suaifan, Mayadah B. Shehadeh, Patrick N.
Okechukwu
PII:
S0223-5234(20)30485-2
DOI:
https://doi.org/10.1016/j.ejmech.2020.112513
EJMECH 112513
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 October 2019
Revised Date: 11 May 2020
Accepted Date: 19 May 2020
Please cite this article as: A.A.M. Mohammed, G.A.R.Y. Suaifan, M.B. Shehadeh, P.N. Okechukwu,
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European Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.112513.
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Design, Synthesis and Antimicrobial Evaluation of Novel
Glycosylated-fluoroquinolones Derivatives
Aya A. M. Mohammed1 | Ghadeer A. R. Y. Suaifan1* | Mayadah B. Shehadeh1 | Patrick N.
Okechukwu2
1*Department of Pharmaceutical Sciences, Faculty of Pharmacy, The University of Jordan,
Amman, Jordan
2*Department of Biotechnology, Faculty of Applied Sciences, UCSI University, Kuala
Lumpur, Malaysia
Corresponding author
Ghadeer A. R. Y. Suaifan,
Department of Pharmaceutical Sciences,
Faculty of Pharmacy,
The University of Jordan,
Amman 11942, Jordan.
Email: gh.suaifan@ju.edu.jo

Abstract
Herein we report the design, synthesis and biological evaluation of structurally modified
ciprofloxacin, norfloxacin and moxifloxacin standard drugs, featuring amide functional
groups at C-3 of the fluoroquinolone scaffold. In vitro antimicrobial testing against various
Gram-positive bacteria, Gram-negative bacteria and fungi revealed potential antibacterial and
antifungal activity. Hybrid compounds 9 (MIC 0.2668 ± 0.0001 mM), 10 (MIC 0.1358 ±
00025 mM) and 13 (MIC 0.0898 ± 0.0014 mM) had potential antimicrobial activity against a
fluoroquinolone-resistant Escherichia coli clinical isolate, compared to ciprofloxacin (MIC
0.5098 ± 0.0024 mM) and norfloxacin (MIC 0.2937 ± 0.0021 mM) standard drugs.
Interestingly, compound 10 also exerted potential antifungal activity against Candida
albicans (MIC 0.0056 ± 0.0014 mM) and Penicillium chrysogenum (MIC 0.0453 ± 0.0156
mM). Novel derivatives and standard fluoroquinolone drugs exhibited near-identical
cytotoxicity levels against L6 muscle cell-line, when measured using the MTT assay.
Keywords
Glucosamine; ciprofloxacin; antibacterial; antifungal; MTT assay; structure-activity
relationship.

1. Introduction
Since the discovery of nalidixic acid, the prototype of quinolone-based antibiotics (Fig. 1A)
[1], extensive research has been conducted around the quinolone basic scaffold to develop
novel derivatives of value as reserve antimicrobial agents with improved antibacterial activity
[1, 2]. Of these, fluoroquinolones, such as norfloxacin and ciprofloxacin (Fig. 1B, 1C), were
developed and considered to be clinically effective against a wide range of pathogenic
microorganisms due to their broad spectrum of activity. Hence, fluoroquinolones are
prescribed for the treatment of various infections including urinary tract, respiratory tract,
gastrointestinal, skin and bone infections [3]. The widespread consumption of
fluoroquinolones such as ciprofloxacin (the most dispensed antibacterial agent globally) has
led to the emergence of resistant bacterial strains [4]. Therefore, medicinal chemists have
often sought to design and synthesize novel fluoroquinolone derivatives.
A
B
C
Fig. 1 Chemical structures of quinolones. (a) Nalidixic acid, (b) Norfloxacin and (c)
Ciprofloxacin.
Recent structure-activity relationship (SAR) studies on the bacterial growth inhibitory
activities of fluoroquinolones have been primarily related to molecular modification at C-6
and C-7 [2, 5]. Additionally, the researchers synthesized various derivatives by introducing
new functional moieties to the secondary amine at the piperazinyl or hexahydropyrolo rings
at C-7. However, this chemimanipulation does not always improve the antimicrobial activity
[6]. Other studies revealed that bioisosteric replacement of C-3 carboxylic acid moiety
enhances potency. Such modification involved amidation [1], esterification to form a prodrug
[7] or conjugation with other compounds such as carbohydrates [8, 9]. Today, available

antifungal agents are limited and are known to cause toxicity to mammalian cells [10]. This
encourages investigation of novel antifungal activity of fluoroquinolones, since these agents
act by inhibiting bacterial DNA gyrase and topoisomerase IV [11, 12] which are also present
in Aspergillus flavus (A. flavus), the fungus causing aspergillosis. Moreover, the emergence
of resistant Candida strains against several antifungal agents (including fluconazole and
ketoconazole) [13, 14] encourages investigating novel synthetic fluoroquinolone derivatives
against various fungal strains.
Given the previous rationale, a new hybrid strategy was adopted for swift development of
novel biologically active fluoroquinolone derivatives. Here, we describe modification of
fluoroquinolone at C-3 by coupling with a glucosamine moiety (a carbohydrate analogue of
N-Acetyl Glucosamine (NAG), one of the bacterial cell-wall constituents) to deliver hybrid
derivatives with improved biological activities (Fig. 2). The present work describes the
synthesis of a new series of derivatives of ciprofloxacin (9 and 12), norfloxacin (10 and 13),
and moxifloxacin (11 and 14). The sugar moiety may act as a carrier in this proposed pro-
drug approach, to enhance the selective uptake of fluoroquinolones into the microbial cells,
hence enhancing selectivity and potency, and lowering cytotoxicity. Moreover, bacterial
glycosylated biogenic proteins control bacterial cells vital functions including cell division,
̕
energy conversion and motility [15, 16]. Accordingly, we synthesized novel glucosamine-
fluoroquinolone conjugates as potential antibacterial/antifungal agents. Synthesized
compounds were screened in vitro for their antimicrobial activity.
Fig. 2 Chemical Structure of D-(+)-Glucosamine hydrochloride.
2. Results and discussion
2.1 Chemistry (Synthesis)
Novel glycosylated fluoroquinolone derivatives were synthesized as depicted in Schemes 1
and 2. Compound 4 was prepared from the commercially available D-(+)-glucosamine
hydrochloride as previously described [17, 18], with slight modifications. The primary amine
of D-(+)-glucosamine was protected as an imine with 4-methoxybenzaldehyde under basic
conditions to afford compound 1 in a good yield. The alcohols of 1 were then esterified using

acetic anhydride to afford compound 2 as a white solid. The imine was selectively hydrolysed
with dilute hydrochloric acid in warm acetone to expose the protonated primary amine in 3.
Neutralization with 1 M aq. Na2CO3 gave the free amine 4 (Scheme 1).
Scheme 1: Synthesis of 1,3,4,6-O-acetylated-D-glucosamine.
To prepare the quinolone coupling partners, ciprofloxacin HCl was converted to the free base
form under basic conditions, followed by acetylation to afford compound 6 using acetic
anhydride to avoid any potential dimerization during the subsequent coupling reaction
(Scheme 2). Similarly, the norfloxacin piperazinyl amino group was protected to afford
compound 7 in good yield. Moreover, the acetyl-derived moxifloxacin (compound 8) was
synthesized by dissolving moxifloxacin HCl in CH2Cl2 and then adding Ac2O and
triethylamine, where the latter acted to free the secondary amine of the side-chain [21].
Fluoroquinolone derivatives 6-8 were treated with ethyl chloroformate and Et3N in CH2Cl2 at
-20 °C to yield the mixed anhydride. Nucleophilic acyl substitution [23, 24] with compound 4
afforded the corresponding glycosylated targets 9-11. The molar equivalents of the reactants
and the durations of the reactions were adjusted according to monitoring by TLC. Work-up
comprised washing the organic layer with water to deactivate unreacted ethyl chloroformate,
followed by washing with aq. HCl and aq. NaHCO3. Trituration with Et2O afforded gummy
products and compounds 9-11 were finally purified by column chromatography. The
synthesized acyl-glycosylated fluoroquinolone derivatives 9-11 chemical structures were
1
confirmed by FTIR, H NMR, 13C NMR, COSY, HMQC, DEPT 135 and mass spectra.

