Discovery of the highly potent Fluoroquinolone-based Benzothiazolyl-4- thiazolidinone hybrids as antibacterials

Article abstract of DOI:10.1111/cbdd.12299

A new series of fluoroquinolone-based benzothiazolyl-4-thiazolidinone hybrids has been yielded via sulfated tungstate-promoted highly accelerated N-formylation at a piperazine residue of ciprofloxacin and norfloxacin entities. The formylated fluoroquinolone moieties were then coupled with substituted 2-aminobenzothiazoles, which were generated from their respective para-substituted amines to form corresponding Schiff base intermediates. The Schiff bases were then treated with thioglycolic acid to equip a new class of 4-thiazolidinones to be analyzed for their antibacterial effects against two Gram-positive (Staphylococcus aureus and Bacillus subtilis) and two Gram-negative (Escherichia coli and Pseudomonas aeruginosa) bacterial strains and were found highly potent with lowest Minimum inhibitory concentrations (MIC), 1-2 μg/mL, that is, more potent than control drugs ciprofloxacin (3.12-6.25 μg/mL). Initial outcomes provided for these novel molecular systems will aid researchers to design and develop new antibacterial drugs. The structural assignments of the new products were done on the basis of FT-IR, ¹H NMR and ¹³C NMR spectroscopy, and elemental analysis. A truly rationalized design of a new class of 4-thiazolidinones revealed potent antibacterial efficacies with lowest MICs 1-2 μg/mL when compared to control drug ciprofloxacin at 3.12-6.25 μg/mL. Combination of electron-withdrawing substituent on the benzothiazole ring and norfloxacin entity furnished anti-Gram-positive effects, as well as combination of electron-releasing substituent with ciprofloxacin entity furnished anti-Gram-negative potency. Two thiazole rings positively enhanced the potency of the final scaffolds.

Full text of DOI:10.1111/cbdd.12299

Received Date : 21-Sep-2013
Revised Date : 06-Jan-2014
Accepted Date : 27-Jan-2014
Article type : Research Letter
Discovery of the highly potent fluoroquinolone based benzothiazolyl-4-
thiazolidinone hybrids as Antibacterials
Rahul V. Patel and Se Won Park
Organic Research Laboratory, Department of Bioresources and Food Science, College of Life
and Environmental Sciences, Konkuk University, Seoul- 143 701, South Korea.
Correspondence: Email: rahul.svnit11@gmail.com, sewpark@konkuk.ac.kr, Tel.: 82 2450
3945, Fax: 82 2 3436 5439.
Short Title: Fluoroquinolone based 4-thiazolidinones as antibacterials
Keywords: N-formylation, Sulfated tungstate, benzothiazole, piperazine, fluoroquinolones,
Schiff base, 4-thiazolidinones, antibacterial activity
Abstract
A new series of fluoroquinolone based benzothiazolo-4-thiazolidinone hybrids has been yielded
via sulfated tungstate promoted highly accelerated N-formylation at a piperazine residue of
ciprofloxacin and norfloxacin entities. The formylated fluoroquinolone moieties were then
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 an
'Accepted Article', doi: 10.1111/cbdd.12299
This article is protected by copyright. All rights reserved.

coupled with substituted 2-aminobenzothiazoles which were generated from their respective
para-substituted amines to form corresponding Schiff base intermediates. The Schiff bases were
then treated with thioglycolic acid to equip a new class of 4-thiazolidinones to be analyzed for
their antibacterial effects against two Gram-positive (S. aureus and B. subtilis) and two Gram-
negative (E. coli and P. aeruginosa) bacterial strains and were found highly potent with lowest
MICs, 1-2 µg/mL, i.e., more potent than control drugs ciprofloxacin (3.12-6.25 µg/mL). Initial
outcomes provided for these novel molecular systems will aid several researchers to design and
develope new anti-bacterial drugs. The structural assignments of the new products were done on
the basis of FT-IR, 1H NMR and 13C NMR spectroscopy and elemental analysis.
Introduction
In latest several years, there has been growing interest to design hybrid drug-like
molecules with the structural features different than existing drugs because the emergence of
resistance among several Gram-positive [1, 2] and Gram-negative [3, 4] bacterial strains can be
considered as the severe therapeutic problem [5]. New hybrid molecules can be regarded to act in
an identical way in terms of mode of action by which the parent drugs act, while in these designs
there may least possibilities to develop drug resistance due to structural variations. Treatment of
patients having infections of multi-drug resistant microbes is often pandemic while patients with
AIDS or cancer are more likely to infect with such debilitating infections [6]. Hence, there is an
immediate need to discover new elements with improved efficiency against resistant microbes as
well as exclusive structural features which may not allow microorganisms to hold up against
their strike.
4-Thiazolidinones involving the ciprofloxacin entity with 1-4-methoxyphenylpiperazine (1)
[7] and N-methyl-3-phenylpiperazine (2) [8] ring combinations has been recently explored and
evaluated their antimicrobial action against a range of Gram-positive and Gram-negative bacteria
and the compounds indicated MICs, >100 µg/mL and >50 µg/mL, respectively. In a individual
effort, norfloxacin based 4-thiazolidinones (3) has been generated and checked for their in vitro
effects against some bacterial strains and the lowest MICs observed were varied from 3.12-6.25
µg/mL [9]. The combination of piperazine moiety and thiazole nucleus along with the presence
of a quinolone ring (4) is recently found to have good antimicrobial potency (3) with MICs, 12.5-
50 µg/mL [10] as compared to control drugs ampicillin (MICs, 6.25-25 µg/mL) and gentamicin
This article is protected by copyright. All rights reserved.

