Design, synthesis, DNA assessment and molecular docking study of novel 2-(pyridin-2-ylimino)thiazolidin-4-one derivatives as potent antifungal agents

Article abstract of DOI:10.1016/j.bioorg.2018.11.050

A series of novel 2-imino-4-thiazolidinone derivatives 4a,b was synthesized through reaction of unsymmetrical thioureas 3a,b with chloroacetic acid. Condensation of 4a,b with aromatic aldehydes 5a-eyielded the corresponding 5-arylidene derivatives 6a-j. In addition, the reaction of 4a,b with 4-arylazo-3-hydroxybenzaldehydes 8a-c furnished the respective mono-arylazo-4-thiazolidinones10a-f. All the newly synthesized compounds were confirmed by their elemental analysis and spectral data. The antifungal activity of the newly synthesized compounds was assessed and the compounds 6d, 6e, 6i, 6j, 9a,b and 10a-frevealedhigher antifungal activity towards Alternaria solani than to the standard Ridomil gold plus. Moreover, the DNA toxicity of 4-thiazolidinone derivatives 6d, 9a, 10b, 10c and 10f on the nucleic acid of Alternaria solani (KT354939) was performed and the results showed qualitatively more than 70% cleavage. Also, compounds 6i, 6j, 9b, 10c and 10f were docked inside the active site of 1ZOYenzyme and suitable binding with the active site of amino acids, were displayed according to their bond lengths, angles and conformational energy.

Full text of DOI:10.1016/j.bioorg.2018.11.050

Accepted Manuscript
Design, synthesis, DNA assessment and molecular docking study of novel 2-
(pyridin-2-ylimino)thiazolidin-4-one derivatives as potent antifungal agents
Nadia Hanafy Metwally, Ibrahim Taha Radwan, Walaa Salah El-Serwy,
Mohamed Ahmed Mohamed
PII:
S0045-2068(18)30342-0
DOI:
https://doi.org/10.1016/j.bioorg.2018.11.050
YBIOO 2658
Reference:
Bioorganic Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
8 April 2018
24 November 2018
27 November 2018
Please cite this article as: N. Hanafy Metwally, I. Taha Radwan, W. Salah El-Serwy, M. Ahmed Mohamed, Design,
synthesis, DNA assessment and molecular docking study of novel 2-(pyridin-2-ylimino)thiazolidin-4-one
derivatives as potent antifungal agents, Bioorganic Chemistry (2018), doi: https://doi.org/10.1016/j.bioorg.
2018.11.050
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Design, synthesis, DNA assessment and molecular docking study of
novel 2-(pyridin-2-ylimino)thiazolidin-4-one derivatives as potent
antifungal agents
Nadia Hanafy Metwally1*, Ibrahim Taha Radwan2, Walaa Salah El-Serwy3
and Mohamed Ahmed Mohamed4
1Chemistry Department, Faculty of Science, Cairo University, Giza, Egypt 12613.
2Basic Science Department, Faculty of Oral and Dental Medicine, Future University,
New Cairo, Egypt.
3Therapeutical Chemistry Department, National Research Center, Giza, Egypt 12622.
4Plant Pathology Research Institute, Agricultural Research Center, Giza, Egypt.
12619.
E-mail address:nhmmohamed@yahoo.com
ABSTRACT: A series of novel 2-imino-4-thiazolidinone derivatives 4a,b was
synthesized through reaction of unsymmetrical thioureas 3a,b with chloroacetic acid.
Condensation of 4a,b with aromatic aldehydes 5a-eyielded the corresponding 5-
arylidene derivatives6a-j. In addition, the reaction of 4a,b with 4-arylazo-3-
hydroxybenzaldehydes
8a-c
furnished
the
respective
mono-arylazo-4-
thiazolidinones10a-f. All the newly synthesized compounds were confirmed by their
elemental analysis and spectral data. The antifungal activity of the newly synthesized
compounds was assessed and the compounds 6d, 6e, 6i, 6j, 9a,b and 10a-
frevealedhigher antifungal activity towards Alternaria solani than to the standard
Ridomil gold plus. Moreover, the DNA toxicity of 4-thiazolidinone derivatives 6d, 9a,
10b, 10c and 10f on the nucleic acid of Alternaria solani (KT354939) was performed
and the results showed qualitatively more than 70% cleavage. Also, compounds 6i, 6j,
9b, 10c and 10f were docked inside the active site of 1ZOYenzyme and suitable
binding with the active site of amino acids,were displayedaccording to their bond
lengths, angles and conformational energy.
KEYWORDS: Iminoarylthiazolidinones, Arylidene iminoarylthiazolidinones,
Antifungal activity, Alternaria solani, DNA toxicity, Docking study.
1