Deprotection with six equivalents of NaOMe in MeOH, according to the the Zemplén
procedure [25], led to the O-deacetylated compounds 12-14.Once the TLC indicated the
complete consumption of the starting material, the reaction was neutralized with amberlite
resin and the aqueous solution of the products was freeze-dried to obtain 12-14 in excellent
1
yields (90-96%). The structures of 12-14 were confirmed by FTIR, H NMR, 13C NMR,
COSY, HMQC, DEPT 135 and mass spectra.

OAc
OAc
OH
OH
O
N
O
O
O
O
O
O
N
O
O
N
O
O
N
O
F
F
N
N
F
F
F
H
OH
H
OH
OH
N
N
N
ii
OAc
O
OH
i
O
N
N
N
OAc
N
N
N
N
OH
N
(9)
(12)
HN
HN
O
O
O
O
OAc
OH
(6)
O
N
HCl
OAc
OH
O
N
O
N
O
N
O
F
O
O
F
OAc
N
OH
N
F
F
H
O
OAc
H
O
OH
OH
iv
OH
v
N
N
ii
+ (4)
N
N
N
HN
OAc
OH
OAc
OH
O
O
O
O
O
O
O
O
N
O
F
F
OAc
N
OH
N
F
F
H
O
OAc
H
OH
O
OH
OH
N
N
OCH3
N
N
OCH3
iii
N
N
N
O
N
O
N
O
O
N
(
11)
)
(14
NH
O
HCl
(i) 5% NaHCO3; (ii) Ac2O,CH3CO2H; (iii)Ac2O, TEA, CH2Cl2; (iv) ClCOOC2H5 , TEA, CH2Cl2; (v) MeOH, NaOMe, amberlite resin
Scheme 2: Synthesis of novel glycosylated-based fluoroquinolones.

2.2 Antibacterial Activity
Target compounds 9-14 were evaluated in vitro for their antimicrobial activity using standard
techniques against Gram-negative and Gram-positive bacteria including: (1) food poisoning
bacteria Eischerichia coli (E. coli) O157:H7; Listeria monocytogenes (L. monocytogenes),
ATCC 19115; Salmonella enterica (S. enterica), ATCC 13312); (2) Other Gram-negative
species (Pseudomonas aeruginosa (P. aeruginosa), ATCC 9627; E. coli, NCTC 11954; E. coli,
ATCC 8739 and clinical E. coli isolate (resistant to nalidixic acid, ciprofloxacin HCl and
norfloxacin antibiotics); (3) Gram-positive (Methicillin-resistant staphylococcus aureus
(MRSA), ATCC 33591 and Methicillin-sensitive staphylococcus aureus (MSSA), ATCC 9253).
MICs of the synthesized compounds were compared with the positive controls; ciprofloxacin
HCl, norfloxacin and moxifloxacin for antibacterial activity, as presented in Table 1.
Corresponding to the broad antimicrobial spectrum of fluoroquinolones, tested compounds
showed a wide-ranging antibacterial profile. Among the derivatives investigated, 9 displayed
potent antibacterial activity against S. enterica, ATCC 13312 and P. aeruginosa, ATCC 9627
(MIC 0.0111 ± 0.0629 mM and 0.0111 ± 0.0177 mM, respectively) whereas 10 displayed potent
antibacterial activity against S. enterica, ATCC 13312 (0.0113 ± 0.0269 mM). Interestingly, 9
was twice as potent as ciprofloxacin parent drug against a E. coli clinical isolate (MIC 0.2668 ±
0.0001 mM and 0.5098 ± 0.0024 mM, respectively). Similarly, 10 (MIC 0.1358 ± 00025 mM)
was approximately twice as potent and 13 (MIC 0.0898 ± 0.0014 mM) was approximately three
times more potent than the corresponding positive control norfloxacin (0.2937 ± 0.0021 mM)
against E. coli clinical isolate. On the contrary, 11 and 14 were less active than their parent
moxifloxacin drug against E. coli clinical isolate (MIC 0.4853 ± 0.0134 mM and 0.3103 ±
0.0007 mM, respectively). Notably, poor or no antibacterial activity was observed for 9 and 10
against the two Gram-positive S. aureus species (ATCC 33591 and ATCC 9253). In addition, 10
and 13 had lower antibacterial activity against E. coli O157:H7 (MIC 0.0453 ± 0.0049 and
0.0599 ± 0.0040 mM, respectively) compared to norfloxacin (MIC <0.0059 ± 0.0093 mM).
2.3 Antifungal activity
Most fungi are resistant to conventional antibacterial drugs. In this study, we aimed to investigate
whether functional group modification at the C3 position induces antifungal properties in parent
fluoroquinolones (ciprofloxacin, norfloxacin and moxifloxacin). Accordingly, the antifungal

activity of hybrid compounds was evaluated against Candida albicans (C. albicans), ATCC
10231 and other spore-forming fungi, including A. flavus, ATCC 9643; Fusarium solani (F.
solani), ATCC 36031; Stachybotrys chartarum (S. chartarum), IBT 7711; Penicillium
chrysogenum (P. chrysogenum),ATCC 10106.
As illustrated in Table 2, ciprofloxacin derivative 12 (MIC 0.2338 ± 0.0071 mM) and
norfloxacin derivatives 10 (MIC 0.3620 ± 0.0099 mM) and 13 (MIC 0.4784 ± 0.0049 mM), and
moxifloxacin derivatives 11 (MIC 0.1618 ± 0.0212 mM) and 14 (0.2069 ± 0.0106 mM)
exhibited effective antifungal properties against A. flavus, compared to their parents
ciprofloxacin (inactive) , norfloxacin (MIC 0.7829 ± 0.0926 mM) and moxifloxacin (MIC
0.2855 ± 0.0085 mM). Similarly, norfloxacin glucosamine hybrids 10 and 13 were found to be
more active antifungal agents against S. chartarum and P. chrysogenum, compared to the parent
norfloxacin. Likewise, ciprofloxacin hybrid 9 (MIC <0.0056 ± 0.0304 mM) had higher
antifungal activity than its parent ciprofloxacin HCl (MIC <0.0106 ± 0.0191 mM) and the
antifungal drug fluconazole (MIC 0.2041 ± 0.0099 mM) against the allergenic, toxic and
pathogenic spore-forming P. chrysogenum.
Remarkably, compounds 10 (MIC 0.0056 ± 0.0014 mM), 11 (MIC 0.0809 ± 0.0098 mM), 13
(MIC 0.0599 ± 0.0007 mM) and 14 (MIC 0.2069 ± 0.0453 mM) had potential antifungal activity
against C. albicans, in comparison to fluconazole (MIC 0.4081 ± 0.0375 mM). Notably, the
acetylated norfloxacin derivative 10 and moxifloxacin derivative 11 were more potent than the
deacetylated analogues 13 and 14. Moreover, ciprofloxacin analogue 9 (MIC <0.0056 ± 0.0304
mM), norfloxacin analogues 10 (MIC 0.0453 ± 0.0156 mM) and 13 (MIC 0.1196 ± 0.0135 mM),
and moxifloxacin analogues 11 (MIC 0.1618 ± 0.0214 mM) and 14 (MIC 0.0017 ± 0.0266 mM)
had better antifungal activity against P. chrysogenum than the standard antifungal drug control
fluconazole (MIC 0.2041± 0.0099 mM).
Notably, compound 14 (MIC 0.0017 ± 0.1181 mM) is 100 times more potent as an antifungal
agent than the reference fluconazole control (MIC 0.1022 ± 0.0007 mM) against the life-
threatening toxic S. chartarum fungi, commonly associated with damp building-related illnesses
(DBRI) which trigger immunologic, neurologic and oncogenic disorders [26, 27]. Furthermore,
10 (MIC 0.0453 ± 0.0191 mM) is approximately twice as active and 13 (MIC 0.1196 ± 0.0099
mM) had a comparable activity against this toxic mould, compared to the control fluconazole,
while ciprofloxacin analogues 9 and 12 were less active against S. chartarum, in comparison to

the control, as shown in Table 2. On the other hand, all tested compounds had lower antifungal
activity against A. flavus and F. solani than did fluconazole.

