5-Adamantan thiadiazole-based thiazolidinones as antimicrobial agents. Design, synthesis, molecular docking and evaluation

Article abstract of DOI:10.1016/j.bmc.2018.08.004

In continuation of our efforts to develop new compounds with antimicrobial properties we describe design, synthesis, molecular docking study and evaluation of antimicrobial activity of seventeen novel 2-{[5-(adamantan-1-yl)-1,3,4-thiadiazol-2-yl]-imino}-5-arylidene-1,3-thiazolidin-4-ones. All compounds showed antibacterial activity against eight Gram positive and Gram negative bacterial species. Twelve out of seventeen compounds were more potent than streptomycin and all compounds exhibited higher potency than ampicillin. Compounds were also tested against three resistant bacterial strains: MRSA, P. aeruginosa and E. coli. The best antibacterial potential against ATCC and resistant strains was observed for compound 8 (2-{[5-(adamantan-1-yl)-1,3,4-thiadiazol-2-yl]-imino}-5-(4-nitrobenzylidene)-1,3thiazolidin-4-one). The most sensitive bacterium appeared to be S. typhimirium, followed by B. cereus while L. monocitogenes and M. flavus were the most resistant. Compounds were also tested for their antifungal activity against eight fungal species. All compounds exhibited antifungal activity better than the reference drugs bifonazole and ketokonazole (3-115 times). It was found that compound 8 appeared again to be the most potent. Molecular docking studies on E. coli MurB, MurA as well as C. albicans CYP 51 and dihydrofolate reductase were used for the prediction of mechanism of antibacterial and antifungal activities confirming the experimental results.

Full text of DOI:10.1016/j.bmc.2018.08.004

Accepted Manuscript
5-Adamantan thiadiazole-based thiazolidinones as antimicrobial agents. De-
sign, synthesis, molecular docking and evaluation
Maria Fesatidou, Panagiotis Zagaliotis, Charalampos Camoutsis, Anthi Petrou,
Phaedra Eleftheriou, Christophe Tratrat, Micheline Haroun, Athina Geronikaki,
Ana Ciric, Marina Sokovic
PII:
S0968-0896(18)31027-7
https://doi.org/10.1016/j.bmc.2018.08.004
BMC 14491
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
30 May 2018
24 July 2018
2 August 2018
Please cite this article as: Fesatidou, M., Zagaliotis, P., Camoutsis, C., Petrou, A., Eleftheriou, P., Tratrat, C., Haroun,
M., Geronikaki, A., Ciric, A., Sokovic, M., 5-Adamantan thiadiazole-based thiazolidinones as antimicrobial agents.
Design, synthesis, molecular docking and evaluation, Bioorganic & Medicinal Chemistry (2018), doi: https://
doi.org/10.1016/j.bmc.2018.08.004
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5-Adamantan thiadiazole- based thiazolidinones as antimicrobial agents. Design, synthesis,
molecular docking and evaluation.
Maria Fesatidoua , Panagiotis Zagaliotisa , Charalampos Camoutsisb , Anthi Petroua , Phaedra Eleftheriouc, Christophe Tratratd ,
Micheline Haround , Athina Geronikakia, Ana Cirice, and Marina Sokovice
a Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotle University of Thessaloniki, 54124, Greece
bSchool of Health Sciences, Department of Pharmacy, Laboratory of Pharmaceutical Chemistry, Universityof Patras, Greece .
cDepartment of Medical Laboratories, School of Health and Care Professions, Alexander Technological Educational Institute of
Thessaloniki, 54700, Greece,
d Department of Pharmaceutical Sciences, College of Clinical Pharmacy, King Faisal University, Al-Ahsa 31982, Saudi Arabia
eMycological Laboratory, Department of Plant Physiology, Institute for Biological Research, Siniša Stanković, University of
Belgrade, Bulevar Despota Stefana 142, 11000, Belgrade, Serbia.
Seventeen novel 2-{[5-(adamantan-1-yl)-1,3,4-thiadiazol-2-yl]-imino}-5-arylidene-1,3-thiazolidin-4-ones were designed, synthesized and evaluated
for antimicrobial activity. All compounds were potent antimicrobial agents. According to molecular docking studies, inhibition of MurB and CYP 51 may
be involved in the mechanism of antibacterial and antifungal activities of most of the compounds.
E.Coli MurB (PDB: 1Q85)
Best compound anti-bacterial activity: MIC 1.65-3.30 .10-2µM
anti-fungal activity: MIC 0.80-3-30 .10-2µM
Compound 8: R= 4-NO2 MIC : 2.20 10-2 µM
Correspondent author: A.Geronikaki. Tel.: +302310997616; fax: +302310997612; e-mail: geronik@pharm.auth.gr

Bioorganic & Medicinal Chemistry
journal homepage: www.els evier.com
5-Adamantan thiadiazole-based thiazolidinones as antimicrobial agents. Design,
synthesis, molecular docking and evaluation.
Maria Fesatidoua , Panagiotis Zagaliotisa , Charalampos Camoutsisb , Anthi Petroua , Phaedra Eleftheriouc
Christophe Tratratd , Micheline Haround , Athina Geronikakia , ,Ana Cirice , and Marina Sokovice
a
Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotle University of Thessaloniki, 54124, Greece
b School of Health Sciences, Department of Pharmacy, Laboratory of Pharmaceutical Chemistry, University of Patras, Greece .
c Department of Medical Laboratories, School of Health and Care Professions, Alexander Technological Educational Institute of Thessaloniki, 54700, Greece,
dDepartment of Pharmaceutical Sciences, College of Clinical Pharmacy, King Faisal University, Al-Ahsa 31982, Saudi Arabia
eMycological Laboratory, Department of Plant Physiology, Institute for Biological Research, Siniša Stanković, University of Belgrade, Bulevar Despota Stefana
142, 11000, Belgrade, Serbia.
ARTICLE INFO
ABSTRACT
Article history:
Received
Received in revised form
Accepted
In continuation of our efforts to develop new compounds with antimicrobial properties we
describe design, synthesis, molecular docking study and evaluation of antimicrobial activity of
seventeen
novel
2-{[5-(adamantan-1-yl)-1,3,4-thiadiazol-2-yl]-imino}-5-arylidene-1,3-
thiazolidin-4-ones. All compounds showed antibacterial activity against eight Gram positive and
Gram negative bacterial species. Twelve out of seventeen compounds were more potent than
streptomycin and all compounds exhibited higher potency than ampicillin. Compounds were
also tested against three resistant bacterial strains: MRSA, P. aeruginosa and E. coli. The best
antibacterial potential against ATCC and resistant strains was observed for compound 8 (2-{[5-
(adamantan-1-yl)-1,3,4-thiadiazol-2-yl]-imino}-5-(4-nitrobenzylidene)-1,3thiazolidin-4-one).
The most sensitive bacterium appeared to be S. typhimirium, followed by B. cereus while L.
monocitogenes and M. flavus were the most resistant. Compounds were also tested for their
antifungal activity against eight fungal species. All compounds exhibited antifungal activity
better than the reference drugs bifonazole and ketokonazole (3-115 times). It was found that
compound 8 appeared again to be the most potent. Molecular docking studies on E.coli MurB,
MurA as well as C. albicans CYP 51 and dihydrofolate reductase were used for the prediction of
mechanism of antibacterial and antifungal activities confirming the experimental results.
Available online
Keywords:
Adamantan
Thiadiazole
Thiazolidinone
Antibacterial
Antifungal
Docking
MurB
MurA
CYP51
Dihydrofolate reductase
2009 Elsevier Ltd. All rights reserved.
Microorganism resistance to antimicrobials threatens the health
of many people throughout the world, since both old and new
1. Introduction
Bacterial infections are a growing problem worldwide causing
morbidity and mortality mainly in developing countries. Some of
the most deadly diseases such as salmonella, diarrhea and
widespread epidemics as well as rheumatic fever and food
poisoning result from bacterial infections.
The action of known antimicrobial drugs deals with inhibition of
limited spectrum of cellular processes which are biosynthesis of
proteins, RNA, DNA, cell wall and folic acid. Despite the
infectious diseases remain a formidable public health threat. It
should be mentioned that systemic fungal infections have
progressively increased during the last decades. As a result, rates
of morbidity and mortality, mainly due to the low effectiveness
of available medications and the development of resistant strains,
rose up. The problem becomes more serious in case of immune-
compromised patients, who are more disposed to opportunistic
fungal infections.
The treatment of such infections is quite difficult due to the
spread of antifungal drug resistance generally in patients who are
often subjected to antimytotic therapy and especially in immuno-
compromised patients (cancer, transplants, AIDs).4
1,2
continued success in discovery inhibitors against such targets
the frequency is low compared to the period of “golden era” of
antibiotic drug discovery.3
Moreover, pathogenic bacteria have developed sophisticated
intrinsic drug resistant mechanisms that are hard agitated in the
microbial metagenome as a natural phenomenon and thus are
difficult to fight.
Correspondent author: A.Geronikaki. Tel.: +302310997616; fax: +302310997612; e-mail: geronik@pharm.auth.gr