(MICs, 12.5-25 µg/mL). Recently, some piperazine based 4-thiazolidinones (5) [11] were
equipped involving various substituted phenyl ring systems with an antimicrobial potency of
4.68-9.37 µg/mL MICs. In addition, several other illustrations persisted in the literature recently
for the synthesis and antimicrobial screening of 4-thiazolidinones having either direct or indirect
substitution of benzothiazole or piperazine entity [12-14] and literature reports verified the
powerful participation of piperazine ring systems in drug design and discovery [15].
All these findings convinced us to rationale a novel class of extremely effective
antibacterial scaffolds (6) based on benzothiazole, quinolone, piperazine and thiazolidinone
compact system (Figure 1) and compounds were having highly potent action against a
mentioned panel of bacterial strains at lowest MICs, 1-2 µg/mL besides fact that 4-thiazolidinone
ring is essential pharmacophore for potent biological action [16, 17]. In order to furnish the
intended molecular designed, the first task was to introduce piperazine entity to the final 4-
thiazolidinone system, hence the N-formylation of the piperazine residue of fluoroquinoline
entity was carried out using sulfated tungstate as a heterogeneous acid catalyst in the transaction
to reveal a key element, Schiff base required for the organization of final scaffold. Researchers
have observed that sulfated tungstate is an efficient catalysts for N-formylation having synthetic
ease and recyclable with preferred properties [18].
Methods and Materials
Ciprofloxain and norfloxacin were gifted from Smruthi Organics Limited, Solapur, India.
Melting points were determined in open capillaries on a Veego electronic apparatus VMP-D and
are uncorrected. FT-IR spectra of synthesized compounds were recorded on a Shimadzu 8400-S
FT-IR spectrophotometer using KBr pellets. Thin layer chromatography was performed on
object glass slides (2 x 7.5 cm) coated with silica gel-G (60F254) and spots were visualized under
1
UV irradiation. H NMR and 13C NMR spectra were recorded on a Varian 400 MHz model
1
spectrometer using DMSO-d6 as a solvent and TMS as internal standard. The H NMR and 13C
NMR chemical shifts were reported as ppm downfield from TMS. The splitting patterns are
designated as follows; s, singlet; br s, broad singlet; d, doublet; dd, doublet of doublets; q,
quartet, m, multiplet.
This article is protected by copyright. All rights reserved.

Preparation of sulfated tungstate
A mixture of chlorosulfonic acid (0.1 mol) in chloroform (75 mL) was kept at 0-5oC in an ice
bath which was fitted with CaCl2 drying tube. To it, while continuing stirring, 0.05 mol of
anhydrous sodium tungstate was added in portion maintaining temperature between 0-5oC. After
the completion of the reaction, the resulted mixture was allowed to stir for another 1.5 h and as a
result a yellowish-white solid was obtained which was filtered, washed several times with
Millipore water until the filtrate was neutral and free from chloride ions as confirmed by the
AgNO3 test. The final crude was dried in an oven for 2 h at 100oC to get final catalyst, sulfated
tungstate [18]. IR (KBr, cm–1): 3451, 1616, 1222, 1089, 961, 654.
1-Cyclopropyl-6-fluoro-7-(4-formylpiperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic
acid (3) and 1-Ethyl-6-fluoro-7-(4-formylpiperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-
carboxylic acid (4)
To a stirred solution of ciprofloxacin (1) or norfloxacin (2) (0.1 mol) and formic acid (0.12 mol),
10 wt% of sulfated tungstate was added and the resulting mixture was heated in an oil bath at
70oC for 8 h (for ciprofloxacin) or 6.5 h (for norfloxacin). The progress of the reaction was
monitored by Thin Layer Chromatography using toluene: acetone (8:2) solvent system as well as
when the full conversion happed the liquid reaction mixture got automatically solidified, which
is the main physical change confirming the completion the reaction. After the completion, the
reaction mixture was allowed to cool and diluted with 20 mL of ethyl acetate and at the same
time upon filtration, the insoluble catalyst was recovered. Combined organic layer (filtrate and
washings) was washed with water (2x10 mL), dried over anhydrous sodium sulfate and pure
product was equipped via evaporation of solvent under reduced pressure [18] as 3: Yield 94%,
m.p. 241-243 °C, IR (KBr, cm–1): 1729 (CHO) and 4: Yield 90%, m.p. 219-222 °C, IR (KBr,
cm–1): 1731 (CHO).
General synthetic procedure for 2–amino–6–substituted benzothiazoles 6a-g
To 100 mL glacial acetic acid, 0.1 mol of appropriate para-substituted amine derivative (5a-g)
and equimolecular amount of potassium thiocyanate was added and the reaction mixture was
kept in an ice bath and cooled. The mixture was allowed to cool at this temperature up to 20 min.
Then, in the reaction mixture, bromine in glacial acetic acid (0.1 mol) was added very slowly to
This article is protected by copyright. All rights reserved.