1. Introduction
In spite of the progress in antimicrobial therapy, diseases caused by different
types of bacteria and fungi are still a breakneck health problem. This is due to the
continual development of resistance to the existing antimicrobial drugs. This led to an
everlasting screening for new biologically active compounds with higher
antimicrobial efficacy and lower cytotoxicity. Alternaria solani is considered as one
of the well known examples of a pathogen, which early blight (EB), a disease
infecting tomato plants. Foliar symptoms initially appear on the oldest leaves as small,
brownish to black lesions. These leaf spots enlarge up to ½ inch (1.3 cm) in diameter
in a characteristically concentric fashion. The area around the spot may become
yellow, as may entire severely affected leaves (Fig. 1).
II
I
III
Fig.1. Tomato early blight disease symptoms: (I) Alternaria solani spore; (II)
Disease symptoms on tomato leaves; (III) concentric lesions on fruits.
Early blight (EB) of the tomato plant is probably one of the most destructive
disease of field crops. This is due to its property to in fest rapidly after seed initiation.
In addition, it destroys foliage and reduces yield typically by ~20%. in some cases, it
has been reported to reduce the yield by 70-80% [1].Infection by the fungus is highly
rapid at 28-30°C in humid conditions. Various fungicides have been developed to
control EB, such as quinone outside inhibitors (QoIs, FRAC group 11), succinate
dehydrogenase inhibitors (SDHIs, FRAC group 7) and others. SDHI fungicides target
2

the succinate dehydrogenase complex, which blocks the citric acid cycle and the
production of ATP required for fungal respiration. The succinate dehydrogenase
(SDH) complex consists of a flavoprotein (SDH subunit A), an iron-sulphur protein
(SDH subunit B) and two membrane-anchoring proteins (SDH subunits C and D).
Succinate dehydrogenase inhibitors bind to a highly conserved active site composed
of residues found in subunits B, C and D, where ubiquinone (coenzyme Q10) is
reduced to ubiquinol [2]. Some commercial SDH fungicides were consisted of
structurally diverse frameworks, including thifluzamide and boscalid which displayed
broad spectrum in antifungal activity, as example thifluzamide has biological effect
on basidiomycetes, particular for diseases caused by Rhizoctonia spp., on rice, wheat,
, papaya, corn, potato, tomato and sugar beet [3]. Also, boscalid has been used as a
broad-spectrum fungicide against mainly on Alternaria atternata [4]. However,
resistance against these fungicides in Alternaria strains [5] make it an advantageous
and interesting task to discover more potent and effective compounds against
Alternaria strainsto prevent this disease. It is well known that 4-thiazolidinone
derivatives are one of the heterocyclic categories which play an important role in
therapeutical chemistry, due to their variety in biological activitysuch as antiviral [6],
anticancer [7], anti-inflammatory [8], antitumor [9], antimicrobial [10], anticonvulsant
[11], antidepressant [12], antioxidant [13]. Moreover, 4-thiazolidinonesare used as
apotent antifungal activity [14-17] and as new class of anti-HIV-1 agents with marked
reverse transcriptase (RT) inhibitory effects [18,19]. A variety of thiazolidinone based
derivatives having different substituent around the core nucleus are being considered
as potential antimicrobial agents [20-24]. Additionally, the combination of 4-
thiazolidinones with other heterocyclic rings showed wide range of biological
activities likethiazolo-linked pyrimidine and thiazolidinone-oxoindoline base
compounds have been used as a potent CA inhibitor [25,26].Based on the above
reports of interest biological activity of 4-thiazolidinoes and in continuation of our
interest in the synthesis of some new bioactive 4-thiazolidinone derivatives [27-36],
we planned to synthesize some novel2-imino-3-aryl-4-thiazolidinone derivatives
which containing pyridine moiety as an effective compound against some types of
fungi especially Alternaria strains.
3