Table 1: Minimum inhibitory concentrations (MIC (mM ±SD)) of target compounds and corresponding positive controls against
different bacterial strains.
Compound
Microorganism
Ciprofloxacin
9
12
Norfloxacin
10
13
Moxifloxacin
11
14
HCl
<0.0052
G –ve*
G -ve
ND
ND
ND
ND
0.0244
0.1810
0.2392
<0.0005
± 0.0044
<0.0005
± 0.0021
<0.0005
± 0.0035
0.0034
ND
0.2069
±0.0644
0.2069
±0.0014
0.8275
±0.0064
0.3103
±0.0007
ND
E. coli NCTC
11954
± 0.0332
<0.0052
± 0.01474
± 0.0541
0.0489
± 0.0070 ± 0.0097
ND
ND
0.1618
±0.0495
0.1618
±0.0038
0.4853
± 0.0134
ND
E. coli ATCC
8739
± 0.0184
<0.0059
± 0.0093
0.2937
G –ve** <0.0052
± 0.0850
0.1779
± 0.0082
0.2668
± 0.0001
0.0111
± 0.0629
0.0111
± 0.0177
ND
0.2338
± 0.0304
0.7015
± 0.0064
ND
0.0453
0.0599
E. coli
± 0.0049 ± 0.0040
O157:H7
E. coli
G –
0.5098
0.1358
± 00025
0.0113
0.0898
± 0.0014
0.0149
ve***
± 0.0024
± 0.0021
<0.0059
± 0.0057
0.0980
± 0.0007
<0.0043
± 0.0104
0.0715
G –ve** <0.0052
± 0.0049
S. enterica
± 0.0269 ± 0.0049
ATCC13312
P. aeruginosa
ATCC 9627
S. aureus
G -ve
<0.0052
± 0.0014
<0.0052
ND
ND
ND
ND
0.3235
0.0518
± 0.0070
0.0244
± 0.0127
<0.0005
± 0.0325
± 0.0071
0.1618
±0.0028
0.2069
G
0.3620
0.4784
ATCC 33591
+ve**** ± 0.0042
± 0.0478
± 0.0113
± 0.0046
±0.0042
±0.0007
G +ve
<0.0052
± 0.0078
ND
ND
ND
ND
0.0244
ND
ND
ND
ND
<0.0005
± 0.0070
<0.0005
ND
ND
S. aureus
± 0.0480
<0.0059
±0.0339
ATCC 9253
L.
G +ve** 0.0005
± 0.0071
<0.0050
±0.0064
0.1034
±0.0035
monocytogenes
ATCC 19115
±
0.0095
*Penicillinase without extended-spectrum β –lactamase.
** Food-poisoning bacterial strain.
*** Fluoroquinolones resistant Escherichia coli clinical isolate.
**** Methicillin-resistant staphylococcus aureus.
ND: Not Detected (No activity observed at the maximum tested concentration).

Table 2: Minimum inhibitory concentrations (MIC (mM ±SD)) of target compounds and corresponding positive controls against
different fungi
.
Compound
Norfloxacin
Microorganism
C. albicans
Fluconazole Ciprofloxacin
(mM) HCl
9
12
10
13
Moxifloxacin
11
14
0.4081 0.0003
ND
ND
<0.0059
± 0.1181
0.7829
0.0056
0.0599
0.0011
0.0809
0.2069
± 0.0375
0.0510
± 0.0297
ND
± 0.0014
0.3620
± 0.0007
0.4784
± 0.0311
0.2855
±0.0098
0.1618
±0.0453
0.2069
0.7116
0.2338
A. flavus
± 0.0021
0.1022
± 0.0078
0.3558
± 0.0071
0.4677
± 0.0926
0.3914
± 0.0099
0.1810
± 0.0049
0.2392
± 0.0085
0.2855
±0.0212
0.3235
±0.0106
0.2069
<0.0106
± 0.0085
0.0850
F. solani
± 0.0099
0.1022
± 0.0127
0.3558
± 0.0028
0.4677
± 0.0136
0.1957
± 0.0578
0.0453
± 0.0092
0.1196
± 0.0149
0.0023
±0.0143
0.3235
±0.0057
0.0017
S. chartarum
P. chrysogenum
± 0.0007
0.2041
± 0.0283
<0.0106
± 0.0191
± 0.0064
<0.0056
± 0.0304
± 0.0028
0.2338
± 0.0057
0.1957
± 0.0191
0.0453
± 0.0099
0.1196
± 0.0481
0.0011
±0.0068
0.1618
±0.1181
0.0017
± 0.0099
± 0.0035
± 0219
± 0.0156
± 0.0135
± 0.0113
±0.0214
±0.0266
ND: Not Detected (No activity observed at the maximum tested concentration).
`

2.4. MTT assay
The cytotoxic activity of compounds (9-14) and the standard fluoroquinolones (ciprofloxacin
HCl, norfloxacin and moxifloxacin) were evaluated using an MTT assay against L6 muscle cell
line, according to Mossman protocol [22]. In brief, novel derivatives exhibited near-identical
cytotoxic profiles against L6 muscle cells, as depicted in Table 3. Glycosylated ciprofloxacin
derivatives 9 and 12 (3.6164 ± 0.0181 µM, 4.0270 ± 0.0100 µM, respectively) had higher IC50
compared to the lead ciprofloxacin HCl (3.3998±0.0083 µM). Similarly, 10 (5.3167 ± 0.0100
µM) and 13 (3.7249±0.0189 µM) had less cytotoxicity, when compared to norfloxacin
(3.4311±0.0078 µM). Whereas, compounds 11 (5.5257 ± 0.6086 µM) and 14 (5.0765 ± 0.0064
µM) had decreased the proliferation of the cells, compared to moxifloxacin (5.7783 ± 0.0120
µM).
Table 3. In vitro cytotoxicity of tested compounds
Compound
IC50 (µM)*
L6- muscle cells
3.3998 ± 0.0083
3.6164 ± 0.0181
4.0270 ± 0.0100
3.4311 ± 0.0078
5.3167 ± 0.0100
3.7249 ± 0.0189
5.7783 ± 0.0120
5.5257 ± 0.6086
5.0765 ± 0.0064
Ciprofloxacin HCl
Compound 9
Compound 12
Norfloxacin
Compound 10
Compound 13
Moxifloxacin
Compound 11
Compound 14
*IC50 values from MTT assays after treatment. The values are
mean ± SD of at least three independent experiments.