Thus, the increase in resistance of bacteria to antibiotics led
the scientific community to focus on developing novel
approaches to antimicrobial therapy. 5-11
(NH). 1H-NMR: (δ ppm, DMSO, 500 MHz): 1.70-2.01 (m, 15Η,
adamant.), 7.01 (s, 2Η, -ΝΗ2).
2.4. Synthesis of N-[5-(1-adamantyl)-1,3,4-thiadiazol-2-yl)]-2-
chloroacetamide
In this study we describe design, synthesis, molecular docking
study and evaluation of antimicrobial activity of seventeen novel
2-{[5-(adamantan-1-yl)-1,3,4-thiadiazol-2-yl]-imino}5-aryliden-
1,3-thiazolidin-4-ones.
To a stirred solution of [(5-adamantan-1-yl)-2-amino-2-yl)]-1,3,4-
thiadiazole (6.3 g, 0.03 mol) in anhydrous dimethyl formamide
(64mL) , anhydrous sodium carbonate (3.2g, 0.08 mol) was added and
then a solution of chloroacetyl chloride (9.0g, 0.080 mol) in anhydrous
DMF (34.9 mL) was added dropwise. The reaction mixture was
allowed to stir for 3 h at room temperature (25oC), then the resulting
solution was poured onto ice water and the precipitate formed was
filtered, washed with water, dried and crystallized from ethanol. Yield:
Adamantane derivatives have been found to interfere with various
enzymes and possess a variety of therapeutic activities, such as anti-
inflammatory, 9-11 anti-viral, 13-15 anti-Parkinson, 16,17 antimicrobial, 18,19
anticancer,14,20,21 CNS22 as well as anti-HIV activity.23,24
Another interesting core is the thiazolidinone ring, also
responsible for numerous pharmacological properties such as
o
93%, m.p. 179-180 C, Rf =0,73(petrol. ether-ethylacetate 1:1). IR
(cm-1, Nujol): 1712 (C=O), 3087 (NH). 1H-NMR: (δ ppm, DMSO- d6,
300 MHz): 1.26 (s, 6H, adamantane) 1.52- 1.57 (d, 9H, adamantane),
3.91 (s, 2H, CH2), 7.46 (s, 1H, NHCO). Anal. Calc. For C14H18ClN3OS
(MW 311.82) C: 53.93, H: 5.77, N: 13.48. Found% C: 53.90,
H:5.81,N:13.52%.
25-28
35,36
29-31
38
32-34
antimicrobial,
antioxidant,
activities.
anti-inflammatory,20,
anticancer,
anti-viral,
and antiarrythmic
37
antidiabetic35
35
4-thiazolidinones were recently found to exhibit
antimicrobial activity, probably acting as inhibitors of MurB,
thus inhibiting bacterial cell wall biosynthesis38.
In addition, thiadiazole is heterocyclic system conferring
antimicrobial,40-42 antifungal43-45 and a range of other activities,46-50
possibly due to the presence of the toxophoric -N-C-S moiety.51
2.5. Synthesis of 2-[5-(adamantan-1-yl)-1,3,4-thiadiazol-2-
yl]imino]-1,3-thiazolidin-4-one
To a solution of N-[5-(adamantan-1-yl)-1,3,4-thiadiazol-2-yl]-
2-chloroacetamide (0.005 mol) ammonium thiocyanate (0.01
mol) in ethanol 96%(20 mL) was added. The mixture was heated
to reflux on a water bath for 1 h and left overnight. The resultant
precipitate was filtered, washed with water and recrystallized
from ethanol. Yield, 72%, m.p. 256-257oC. IR (cm-1, Nujol):
3360 (νΝ-Η), 1740 (C=O) (strong absorption), 1620 (aromat.).
1H-NMR: (δ ppm, DMSO-d6, 300 MHz): 1.75 (s, 6H, adamant), 1.99-
2.06 (d, 9H, adamant), 4.07 (s, 2H, CH2 thiazolid.), 12.20 (s, 1H,
NHCO). Anal.calc. for C15H18N4OS2 (MW 334.45) C: 53.86; H: 5.42;
N: 16.75%. Found: C: 53.83; H:.5.40; N:16.80%.
In general, thiadiazoles are a member of the big family of
imidazole and triazole synthetic antifungal drugs designed to
inhibit the enzyme cytochrome P450 14α-demethylase as well as
the conversion of lanosterol to ergosterol, which is required in
fungal cell membrane synthesis.
Several thiazolidinone derivatives were designed by
introducing different arylidene substituents at the 5 position of
the thiazolidinone moiety, which according to our previous
52
observations
can be useful to encompass certain physico-
chemical properties such as hydrophobic and steric.
2.6. General procedure for synthesis of 2-{[5-(adamantan-1-yl)-
1,3,4-thiadiazol-2-yl]-imino}-5-arylidene-1,3-thiazolidin-4-
ones46
2. Methods
2.1. Materials
To a well-stirred solution of 2-[5-(adamantan-1-yl)-1,3,4-
thiadiazol-2-yl]imino}-1,3-thiazolidin-4-one (0.001 mol, 0.33 g)
in acetic acid (10 mL) buffered with sodium acetate (0.002 mol,
0.16g) the appropriate aromatic aldehyde (0.0015 mol) was
added. The solution was heated to reflux for 4 h and then poured
onto ice-cold water. The precipitate formed was filtered, washed
with water and the resulting crude product purified by
recrystallisation from dioxane.
All the chemicals used were of analytical grade and
commercially available.
2.2. Synthesis of 1-(adamantyl-5-carboxy)thiosemicarbazide
To a stirred solution of thiosemicarbazide (0.91g, 0.01 mole)
in dry pyridine (15mL) at -5OC
a
solution of 1-
adamantanecarbonyl chloride (2.0g, 0.01 mole) in dry benzene
(15 mL) was added. Stirring was continued for 0.5h at -5oC and
then overnight at room temperature (25oC). The solvent was
evaporated, water was added to the residue and the precipitate
was filtered and crystallized from CHCl3-CH3OH to give 1.9g
(75%) of the thiosemicarbazide. M.p.210-211 oC. Anal. Calc. for
C12H19N3OS (MW 253.36): C: 56.88; H: 7.55; N: 16.59. Found (%):
C: 56.85; H: 7.52; N: 16.54%.
2.6.1. 2-{[5-(Adamantan-1-yl)-1,3,4-thiadiazol-2-yl]-imino}
(benzylidene)-1,3-thiazolidin-4-one (1)
-5-
Yield: 84%, m.p. 213-214°C (dioxane), Rf = 0.73 (toluene:-
ethanol 8:2), IR: (cm-1, Nujol): 1457 , 1653 (arom), 1717 (C=O),
1
3360 (NH). H-NMR: (δ ppm, DMSO-d6, 300 MHz): 8.89 (s, 1H, -
NH) , 7.94 (t, 1H, C4) , 7.86 – 7.89 (t, 2H, C3, C5) , 7.46 – 7.49
(d, 2H C2, C6) , 7.12 (s, 1H, -CH=) , 1.56 – 2.29 (m, 15H,
adamantane). Anal.calc. for C22H22N4OS2 (MW 422.56) C: 62.53; H:
5.25; N:13.26%. Found:C: 62.49; H:5.29 ; N:13.20%
2.3. Synthesis of 5-adamantyl-2-amino-1,3,4-thiadiazole
To well stirred and cooled (<5OC) concentrated sulfuric acid (10
mL), 1-(adamantyl-5-carboxy)thiosemicarbazide (1.1 g, 0.005
mole) was added in small portions. After the final addition the
mixture was stirred for a further 15 min and was then allowed to
reach ambient temperature (25oC), left stirring for a further 30 min
and then poured cautiously onto crushed ice. The reaction mixture
was alkalized to pH 8 with aqueous ammonia and the precipitated
product was filtered off, washed with water and recrystallized from
2.6.2. 2-{[5-(Adamantan-1-yl)-1,3,4-thiadiazol-2-yl]-imino}-5-(4-
hydroxy-benzylidene)-1,3-thiazolidin- 4-one (2)
Yield: 49%, m.p. 258-259°C (dioxane), Rf = 0.71 (toluene-EtOH
7:3), IR: (cm-1, Nujol): 1570 (arom) , 1708 (C=O), 3089 (NH).
1H-NMR: (δ ppm, DMSO-d6, 300 MHz): 1.77 (s, 6H, adam.), 2.03-
2.07 (d, 9H, adam), 6.95-6.98 (d, 2H, C3, C5), 7.51-7.53 (d, 2H, C2,C6),
7.67 (s, 1H, C-CH=), 10.22 (s, 1H, -OH), 12.60 (s, 1H, NHCO). 13C-
CH3OH-CH2Cl2. Yield 87%. M.p. 200-201 C. IR (cm-1, KBr): 3050
o
Correspondent author: A.Geronikaki. Tel.: +302310997616; fax: +302310997612; e-mail: geronik@pharm.auth.gr