maintain the temperature of the reaction mixture below 10 ºC and was stirred at room
temperature for 2 to 4 h to obtain the hydrobromide (HBr) salt. The salt was then filtered,
washed with acetic acid, dried in a vacuum oven and basified to pH 11.0 with NH3·H2O. The
solid precipitate thus formed was filtered, washed with water and dried in a vacuum oven to
yield the intermediates 6a-g. TLC technique involving toluene: acetone (V/V, 8:2) solvent
system was used to monitor the reaction progress [19, 20].
General synthetic procedure for Schiff base: 7a-8g
To a solution of N-formylated fluoroquinolone (3 or 4, 0.005 mol) in absolute ethanol (25 mL),
containing a catalytic amount of piperidine, equimolecular amount of the appropriate 2–amino–
6–substituted benzothiazoles 6a-g was added. The reaction mixture was refluxed for 7-14 h. It
was then cooled at room temperature, poured into crushed ice, filtered, washed, dried and
recrystallized from chloroform to yield 7a-8g [21]. For example, 7a: Yield: 72%. M.p. 267-268
oC; IR (KBr, cm–1): 3031 (C–H str., Ar.), 2825 (O-H str., carboxylic), 1731 (C=O str,
carboxylic), 1656 (C=N, benzothiazole, str.), 1621, 1614 1610 (N=CH and C=O str.), 1027 (C-N
1
str. piperazine). H NMR (400 MHz, DMSO–d6): δ 14.58 (s, 1H, COOH), 8.62 (s, 1H, H-2,
ciprofloxacin), 8.31 (s, 1H, N=CH), 7.81 (d, J = 13.2 Hz, 1H, H-5, ciprofloxacin), 7.52 (d, J =
7.5 Hz, 1H, H-8, ciprofloxacin), 7.10-7.00 (m, 3H, Ar-H), 3.87 (br s, 4H, piperazine ring), 3.71–
3.75 (m, 1H, cyclopropyl), 3.41 (br s, 4H, piperazine ring), 1.20–1.36 (m, 4H, cyclopropyl); 8a:
Yield: 76%. M.p. 241-243 oC; IR (KBr, cm–1): 3025 (C–H str., Ar.), 2833 (O-H str., carboxylic),
1729 (C=O str, carboxylic), 1651 (C=N, benzothiazole, str.), 1618, 1611 1608 (N=CH and C=O
str.), 1033 (C-N str. piperazine). 1H NMR (400 MHz, DMSO–d6): δ 14.50 (s, 1H, COOH), 8.42
(s, 1H, N=CH), 8.77 (s, 1H, H-2, norfloxacin), 8.22 (d, J = 12.7 Hz, 1H, H-5, norfloxacin), 7.81
(d, J = 7.5 Hz, 1H, H-8, norfloxacin), 7.09-6.92 (m, 3H, Ar-H), 4.31 (q, 2H, J = 6 Hz, CH2CH3),
3.79 (br s, 4H, piperazine ring), 3.45 (br s, 4H, piperazine ring), 1.33 (t, 3H, J = 6.6 Hz, CH3).
General synthetic procedure for 4-Thiazolidinones: 2-(4-(4-methoxyphenyl)piperazin-1-yl)-3-
(6-substituted-benzo[d]thiazol-2-yl)thiazolidin-4-one (9a-10g)
To a mixture of thioglycolic acid (6, 0.005 mol) in dry benzene (25 mL), an appropriate quantity
of Schiff base (0.003 mol, 7a-8g) was added and the reaction mixture was refluxed for 11-18 h.
Water formed during the reaction was removed azeotropically by Dean-Stark apparatus. Progress
This article is protected by copyright. All rights reserved.