2.5. Structure-Activity Relationship
Modifications on fluoroquinolone 3-carboxylic acid group are rarely attempted due to the
importance of C-3 carboxylate pharmacophore in the binding of the drugs to DNA
topoisomerases. Dax cited the fusion of an isothiazolone ring, which serves as a carboxylic acid
mimic, to afford derivatives with higher antibacterial activities [28]. Furthermore, the
incorporation of aldehyde groups or labile carboxylate esters to quinolones can produce
derivatives with potential antibacterial activity, following their conversion in vivo to the
corresponding carboxylic acids [28]. Similarly, Bartzatt et al. synthesized ciprofloxacin ester
analogues with modified hydrophobicity and polar surface area to improve tissue penetration, yet
maintaining strong repression of penicillin-resistant E. coli [29, 30]. Accordingly,
chemimanipulation of the fluoroquinolone C-3 position is challenging and requests a further
investigation. The inspired use of carboxylic acid isosteres has redefined traditional quinolone
structure-activity relationships. Following carbohydrate-based drug discovery, commercially
available fluoroquinolones were modified by amidation with glucosamine moieties (acetylated
and deacetylated) with different lipophilicity. The data presented here show that amidation of
the fluoroquinolones with glucosamines altered the antimicrobial activity of the parent drugs.
Moreover, amidation of ciprofloxacin and norfloxacin with glucosamines (acetylated and
deacetylated) generated antibacterial activity against a quinolone-resistant E. coli clinical isolate.
However, amidation of moxifloxacin with glucosamine lowered its growth inhibitory activity
against this pathogen. Similarly, incorporation of glucosamine diminished the antibacterial
activity of moxifloxacin against S. aureus ATCC 9253 and S. enterica ATCC13312. The
molecular mechanism by which the tested compounds exert their antimicrobial activity remains
to be determined and could be attributed to several factors such as steric effects, electronic
effects, solubility effects, permeability/uptake, affinity to target enzyme, molecular masses, etc.
Remarkably, norfloxacin conjugated with the O-acetylated glucosamine moiety (10) gave
comparable or better antifungal activity, when compared to their deacetylated analogue (13)
which may be attributed to its higher lipophilicity. These results are consistent with other studies
in which an increase in the lipophilic character of fluoroquinolone analogues led to
enhancements in antimycobacterial activity [31], due to the increased penetration and consequent
concentration in the macrophages [32, 33].
3. Conclusion

A set of novel glycosylated ciprofloxacin, norfloxacin and moxifloxacin hybrids featuring amide
functional groups at C-3 of the fluoroquinolone scaffold were successfully synthesized through
various synthetic routes. Synthesized derivatives were in vitro evaluated for their antibacterial
and antifungal activity. Hybrid compounds 9, 10 and 13 were confirmed to be potential
antimicrobial agents against the resistant clinical E. coli. The observed antifungal activity of
hybrids 10 and 13 against C. albicans, S. chartarum and P. chrysogenum and compounds 10, 11,
12 and 13 against A. flavus are probably triggered by the functional group conversion at the C-3
position. Moreover, the cytotoxic activity of the synthesized compounds was evaluated against
L6 muscle cell line in vitro using the MTT assay and showed near-identical cytotoxic profiles to
the corresponding parent analogue. Accordingly, it could be proposed that the attachment of
glucosamine via an amide linkage on the 3-position of fluoroquinolone core promoted the
antibacterial and antifungal properties of the parent fluoroquinolones. Based on these results,
glycosylated fluoroquinolone analogues could be identified as preferred hits, leading into further
investigations, whereas detailed evaluation of the effect of these substituents on standard
fluoroquinolones linked to microbial transporters remains to be determined.
4. Experimental
4.1 Materials and Equipment
Ciprofloxacin hydrochloride, norfloxacin and moxifloxacin hydrochloride were obtained from
Dar Al Dawa, Jordanian Pharmaceutical Manufacturing Company and Al Taqaddom
Pharmaceutical Industries, respectively. D-(+)-Glucosamine hydrochloride, solvents and other
reagent grade chemicals were purchased from Sigma-Aldrich and used without any further
purification. TLC was performed on pre-coated TLC sheets ALUGRAM Xtra SIL G/UV254 and
compounds were visualized under UV light. The melting points were recorded with the Stuart
melting point apparatus (SMP3, UK) and Differential Scanning Calorimetry (DSC) (Mettler
Toledo, Switzerland). FTIR spectra were recorded using Shimadzu FTIR spectrophotometer
1
(Japan). H NMR,13C NMR, COSY, DEPT 135 and HQMC spectra were acquired using Bruker
Advance III spectrometers (500 MHz) (Switzerland) with deuterated solvents. Chemical shifts
are reported in parts per million (δ) relative to tetramethylsilane (TMS) as an internal standard.
1
Significant H NMR data were tabulated in the following order: the number of proton(s) and
multiplicity (s, singlet; d, doublet; t, triplet; q, quartet; and m, multiplet); J values are in Hertz

(Hz). High-resolution mass spectra (HRMS) were recorded (in positive or negative mode) using
the electrospray ion trap (ESI) technique by collision-induced dissociation on a Bruker Apex IV
(7 T) instrument (Germany). Samples were dissolved and infused using a syringe pump with a
flow rate of 120 µL min-1. External calibration was conducted using the arginine cluster in the
mass range of 175-871 Da. Mass error: 0.00-0.50 mDa.
4.2. Chemical synthesis
4.2.1. 2-Deoxy-2-(4-methoxybenzylideneamino)-D-glucopyranose (1)
4-Methoxybenzaldehyde (12 mL, 93 mmol) was added dropwise to D-(+)-glucosamine
hydrochloride (20 g, 93 mmol) in aq. NaOH (1.0 M, 96 mL). Stirring was continued at room
temperature until complete crystallization indicated the end of the reaction. The crystals were
filtered, washed with cold water (100 mL), washed with EtOH:Et2O (1:1, 100 mL) and dried in a
desiccator under vacuum overnight to obtain 1 (15.2 g, 55%) as a white powder: mp 164-168 °C
1
(Lit. [17]: mp165-166 °C); IR νmax: 1650 (N=C), 3200 cm-1 (O-H); H NMR (DMSO-d6, 500
MHz) δH 2.80 (1H, t, J = 8.4 Hz, CH), 3.13-3.18 (1H, m, CH), 3.22-3.25 (1H, m, CH), 3.41-3.43
(1H, m, CH), 3.45-3.52 (1H, m, CH), 3.73 (1H, dd, J = 5.4, 10.4 Hz, CH), 3.81 (3H, m, OCH3),
4.53 (1H, t, J = 5.7 Hz, OH), 4.70 (1H, t, J = 7.2 Hz, OH), 4.80 (1H, d, J = 5.5 Hz, OH), 4.91
(1H, d, J = 5.2 Hz, OH), 6.50 (1H, d, J = 5.5 Hz, CH), 6.99 (2H, d, J = 8.5 Hz, Ar-H), 7.70 (2
H, d, J = 8.6 Hz, Ar-H), 8.12 (1H, s, N=CH).
4.2.2. 1,3,4,6-Tetra-O-acetyl-2-deoxy-2-(4-methoxybenzylidene(amino)-ß-D-glucopyranose (2)
Compound 1 (15.1 g, 51 mmol) was added portionwise to a cold solution of pyridine (83 mL)
and Ac2O (45 mL). Stirring at 0 °C was continued for 1 h, followed by 2-d stirring at room
temperature. The reaction was monitored using TLC (n-hexane: EtOAc). Ice and cold water (300
mL) were added to induce precipitation. The precipitate was filtered, washed with cold water
(185 mL) and dried in a desiccator under vacuum overnight, yielding 2 (16.6 g, 70%) as a white
powder; mp 179-183.1 °C (Lit. [17]: mp 180-182 °C), IR νmax 1750 (C=O), 1650 cm-1 (C=N); 1H
NMR (DMSO-d6, 500 MHz) δH 1.82 (3H, s, CH3CO), 1.99 (6H, s, 2 × CH3CO), 2.48 (3H, s,
CH3CO), 3.45 (1H, m, H-2), 3.77 (3H, s, CH3O), 3.95-4.04 (1H, m, H-5), 4.20-4.40 (2H, m, H-