NMR (500 MHz, DMSO-d6): 28.44(3C), 31.78, 36.85(3C),
42.91(3C), 115.88(2C), 116.21, 126.12, 130.79(2C), 143.55,
158.32, 158.64, 159.21, 168.34, 173.28. Anal. Calcd. for
C22H22N4O2S2 (MW 438.56) C: 60.25; H: 5.06; N: 12.78%.
Found%: C: 60.17; H: 5.01; N: 12.70%.
7.22 (s, 1H, -CH=) , 1.01 – 2.49 (m, 15H, adamantane). 13C-NMR
(500 MHz,DMSO -d6): 28.43(3C), 31.72, 36.81(3C), 42.91(3C),
116.01, 122.88, 123.02, 129.34, 134.23, 136.10, 143.66, 147.82,
158.13, 159.12, 168.34, 173.22. Anal. Calcd. for C22H21N5O3S2
(MW 467.56) C: 56.51; H: 4.53; N: 14.98%. Found (%):C: 56.50; H:
4.55; N: 15.00%.
2.6.3. 2-{[5-(Adamantan-1-yl)-1,3,4-thiadiazol-2-yl]-imino}- 5-
(4-methoxy-benzylidene)1,3-thiazolidin-4-one (3)
2.6.8.2-{[5-(Adamantan-1-yl)-1,3,4-thiadiazol-2-yl]-imino}-5-(
4-nitrobenzylidene)-1,3thiazolidin-4-one (8)
Yield: 62%, m.p. 222-223°C (dioxane), Rf = 0.74 (toluene-EtOH
7:3), IR: (cm-1, Nujol): 1654 (arom) , 1717 (C=O), 3360 (NH).
1H-NMR: (δ ppm, DMSO-d6, 300 MHz): 8.79 (s, 1H, -NH) , 7.81-
7.83 (d, 2H, C2, C6) , 7.15 – 7.17 (d, 2H, C3, C5) , 7.11 (s, 1H, -
CH=), 3.78 (s, 3H, -OCH3), 1.56 – 2.27 (15H, adamantane). 13C-
NMR (500 MHz, DMSO-d6): 28.12(3C), 31.75, 36.15(3C),
42.92(3C), 55.93, 114.59(2C), 116.67, 127.93(2C), 130.96(2C),
142.33, 158.81, 158.96, 159.17, 168.32, 174.21. Anal. Calcd. for
C23H24N4O2S2 (MW 452.59) C: 61.04; H: 5.34; N: 12.38%.
Found%: C: 61.00; H: 5.31; N: 12.35%.
Yield: 72%, m.p. 306-307°C (dioxane), Rf = 0.76 (toluene-EtOH
8:2), IR: (cm-1, Nujol): 1559 (NO2),1654 (arom) 1717 (C=O),
1
3360 cm-1 (NH). H-NMR: (δ ppm, DMSO-d6 , 300 MHz): 8.68 (s,
1H, -NH), 8.10-8.12 (d, 2H, C3, C5), 7.75-7.77 (d, 2H, C2, C6) ,
7.21 (s, 1H, -CH=) , 1.05 – 2.49 (m, 15H, adamantane). 13C-NMR
(500 MHz, DMSO3-d6): 28.43(3C), 31.72, 36.81(3C), 42.93(3C),
116.11, 123.11(2C), 129.37(2C), 140.92, 143.73, 147.11, 158.23,
159.16, 168.35, 173.35. Anal. Calcd. for C22H21N5O3S2 (MW
467.56) C: 56.51; H: 4.53; N: 14.98%. Found (%):C: 56.53; H: 4.51;
N: 14.97%.
2.6.4. 2-{[5-(Adamantan-1-yl)-1,3,4-thiadiazol-2-yl]-imino}- 5-
(3-methoxy-4-hydroxy-benzylidene)-1,3-thiazolidin-4-one (4)
2.6.9.2-{[5-(Adamantan-1-yl)-1,3,4-thiadiazol-2-yl]-imino}-5-(4-
dimethylaminobenzylidene)-1,3-thiazolidin- 4-one (9)
Yield: 62%, m.p. 264-266°C (dioxane), R = 0.46 (petroleum ether:
f
ethyl acetate 1:1), IR: (cm-1, Nujol): 1600 (arom), 1711 (C=O), 3089
(NH). 1H-NMR: (δ ppm, DMSO-d6 300 MHz): ): 1.77 (s, 6H, adam),
2.03-2.08 (d, 9H, adam.), 3.85 (s, 3H, -OCH3), 7.00-7.03 (d, 1H, C5),
7.15-7.17 (d, 1H, C6), 7.29 (s, 1H, C2), 7.72 (s, 1H, -CH=), 9.95 (s, 1H,
-OH), 12.69 (s, 1H, NHCO). 13C-NMR (500 MHz, DMSO-d6):
28.44(3C), 31.78, 36.85(3C), 42.91(3C), 56.87, 111.85, 116.21,
116.93, 123.00, 128.51, 142.96, 148.11, 149.26, 158.32, 158.64,
168.41, 173.25.Anal. Calcd. for C23H24N4O3S2 (MW 468.59) C:
58.95; H:5.16; N: 11.96%.Found%: C: 58.86.; H: 5.20; N:11.90%.
Yield: 75%, m.p. 292-293°C (dioxane), Rf = 0.76 (toluene-
EtOH 8:2), IR: (cm-1, Nujol): 1654 (arom), 1717 (C=O), 3360
1
(NH). H-NMR: (δ ppm,, DMSO-d6 300 MHz): 9.34 (s, 1H, -NH) ,
7.23 – 7.25 (d, 2H, C2, C5) , 7.11 – 7.13 (d, 3H, C3, C4, -CH=) ,
2.39 (s, 6H, -CH3), 1.56-2.29 (m, 15H, adamantane). Anal. Calcd.
for C24H27N5OS2 (MW 465.63) C: 61.90; H: 5.84; N: 15.04%. Found
(%):C: 61.89; H: 5.80; N: 15.00%.
2.6.10. 2-{[5-(Adamantan-1-yl)-1,3,4-thiadiazol-2-yl]-imino}-5-
(4-fluorobenzylidene)-1,3-thiazolidin-4-one (10)
Yield: 66%, m.p. 123-124°C (dioxane), Rf = 0.75 (toluene-EtOH
8:2), IR: (cm-1, Nujol): 1653 (arom), 1717 (C=O), 3360 (NH). 1H-
NMR: (δ ppm, DMSO-d6, 300 MHz): 7.86 – 7.89 (dd, 2H, C3, C5),
7.46 – 7.49 (t, 2H C2, C6), 7.12 (s, 1H, -CH=), 1.56 – 2.29 (m,
15H, adamantane). 13C-NMR (500 MHz, DMSO-d6): 28.11(3C),
31.76, 36.15(3C), 42.92(3C), 115.66(2C), 116.85, 130.96(2C),
131.57, 142.31, 158.81, 159.27, 160.88, 168.32, 174.13. Anal.
Calcd. for C22H21FN4OS2 (MW 440.55) C: 59.98; H: 4.80; N: 12.72%.
Found (%):C: 59.96; H: 4.83; N: 12.75%.
2.6.5. 2-{[5-(Adamantan-1-yl)-1,3,4-thiadiazol-2-yl]-imino}-5-(
4-methylbenzylidene)-1,3thiazolidin-4-one (5)
Yield: 58%, m.p. 217-218°C (dioxane), Rf = 0.74 (toluene-EtOH
8:2), IR: (cm-1, Nujol): 1657 (arom) 1718 (C=O), 3360 (NH). 1H-
NMR: (δ ppm, DMSO-d6 300 MHz): 9.34 (s, 1H, -NH) , 7.23 –
7.25 (d, 2H, C2, C5) , 7.11 – 7.13 (d, 3H, C3, C4, -CH=) , 2.39 (s,
3H,-CH3), 1.56-2.29 (m, 15H, adamantane). 13C-NMR (500 MHz,
DMSO-d6): 21.33, 28.36(3C), 31.66, 36.81(3C), 42.95(3C),
116.88, 128.43(2C), 128.66(2C), 132.03, 137.22, 142.83, 158.80,
159.16, 168.31, 173.74. Anal. Calcd. for C23H24N4OS2 (MW
436.59) C: 63.27; H: 5.54; N: 12.83%.Found%: C: 63.23; H:
5.50; N: 12.80%.
2.6.11. 2-{[5-(Adamantan-1-yl)-1,3,4-thiadiazol-2-yl]-imino}-5-
(2,6-difluorobenzylidene)-1,3-thiazolidin-4-one (11)
Yield: 51%, m.p. 239-240°C (dioxane, Rf = 0.78 (toluene-EtOH
8:2), IR: (cm-1, Nujol): 1654 (arom), 1717 (C=O), 3360 (NH). 1H-
NMR: (δ ppm, DMSO-d6 , 500 MHz): 9.14 (s, 1H, - NH) , 7.40 (m,
1H, C4) , 7.18 (s, 1H, -CH=), 6.84 (t, 2H, C3, C5), 1.57 – 2.31 (m,
15H, adamantane). 13C-NMR (500 MHz, DMSO-d6): 28.44(3C),
31.74, 36.85(3C), 42.91(3C), 111.37(2C), 112.98, 116.32,
129.77, 143.55, 158.02, 158.36(2C), 159.21, 168.24, 173.75.
Anal. Calcd. for C22H20F2N4OS2 (MW 458.55) C: 57.62; H: 4.39; N:
12.22%. Found (%):C: 57.61; H: 4.43; N: 12.25%.
2.6.6. 2-{[5-(Adamantan-1-yl)-1,3,4-thiadiazol-2-yl]-imino}-5-(
2-nitrobenzylidene)-1,3-thiazolidin-4-one (6)
Yield: 71%, m.p. 239-240°C (dioxane), Rf = 0.77 (toluene-EtOH
8:2), IR: (cm-1, Nujol): 1559 (νNO2), 1654 (arom) 1717 (C=O),
1
3360 (NH). H-NMR: (δ ppm, DMSO-d6, 300 MHz): 8.84 (s, 1H, -
NH) , 8.20 (d, 1H, C5) , 7.92 (t, 1H, C4) , 7.78 (d, 1H, C2) , 7.71
(d, 1H, C3) , 7.51 (s, 1H, -CH=) , 1.56 – 2.29 (m, 15H,
adamantane). 13C-NMR (500 MHz, DMSO3-d6): 28.43(3C),
31.72, 36.81(3C), 42.92(3C), 116.41, 123.66, 127.12(2C),
128.95, 134.81, 143.65, 147.22, 158.12, 159.11, 168.34, 173.68.
Anal. Calcd. for C22H21N5O3S2 (MW 467.56) C: 56.51; H: 4.53; N:
14.98%. Found (%):C: 56.48; H: 4.50; N: 14.94%.
2.6.12.2-{[5-(Adamantan-1-yl)-1,3,4-thiadiazol-2-yl]-imino}-5-
(4-bromobenzylidene)-1,3-thiazolidin-4-one (12)
Yield: 39%, m.p. 270-271°C (dioxane), Rf = 0.64 (petroleum
ether: ethylacetate 1:1), IR: (cm-1, Nujol): 1718 (C=O), 3067
1
(NH). H-NMR: (δ ppm, DMSO-d6, 300 MHz): 1.66-1.97 (m, 15H,
2.6.7.2-{[5-(Adamantan-1-yl)-1,3,4-thiadiazol-2-yl]-imino}-5-(
3-nitrobenzylidene)-1,3-thiazolidin-4-one (7)
adam.), 7.51-7.53 (d, 2Η, C3, C5), 7.66 (s, 1H, -CH=), 7.69-7.72 (d,
2H, C2, C6), 11.97 (s, 1H, NHCO). 13C-NMR (500 MHz, DMSO-
d6): 28.44(3C), 31.78, 36.85(3C), 42.91(3C), 116.11, 122.13,
128.56(2C), 131.79(2C), 133.22, 143.55, 158.32, 159.21, 168.34,
173.85. Anal. Calcd. for C23H21BrN4OS2 (MW 501.46) C: 52.69;
H: 4.22; N: 11.17%.Found%: C: 52.75; H: 4.25; N: 11.14 %.
Yield: 69%, m.p. 257-258°C (dioxan), Rf = 0.76 (toluene-EtOH
8:2), IR: (cm-1, Nujol): 1559 (NO2),1653 (arom) 1717 (C=O),
1
3360 (NH). H-NMR: (δ ppm, DMSO-d6, 300 MHz): 9.29 (s, 1H, -
NH) , 8.94 (s, 1H, C2) , 8.30-8.32 (d, 2H, C4, C6) , 7.85 (t, 1H, C5) ,
Correspondent author: A.Geronikaki. Tel.: +302310997616; fax: +302310997612; e-mail: geronik@pharm.auth.gr