of the reaction the reaction was checked by TLC using benzene/ether as eluent. After the
completion of reaction benzene was removed by distillation to give solid, which was dissolved in
methanol (20 mL). This solution was warmed and treated with sodium bicarbonate solution to
remove unreacted acid. The solid obtained was filtered, washed with ether and purified by
crystallization from methanol to get desired products 9a-10g [21]. For example, 1-cyclopropyl-
6-fluoro-7-(4-(3-(6-fluorobenzo[d]thiazol-2-yl)-4-oxothiazolidin-2-yl)piperazin-1-yl)-4-oxo-
1,4-dihydroquinoline-3-carboxylic acid (9a): Yield 68%, m.p. 280-282 °C, IR (KBr, cm–1):
3045 (C–H str., Ar.), 2821 (O-H str., carboxylic), 1729 (C=O str, carboxylic), 1659 (C=N,
benzothiazole, str.), 1631 (C=O, thiazolidin-4-one), 1620, 1614 (C=O str.), 1021 (C-N str.
piperazine), 638 (CS). 1H NMR (400 MHz, DMSO–d6): δ 14.46 (s, 1H, COOH), 8.67 (s, 1H, H-
2, ciprofloxacin), 7.90 (d, J = 13.2 Hz, 1H, H-5, ciprofloxacin), 7.56 (d, J = 7.5 Hz, 1H, H-8,
ciprofloxacin), 7.06-6.95 (m, 3H, Ar-H), 5.39 (s, 1H, CH, thiazolidin-4-one), 3.84 (br s, 4H,
piperazine ring), 3.73–3.78 (m, 1H, cyclopropyl), 3.53 (s, 2H, CH2, thiazolidin-4-one), 3.37 (br s,
4H, piperazine ring), 1.17–1.32 (m, 4H, cyclopropyl). 13C NMR (100 MHz, DMSO–d6): δ 177.2
(1C, C-4, ciprofloxacin), 171.1 (1C, C=O, thiazolidin-4-one), 167.6 (1C, COOH), 156.8 (1C, C–
2, benzothiazole), 147.1 (1C, C-2, ciprofloxacin), 142.6 (1C, C-6, ciprofloxacin), 141.3 (1C, C-7,
ciprofloxacin), 138.1 (1C, C-8, ciprofloxacin), 133.7-117.5 (7C, Ar–C), 113.6, 112.9 (2C, C-3
and C-5, ciprofloxacin), 105.8 (1C), 65.9 (1C, CH, thiazolidin-4-one), 51.4 (2C, piperazine),
41.7 (2C, piperazine), 38.7 (1C, CH, cyclopropyl), 34.7 (1C, CH2, thiazolidin-4-one), 8.6 (2C,
CH2, cyclopropyl). Anal Calcd for C27H23F2N5O4S2: C, 55.56; H, 3.97; N, 12.00; Found: C,
55.34; H, 4.14; N, 11.89; 1-ethyl-6-fluoro-7-(4-(3-(6-fluorobenzo[d]thiazol-2-yl)-4-
oxothiazolidin-2-yl)piperazin-1-yl)-4-oxo-1,4-dihydroquinoline-3-carboxylic acid (10a):
Yield 70%, m.p. 257-258 °C, IR (KBr, cm–1): 3038 (C–H str., Ar.), 2835 (O-H str., carboxylic),
1733 (C=O str, carboxylic), 1656 (C=N, benzothiazole, str.), 1626 (C=O, thiazolidin-4-one),
1623, 1616 (C=O str.), 1022 (C-N str. piperazine), 646 (CS). 1H NMR (400 MHz, DMSO–d6): δ
14.55 (s, 1H, COOH), 8.72 (s, 1H, H-2, norfloxacin), 8.13 (d, J = 12.7 Hz, 1H, H-5,
norfloxacin), 7.71 (d, J = 7.5 Hz, 1H, H-8, norfloxacin), 7.11-6.94 (m, 3H, Ar-H), 5.51 (s, 1H,
CH, thiazolidin-4-one), 4.34 (q, 2H, J = 6 Hz, CH2CH3), 3.83 (br s, 4H, piperazine ring), 3.50 (s,
13
2H, CH2, thiazolidin-4-one), 3.41 (br s, 4H, piperazine ring), 1.41 (t, 3H, J = 6.6 Hz, CH3). C
NMR (100 MHz, DMSO–d6): δ 176.2 (1C, C-4, norfloxacin), 172.3 (1C, C=O, thiazolidin-4-
one), 166.9 (1C, COOH), 158.3 (1C, C–2, benzothiazole), 153.1 (1C, C-2, norfloxacin), 148.3
This article is protected by copyright. All rights reserved.