2. Results and discussion
2.1.Synthetic Chemistry
Unsymmetrical 1,3-diarylthioureas 3a,b as starting materials were prepared by
the reaction of 2-amino-6-picoline (1) with isothiocyanate derivatives 2a,b in
dichloromethane under stirring at room temperature. The IR spectrum of the isolated
product 3b displayed a characteristic absorption bands at υmax = 3194 and 3113 cm-1
corresponding to the two NH groups and another absorption band at υmax = 1256 cm-1
1
for C=S stretch. Its H NMR spectrum provided the position of methyl protons at δ
2.19 ppm, besides the two D2Oexchangeable signals appeared at δ = 10.76 and 14.16
ppm, respectively assigned to the NH protons. Also, mass spectrum showed a
corrected molecular ion peak at m/z = 322 (M+, 55.5%) beside some other abundant
fragments, that established the molecular formula C13H12BrN3S. Elemental analysis
together with mass spectrum are in agreement with the proposed structure 3a,b.
Cyclization of compounds 3a,b with chloroacetic acid in glacial acetic acid in the
presence of anhydrous sodium acetate provided yellow crystals 4a,b. The structure of
the isolated products 4a,b was established by using 1H NMR spectra which revealed a
characteristic singlet signal at δ ~ 4.0 ppm assigned to the methylene (-CH2-) protons.
13
Moreover, C NMR for compound 4b confirmed the proposed structure due to the
appearance of a characterized signal at δ = 33.76 ppm due to the active methylene
carbon, besides the other expected signals due to the aromatic carbons. Analytical and
spectral data of the prepared compounds 4a,b were in agreement with the proposed
structure. Mass spectrum of 4b showed a corrected molecular ion peak at m/z = 362
(M+, 63.7%) which deduce the formula C15H12BrN3OS.
4

Scheme 1
Condensation of 4-thiazolidinones 4a,b with various aromatic aldehydes 5a-e
in refluxing glacial acetic acid in the presence of anhydrous sodium acetate afforded
the respective arylidene-4-thiazolidinone derivatives 6a-j. Structures of 6a-jwere
inferred from their analytical and spectral data. Thus, IR spectrum of compound 6has
example showed absorption band υmax = 1710 cm-1 corresponding to the carbonyl
1
group (C=O). Its H NMR revealed a singlet signal at δ = 7.64 ppm attributable to
vinylic (CH=) proton in addition to the other expected signals corresponding to
aromatic protons. Also, mass spectrum of 6h showed a corrected molecular ion peaks
at m/z = 503(M+, 38.3%) and m/z = 505 (M++2, 11.5%) which is consistent with the
formula C22H15ClFN3OS. Moreover, compounds 6a-j were synthesized as one pot
synthesis via refluxing 1,3-unsymmetrical thioureas 3a,b with aromatic aldehydes 5a-
f and chloroacetic acid in glacial acetic acid in the presence of anhydrous sodium
acetate. Condensation of compounds 4a,b with 4-arylazo-3-hydroxybenzaldehydes
8a-c which prepared from coupling reaction of 3-hydroxybenzaldehyde with
o
aryldiazonium salts 7a-c in ethanol containing sodium acetate at 0-5 C afforded the
isolated products 10a-f. The structure of the synthesized compounds 10a-f was
determined by elemental analysis and spectral data. The IR spectrum of 10a showed
5

absorption band at υmax = 3408 cm-1 corresponding to the hydroxyl group, in addition
to the two absorption bands at υmax = 1710 and 1661 cm-1 due to the carbonyl (C=O)
Scheme 2
and imino (C=N) groups, respectively. The 1H NMR spectrum of the isolated product
10a displayed a singlet signal exchangeable D2O at δ = 9.83 ppm assigned to the
hydroxyl proton (OH). In addition to the expected protons for aryl and vinylic
protons. Elemental analysis together with mass spectrum are confirmed the proposed
structure of 10a. Based on the above collective data for 10a, the formation of
diarylazo derivatives 11a-fwas ruled out. An alternative route for the formation of
compounds 10a-f chemically was observed, thus the condensation of compounds 4a,b
with 3-hydroxybenzaldehyde in glacial acetic acid containing anhydrous sodium
acetate afforded the corresponding 5-arylidene derivatives 9a,b. The structure of the
isolated products 9a,b was determined by elemental analyses and spectral data. The
IR spectrum of 9a showed an absorption band at υmax = 3408 cm-1 corresponding to
the hydroxyl group, in addition to an absorption band at υmax = 1702cm-1 due to the
6

carbonyl (C=O) group. Its 1H NMR spectrum displayed a singlet signal exchangeable
D2O at δ = 9.82 ppm assigned to the hydroxyl proton (OH). In addition to the
expected protons for aromatic and vinylic protons. Also, mass spectrum exhibited a
molecular ion peak at m/z = 387 (M+, 99.1%) which was consistent the molecular
formula C22H17N3O2S.Coupling of compounds 9a,b with arenediazonium salts 7a-c in
N,N-dimethylformamide in the presence of sodium hydroxide at 0-5oC afforded the
corresponding arylazo derivatives which are identical in all aspects (TLC, melting
point and mixed melting point) with compounds 10a-f (Scheme 3).
Scheme 3
7