6α, H-6β), 4.97 (1H, t, J = 9.7 Hz, H-4), 5.45 (1H, t, J = 9.7 Hz, H-3), 6.07 (1H, d, J = 8.2 Hz,
H-1), 6.99 (2H, d, J = 8.6 Hz, Ar-H), 7.66 (2 H, d, J = 8.6 Hz, Ar-H), 8.29 (1H, s, N=CH).
4.2.3. 1,3,4,6-Tetra-O-acetyl-ß-D-glucosamine hydrochloride (3)
Compound 2 (16.5 g, 35 mmol) was added to a mixture of aq. HCl (5.0 M, 8.3 mL) and warm
acetone (150 mL), leading to the formation of a white precipitate after a few min. The mixture
was cooled. Stirring was resumed for 2 h after adding Et2O (150 mL). Filtration and washing
with Et2O (50 mL) afforded 3 (12.4 g, 92 %) as a white precipitate; mp 292-230 °C (Lit. [17]:
mp 235 °C), IR νmax: 2800 (N+H3), 1750 cm-1 (C=O) ; 1H NMR (DMSO-d6, 500 MHz) δH 2.00
(3H, s, CH3CO), 2.04 (6H, s, 2 × CH3CO), 2.12 (3H, s, CH3CO), 3.55 (2H, t, J = 9.5 Hz, H-2),
3.99-4.05 (2H, m, H-5, H-6), 4.20 (1H, dd, J = 4.1, 12.4 Hz, H-6), 4.95 (1H, t, J = 9.6 Hz, H-4),
5.37 (1H, t, J = 9.7 Hz, H-3), 5.94 (1H, d, J = 8.6 Hz, H-1), 8.87 (3H, s, N+H3).
4.2.4. 1,3,4,6-Tetra-O-acetyl-ß-D-glucosamine (4)
Compound 3 (6.0 g, 16 mmol) was dissolved in CH2Cl2 (85 mL). Aq. Na2CO3 (1.0 M, 85 mL)
was added and the solution was stirred for 30 min. Extraction with CH2Cl2, washing with water,
drying over anhydrous Na2SO4 and evaporation afforded 4 (4.7 g, 84%) as a white powder; mp
1
138-142 °C (Lit. [18]: mp.138 °C); H NMR (DMSO-d6, 500 MHz) δH 1.65 (2H, s, NH2), 2.00
(9H, s, 3 × CH3CO), 2.12 (3H, s, CH3CO), 2.76 (1H, dd, J = 8.8, 9.7 Hz, H-2), 3.96-3.98 (2H, m,
H-5, H-6), 4.17 (1H, dd, J = 5.1, 13.0 Hz, H-6), 4.82 (1H, t, J = 9.6 Hz, H-4), 5.06 (1H, t, J = 9.6
Hz, H-3), 5.55 (1H, d, J = 8.5 Hz, H-1).
4.2.5. 1-Cyclopropyl-6-fluoro-4-oxo-7-(piperazin-1-yl)quinoline-3-carboxylic acid (5)
Ciprofloxacin HCl (8.00 g, 21.7 mmol) was dissolved in water (267 mL), followed by the
addition of excess aq. NaHCO3 (5%, 300 mL). The precipitate was filtered, washed with cold
water and dried in a desiccator under reduced pressure to produce ciprofloxacin 5 (6.8 g, 95%) as
1
a white solid: mp 253-255 °C (Lit. [19]; m.p. 255-257 °C); H NMR (DMSO-d6, 500 MHz) δH
1.14 (2H, m, cyclopropane 2,3-H2), 1.28 (2H, q, J = 6.2 Hz, cyclopropane 2,3-H2), 2.87 (4H, m,
2 × NCH2), 3.20 (4H, m, 2 × NCH2), 3.78 (1H, m, cyclopropane 1-H), 7.49 (1H, d, J = 7.4 Hz,
H-8), 7.85 (1H, d, J = 13.4 Hz, H-5), 8.62 (1H, s, H-2).

4.2.6. 7-(4-Acetylpiperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid (6)
Ac2O (0.5 mL) was added to ciprofloxacin 5 (1.5 g, 4.5 mmol) in CH3CO2H (12.0 mL). The
reaction was warmed to no more than 35°C for 1 h and left for a few min to crystallize at room
temperature. The crystals were filtered, washed with Et2O and dried overnight in a desiccator to
afford 6 (0.94 g, 56%) as a white solid; mp 253-256 °C (Lit. [20] mp 255 °C); IR νmax 1716 cm-1
(C=O); 1H NMR (DMSO-d6, 500 MHz) δH 1.15 (2H, m, cyclopropane 2,3-H2), 1.27 (2H, q, J =
6.2 Hz, cyclopropane 2,3-H2), 2.02 (3H, s, CH3CO), 3.32 (4H, m, 2 × NCH2), 3.63 (4H, m, 2 ×
NCH2), 3.77 (1H, m, cyclopropane 1-H), 7.53 (1H, d, J = 7.4 Hz, H-8), 7.89 (1H, d, J = 13.2 Hz,
H-5), 8.61 (1H, s, H-2), 15.02 (1H, s, COOH).
4.2.7. 7-(4-(Acetyl)-piperazin)-1-ethyl-6-fluoro-4-oxo-1,4-dihydroquinoline-3-carboxylic acid
(7)
Ac2O (1.8 mL, 19.5 mmol) was added dropwise to a solution of norfloxacin (4.0 g, 13 mmol) in
glacial acetic acid (150 mL). The reaction was warmed to no more than 35°C for 1 h and left for
a few minutes to crystallize at room temperature. The crystals were filtered, washed with Et2O
and dried overnight in a desiccator to afford 7 (3.1 g, 69%) as a white powder; mp 297-302 °C
(Lit. [20, 21] 300 °C); IR νmax 1722 cm-1 (C=O); 1H NMR (DMSO-d6, 500 MHz) δH 1.37 (3H, t,
J = 6.9 Hz, CH2CH3), 2.02 (3H,s, CH3CO), 3.35 (4H, m, 2 × NCH2), 3.61 (4H, m, 2 × NCH2),
4.54 (2H, q, J = 6.9 Hz, CH2CH3), 7.16 (1H, s, H-8), 7.90 (1H, d, J = 12.0 Hz, H-5), 8.92 (1H, s,
NCH=C), 15.27 (1H, s, COOH).
4.2.8. 7-(1-Acetylhexahydro-1H-pyrrolo[3,4-b]pyridin-6(2H)-yl)-1-cyclopropyl-6-fluoro-8-
methoxy-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (8)
Moxifloxacin hydrochloride (4.85 g, 11 mmol) was dissolved in CH2Cl2 (30 mL). Ac2O (1.6 mL,
16.5 mmol) was added along with Et3N (3.1 mL, 22 mmol). The reaction was slightly warmed
while stirring was continued until TLC (chloroform (CHCl3): methanol (MeOH)) indicated the
end of the reaction. The solvent was evaporated under reduced pressure, MeOH was added and
the reaction mixture was left standing overnight. Evaporation and trituration with Et2O afforded
8 (quant., [21]) as a white solid; IRvmax 1746.86 cm-1 (C=O); 1H NMR (DMSO-d6, 500 MHz) δH
0.77, 1.08 and 1.25 (4H, m, cyclopropyl 2,3-H4), 1.39 (2H, m, CH2), 1.83 (4H, m, CH2), 2.11