(1C, C-6, norfloxacin), 146.0 (1C, C-7, norfloxacin), 139.1 (1C, C-8, norfloxacin), 133.9-115.3
(7C, Ar–C), 111.0, 107.3 (2C, C-3 and C-5, norfloxacin), 104.7 (1C), 66.1 (1C, CH, thiazolidin-
4-one), 51.4 (2C, piperazine), 48.7 (1C, CH2CH3), 45.3 (2C, piperazine), 34.5 (1C, CH2,
thiazolidin-4-one), 15.3 (1C, CH3). Anal Calcd for C26H23F2N5O4S2: C, 54.63; H, 4.06; N, 12.25;
Found: C, 54.78; H, 3.89; N, 12.46. All the remaining final derivatives (9b-9g and 10b-10g)
were obtained following the similar method as 9a.
Methods for pharmacological activity evaluations
The synthesized derivatives (9a-10g) were examined for antimicrobial activity against
Staphylococcus aureus, Bacillus subtilis, E. coli, P. aeruginosa bacteria and Candida albicans
fungi. Ciprofloxacin, norfloxacin and fluconazole were used as control drugs for antibacterial
and antifungal activity, respectively using method described previously [22] as agar streak
dilution method measuring MICs. A stock solution of the synthesized compound (100 µg/mL) in
dimethylsulfoxide was prepared and graded quantities of the test compounds were incorporated
in a specified quantity of molten sterile agar, i.e. nutrient agar for evaluation of antibacterial and
sabouraud dextrose agar for antifungal activity, respectively. The medium containing the test
compound was poured into a petri dish at a depth of 4-5 mm and allowed to solidify under
aseptic conditions. A suspension of the respective microorganism of approximately 105 CFU/mL
was prepared and applied to plates with serially diluted compounds with concentrations of the
various ranges in µg/mL in dimethylsulfoxide and incubated at (37 1) °C for 24 h (bacteria) or
48 h (fungi). The lowest concentration of the substance that prevents the development of visible
growth is considered to be the MIC value as described earlier [23]. Zone of inhibitions were
evaluated using paper disc diffusion technique [24].
Results and Discussion
Chemistry
Synthetic work performed in the existing research is defined in Scheme 1. The solvents
and reagents were used as obtained or were dried before use as required. Anhydrous sodium
tungstate was treated with chlorosulfonic acid in chloroform to furnish a yellowish white
heterogeneous solid acid catalyst, sulfated tungstate in almost pure form to be used further for
the N-formylation of fluoroquinolone moieties. The appropriate formation of the catalyst was
This article is protected by copyright. All rights reserved.

verified upon recording FT-IR spectrum which was in precise compliance with the literature
data. In continuous synthesis, fluoroquinolone moieties (1 and 2) were treated with formic acid
in the presence of 10wt% catalyst and after refluxing for 8 h (for ciprofloxacin) or 6.5 h (for
norfloxacin) on oil bath at 70oC, the reaction mass got solidified, and this physical change
confirmed the complete transformation to N-formylated piperazine residue (3 and 4) as
confirmed by operating TLC also. The FT-IR band at 1728 cm-1 and 1731 cm-1 for 3 and 4,
respectively confirmed the formation of aldehyde group. The 2-amino-6-Substituted
benzothiazoles (6a-g) were synthesized by reacting aryl amines (5a-g) with KSCN in by a
known preparation method. Intermediates 6a-g were then reacted with N-formylated
fluoroquinolones (3 and 4) in the presence of piperidine organocatalyst to produce the
corresponding Schiff base derivatives 7a-g (ciprofloxacin) and 8a-g (norfloxacin) in 67-81% of
yields. The FT-IR spectrum of Schiff base derivatives (7a and 8a) exposed a band at 1614 cm-1
and 1611 cm-1, respectively, confirming the correct formation of an azomethane functionality
while a band for CHO group is not observed, whereas, in their 1H NMR spectra, the proton atom
1
of CHO resonated at 8.31 ppm and 8.41 ppm, respectively. All other FT-IR and H NMR data
were in complete accordance with the proposed Schiff base structures. Hence, the reaction
sequence was proceeded further to acquire a final 4-thiazolidinone scaffold based on
ciprofloxacin (9a-9g) and norfloxacin (10a-10g) core in 55-68% and 60-70% of yields,
respectively via refluxing appropriate Schiff base (7a-8g) with thioglycolic acid in dry benzene.
These azeotropic reactions were carried out in a Dean-Stark apparatus in order to eliminate water
formed during the reaction. All the FT-IR and 1H NMR spectral data of compounds 9a-10g were
in accordance with assumed structures, for example carbonyl functional group of 4-
thiazolidinone ring revealed its corresponding FT-IR band at 1631 cm-1, while its C-S bonding
appeared at around 638 cm-1 for compound 9a. Additionally, a carboxylic acid group of
fluoroquinolone entity indicated corresponding bands at 2821 cm-1 and 1729 cm-1 for COOH and
C=O groups, respectively, and a band at 1021 cm-1 assigned due to C-N stretching of piperazine
moiety for 9a. Furthermore, CH and CH2 protons of 4-thiazolidinone ring resonated at 5.39 ppm
1
and 3.53 ppm as a singlet, respectively, in the H NMR spectra of 9a, while all proton atoms
belonging to the fluoroquinolone entity resonated in the range 8.67-7.56 ppm. Multiplets of
cyclopropyl ring appeared in the range of 3.73-3.78 ppm and 1.17-1.32 ppm. In the 13C NMR
spectra of 9a, C=O functionality of 4-thiazolidinone ring appeared at 171.1 ppm, while CH and
This article is protected by copyright. All rights reserved.