Pseudomonas aeruginosa (ATCC 27853), Salmonella
typhimurium (ATCC 13311), as well as Gram-positive bacteria:
Listeria monocytogenes (NCTC 7973), Bacillus cereus (clinical
isolate), Micrococcus flavus (ATCC 10240), and Staphylococcus
aureus (ATCC 6538) were used. The organisms were obtained
from the Mycological Laboratory, Department of Plant
Physiology, Institute for Biological Research ‘Siniša Stankovic,
Belgrade, Serbia
2.6.13.2-{[5-(Adamantan-1-yl)-1,3,4-thiadiazol-2-yl]-imino}-5-
(3-chlorobenzylidene)-1,3- thiazolidin-4-one (13)
Yield: 56%, m.p. 248-250°C (dioxane), R = 0.84 (petroleum ether:
f
ethylacetate 1:1), IR: (cm-1, Nujol): 1615 (arom), 1696 (C=O), 3092
(NH). 1H-NMR: (δ ppm, DMSO-d6 300 MHz): 1.72 (s, 6H, adam.),
1.98-2.02 (d, 9H, adam.), 7.58-7.72 (m, 5H, C2,C5, C4,C6, -CH=), 12.87
(s, 1H, NHCO). 13C-NMR (500 MHz, DMSO-d6): 28.43(3C),
31.75, 36.85(3C), 42.91(3C), 116.17, 126.38(2C), 128.11,
130.55, 134.21, 136.65, 142.13, 158.80, 159.17, 168.32, 174.05.
Anal. Calcd. for C22H21ClN4OS2 (MW 457) C: 57.82; H: 4.63; N:
12.26%. Found%:C: 57.79; H: 4.65; N: 12.21%.
The minimum inhibitory (MIC) and minimum bactericidal
(MBC) concentrations were determined by the microdilution
method. Briefly, fresh overnight culture of bacteria was adjusted
by the spectrophotometer to a concentration of 1×105 CFU/mL.
Dilutions of inocula were cultured on solid medium to verify the
absence of contamination and check the validity of the inoculum.
Tested compounds were dissolved in 5% DMSO and added in
broth Triptic Soy broth (TSB) medium (100 µL) with bacterial
inoculum (1.0×104 CFU per well) to achieve the wanted
concentrations (0.001-1.0 mg/ml) in dilution order. The
microplates were incubated for 24 h at 370C. The MIC of the
samples was detected following the addition of 40 µL of
iodonitrotetrazolium chloride (INT) (0.2 mg/mL) and incubation
at 37°C for 30 min. The lowest concentration that produced a
significant inhibition of the growth of the bacteria in comparison
with the positive control was identified as the MIC. The optical
density of each well was measured at a wavelength of 655 nm by
Microplate manager 4.0 (Bio-Rad Laboratories) and compared
with a blank and the positive control. The minimum inhibitory
concentrations (MICs) obtained from the susceptibility testing of
various bacteria to tested extracts were determined also by a
colorimetric microbial viability assay based on reduction of a
INT color and compared with positive control for each bacterial
strains. MBC was determined by serial sub-cultivation of 10 µL
into microplates containing 100 µL of TSB. The lowest
concentration that shows no growth after this sub-culturing was
read as the MBC indicating 99.5% death of the original
inoculum. Standard drugs, namely streptomycin and ampicillin
were used as positive controls. Five % of DMSO was used as
negative controls. All experiments were performed in duplicate
and repeated three times.
2.6.14. 2-{[5-(Adamantan-1-yl)-1,3,4-thiadiazol-2-yl]-imino}-5-
(4-chlorobenzylidene)-1,3- thiazolidin-4-one (14)
Yield: 59%, m.p. 263-264°C (dioxane), Rf = 0.67 (toluene-ethanol
1
8:2), IR: (cm-1, Nujol): 1578 (arom), 1718 (C=O), 3110 (NH). H-
NMR: (δ ppm, CDCl3, 300 MHz): 1.74 (s, 6H, adam.), 1.99-2.05 (d,
9H, adam.), 7.63-7.65 (m, 4H, C2,C3, C5,C6), 7.73 (s, 1H, -CH=), 12.82
(s, 1H, NHCO). 13C-NMR (500 MHz, CHCl3-d6): 28.49(3C),
31.15, 36.85(3C), 42.91(3C), 116.03, 128.72(2C), 129.27 (2C),
133.01, 133.68, 142.95, 158.30, 159.15, 168.34, 173.39. Anal.
Calcd. for C22H21ClN4OS2 (MW 457) C: 57.82; H: 4.63; N:
12.26%.Found%:C: 57.85; H: 4.58; N: 12.31 %.
2.6.15. 2-{[5-(Adamantan-1-yl)-1, 3, 4-thiadiazol-2-yl]-imino}-5-
(2,3-dichlorobenzylidene)-1,3- thiazolidin-4-one (15)
Yield: 67%, m.p. 242-243°C (dioxane), Rf = 0.75 (toluene-ethanol
1
8:2), IR: (cm-1, Nujol): 1654 (arom), 1717 (C=O), 3360 (NH). H-
NMR: (δ ppm, CDCl3, 300 MHz): 7.62 (s, 1H, C4), 7.15 (d, 2H, C5,
C6), 7.12 (s, 1H, -CH=), 1.56 – 2.27 (m, 15H, adamantane). 13C-NMR
(500 MHz, CHCl3-d6): 28.77(3C), 32.41, 36.95(3C), 42.98(3C),
116.49, 125.34, 128.12(2c), 130.02, 134.52, 142.66, 143.19,
158.77, 159.34, 168.31, 174.18. Anal. Calcd. for C22H20Cl2N4OS2
(MW 491.55) C: 53.75; H: 4.10; N: 11.40%. Found (%):C: 53.73; H:
4.12;N: 11.44%.
2.6.16. 2-{[5-(Adamantan-1-yl)-1,3,4-thiadiazol-2-yl]-imino}-5-
(2,4-dichlorobenzylidene)-1,3- thiazolidin-4-one (16)
The antibacterial assay was carried out by the microdilution
method as previously reported.53,54 All experiments were
performed in duplicate and repeated three times.
Yield: 46%, m.p. 219-220°C (dioxane), R = 0.84 (toluene-ethanol 8:2),
f
IR: (cm-1, Nujol): 1654 (arom), 1717 (C=O), 3360 (NH). 1H-NMR: (δ
ppm, CDCl3,300MHz):8.94 (s, 1H, -NH), 7.62(s, 1H, C3), 7.15 (d, 2H,
C5, C6), 7.12 (s, 1H, -CH=), 1.56 – 2.18 (m, 15H, adamantane). 13C-
NMR (500 MHz, CHCl3-d6): 28.15(3C), 32.12, 36.96(3C),
42.74(3C), 116.45, 125.31, 126.72 128.33(2c), 129.55, 130.11,
136.42, 143.08, 158.73, 159.24, 168.25, 173.97. Anal. Calcd. for
C22H20Cl2N4OS2 (MW 491.55) C: 53.75; H: 4.10; N: 11.40%. Found
(%):C:53.80; H:4.00; N:11.38%.
3.2. Antifungal activity
For the antifungal bioassays, eight fungi were used:
Aspergillus niger (ATCC 6275), Aspergillus ochraceus (ATCC
12066), Aspergillus fumigatus (1022), Aspergillus versicolor
(ATCC 11730), Penicillium funiculosum (ATCC 36839),
Penicillium ochrochloron (ATCC 9112), Trichoderma viride
(IAM 5061), and Candida albicans (human isolate). The
organisms were obtained from the Mycological Laboratory,
Department of Plant Physiology, Institute for Biological
Research ‘‘Siniša Stankovic,’’ Belgrade, Serbia. All experiments
were performed in duplicate and repeated three times.55,56
2.6.17. 2-{[5-(Adamantan-1-yl)-1,3,4-thiadiazol-2-yl]-imino}-5-
(2,6-dichlorobenzylidene)-1,3- thiazolidin-4-one (17)
Yield: 51%, m.p. 223-224°C (dioxane), Rf = 0.78 (toluene-
ethanol 8:2), IR: (cm-1, Nujol): 1654 (arom), 1717 (C=O), 3360
1
(NH). H-NMR: (δ ppm, DMSO-d6 300 MHz): 7.54 (d, 2H, C3, C5),
7.22 (s, 1H, -CH=) , 1.60 – 2.29 (m, 15H, adamantane). Anal. Calcd.
for C22H20Cl2N4OS2 (MW 491.55) C: 53.75; H: 4.10; N: 11.40%.
Found (%):C: 53.75; H: 4.11; N: 11.41%.
The micromycetes were maintained on malt agar and the
cultures were stored at 4° C and sub-cultured once a month. The
antifungal assay was carried out by modified microdilution
technique. The fungal spores were washed from the surface of
agar plates with sterile 0.85% saline containing 0.1% Tween 80
(v/v). The spore suspension was adjusted with sterile saline to a
concentration of approximately 1.0×105 in a final volume of 100
µL per well. The inocula were stored at 4° C for further use.
Dilutions of the inoculum were cultured on solid malt agar to
verify the absence of contamination and to check the validity of the
3. Biological assays
3.1. Antibacterial activity
The following Gram-negative bacteria: Escherichia coli
(ATCC 35210), Enterobacter cloacae (clinical isolate),
Correspondent author: A.Geronikaki. Tel.: +302310997616; fax: +302310997612; e-mail: geronik@pharm.auth.gr