(3H, s, CH3CO), 2.22 (1H, m, CH), 3.22-4.06 (9H, m, CH2, CH, OCH3) 7.73 (1H, d, J =13.8 Hz,
H-5), 8.72 (1H, s, NCH=C), 14.96 (1H, bs, COOH).
4.2.9. 7-(4-(Acetylpiperazin-1-yl)-1-cyclopropyl-6-fluoro-4-oxo-N-(1,3,4,6-tetra-O-acetyl-2-
deoxy-D-glucopyranose-2-yl)-1,4-dihydroquinoline-3-carboxamide (9)
Ethyl chloroformate (0.5 mL, 5.4 mmol) was added dropwise to Et3N (0.8 mL, 5.4 mmol) and
compound 6 (2 g, 5.4 mmol) in cold CH2Cl2 (150 mL, -20 °C). The stirred solution was
monitored by TLC (CHCl3: MeOH) until formation of the mixed anhydride was complete.
Compound 4 (1.88 g, 5.4 mmol) in cold CH2Cl2 (30 mL, -20 °C) was then added dropwise and
the mixture was stirred at -20 °C for 5 h and at room temperature for 2 d. The solution was
washed with water (180 mL), aq. HCl (1.0 M, 180 mL), aq. NaHCO3 (1.0 M, 180 mL) and water
(180 mL). The organic layer was dried over anhydrous Na2SO4. The gummy evaporation residue
was triturated with Et2O and purified using silica gel column chromatography (CHCl3 : MeOH)
to afford 9 (0.50 g, 15%) as a white solid; m.p 151-153 °C; IR νmax; 1741 (C=O amide), 1654
1
cm-1 (C=O); H NMR (CDCl3, 500 MHz) δH 1.14 (2H, m, cyclopropyl 2,3-H2), 1.39 (2H, m,
cyclopropyl 2,3-H2), 1.92 (3H,s, CH3CO), 1.99 (3H, s, CH3CO), 2.01 (3H, s, CH3CO), 2.06
(3H,s, CH3CO), 2.13 (3H, s, CH3CO), 3.20 (2H, s, NCH2), 3.26 (1H, m, cyclopropyl 1-H), 3.41
(2H, s, NCH2), 3.66 (2H, s, NCH2), 3.82 (2H, s, NCH2), 3.89 (1H, m Gluc H-5), 4.11 (2H, m,
Gluc H-2, Gluc H-6), 4.30 (1H, m, Gluc H-6), 5.10 (1H, t, J = 8.9 Hz, Gluc H-4), 5.62 (1H, t, J =
8.9 Hz, Gluc H-3), 6.11 (1H, d, J = 6.2 Hz, Gluc H-1),7.28 (1H, bs, H-8), 7.99 (1H, d, J = 11.5
Hz, H-5), 8.75 (1H, s, NCH=C), 10.2 (1H, d, J = 8.2 Hz, CONH); 13C NMR (CDCl3, 125 MHz)
8.20 (cyclopropane 2,3-C2), 20.65-21.30 (5 × CH3CO), 34.84 (cyclopropane C-1), 41.12 (NCH2),
46.18 (NCH2), 49.54 (NCH2), 50.49 (NCH2), 53.74 (Gluc 2-C), 61.83 (Gluc 6-C), 68.41 (Gluc 4-
C), 72.05 (Gluc 3-C), 72.64 (Gluc 5-C), 91.84 (Gluc 1-C), 105.02 (C-8), 110.61 (Cq), 112.97 (C-
5), 121.00 (Cq), 138.41 (Cq), 146.00 (Cq), 147.11 (NCH=C), 152.00-154.00 (2 × Cq), 165.49,
169.07, 169.61, 170.09, 170.70, 175.30 (5 × CH3CO, CONH); MS (ESI +) m/z 666.23835 [M +
Na – MeCO2H]+ (12C3013C1H35FN4NaO10 requires 666.22685), 665.23717 [M + Na – MeCO2H]+
(12C31H35FN4NaO10 requires 665.22350).
4.2.10. 7-(4-Acetylpiperazin-1-yl)-1-ethyl-6-fluoro-4-oxo-N-(1,3,4,6-tetra-O-acetyl-2-deoxy-D-
glucopyranose-2-yl)-1,4-dihydroquinoline-3-carboxamide (10)

Ethyl chloroformate (0.2 mL, 2.1 mmol) was added drop-wise to Et3N (0.3 mL, 2.1 mmol) and 7
(0.500 g, 1.39 mmol) in cold CH2Cl2 (250 mL, -20 °C). The stirred solution was monitored by
TLC until formation of the mixed anhydride was complete. Then 4 (0.7250 g, 2.025 mmol) in
cold CH2Cl2 (30 mL, -20 °C) was added dropwise. The mixture was stirred at -20 °C for 5 h and
at room temperature for 2 d. The mixture was washed with water (300 mL), aq. HCl (1.0 M, 300
mL), aq. NaHCO3 (1.0 M, 300 mL) and water (300 mL). The organic layer was dried over
anhydrous Na2SO4. The gummy evaporation residue was triturated with Et2O and purified using
silica gel column chromatography (CHCl3: MeOH) to afford 10 (0.65 g, 47%) as a white
powder; mp 135-139 °C; IR νmax; 3226 (NH), 1650 cm-1 (C=O); 1H NMR (CDCl3, 500 MHz) δH
1.50 (3H, t, J = 7.2 Hz, CH2CH3), 1.91 (3H, s, CH3CO), 1.97 (3H, m, CH3CO), 2.01 (3H, s,
CH3CO), 2.05 (3H, s, CH3CO), 2.11 (3H, s, CH3CO), 3.17 (2H, m, NCH2), 3.23 (2H, m, NCH2),
3.65 (2H, m, NCH2), 3.80 (2H, m, NCH2), 3.90 (1H, m, Gluc H-5), 4.10 (2H, m, Gluc H-2, Gluc
H-6), 4.22 (2H, q, J = 7.2 Hz, CH2CH3), 4.31 (1H, dd, J = 4.5, 12.3 Hz, Gluc H-6), 5.10 (1H, t, J
= 9.6 Hz, Gluc H-4), 5.58 (1H, t, J = 9.6 Hz, Gluc H-3), 6.10 (1H, d, J = 8.6 Hz, Gluc H-1), 6.75
(1H, d, J = 6.7 Hz, H-8), 8.02 (1H, d, J = 13.0 Hz, H-5), 8.60 (1H, s, NCH=C), 10.25 (1H, d, J =
8.4 Hz, CONH); 13C NMR (CDCl3, 500 MHz) δC 14.41 (NCH2CH3), 20.63-21.28 (5 × CH3CO),
41.09 (2 × NCH2), 46.13 (2 × NCH2), 49.25 (NCH2CH3), 53.55 (Gluc C-2), 61.83 (Gluc C-6),
68.40 (Gluc C-4), 72.10 (Gluc C-3), 72.62 (Gluc C-5), 92.15 (Gluc C-1), 104.06 (C-8), 110.80
(Cq), 113.23 (C-5), 123.02 (Cq), 136.44 (Cq), 144.79 (Cq), 146.92 (NCH=C), 152.00-154.00 (2 ×
Cq), 165.58, 169.06, 196.60, 170.56, 170.68, 175.15 (5 × CH3CO, CONH); MS (ESI +) m/z
716.2748 [M + Na]+ (12C2913C3H39FN4NaO12 requires 716.2547), 715.2686 [M + Na]+
(12C3013C2H39FN4NaO12 requires 715.2513), 714.2661 [M + Na]+ (12C3113C1H39FN4NaO12
requires 714.2480), 713.2637 [M + Na]+ (12C32H39FN4NaO12 requires 713.2446), 672.2551 [M +
Na – H2CCO]+ (12C2913C1H37FN4NaO11 requires 672.2374), 671.2500 [M + Na – H2CCO]+
(12C30H37FN4NaO11 requires 671.2341).
4.2.11.
7-(1-Acetylhexahydro-1H-pyrrolo[3,4-b]-pyridin-6(2H)-yl)-1-cyclopropyl-6-fluoro-8-
methoxy-4-oxo-N-(1,3,4,6-tetra-O-acetyl-2-deoxy-D-glucopyranose-2-yl)1,4-dihydroquinoline-3-
carboxamide (11)
Ethyl chloroformate (0.266 mL, 2.8 mmol) was added to Et3N (0.291 mL, 2.1 mmol) and 8 (1.0
g, 2.3 mmol) in cold CH2Cl2 (40 mL, -20 °C). The stirred solution was monitored by TLC until