CH2 carbon atoms were appeared at 65.9 ppm and 34.7 ppm, respectively, likewise, peaks of
carbon atoms for fluoroquinolone ring seen in the range 177.2 ppm to 112.9 ppm, while
cyclopropyl carbon atoms at 38.7 ppm and 8.6 ppm. We have previously studied the synthesis of
1
some benzothiazole and piperazine based systems and in the current investigation, the H NMR
and 13C NMR data of the final compounds 9a-10g were in accordance with those we have
observed previously. The purity of the produced substances was monitored by TLC and
determined by elemental analysis.
Pharmacology
Antimicrobial screening for the targeted compounds (9a-10g) has been performed against
two Gram-positive bacsteria (S. aureus and B. subtilis), two Gram-negative bacteria (E. coli and
P. aeruginos) as well as against C. albicals fungal strain. The results of such investigation are
described in Table 1. Overall, the final scaffolds expressed a highly potent action against
bacterial strains (1-2 µg/mL), however dealt moderately with fungi (>32 µg/mL). However, we
have accessed antibacterial potency of Schiff base derivatives of ciprofloxacin (7a-g) and
norfloxacin derivatives (8a-g), in which lowest MICs were observed in the range 4-16 µg/mL
and we have noticed similar pattern of activity among potent analogues in case of substitution of
various benzothiazoles with different functional groups, hence suggesting that substituted
benzothiazoles are essential for the significant activity against particular bacteria, however
thiazolidinone nucleus is the key for increased potency. From the bioassay results of these
intermediate Schiff bases, it is confirmed that heterocyclization to 4-thiazolidinone nucles lead to
more potent antibacterial agents with decreased MIC level (1-4 µg/mL) against each of the
bacterial strain studied presently. The bioassay data demonstrated that these 4-thiazolidinones
were more efficacious towards Gram-positive strains at lowest MICs, 1-2 µg/mL in comparison
to their durability against Gram-negative bacteria with 2-4 µg/mL of MICs. The most appealing
outcomes were acquired by the replacement of electron donating alkoxy functional group (9f,
10f) to the benzothiazole moiety. Overall, among the electron donating substituent, antibacterial
potency falls in the order of OC2H5>NHCOCH3>OCH3>CN while in case of substation of
electron withdrawing groups these order gone to Br>F>NO2. In which, existence of electron
withdrawing substituent showcased potent anti Gram-positive bacterial effects while electron
donating groups were found highly active against Gram-negative bacterial strains. Likewise,
This article is protected by copyright. All rights reserved.

alteration of fluoroquinoline ring had significant effects on the biological potency of resultant
analogues, as ciprofloxacin based derivatives appealed higher inhibitory action against Gram-
negative strains, as a ciprofloxacin based derivatives with an ethoxy functional group (9f)
appeared with 2 µg/mL of MIC level and 22 mm as well as 23 mm of inhibitory zone against E.
coli and P. aeruginosa, respectively, i.e. more potent than ciprofloxacin (6.25 µg/mL). In
addition, fluorinated derivative (9a) with ciprofloxacin core indicated 4 µg/mL of MIC against
both the Gram-negative strains with 19 mm and 21 mm of zone of inhibition in addition to the
analogue 9g with 4.0 µg/mL of MIC and 22 mm of inhibitory zone, which were more efficacious
than ciprofloxacin control (6.25 µg/mL).
Norfloxacin derivatives expressed significant inhibition of Gram-positive ones, for
example, derivative 10b with the electron withdrawing bromine atom to the benzothiazole ring
demonstrated excellent potency at 1 µg/mL and 25 mm of inhibitory zone against the Gram-
positive bacteria S. aureus, while another several norfloxacin based derivatives with fluorine
(10a), bromine (10b), ethoxy (10f) and acetamido (10g) functional groups exerted 2 µg/mL of
MIC and 26-28 mm of inhibitory zones against B. cereus. The potency of these compounds was
better than control drug ciprofloxacin (3.12 µg/mL) against S. aureus and B. cereus. Overall,
SARs demonstrated that multiplicity of the thiazole ring system in the form of benzothiazole ring
and thiazolidinone ring positively enhanced the activeness of the resultant compounds. With the
exemption, a ciprofloxacin derivative 9b with bromine atom substitution appeared with 2 µg/mL
of MIC against Gram-positive strain S. aureus and was also more potent than control drug.
Overall, The most potent compounds against mentioned bacterial strains were 9f from
ciprofloxacin series and 10a, 10b, 10f and 10g from norfloxacin series; hence it was necessary to
inspect their cytotoxic nature. Therefore, in vitro cytotoxicity of these compounds was
determined by incubating Vero mammalian cellular suspensions at 32 µg/mL of concentration
level, where the maximum activity level was tested (MICs), using neutral red dye method [25].
All five compounds were well tolerated by Vero cells at this concentration and expressing the
fact that the CC50 level for all five derivatives should >32 µg/mL, indicating a selectivity of
antibacterial action. Various left over derivatives were succeeded to exhibit 4-8 µg/mL of MICs
against the mentioned panel of bacterial strains, while some were found inactive, for example
derivatives with cyano substituent with MICs, ≥32 µg/mL. Similarly, all the final derivatives
This article is protected by copyright. All rights reserved.