inoculum. MIC determinations were performed by a serial dilution
technique using 96-well microtiter plates. The examined compounds
were diluted in 5% of DMSO (0.001-1.0 mg/ml) and added in broth
Malt medium (MA) with inoculum. The microplates were incubated
at rotary shaker (160 rpm) for 72 h at 28° C. The lowest
concentrations without visible growth (at the binocular microscope)
were defined as MICs. The fungicidal concentrations (MFCs) were
determined by serial subcultivation of 2 µL of tested fractions
dissolved in medium and inoculated for 72 h, into microtiter plates
containing 100 µL of broth per well and further incubation 72 h at
28° C. The lowest concentration with no visible growth was defined
as MFC indicating 99.5% killing of the original inoculum. The
fungicides bifonazole and ketoconazole were used as positive
controls (1-3500 µg/mL). Three independent experiments were
performed in duplicate.
For the estimation of the probable mechanism of antifugal activity,
all the synthesized compounds were docked in the active site of
Dihydrofolate reductase 4HOF and CYP51 5V5Z of C. albicans
(docking box centers: x=-0.895, y=0.131, z=32.109 and x=-47.731,
y=-13.422, z=22.982, respectively). The human lanosterol 14a-
demethylase CYP51 (3LD6), was also used for estimation of the
selectivity of the compounds against the fungal enzyme. The
docking box was also set at 50×50×50 Å and centered at x=39.272,
y=8.184 and z=3.545. All selected structures were in complex with
inhibitors. Docking of these inhibitors to their enzyme structures was
performed for verification of the method (docking results at the
supplementary files, Fig. 2S and 3S). The reference compound
ketoconazole was also docked in the active site of 5V5Z structure.
Molecular Docking Studies of all compounds in E.coli MurB
2Q85, MuA (3ΚR6) and dihydrofolate reductase (4HOF) and
CYP51 (5V5Z) of C. albicans were performed using the software
Autodock 4.2. For the simulation, default values of quaternation,
translation and torsion steps were applied. The Lamarckian Genetic
Algorithm with default parameters was applied for minimization.
The number of docking runs was 100. The graphical depictions of
all ligand-protein complexes were consummated by Discovery
studio visualizer version 4.0(BIOVIA, San Diego, CA, USA) and
LigandScout 4.1.1. For the preparation of ligand structures, 2D
structure was sketched in Chem Draw 12.0 and converted to 3D
structure, mol2 format, for each ligand. Hydrogens were added to
the structures and used for docking. All procedures were performed
according to previous papers.59,60 The online service of Molecular
Docking Server, also based on AutoDock, was used for MurB
enzymes of Pseudomonas aeruginosa (4JAY), Listeria
monocytogenes (3TX1) and Staphylococcus aureus (1HSK) as
previously described in our paper.61
3.3. Statistical analysis
All the assays were carried out in triplicate and the results are
expressed as mean values and standard deviation (SD). The results
were analyzed using one-way analysis of variance (ANOVA) followed
by Tukey’s HSD Test with α = 0.05. This treatment was carried out
using SPSS v. 18.0 programs.
3.4. Docking studies
MurB, a common target of thiazolidinones was chosen for
molecular docking studies in order to estimate the probable
mechanism of antibacterial action. Among the available
structures of MurB in the Protein Data Bank, the structure 2Q85
of E.coli MurB with its inhibitor, (5Z)-3-(4-chlorophenyl)-4-
hydroxy-5-(naphthalen-1-ylmethylidene)furan-2-one, was chosen
for docking analysis of the compounds. The MurB structures of
Pseudomonas aeruginosa (4JAY and 4JB1), Listeria
monocytogenes (3TX1) and Staphylococcus aureus (1HSK) are
also available in the Data Bank and were also used for docking
analysis of the most potent compounds. However, the structures
3TX1 and 1HSK correspond to enzymes crystallized without
substrate or inhibitor and may not be the most appropriate for
docking of potential inhibitors. Since conformational changes
usually happen upon interaction of enzymes with substrates or
inhibitors, structures derived from such complexes usually better
present the domains needed for binding of the inhibitor.57,58 The
structure 4JAY of Pseudomonas aeruginosa MurB is derived
from a complex of the enzyme with each co-enzyme FAD and
NADPH which occupies the binding site of substrate or inhibitor
and can be considered as appropriate for inhibitor screening.
During enzyme preparation the inhibitor and the NADPH were
abstracted from the structures of 2Q85 and 4JAY respectively.
The co-enzyme, FAD, was maintained in all structures. The
docking box was 50x50x50 Å in all cases. For the determination
of the docking box center of the E.coli MurB (2Q85), the oxygen
of -OH of the side chain of the catalytic amino acid Ser228 was
used (x=7.534, y=-3.428 and z=11.525). The docking box was
centered at x=47.65, y=-11.56 and z=14.08 for 4JAY, at x=-
26.78, y=26.31 and z=-3.51 for 3TX1 and at x=180.81, y=148.25
and z =164.78 for 1HSK. The area of substrate/inhibitor binding
and the co-enzyme FAD were always included in the docking
box. The known MurB inhibitor Naphthyl Tetronic Acid was also
docked for comparison reasons (Fig. 1S, supplementary files).
Docking of some compounds at the E.coli MuA (3ΚR6) was also
performed. The docking box was set at 50ꢀ×ꢀ50ꢀ×ꢀ50 Å and was
centered at x=60.1366, y=46.2431 and z=148.6266 (center of the
bound inhibitor fosfomycin) .
4. Results and discussion
4.1. Prediction of Toxicity
As prediction of toxicity of a compound is a very important step
in the design of new drug candidates, the in silico toxicity study
can be used since it is a more rapid and less expensive process
than in vivo toxicity testing in animals and in vitro testing in cell
lines. Besides, it can help to significantly reduce the number of
animals used in the experimental assays. There are several online
programs using in silico models to access toxicity, that predict
average lethal dose, carcinogenicity, mutagenicity etc.
Two computer programs, ToxPredict (OPEN TOX) and
PROTOX, were used in this work .62-64
The ToxPredict program predicts the probability of
carcinogenicity of the compounds in various organisms, as well
as the probability of mutagenesis using an in silico model
corresponding to the Ames test. The results are presented in
Table 1S (Supplementary material). It should be mentioned that
the accuracy of prediction increases as the confidence values
rises. In particular, reliable estimates are considered to be more
than 0.025.
The PROTOX program [58], predicts the average lethal dose
(LD50) in rodents. According to this program all chemical
compounds can be classified into six GHS (Globally Harmonized
System of Classification and Labeling of Chemicals) categories,
65
depending on the toxicity of the compounds and the LD50
values (Table 2S).
Category I: LD50 ≤ 5 mg/kg
Correspondent author: A.Geronikaki. Tel.: +302310997616; fax: +302310997612; e-mail: geronik@pharm.auth.gr

S
a
N
N
N
Category II: 5 < LD50 ≤ 50 mg/kg
Category III: 50 < LD50 ≤ 300 mg/kg
Category IV: 300 < LD50 ≤ 2000 mg/kg
Category V: 2000 < LD50 ≤ 5000 mg/kg
Category VI: LD50 > 5000 mg/kg
CONHNHCNH2
NH2
S
N
N
N
b
+
ClCOCH2Cl
NHCOCH2Cl
NH2
S
S
O
N
HN
N
N
N
N
c
+
NH4SCN
NHCOCH2Cl
N
S
S
S
All compounds tested were in category IV.
O
O
R
R
HN
HN
N
N
N
d
CHO
+
4.2. Chemistry
N
S
N
S
S
S
1-17
The general method employed to prepare the final compounds
is shown in scheme 1. 5-Adamantyl-2-(1,3,4-thiadiazole)imino-
5-arylidene-4-thiazolidinones were synthesized starting from 5-
adamantyl-1,3,4-thiadiazole, prepared from the reaction of 1-
adamantyl-5-carbonylchloride with thiosemicarbazide and
subsequent cyclization of the intermediate 1-(1-adamantyl-5-
carboxy)thiosemicarbazide in cold sulfuric acid (Scheme 1). 5-
Adamantyl-1,3,4-thiadiazolyl-2-chloroacetamide, obtained by
Com.
R
Com.
10
R
1
2
3
4
5
6
7
8
9
H
4-F
4-OH
11
2,6-di-F
4-Br
4-OMe
12
4-OH, 3-OMe
4-Me
13
3-Cl
reacting of
5-adamantyl-1,3,4-thiadiazolyl-2-amine with
14
4-Cl
chloroacetyl chloride, underwent heterocyclization in the
presence of ammonium thiocyanate in refluxing ethanol leading
to 2-{[5-(adamantan-1-yl)-1,3,4-thiadiazol-2-yl]imino}-1,3-thiazoli-
din-4-one. The 2-{[(5-adamantan-1-yl)-1,3,4-thiadiazol-2-yl]-imino}-
5-arylidene-1,3-thiazolidin-4-ones were obtained by heating to reflux
the previous thiazolidinone with the appropriate aldehydes in buffered
glacial acetic acid. Overall the reactions proceeded smoothly in good
yields. Yields, physical properties and molecular formulas are given in
the experimental part. Structures of all compounds were confirmed
2-NO2
15
2,3-di-Cl
2,4-di-Cl
2,6-di-Cl
3-NO2
16
4-NO2
17
N(CH3)2
Scheme 1. Synthesis of 2-{[5-(adamantan-1-yl)-1,3,4-
thiadiazol-2-yl]-imino}-5-arylidene-1,3-thiazolidin-4-ones (a:
conc.H2SO4, <5oC(15 min), r.t.30 min; b: DMF, 3h, r.t.;
c:NH4SCN, EtOH reflux 1h, r.t. overnight; d: gl.CH3COOH,
CH3COONa, refl.4h)
1
spectroscopically (IR, H-NMR, 13C-NMR, MS) and by elemental
analyses.
The mechanism of cyclization with formation of
thiazolidinone was mentioned in our previous paper.46 The IR
spectra were in agreement with the proposed structures, showing
a sharp band in the region 1680–1720 cm–1 (NHC=O).The
substitution position in the cyclocondensation step and the
tautomeric structure of 2-imino-5-arylidene-4-thiazolidinones (1-
Thus, the most sensitive bacterium appeared to be S.
typhimirium, followed by B. cereus while L. monocitogenes and
M. flavus were the most resistant. Compounds (1), (2), (6), (7),
(8), (12), (14) exhibited good activity against S. typhimirium with
MIC at 1.40-3.40 × 10-2 microM and MBC at 3.30-6.60 x 10-2
microM, while some of them (2,6,7,8,14) with MIC and MBC at
2.14-3.40 and 4.28-6.60 × 10-2 microM respectively, were also
potent against P. aeruginosa. Good activity was observed for
compounds (8) and (11) against En. cloacae, as well as for
compound (8) against E. coli. On the other hand (6-8) and (12)
appeared to be active against the Gram positive bacterium B.
cereus, while (7) and (8) were also potent against the most
resistant bacterium L. monocytogenes as well as against M. flavus
and S. aureus, (7) being more active than ampicillin and in some
cases even than streptomycin. Compound (7) appeared to be
about 29 times more active than ampicillin and 20 times than
streptomycin against L. monocytogenes.
1
17) were determined through the analysis of IR and H NMR
spectroscopic data. Thiazolidinones show, in their 1H NMR
spectra, a NH proton at 12.00-12.87 ppm, in accordance with a
lactam proton and not with an imine proton (expected around
9.70 ppm).66-68
5. Biological evaluation
5.1. Antimicrobial activity
The antimicrobial activity of the synthesized compounds was
evaluated using the microdilution method for determining the
minimal inhibitory and minimal bactericidal/fungicidal
concentrations.
In particular, for the Gram-positive bacteria the range of MIC
and MBC was 1.65-29.50 × 10-2microM and 2.14-82.2 × 10-2
microM respectively, whereas for Gram-negative bacteria the
MIC and MBC ranged from 1.40-29.50 × 10-2 microM, and 1.80
to 59.0 × 10-2 microM respectively. It seems that the tested
compounds are more potent against Gram negative bacteria than
against Gram positive.
It can be noticed that compounds 1, 2, 3, 4, 5, 6, 7, 8, 9, 11, 16
and 17 showed better antibacterial potential than both antibiotics
tested, and all compounds exhibited higher activity then
ampicillin (Table 1).
Results of antibacterial activity of adamantane thiadiazole –
based thiazolidinones. (1)-(17) are shown in Table 1. The
antibacterial potential can be presented as follows: 8 > 6 > 7 > 11
> 1 > 2 > 3 > 17 > 5 > 4 > 9 > 16 > 12 > 15 > 13 > 10 > 14.
The best antibacterial activity is achieved for compound (8),
with MIC at 1.65-3.30 × 10-2 mmol/mL and MBC at 3.30-6.60×
10-2 microM. Compound (14) showed the lowest antibacterial
efficacy with MIC and MBC at 3.30-32.8 × 10-2 microM and
6.60-82.20 × 10-2 microM, respectively. It should be mentioned
that bacteria in general showed different sensitivities to
compounds tested.
A structure-activity relationship study revealed that the presence of
a nitro group in general is favorable for antibacterial activity. At the
same time the position and not onlythe nature of the substituent on the
Correspondent author: A.Geronikaki. Tel.: +302310997616; fax: +302310997612; e-mail: geronik@pharm.auth.gr