formation of the mixed anhydride was complete. Then 4 (0.945 g, 3.45 mmol) in cold CH2Cl2
(30.0 mL, -20 °C) was added dropwise. The mixture was stirred at -20 °C for 5 h and then left to
stir at room temperature for 2 d. The solution was washed with water (75 mL), aq. HCl (1.0 M,
75 mL), aq. NaHCO3 (1.0 M , 75 mL) and water (75 mL). The organic layer was dried over
anhydrous Na2SO4, The evaporation residue was triturated with Et2O to afford solid material and
purified using silica gel column chromatography (CHCl3 : MeOH) to afford 11 (196 mg, 11%) as
a white powder; mp 150-154 °C; IR νmax: 2829 (NH), 1724 (C=O), 1583 cm-1 (C=O, amide); 1H
NMR (DMSO-d6, 500 MHz) δH 0.81, 1.03, 1.13 1.25 (4H, m, cyclopropane 2,3-H4), 1.60 (2H, m,
CH2), 1.87 (2H, m, CH2), 1.98 (3H, s, CH3CO), 2.05 (3H, s, CH3CO) 2.08 (3H, s, CH3CO), 2.12
(3H, s, CH3CO), 2.18 (3H, s, CH3CO), 2.21 (1H, m, CH), 3.24-4.17 (13H, m, 3 × CH2, -OCH3,
CH, Glu-H-2, Glu-H-5, Glu-H-6), 4.36 (1H, dd, J = 4.6, 9.4 Hz, Gluc H-6), 5.16 (1H, t, J = 9.4
Hz, Gluc H-4), 5.29 (1H, m, CH), 5.63 (1H, t, J = 9.6 Hz, Gluc H-3), 6.17 (1H, d, J = 8.4 Hz,
Gluc H-1), 7.81 (1H, d, J = 13.6 Hz, H-5), 8.77 (1H, s, NCH=C), 10.29 (1H, d, J = 7.8 Hz,
13
CONH); C NMR (DMSO-d6, 500 MHz) δC 3.75 (CH2), 5.64 (CH2), 15.92 (CH3CO), 16.02
(CH3CO), 16.22 (CH3CO), 16.85 (CH3CO), 17.47 (CH3CO), 19.96 (CH2), 20.45 (CH2), 30.28
(CH), 35.00 (CH), 37.36 (CH2), 43.25 (CH2), 45.40 (CH), 51.27 (CH2), 50.65 (Glu-2), 56.27 (-
OCH3), 57.10 (Gluc C-6), 67.56 (Gluc C-3), 63.67 (Gluc C-4), 67.85 (Gluc C-5), 87.34 (Gluc C-
1), 103.75 (C-5), 105.17 (Cq), 117.39 (Cq), 129.03 (Cq), 136.52 (Cq), 144.48 (NCH=C), 161.80,
164.34, 164.89, 165.33, 165.82, 165.96 (5 CH3CO, CONH) ; MS (ESI +) m/z 773.3047 [M+H]+
(C37H46FN4O13 requires 773.3045), 795.2846 [M+Na]+ (C37H45FN4NaO13 requires 795.2865),
796.28873 [M + Na + H] (12C3613C1H46FN4NaO13 requires 796.2898.
3.2.12. 7-(4-Acetylpiperazin-1-yl)-1-cyclopropyl-N-(2-deoxy-D-glucopyranose-2-yl)-6-fluoro-4-
oxo-1,4-dihydroquinoline-3-carboxamide (12)
NaOMe (0.1 g, 2.1 mmol) was added portionwise to 9 (0.30 g, 0.35 mmol) in MeOH (30 mL).
The mixture was stirred for 2 h. The solution was neutralized using Amberlite resin, filtered,
evaporated and redissolved in water. The solution was freeze-dried to obtain 12 (0.20 g, 90%) as
a white powder; mp 154-165 °C; IR νmax 3289 (CONH), 1621 (CONH) cm-1; 1H NMR (DMSO-
d6, 500 MHz) δH 1.06 (2H, m, cyclopropane 2,3-H2), 1.26 (2H, m, cyclopropane 2,3-H2), 2.02
(3H, s, CH3CO), 3.12 (1H, m, Gluc H-4), 3.15-3.19 (4 H, m, 2 × NCH2), ), 3.26 (1H, m,
cyclopropane 1-H), 3.29 (1H, m, Gluc H-6), 3.47-3.53 (2H, m, Gluc H-3, Gluc H-5), 3.62 (4 H,

m, 2 × NCH2), 3.73-6.54 (7H, m, Gluc H-6, Gluc H-2, Gluc H-1, 4 × OH), 7.46 (1H, bs, H-8),
13
7.82 (1H, d, J = 13.4 Hz, H-5), 8.59 (1H, bs, NCH=C), 9.92 (1H, d, J = 8.5 Hz, CONH); C-
NMR (DMSO-d6, 500 MHz) δC 7.76 (cyclopropane 2,3-C2), 21.37 (CH3CO), 39.99
(cyclopropane C-1), 50.23 (3H, s, CH3CO), 49.98 (4 × NCH2), 54.49 (Gluc C-2), 61.59 (Gluc C-
6), 71.65 (Gluc C-4), 71.78 (Gluc C-3), 72.72 (Gluc C-5), 91.27 (Gluc C-1), 106.87 (C-8),
110.76 (Cq), 117.85 (C-5), 121.53 (Cq), 138.80 (Cq), 144.37 (Cq), 147.24 (NCH=C), 147.61 (Cq),
152.13 (Cq-quat), 164.44, 168.86, 174.64 (2 × CH3CO, CONH); MS (ESI +) m/z 557.2018 [M +
Na]+ (C25H31N4NaO8 requires 557.2024).
4.2.13. 7-(4-Acetylpiperazin-1-yl)-N-(2-deoxy-D-glucopyranose-2-yl)-1-ethyl-6-fluoro-4-oxo-
1,4-dihydroquinoline-3-carboxamide (13)
NaOMe (0.200 g, 2.58 mmol) was added portionwise to 10 (0.40 g, 0.46 mmol) in MeOH (50
mL). After 2 h, TLCindicated the formation of product. The solution was then neutralized using
Amberlite resin, filtered, evaporated and redissolved in water. The solution was freez dried to
obtain 12 (0.20 g, 96%) as a pale yellow powder; mp 170-172 °C; IR νmax 3315 (CONH), 1540
(CONH) cm-1; 1H-NMR (DMSO-d6, 500 MHz) δH 1.35 (3H, t, J = 7.1 Hz, CH2CH3), 2.01 (3H, s,
CH3CO), 3.19-3.70 (8H, m, 2 × NCH2, OH, Gluc H-3, Gluc H-4, Gluc H-5), 3.60 (6H, m, 2 ×
NCH2, 2 × Gluc H-6), 3.81 (1 H, m, Gluc H-2), 4.44 (2H, q, J = 7.1 Hz, CH2CH3), 4.50 (1H, m,
OH), 4.99 (1H, m, Gluc H-1), 7.08 (1H, bs, H-8), 7.85 (1H, d, J = 13.5 Hz, H-5), 8.72 (1H, s,
NCH=C), 9.97 (1H, d, J = 8.6 Hz, CONH); 13C-NMR (DMSO-d6. 500 MHz) δC 14.85
(CH2CH3), 21.62 (CH3CO), 40.81(NCH2), 45.49 (CH2CH3), 48.54, 49.69, 50.06 (3 × NCH2),
54.31 (Gluc), 61.00 (Gluc C-6), 71.57 (Gluc), 71.62 (Gluc), 74.84 (Gluc), 91.08 (Gluc), 106.12
(C-8), 111.67 (C-5), 147.53 (NCH=C), 165.85 (CH3CO); (ESI +) m/z 545.2181 [M + Na]+
(C24H31FN4NaO7 requires 545.2024), 527.2050 [M + Na – H2O]+ (C24H29FN4NaO7 requires
527.1919).
4.2.14. 7-(1-Acetylhexahydro-1H-pyrrolo[3,4-b]-pyridin-6(2H)-yl)-1-cyclopropyl-N-(2-deoxy-D-
glucopyranose-2-yl)-6-fluoro-8-methoxy-4-oxo- 1,4-dihydroquinoline-3-carboxamide (14)
NaOMe (0.21 g, 3.9 mmol) was added portionwise to 11 (0.50 g, 0.65 mmol) MeOH (30 mL).
After 2 h, TLC indicated the formation of product. The solution was neutralized using Amberlite
resin. The evaporation residue was recrystallized from MeOH/Et2O to afford 14 (0.40 g, 90%) as