exhibited ≥32 µg/mL of MICs against C. albicans fungi and were recognized sedentary in
biological action.
Conclusion
This work devoted to the development of new highly potent active antibacterial agents
based on fluoroquinolone moiety, benzothiazole and thiazolidinone compact system. On the
whole, the results of the in vitro pharmacological activities are very motivating, as out of novel
14 compounds tested, 11 exhibited highly potent antibacterial action at MICs 1-4 µg/mL when
compared to control drug ciprofloxacin at 3.12-6.25 µg/mL. It was observed that multiplicity of
the thiazole ring in the form of benzothiazole and thiazolidinone entities approached high
potency. Moreover, a first effort to introduce fluoroquinolone residue at C-1 position of
thiazolidinone ring was found essential to contribute excellent activity when compared to other
equivalent system investigated previously. Comparing the bioassay results of intermediate Schiff
base and heterocyclized 4-thiazolidinones, it was demonstrated that insertion of substituted
benzothiazoles are essential for the significant activity agiants particular bacteria and formation
of thiazolidinone nucles is the key for increased potency. Such a novel class of compounds can
be outlined as new active leads pertaining a powerful incentive for further research in this area.
Particularly, drug design derived in the current work lead to a privileged structure of drug-like
molecules to attain more effective derivatives in ongoing studies.
Supplementary Material
Full characterization data for the final compounds 9b-9g and 10b-10g as well as biological
activity method and results in terms of MICs in µM values for all final compounds are presented
in the supplementary material associated with this article.
Acknowledgements
This article was supported by the SMART Research Professor Program of Konkuk
University, Seoul, South Korea.
Conflict of Interest
There is no conflict of interest.
This article is protected by copyright. All rights reserved.

References
Rice, L.B. (2006) Antimicrobial resistance in gram-positive bacteria. Am J Med 119: S11-
1.
2.
3.
4.
5.
6.
7.
8.
9.
S19
Cornaglia, G. (2009) Fighting infections due to multidrug-resistant Gram-positive
pathogens. Clin Microbiol Infect 15: 209-211.
Engel, L.S. (2009) Multidrug-resistant Gram-negative bacteria: Trends, risk factors, and
treatments, Emerg Med 41: 18-27.
Torres, J.A., Villegas, M.V., Quinn, J.P. (2007) Current concepts in antibiotic-resistant
gram-negative bacteria. Expert Rev Anti Infect Ther 5: 833-843.
Levy, S.B., Marshall, B. (2004) Antibacterial resistance worldwide: causes, challenges and
responses. Nature Med 10: S122-S129.
World
Health
Organization,
Bacterial
Infections.
Available
at
http://www.who.int/vaccine_research/diseases/soa_bacterial/en/index3.html
Patel, N.B., Patel, S.D. (2010) Synthesis and antimicrobial study of fluoroquinolone based
4-thiazolidinones. Med Chem Res 19: 757-770.
Patel, N.B., Patel, S.D. (2009) Synthesis and antimicrobial studies of some 4-
Thiazolidinone containing Fluoroquinolones analogous. Der Pharm Chem 1: 199-209.
Kumar, V., Kumar, A., Sharma, S., Singh, N.P. (2011) Synthesis and antimicrobial activity
of thiazolidinone norfloxacin hybrids. Ind J Chem 50B: 1496-1503.
10. Patel, R.V., Patel, J.K., Nile, S.H., Park, S.W. Copper (II) triflate promoted
multicomponent catalytic clubbing of piperazinyl-thiazoloquinolines and
thiazolocoumarins as antimicrobials. Arch Pharm Chem Life Sci 346: 221-231.
11. Shingade, S.G., Bari, S.B. (2013) Synthesis and antimicrobial screening of 4-thiazolidinone
and 2-azetidinone derivatives of piperazine. Med Chem Res 22: 699–706.
12. Nagarajan, A.S., Kamalraj, S., Muthumary, J., Reddy, B.S.R. (2009) Synthesis of
biologically active benzothiazole substituted thiazolidinone derivatives via cyclization of
unsymmetrical imines. Ind J Chem 48B: 1577-1582.
13. Chavan, A.A., Pai, N.R. (2007) Synthesis and antimicrobial screening of 5-arylidene-2-
imino-4-thiazolidinone. ARKIVOC xvi: 148-155.
This article is protected by copyright. All rights reserved.