Table.1. Antibacterial potential of tested compounds (MIC/MBC × 10-2 microM) and calculated Free binding Energy
(Kcal/mol) to E.coli MurB (2Q85) and E.coli MurA (3KR6)
O
R
HN
N
N
N
S
S
MIC/MBC × 10-2 microM
Free Binding
Energy
(Kcal/mol)
Com/ds
8
R
S.a.
1.65
B.c.
1.65
L.m.
3.30
M.f.
2.20
S.t.
En.cl.
P.a.
2.20
E.c.
2.20
2Q85
3KR6
4-NO2
MIC
1.65
1.80
-13.55
MBC
3.30
3.30
6.60
3.30
3.30
6.60
4.40
3.30
6
2-NO2
3-NO2
2,6-di-F
H
MIC
MBC
3.22
3.22
6.44
3.22
3.22
3.22
3.22
3.22
3.22
-12.85
-12.46
-9.87
6.44
6.44
6.44
6.44
6.44
6.44
6.44
7
MIC
MBC
4.18
2.14
4.20
1.70
4.18
2.14
4.18
2.14
4.18
-9.35
8.36
2.14
8.36
4.28
8.16
4.28
8.16
11
1
MIC
MBC
4.36
4.36
2.18
4.36
4.36
2.18
4.36
4.36
8.72
8.72
4.36
8.72
8.72
4.36
8.72
8.72
MIC
4.73
9.47
9.47
9.47
2.36
9.47
9.47
9.47
-9.74
MBC
9.47
18.94
18.94
18.94
4.72
18.94
18.94
18.94
2
4-OH
MIC
MBC
6.80
3.40
28.40
14.20
3.40
3.40
3.40
9.10
-9.01
-8.72
-8.27
-8.22
14.20
6.80
56.80
56.80
6.80
14.20
6.80
14.20
3
4-OMe
2,6-di-Cl
4-Me
MIC
MBC
4.48
9.95
4.48
9.95
4.48
9.95
9.95
9.95
8.96
19.90
8.96
19.90
8.96
19.90
19.90
19.90
17
5
MIC
MBC
4.07
4.07
2.14
4.07
4.07
2.14
4.07
4.07
-10.44
4.06
32.58
32.58
32.58
16.30
32.58
32.58
32.58
MIC
11.46
11.46
5.93
11.46
5.93
11.46
11.46
11.46
MBC
22.72
22.72
11.46
22.72
11.46
22.72
22.72
22.72
4-OH,
3-OMe
4
9
MIC
MBC
3.20
26.80
3.20
13.40
13.40
53.60
13.40
26.80
3.20
13.40
3.20
13.40
3.20
13.40
13.40
26.80
-8.01
-7.22
4-N(Me)2
2,4-di-Cl
4-Br
MIC
MBC
13.91
13.91
6.95
13.91
6.95
13.91
6.95
13.91
27.82
27.82
13.90
27.82
13.90
27.82
13.90
27.82
16
12
15
13
MIC
MBC
2.03
16.29
16.29
16.29
8.15
16.29
16.29
16.29
-7.15
-7.03
-6.88
-6.74
4.06
32.58
32.58
32.58
16.30
32.58
32.58
32.58
MIC
MBC
6.00
1.40
25.00
25.00
1.40
6.00
6.00
8.00
-8.51
-5.1
25.00
6.00
50.00
50.00
6.00
12.50
12.50
12.50
2,3-di-Cl
3-Cl
MIC
MBC
24.43
24.43
24.43
24.43
12.21
24.43
12.21
24.43
48.86
48.86
48.86
48.86
24.42
48.86
24.42
48.86
MIC
8.80
6.60
13.70
6.60
6.60
13.70
6.60
6.60
-8.29
MBC
13.70
13.70
54.80
27.40
13.70
27.40
13.70
27.40
10
14
4-F
MIC
MBC
14.75
29.50
29.50
29.50
29.5
14.75
29.50
29.50
-6.25
-5.74
29.5
59.00
59.00
59.00
59.00
29.50
59.00
59.00
4-Cl
MIC
13.70
13.70
27.40
32.80
3.30
13.70
3.30
27.40
MBC
54.80
27.40
82.20
54.80
6.60
27.40
6.60
54.80
Amp.
MIC
MBC
MIC
MBC
24.80
37.20
17.20
34.40
24.8
37.2
4.30
8.60
37.20
74.40
25.80
51.60
24.80
37.20
8.60
24.80
49.20
17.20
34.40
24.80
37.20
4.30
74.40
124.0
17.20
34.40
37.20
49.20
17.20
34.40
Strept.
17.20
8.60
B.c.-B.cereus, M.f.-M.flavus, S.a.-S.aurues, l.m.L.monocytogenes, E.c.-E.coli, En.c.-En.cloacae, P.a.-P.aeruginosa, S.t.-S.typhimurium. Relative standard
deviations were all < 2.0. Amp.: Ampicillin, Strept.: Streptomycin. Colors for MIC: pink:1.40-5.00, orange:5.01-10.00, green:10.01-20.00, light blue:20.01-
25.00, dark blue:>25.00. Colors for Free Binding Energy: pink:<-9.80, orange: -9.79 to -8.50, green: -8.49 to -7.00, light blue:-6.99 to 6.50, dark blue:-6.49 to -
5.50, grey:>-5.50
Correspondent author: A.Geronikaki. Tel.: +302310997616; fax: +302310997612; e-mail: geronik@pharm.auth.gr

Table 2. Antibacterial potential of tested compounds toward
The most sensitive bacterial species is S. typhiumurium, while
M. flavus was the most resistant among ATCC strains, and
MRSA was the most resistant among all tested bacteria (ATCC
and clinical isolates).
resistant strains of bacteria (MIC/MBC × 10-2 microM).
Comp.
R
MRSA
P.a.
E.c.
Ampicillin exhibited an inhibitory potential at 24.8-74.4 × 10-2
microM and bactericidal at 37.2-124.0 × 10-2 microM but no
bacteriostatic or bactericidal effects were observed against
MRSA. Streptomycin possessed MIC at 4.30-17.20 × 10-2
microM and MBC at 8.60-34.40 × 10-2 microM, but no
bactericidal effect was observed against MRSA.
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
MIC
MBC
4.73
8.14
4.73
8.14
2.36
4.72
5.53
11.06
11.47
22.94
17.50
35.00
5.35
10.7
0.60
0.80
7.51
15.02
29.54
59.08
39.30
78.60
18.32
36.64
2.03
1
3
H
11.06
22.12
11.47
22.94
17.50
35.00
10.7
21.4
0.80
1.65
15.05
30.10
29.54
59.08
39.30
78.60
18.32
36.64
8.14
11.06
22.12
11.47
22.94
17.50
35.00
5.35
10.7
0.80
0.80
15.05
30.10
29.54
59.08
39.30
78.60
18.32
8.14
4-OMe
4-Me
5
6
2-NO2
3-NO2
4-NO2
N(CH3)2
Structure-activity relationship studies revealed that the
presence of nitro group in position 4 of benzene ring (8) favours
the activity against resistant strains. But in general substitution in
benzene ring of the parent compound (1) decrease the activity
towards resistant strains. It was observed that this depends not
only on the nature of substituent but also on its position in
benzene ring. Thus, 2,6-dichloro- substitution is more favourable
than 2,4-dichloro- and much more favourable than 2,3-dichloro-.
The same was found for nitro derivatives (4-NO2 >> 2-NO2),
while fluoro- derivatives appeared to be the less active against
resistant bacteria strains.
7
8
9
10
11
15
16
17
4-F
2,6-di-F
2,3-di-Cl
16.28
16,28
6.11
2,4-di-Cl
2,6-di-Cl
The antifungal potential of tested compounds is presented in
Table 3 and follows the order: 8 > 1 > 4 > 12 > 17 > 3 > 7 > 10 >
16 > 13 > 14 > 2 > 9 > 15 > 5 > 11 > 6. Compound (8) showed
the best antifungal potential, as in the case of antibacterial
potential. Inhibitory activity was achieved at 0.60-1.65 × 10-2
microM, and fungicidal at 0.80-3.30 × 10-2 microM. The lowest
antifungal effect was observed for compound (6), with MIC and
MFC at 32.11-48.17 × 10-2 microM and 64.22-96.34 × 10-2
microM, respectively.
16.28
6.11
12.22
4.06
3.05
6.1
12.22
MIC
MBC
MIC
-
-
124.0
-
124.0
-
Ampicillin
25.80
-
12.90
25.80
25.80
51.60
Streptomycin
MBC
Ketoconazole showed antifungal potential at MIC 37.60-380.00 ×
MRSA-Methicillin resistant Staphylococcus aureus, P.a-Pseudomonas
aeruginosa, E.c.- Escherichia coli.
-2
10-2 and MFC 94.00-475.00 × 10 microM respectively, while
-2
bifonazole showedMIC at 32.00-64.00 × 10 and MFC at 48.30-80.00
× 10-2 microM respectively. From the results obtained it can be seen
that all tested compounds exhibited higher antifungal potential than
both antimycotics tested (Table 3.).
Relative standard deviations were all < 1.2
benzene ring also influences the activity. Thus presence of a
substituent at the 4-position is most favorable followed by 2-NO2 (6)
and 3-NO2 (7) respectively. Introduction to the unsubstituted derivative
(1) of OH (2) and OMe (3) groups in position 4 of the benzene ring led
to a slight decrease in activity of these derivatives, while introduction
of a 4-Me (5) group decreased the activity further. Introduction of a
methoxy group at position 3 of compound (2) also had a negative
effect on the activity (compound 4). As far as halogen derivatives are
concerned, in general, monosubstitution is not favorable, for fluoro-,
bromo- or chloro- derivatives. However, 2,6-fluoro- (11) substitution
exhibited a completely different effect on antibacterial activity. Among
the dichloro- substituted derivatives the order of activity was found to
be 2,6-Cl > 2,4-Cl > 2,3-Cl, showing that in case of dihalogen
substitution, position 6 is more favorable compared to others.
The most sensitive fungal species are T. viride and A.
ochraceus while the A. fumigatus appeared to be the most
resistant one (Table 3).
Structure-activity relationship studies revealed that, while the
presence of nitro group in position 4 (8) of benzene ring is beneficial
for antifungal activity, 2-NO2 (6) substitution is deleterious while the
3-NO2 derivative (7) exhibited moderate activity. Derivative (1)
lacking any substituent on the benzene ring appeared to be the second
most active compound against fungi, while introduction of other
substituents led to decreases in activity. Thus, introduction of bromine
into position 4 of the benzene ring resulted in the third most active
compound (12) while introduction of a 4-chloro- substituent
remarkably decreased the activity. Among the dichloro- derivatives a
beneficial effect was observed for 2,6-disubstitution compared to 2,4-
and 2,3-substitution.
The antibacterial capacity of the tested compounds towards
resistant strains of bacteria (MRSA, Pseudomonas aerugniosa and
Escherichia coli) and follows the order: 8 > 1 > 17 > 16 > 7 > 3 > 5
> 9 > 15 > 6 > 10 > 11 (Table 2).
As far as the monochloro-derivatives are concerned it seems that
compound (13) substituted in position 3 is a little more active than that
substituted in position 4 (14). 4-Fluoro- substitution appeared to be
more favourable than 2,6-difluoro- substitution. Again, no correlation
of activity with lipophilicitywas observed.
Compound (8) again showed the best antibacterial potential, the same
as against ATCC strains, with MIC at 0.60-0.80 × 10-2 microM and
MBC at 0.80-1.65 × 10-2 microM, respectively
Summarising the results above, it is apparent that compound
(8) possesses the highest antibacterial and antifungal potential
among all compounds synthesized.
The lowest antibacterial activity is observed for compound (11),
with MIC at 39.30 × 10-2 microM and MBC at 78.60 × 10-2
microM. All tested compounds expressed higher antibacterial
potential than both antibiotics tested (Table 2).
Correspondent author: A.Geronikaki. Tel.: +302310997616; fax: +302310997612; e-mail: geronik@pharm.auth.gr