a white powder; mp 156-169 °C; IR νmax 3274 (OH) 2934 (NH), 1643 (CONH), 1536 cm-1 (C=O,
1
amide); H NMR (CDCl3, 500 MHz) δH 0.74, 0.90, 0.99, 1.15 (4H, m, cyclopropane 2,3-H4),
1.41, 1.73 (4H, m, 2 × CH2), 2.01 (3H, s, CH3CO), 2.13 - 2.23 (1H, m, CH), 3.11- 4.01 (17H, m,
3 x× CH2, CH, Gluc H-2, Gluc H-4, Gluc H-5, 2 × Gluc H-6, -OCH3, 2 × OH), 4.32-4.55 (2H, m,
Gluc H-3, OH), 4.99 (2H, m, Gluc H-1, CH), 6.55 (1H, bs, OH), 7.55 (1H, dd, J = 7.5, 11.0 Hz,
H-5), 8.57 (1H, s, NCH=C), 10.00 (1H, d, J = 8.6 Hz, CONH); 13C-NMR (CDCl3, 500 MHz) δC
8.59 (CH2), 10.23 (CH2), 21.63 (CH3CO), 23.82, 24.43, 24.92, 25.07 (2 × CH2), 35.23 (CH),
39.82 (CH), 41.66 (CH2), 48.07 (CH2), 48.47 (CH2), 50.10 (CH), 54.20 (Gluc), 56.07 (Gluc C-
6), 61.42 (OCH3), 71.40 (Gluc), 71.54 (Gluc), 72.45 (Gluc), 91.01 (Gluc C-1), 106.82 (C-5),
110.24 (Cq), 120.50 (Cq), 136.00 (Cq), 142.00 (Cq), 154.20 (Cq), 149.49 (NCH=C), 164.39,
170.33, 174.28 (CH3CO, CONH)); MS (ESI +) m/z 627.2576 [M + Na]+ (C29H37FN4NaO9
requires 627.2442).
4.3. In vitro antimicrobial testing
Target compounds 9-14 were evaluated in vitro for their antimicrobial activities against Gram-
negative bacteria E. coli, O157:H7; S. enterica, ATCC 13312; L. monocytogenes, ATCC 19115;
P. aeruginosa, ATCC 9627; E. coli, NCTC 11954; E. coli, ATCC 8739 and clinical E. coli
isolate (Resistant to nalidixic acid, ciprofloxacin HCl and norfloxacin and acquired from
Biolab® (Amman-Jordan)); Gram-positive bacteria (MRSA, ATCC 33591 and MSSA, ATCC
9253); non-spore-forming fungi C. albicans, ATCC 10231) and spore-forming fungi A. flavus,
ATCC 9643; F. solani, ATCC 36031; S. chartarum provided by Professor Naresh Magan
(Cranfield University, UK)) and P. chrysogenum, ATCC 10106. All media were prepared under
sterile conditions following manufacturers’ instructions. Bacterial broth cultures stocks were
cultivated in the appropriate medium at 37 °C for 24 h before testing. 0.5 McFarland standard
were used to approximate visually the concentration of cells in a suspension.
Fungal conidia were attained from fungi stock previously cultivated on malt extract agar at 30 °C
for 7 d. Fungal conidia were transferred into normal saline (3.0 mL) with a cotton swap
previously impregnated in Tween 20. Then, conidial suspensions were vortexed vigorously for
20 sec, to prevent spore clumping and were left standing at RT for 15 min. The upper layer then
was transferred into a sterile falcon tube and adjusted to 0.5 McFarland at 530 nm to yield 4.38-
10.00 × 106 suspensions of sporangiospores (A. flavus, 5.00 × 106; F. solani, 4.38 × 106; S.

chartarum, 7.50 × 106; P. chrysogenum, 10.00 × 106). Mean spore counts from three trials were
determined using a hemocytometer. 1:50 Dilutions of the stock suspensions with malt extract
broth gave the final working suspension.
The minimal inhibitory concentrations (MICs) against study pathogens were determined for each
of the target compounds using the micro-broth dilution method. MIC was defined as the lowest
concentration of the tested compound showing no microbial viability. A stock solution of the
target compounds in DMSO was prepared, then 100 µL was transferred into each well of 96-well
plate having 100 µL of the appropriate broth, followed by two-fold serial dilution in subsequent
wells. 10 µL of the microbial suspension (106 strength) was added to each well, incubated at
37°C for 24 h and the volume was adjusted to 200 µL with appropriate broth in each well. All
experiments were conducted in triplicate. The absorbance of light at 625 nm was measured using
an Epoch spectrophotometer. Ciprofloxacin HCl, norfloxacin and moxifloxacin HCl were used
as positive controls and negative control (DMSO) samples, along with proper sterility and
growth controls, were prepared and evaluated.
The MIC protocol for the spore-forming fungi was similar to the bacterial protocol; however,
MICs were recorded after 48 h of incubation at 30°C and absorbance was measured at 405 nm.
Stock solutions of compounds had a 4.0 mg mL-1 concentration. Fluconazole was used as a
positive control; DMSO was used as negative control along with proper sterility and growth
controls. Experiments were conducted in triplicate.
4.4. Cell viability testing with MTT [3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium
bromide] assay
L6 rat muscle myoblast cells (ATCC CRL-1458) were cultured as a monolayer under 95% air,
5% CO2 at 37°C in glucose-DMEM supplemented with 10% FBS, 1 mM L-glutamine, 100 U
mL-1 penicillin and 0.1 mg mL-1 streptomycin. Cells were sub-cultured at a ratio of 1:20. The
cytotoxicity study was carried out according to the Mossman protocol [22] using L6 rat muscle
cells with a final density of 2 × 104 cells mL-1. Cells were suspended (20 µL well-1) and seeded
into a 96-well plate at a density of 2 × 104 cells/well and incubated at 37°C, 5% CO2, 95% air,
and 100% relative humidity to allow attachment. After 24 h, cells were treated with different
concentrations of glycosylated fluoroquinolones 9-14, standard fluoroquinolones (ciprofloxacin

HCl, norfloxacin and moxifloxacin) and DMSO (0.1%) as a negative control. Aliquots of novel
derivatives and standards (100 µL) at different concentrations were added to the appropriate
wells and the plates were incubated for 24 h. The medium was then replaced in the dark with 100
ꢀL of fresh medium/well containing 0.5 mg mL-1 MTT (filtered with 0.22 µm nylon) and
incubated for another 4 h. The supernatant was removed and 100 ꢀL of 100% DMSO was added
to each well. Positive-control wells contained cells in complete media without analogues or
drugs. The absorbance was read at 570 nm using the FLUO star Omega microplate reader. The
cytotoxic effect of the analogues was expressed as cell viability using the following formula:
Mean OD of the test sample
%Cell viability =
X 100%
Mean OD of the vehicle control (negative control)
The tests were conducted in triplicate and IC50 values were calculated using dose-effect curves
and expressed as a concentration (μM) of the drug.
Declaration of competing interest
Disclosures related to glycosylated 3-substituted fluoroquinolone derivatives, preparation
methods thereof, and their use in the treatment of antimicrobial infections have been registered
for patents (Reg. No. P/JO/2019/000097) and (Reg. No. PCT/JO2019/050011).
Acknowledgment
The authors would like to acknowledge the Deanship of the Scientific Research for financial
support [Grant numbers 2016, 2213].
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