14. Pareek, D., Chaudhary, M., Pareek, P.K., Kant, R., Ojha, K.G., Pareek, R., Iraqia, S.M.U.,
Pareeka, A. (2010) Synthesis of some bioactive 4-thiazolidinone derivatives incorporating
benzothiazole moiety. Der Chem Sin 1: 22-35.
15. Patel, R.V., Park, S.W. (2013) An evolving role of piperazine moieties in drug design and
discovery. Mini Rev Med Chem 13:1579-1601.
16. Verma, A., Saraf, S.K. (2008) 4-Thiazolidinone – A biologically active scaffold. Eur J Med
Chem 43: 897-905.
17. Jain, A.K., Vaidya, A., Ravichandran, V., Kashaw, S.K., Agrawal, R.K. (2012) Recent
developments and biological activities of thiazolidinone derivatives: a review. Bioorg Med
Chem 20: 3378-3395.
18. Pathare, S.P., Sawant, R.V., Akamanchi, K.G. (2012) Sulfated tungstate catalyzed highly
accelerated N-formylation. Tetrahedron Lett 53: 3259–3263.
19. Sawhney, S.N., Boykin, D.W. (1979) Transmission of substituent effects in heterocyclic
systems by carbon-13 nuclear magnetic resonance: Benzothiazoles. J Org Chem 44: 1136–
1142.
20. Patel, R.V., Patel P.K., Kumari P., Rajani, D.P., Chikhalia, K.H. (2012) Synthesis of
benzimidazolyl–1,3,4–oxadiazol–2ylthio–N–phenyl(benzothiazolyl)
acetamides
as
antibacterial, antifungal and antituberculosis agents. Eur J Med Chem 53: 41-51.
21. Patel, D.K., Kumari P., Patel, N.B. (2012) Synthesis and biological evaluation of some
thiazolidinones as antimicrobial agents. Eur J Med Chem 48: 354-362.
22. Hawkey, P.M., Lewis, D.A. (1994) Medical bacteriology– a practical approach. Oxford
University Press, United Kingdom, 181–194.
23. Patel, R.V., Kumari P., Rajani, D.P., Chikhalia, K.H. (2012) A new class of 2-(4-
cyanophenyl amino)-4-(6-bromo-4-quinolinyloxy)-6-piperazinyl(piperidinyl)-1,3,5-triazine
analogues with antimicrobial/antimycobacterial activity. J Enzyme Inhib Med Chem 27:
370-379.
24. S.H. Gillespie, Medical microbiology-Illustrated, Butterworth Heinemann Ltd., United
Kingdom, (1994) 234-247.
25. Borenfreund, E., Puerner, J.A. (1985) A simple quantitative procedure using monolayer
culture for toxicity assays. J Tissue Cult Methods 9: 7-9.
This article is protected by copyright. All rights reserved.

Tables
Table 1 In vitro antimicrobial activity of compounds 9a-10g
R
MICs in µg/mL (µM)
(Compd.)
32 µg/Disc
S. aureus
B. subtilis
E. coli
P. aeruginosa
F (9a/10a)
Br
8.0(13.71)/4.0(7.00)
2.0(3.10)/1.0(1.58)
16.0(27.41)/2.0(3.50)
4.0(6.21)/4.0(6.32)
4.0(6.85)/8.0(14.00)
4.0(6.85)/8.0(14.00)
8/0(12.41)/16.0(25.30)
16.0(24.82)/32.0(50.59)
(9b/10b)
NO2
>32.0(>52.40)/8.0(13.36) >32.0(>52.40)/2.0(3.34)
32.0(52.40)/8.0(13.36)
32.0(52.40)/8.0(13.36)
(9c/10c)
CN
>32.0 (>54.17)/ >32.0
(>55.30)
>32.0 (>54.17)/ >32.0
(>55.30)
>32.0 (>54.17)/ >32.0
(>55.30)
>32.0 (>54.17)/ >32.0
(>55.30)
(9d/10d) #
OCH3
16.0(26.86)/ >32.0
(>54.83)
16.0(26.86)/ >32.0
(>54.83)
8.0(13.43)/ >32.0
(>54.83)
8.0(13.43)/ >32.0 (>54.83)
(9e/10e)
OC2H5
(9f/10f)
NHCOCH3
(9g/10g)
Cip.
8.0(13.12)/4.0(6.69)
8.0(13.12)/2.0(3.35)
2.0(3.28)/4.0(6.69)
2.0(3.28)/4.0(6.69)
4.0(6.42)/8.0(13.10)
6.25(18.86)
8.0(12.85)/4.0(6.55)
3.12(9.42)
4.0(6.42)/2.0(3.28)
3.12(9.42)
8.0(12.85)/16.0(26.20)
6.25(18.86)
(30
µg/Disc)
Nor.
3.12(9.77)
1.56(4.89)
0.78(2.44)
1.56(4.89)
(30
µg/Disc)
#: Value for both compounds; Cip.: Ciprofloxacin; Nor.: Norfloxacin; Flu.: Fluconazole.
* Each value is the mean of three independent experiments.
This article is protected by copyright. All rights reserved.

Figures and Schemes
Figure 1 Rationalization of work
This article is protected by copyright. All rights reserved.

Scheme 1. Synthesis of fluoroquinolone based benzothiazolo-4-thiazolidinones (9a-10g)
Scheme 1. Synthesis of fluoroquinolone based benzothiazolo-4-thiazolidinones (9a-10g)
This article is protected by copyright. All rights reserved.