Table 3. Antifungal potential of tested compounds (MIC/MFC × 10-2 microM).
Co
mp.
A.fum.
A.v.
A.o.
A.n.
T.v.
P.f.
P.o.
C.a.
P.v.c
R
H
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
MIC
MFC
4,75
9,50
9,50
19,00
7.1
4,75
9,50
7.1
9,50
19,00
14.2
4,75
9,50
4,75
9,50
4,75
9,50
9.500
19.00
1
2
14.2
7.1
7.1
14.2
28.4
56.8
4-OH
28.4
14.2
14.2
9,98
19,96
1.7
28.4
14.2
28.4
28.4
9,98
9,98
17,86
35,72
6.6
17,86
35,72
1.7
9,98
9,98
9.98
3
4-OMe
19,96
13.3
19,96
3.3
19,96
6.6
19,96
6.6
19.96
13.3
26.6
4-OH, 3-
OMe
4
26.6
13.3
13.3
18,35
36,70
32,11
64,22
10,70
21,40
1.23
2.46
15,05
30,10
9,09
18,18
39,3
78,6
1.56
3.12
6.8
26.6
6.6
13.3
13.3
36,69
73,38
32,11
64,22
10,70
21,40
0.80
18,35
36,70
32,11
64,22
10,70
21,40
0.80
18,35
36,70
48,17
96,34
21,40
21,40
1.65
36,69
73,38
32,11
64,22
5,35
18,35
36,70
48,17
96,34
10,70
21,40
1.23
18,35
36,70
32,11
64,22
21,40
42,80
0.80
18.35
36.70
32.11
64.22
10.70
21.40
1.65
5
4-Me
2-NO2
6
7
3-NO2
10,70
0.60
8
4-NO2
1.60
1.60
3.30
0.80
1.65
1.60
3.30
30,10
60,20
9,09
15,05
30,10
9,09
15,05
30,10
18,18
36,36
39,3
30,10
60,20
18,18
36,36
39,3
15,05
30,10
9,09
15,05
30,10
9,09
30.10
60.20
18.18
36.36
39.30
78.60
9
N(CH3)2
4-F
10
11
12
13
14
15
16
17
18,18
39,3
18,18
39,3
18,18
39,3
18,18
39,3
2,6-di-F
4-Br
78,6
78,6
78,6
78,6
78,6
78,6
12.5
6.25
6.25
3.12
12.5
6.25
20.0
25.0
27.4
54.7
27.4
54.7
25.0
12.5
12.5
12.5
25.0
12.5
13.7
6.8
13.7
6.8
13.7
6.8
3-Cl
27.4
13.7
13.7
6.8
27.4
13.7
27.4
13.7
13.7
6.8
13.7
6.8
13.7
13.7
4-Cl
27.4
13.7
13.7
16,29
32,58
8,14
16,28
9,20
18,40
38.0
95.0
48.0
80.0
39.4
13.7
27.4
27.4
16,29
32,58
8,14
16,29
32,58
16,28
32,56
6,10
16,29
32,58
8,14
16,29
32,58
8,14
32,58
65,16
16,28
32,56
6,10
16,29
32,58
16,28
32,56
6,10
16.29
32.58
8.14
2,3-di-Cl
2,4-di-Cl
2,6-di-Cl
Ketoconazole
Bifonazole
16,28
6,10
16,28
6,10
16,28
12,20
24,40
475.0
570.0
64.0
16.28
12.20
24.40
37.60
94.00
32.20
48.30
12,20
38.0
12,20
285.0
380.0
48.0
12,20
38.0
12,20
38.0
12,20
380.0
380.0
48.0
37.6
94.0
32.2
48.3
95.0
95.0
95.0
48.0
48.0
64.0
64.0
64.0
64.0
80.0
80.0
64.0
A.fum.-A.fumigatus, A.v.-A.versicolor, A.o.-A.ochraceus, A.n.-A.niger, T.v.-T.viride, P.f.-P.funiculosum, P.o.-P.ochrochloron, C.a.-C.albicans, P.v.c.-
P.cyclpoium var verucosum.
Relative standard deviations were all < 2.20, except for antimycotics < 4.50
6. Docking studies
of Gln119 and Arg326 (distance 2.24 Å and 2.29 Å respectively),
one between the nitrogen of the thiazole ring of (8) and the
hydrogen of the side chain of Arg213 (distance 2.37 Å) and the
last between the oxygen atom of the thiazole carboxyl group of
(8) and the hydrogen of the side chain of Ser228 (distance 2.01
Å). The benzene core shows hydrophobic interactions with
Asn50, Pro110, Ala226, Glu324, Ile121, Ile109 and Ser49
(Figure 1), while the adamantane ring forms hydrophobic
contacts with Lys216, Gln287, Met212, Tyr124, Tyr189 and
Leu217 (Figure 1).
6.1. Docking to E.coli MurB
Based on the literature, thiazolidinones are known to act via
MurB inhibition.39 Thus, theoretical binding studies of the
compounds at the active site of E. coli MurB (PDB code: 2Q85)
were performed. For the determination of the docking box center,
the oxygen of -OH of the side chain of Ser228 was used. Ser228
is a catalytic residue that plays an important role for the activity
of the enzyme because it takes part in the proton transfer to an
enol intermediate that is formed during the second reduction
step.69 The Free binding energy of the compounds correlates well
with the biological activity to E.coli and relatively well with the
activity to other microorganisms (Table 1).
The least active compound 14 showed only a few hydrophobic
interactions with the residues Ile121, Ile118, Tyr124, Pro110, Leu217
and Tyr18 (Figure 2). Furthermore steric interactions are observed that
destabilize the complex and decrease dramatically the inhibitoryaction
of the compound
The interacting amino acids of all compounds with E.coli
MurB are shown at Table 4.
As observed by the results of Table 1, the compounds exhibit
similar activity against different bacteria in many cases.
Structural and sequence similarity at the active site of MurB can
explain this observation. Structural alignment of E.coli MurB
The docking pose of the most active compound (8) showed
four favorable hydrogen bonds, the first two between the oxygen
atom of the nitro group of (8) and the hydrogens of the side chain
Correspondent author: A.Geronikaki. Tel.: +302310997616; fax: +302310997612; e-mail: geronik@pharm.auth.gr

(2Q85) with the MurB enzymes of Pseudomonas aeruginosa
(4JAY), Listeria monocytogenes (3TX1) and Staphylococcus
aureus (1HSK) indicated 43.88% identity with 4JAY, 22.59%
with 3TX1 and 25.68% with 1HSK with conservation of
important aminoacids such as Gly122, Tyr 124, Arg213 and
Ser218 in all cases. Structural and amino acid alignments are
shown in supplementary material, Fig 4S-6S.
Table 4. Calculated Binding affinity score and important amino
acids for interaction with E. Coli Mur B 2Q85
Free binding
energy
Binding
affinity
N
1
Hb
Residues
-9.74
-9.01
-8.72
-8.01
-8.22
-12.85
-12.46
-13.55
-7.22
-6.25
-9.87
-7.03
-6.74
-5.74
-6.88
-7.15
-8.27
-27.13
-25.11
-25.55
-22.79
-23.47
-36.79
-37.55
-39.11
-21.58
-18.55
-29.16
-20.42
-19.72
-14.88
-20.13
-21.49
-23.84
3
Gly122, Ser228, Asn232
Figure 1. (a1-2) Docked conformation of the most active compound 8 in
E.coli MurB (2Q85).
2
3
3
2
2
2
4
4
4
2
1
3
1
1
-
Tyr124, Tyr189, Ser228
Arg213, Ser228
4
Gly122, Ser228
5
Arg213, Ser228
Tyr124,
Arg158,
6
Arg213, Ser228
Gly122, Tyr124, Tyr189,
Ser228
7
Gln119,
Arg326,
8
Arg213, Ser228
9
Arg213, Ser228
Gly122
10
11
12
13
14
15
16
17
Arg326, Arg213, Ser228
Tyr189
Tyr189
-
1
2
2
Gly122
Arg213, Ser228
Arg213, Ser228
Figure 2. (a1-2) Docked conformation of the less active compound 14 in
E.coli MurB(2Q85).
Hb: Hydrogen bond interactions
Table 5: Calculated Free Binding Energy and important amino
acids for interaction of compound 8 with MurB Pseudomonas
aeruginosa, P.a., (4JAY), Listeria monocytogenes, L.m., (3TX1)
and Staphylococcus aureus, S.a., (1HSK).
Docking analysis of the most active compound 8 to the
structures 4JAY, 3TX1 and 1HSK revealed increased probability
of stable binding with the enzymes with Estimated Free binding
Energies varying between -13.40 and -14.55 kcal/mole,
Hydrogen bond formation, polar, hydrophobic and pi-pi
interactions with amino acids of the active site stabilize the
complexes (Table 5, Figures 4S, 5S of the supplementary files).
Free binding
Microorganism
energy
Hb
Residues
(structure)
(kcal/mol)
Gln297, Gln298, Leu228,
Trp277
The docking of compound 8 at the MurB enzyme of
Pseudomonas aeruginosa (4JAY) is shown at the Figure 3. Two
hydrogen bonds are formed with Gln297 and Gln298.
Hydrophobic interactions between Leu228 and the adamantine
moiety are also observed, while Trp277 participates in pi-pi
interactions with the phenyl ring of the compound (Figure 3).
P.a. (4JAY)
-14.55
2
Ser220, Arg224, Tyr169,
His253
L.m. (3TX1)
S.a. (1HSK)
-13.40
-13.58
2
1
Arg242, Tyr187, Phr247
Hb: Hydrogen bond interactions
Correspondent author: A.Geronikaki. Tel.: +302310997616; fax: +302310997612; e-mail: geronik@pharm.auth.gr

When these three compounds were excluded, application of
MyCurveFit revealed a correlation that can follow an exponential
equation with R2=0.9234, aR2=0.9025 and p=0.000001993
(Figure 4B).
Figure 4. Best curves describing the correlation between in vitro
anti-bacterial properties (MICx10-2 microM) and calculated Free
binding Energy to E.coli 2Q85 of A) all the compounds, B) of the
compounds after removing the results of the compounds 12, 13
and 17.
The observation supported the assumption that these three
compounds may act via a different mechanism. Since MurA is
also a drug target for antibacterial agents, docking analysis of
these three compounds 17, 12 and 13, and of compounds 7 and
15 to E.coli MurA (structure 3KR6) was performed. The results
are also shown in Table 1. It can easily be seen that the calculated
Free binding Energy of the compounds 17, 12 and 13 to E.coli
MurA correlates well with their in vitro activity. The calculated
Free binding Energy to MurA of the compounds 7 and 15 which
were chosen for comparison reasons was low compared to the in
vitro activity, indicating that these compounds probably act as
MurB inhibitors. Total lack of activity against MurA was
predicted for compound 15 with calculated free binding Energy>-
5.5 kcal/mole.
The results of the docking studies and the reference drug
(ketoconazole) are given in the Table 6.
As shown in Table 6, interactions with Arg120 of E.coli MurA
were observed in all cases with the exception of compound 15 for
which no interactions with amino acids of the active site were
observed.
Figure 3. Docking of compound 8 to the MurB of Pseudomonas
aeruginosa (4JAY)
The docking of compound 8 to the MurB of t Listeria
monocytogenes (3TX1) and of Staphylococcus aureus (1HSK)
are shown in Figure 7S and 8S of the supplementary material.
The low values of the calculated Free Energy for Binding of
the most active compound, 8, to MurB enzyme of E.c., P.a., L.m.
and S.a. indicate that inhibition of MurB may explain the
antibacterial activity in all tested bacteria.
The form of correlation between in vitro antibacterial activity
to E.coli (MIC) and the Free binding Energy to E.coli MurB was
further investigated using MyCurveFit online program. The
application exported a curve fit with R2=0.692, aR2=0.5891 and
p=0.00441 which corresponds to a fourth order polynomial
regression presented in Figure 4A. Observation of the curve, as
well as the Table 1, revealed that three compounds, 17, 12 and
13, were mostly responsible for the poor correlation. All three
compounds exhibited better in vitro activity than expected
according to their calculated Free binding Energy.
Docking of the most potent MurA inhibitor, compound 17 is
shown in figure 5. The compound is oriented with the phenyl ring in
vicinity to Gly114, where fosfomycin is also placed. Hydrogen bond
Correspondent author: A.Geronikaki. Tel.: +302310997616; fax: +302310997612; e-mail: geronik@pharm.auth.gr

interactions with Arg91 and Arg120 stabilize the complex while
Arg397 and Phe328 participate in ion-pi and pi-pi interactions with
the phenyl group and thiazolyl ring respectively.
6.2. Docking to lanosterol 14α-demethylase of C. albicans
(CYP51)
Most antifungals target to the inhibition of the biosynthesis of
ergosterol, a main component of the cytoplasmic membrane of these
microorganisms. Several categories of compounds have been found to
inhibit different enzymes of this pathway such as the squalene epoxidase,
ERG1, (allylamides and thiocarbamates), ERG2 and ERG24-27
(morpholines), ERG4 (polyenes), 14alanosterol demethylase, ERG11
(azoles)69. Moreover, according to previous research, several thiazolyl,
thiazolydinone derivatives have exhibited increased probability to act as
14alanosterol demethylase inhibitors70. Based on these, Candida albicans
ERG11 enzyme (CYP51), available in the PDB database, PDB ID:5v5z,
was chosen for Docking analysis, as a first efford to explore the probable
mechanism of anti-fungal activity of the compounds. C. albicans
Dihydrofolate reductase (PDB ID: 4HOF) was also used for docking
analysis.71
Table 6: Calculated Free Binding Energy, Binding affinity score
and important amino acids for interaction with E. coli MurA:
3ΚR6
Free binding
Binding
Comp.
Hb
Residues
Arg120, Lys22
Arg120
energy
affinity score
7
-9.35
-8.51
-8.29
-5.10
-10.44
-32.76
-31.38
-30.24
-16.45
-36.23
2
12
2
13
1
Arg120
15
-
-
17
2
Arg120, Arg91
Hb: Hydrogen bond interactions
Table 7: Estimated binding Energy (kcal/mol) to
Dihydrofolate reductase (DHFR, PDB ID:4HOF) and lanosterol
14alpha-demethylase (CYP51, PDB ID:5V5Z) of Candida
Albicans and important amino acid residues for interaction of the
compounds with CYP51 of C. albicans, 5V5Z
Free binding Energy
(kcal/mol)
Residues
of
CYP51
DHFR
4HOF
CYP51
5V5Z
Comp.
1
Hb
5V5Z
Tyr118,
Tyr132
-8.50
-13.54
2
-5.82
-7.55
2
3
-8.69
1
1
Tyr132
Tyr132
-10.55
Tyr118,
Ser312
-8.24
4
-12.47
2
-5.14
-4.21
5
6
-7.21
-6.52
-
-
-
-
Tyr95,
-7.19
7
-10.21
2
Gly472
-8.74
-6.01
-7.15
-5.11
8
9
-14.85
-8.13
-9.84
-7.02
1
1
1
-
Tyr118
Ser312
Gly472
-
10
11
Tyr118,
Tyr311
-8.13
12
-11.95
2
-5.92
-6.76
13
14
15
16
-8.77
-8.13
-7.33
-9.62
1
-
Tyr311
-
-5.18
-7.12
-
-
1
Tyr132
Tyr118,
Tyr132
17
-7.88
-6.75
-11.23
-8.23
2
1
Ketoc.
Tyr64
Figure 5 a. Docking of compound 17 (yellow) to E.Coli MurA
(3KR6). Fosfomycin (red) is also shown for comparison. b.
Interactions of the compound with amino acids of the binding
site. Hydrogen bonds are shown in green. Pi-ion interactions are
shown in orange and pi-pi interaction in dark pink.
Ketoc.: Ketoconazol, Hb: Hydrogen bond interactions
Correspondent author: A.Geronikaki. Tel.: +302310997616; fax: +302310997612; e-mail: geronik@pharm.auth.gr

The calculated Free Binding Energy to CYP51 varied
between -6.52 kcal/mole for compound 6 which exhibited the
lowest in vitro activity and -14.85 kcal/mole for the most active
compound 8. Interestingly, the calculated Free Energy follows
the same order as the experimentally measured MIC, 8< 1 < 4 <
12 < 17 < 3 < 7 < 10 < 16 < 13 < 2 < 14 = 9 < 15 < 5 < 11 < 6,
with a reverse in the positions of compounds 2 and 14, which
strongly supports the suggestion that CYP51 inhibition is
involved in the anti-fungal action of the compounds. Higher Free
binding Energies (low inhibitory action) were calculated for
docking of the compounds to the human enzyme.
Energy for the eleven more active compounds also follows the
order of the experimentally measured biological activity.
7. Conclusion
The range of seventeen newly designed and synthesized 2{[5-
(adamantan-1-yl)-1,3,4-thiadiazol-2-yl]-imino}-5-arylidene-1,3-
thiazolidin-4-ones exhibited a remarkable inhibition of the
growth of a wide spectrum of Gram-positive, Gram-negative
bacteria and fungi. All compounds except of (10) and (15)
exhibited better or comparable antibacterial activity compared to
the reference drug ampicillin (up to 15 fold). Furthermore, some
of the compounds showed better or comparable potency
compared to streptomycin with the most potent among them
being compound 8 (15 and 7 fold more active than ampicillin and
streptomycin respectively). It was observed that, among the
Gram-negative bacteria, the most sensitive to the tested
compounds was S. typhimirium, while E.colι was the most
resistant one. Regarding the Gram-positive bacteria, the most
sensitive one was S. aureus, while L .monocytogenes was found
to be the most resistant bacterium.
According to the docking results (Table 7), the synthesized
compound as well as the reference compound, ketoconazole, bind
in a similar way to CYP51Ca. For compound 8 a hydrogen bond
interaction was detected between the H of the side chain of
Tyr118 of CYP51Ca and S of the thiazole ring of the
compound(distance 2.76 Å). Moreover, hydrophobic interactions
between Tyr132, Ile131 and Leu376 and the adamantane ring of
the compound 8 were detected. During the docking study, the
heme group of the protein exhibited positive interactions with the
heterocyclic rings of the compound 8, as well as with the benzene
ring of ketoconazole (figure 6)
As far as fungi were concerned, the tested compounds possess
excellent activity against all the fungal species tested, being 3-
115 times more active than ketoconazole and 1.7-37 times more
active than bifonazole with the exception of compounds (6) and
(11). The most promising was compound (8), followed by the
parent structure (1).
The most sensitive fungi to compounds tested were found to
be T. viride and A. ochraceus while the A. fumigatus appeared to
be the most resistant one It can be observed that the growth of
both Gram-negative and Gram-positive bacteria and fungi
responded differently to the tested compounds, which indicates
that different substituents may lead to different modes of action
or that the metabolism of some bacteria/fungi was better able to
overcome the effect of the compounds or adapt to it.
Docking analysis to E.coli MurB and MurA indicated a
probable involvement of MurB inhibition in the anti-bacterial
mechanism of most compounds and a probable involvement of
MurA inhibition at the mechanism of action of compounds 12, 13
and 17.
Docking analysis to 14a-lanosterol demethylase (CYP51) and
tetrahydrofolate reductase of Candida albicans indicated a
probable implication of CYP51 reductase at the anti-fungal
activity of the compounds and a secondary involvement of
dihydrofolate reductase inhibition at the mechanism of action of
the most active compounds.
Acknowledgments
We would like to acknowledge the contribution of Prof.L.
Harwood, (ReadingUniversity, UK) in correction of the
manuscript language.
Figure 6: Docked conformation of the most active compound 8
(A) and ketoconazole (B) in lanosterol 14alpha-demethylase of
C. albicans (CYP51ca) (5V5Z).
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Phaedra Eleftheriou
,
Panagiotis Zoumpoulakis, Charalmpos Fotakis, Ana
Ćirić, Jasmina Glamočlija, Marina Soković.
MedChemComm, 2017, 8, 2142 – 2154
Correspondent author: A.Geronikaki. Tel.: +302310997616; fax: +302310997612; e-mail: geronik@pharm.auth.gr

Highlights
1. Novel adamantanyl thiadiazolyl thiazolidinones with
antimicrobial activity.
2. Molecular docking studies on E.coli MurB and MurA
enzymes
3. Docking studies on dihydrofolate reductase and CYP51
enzymes of C. albicans
Seventeen novel 2-{[5-(adamantan-1-yl)-1,3,4-thiadiazol-2-yl]-imino}-5-
arylidene-1,3-thiazolidin-4-ones were designed, synthesized and evaluated
for antimicrobial activity. All compounds were potent antimicrobial agents.
According to molecular docking studies, inhibition of MurB and CYP 51
may be involved in the mechanism of antibacterial and antifungal activities of
most of the compounds.
E.Coli MurB (
Best compound anti-bacterial activity: MIC 1.65-3.30 .10-2µM
anti-fungal activity:
MIC 0.80-3-30 .10-2µM
Compound8
Correspondent author: A.Geronikaki. Tel.: +302310997616; fax: +302310997612; e-mail: geronik@pharm.auth.gr