2.2. Biological evaluation
2.2.1. Antifungal activity
The antifungal activity of the prepared compounds 4a,b, 6a-j, 9a,b and 10a-f
against cultures of five fungal strains Aspergillusniger (KU681408), Aspergillus
flavus(KU681428), Alternaria solani(KT354939), Aspergillus ochraceous
(DQ087531) and Macrophominaphaseolina (AM410964) were investigated by agar
dilution assay [37].Where, the concentration of the compounds under investigation
were prepared at10 and 100 µg/ml. The results of in-vitro antifungal screening all the
novel compounds which expressed in inhibition zone diameter are listed in Table 1.
Both Ridomil gold plus and Mancozeb used as positive antifungal control for
Alternaria solani. Qualitatively, compounds 6d, 6e, 6i, 6j, 9a, 9b, 10a, 10b and 10c
gave higher potency than the positive controlwhereas 10d, 10e and 10f represented
moderate potency rather than compounds 4a, 4b, 6a, 6b, 6c, 6f, 6g and 6h revealed
very lower activity.
2.2.2. Mechanism of action and the effect of the isolated compounds 6d, 9a, 10b,
10c and 10f on the nucleic acid of the pathogenic fungus Alternariasolani.
DNA damage study was performed according to the protocol of Kasibhatla
and Gustava [38]. The fungal DNA amplified by PCR was treated with (100 µg/ml)
from each of the synthesized compounds 6d, 9a, 10b, 10c and 10f, also each sample
was incubated for one hour. DNA without treatment served as a control. After
treatment DNA was separated by electrophoresis using a 1 % agarosegel at 75 V for
30 min. The gel was stained with ethidiumbromide and then subjected to UV
irradiation to visualize the DNA bands.
2.2.3. Effect of 4-thiazolidinone derivatives 6d, 9a, 10b, 10c and 10f on nucleic
acid of Alternariasolani
In order to analyze the effect of 4-thiazolidinone derivatives 6d, 9a, 10b, 10c
and 10f on the nucleic acid of Alternaria solani (KT354939), we set up a gel
electrophoresis analysis. The results showed the interact DNA bands with the
untreated DNA (control), where no significant damage occurred. In contrast, DNA
treated with the selected 4-thiazolidinone derivatives showed substantial alteration in
8

electrophoretic migration as shown in the figure 2. These results suggest that the
antifungal effect of 4-thiazolidinone derivatives may occur by direct chemical damage
to DNA. Thus, 4-thiazolidinones may act on the cell membrane of the pathogenic
Alternaria solani, penetrating it, followed by damage and inhibition of DNA
replication.
Table 1. Antifungal activity for compounds 4a,b, 6a-j, 9a,b and 10a-f.
Aspergillus- Aspergill- Aspergillus- Alternarisolani
comp.
Macrophomia-
phasolina
Flavous
usniger
ochraceous
fungi
4a
4b
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
9a
9b
10a
10b
10c
NA
NA
19
45
40
91
93
14
17
22
94
93
90
92
88
89
95
81
81
95
NA
24
15
20
38
44
95
93
15
24
29
89
95
94
93
86
92
92
86
86
95
NA
17
27
24
42
49
83
84
12
20
18
90
92
94
95
91
93
94
88
82
90
NA
10
19
25
50
51
86
88
11
14
58
95
94
90
95
93
94
95
92
89
95
NA
NA
NA
10
24
40
71
83
NA
NA
18
87
90
84
88
87
81
95
85
10d
10e
10f
84
84
NA
DMSO
(negative
control)
Ridomil
gold plus
(positive
control)
Mancozeb
(positive
controle
77
70
9

Activity color key
Higher activity (more
than 85%)
color
Moderate
activity
(between 70-84 %)
Low activity (less than 70
%)
Fig. 2. Gel electrophoresis analysis of different synthetic compounds on DNA. After
electrophoresis, the 1 % agarose gel was stained with ethidium bromide. Lane 1, DNA
size marker; lane 2, control DNA non-treated with compounds; lanes 3 to 7, DNA
treated with compounds 6d, 9a, 10b, 10c and 10f, respectively.
2.2.4. Structure–activity relationship (SAR) studies
Based on the results, mentioned in Table 1, it was found that the starting 2-imino-3-
aryl-4-thiazolidinone derivatives 4a,b showed noactivity against Aspergillusflavus
and Macrophominaphaseolina. The results also showed low activity against the other
type of antifungal as well as the 5-benzylidene-4-thiazolidinone 6a, which bearing the
phenyl group. Introducing the withdrawing groups in aryl group at position-5 like
compounds 6b,c which contain the fluro and chloro groups improved the activity
against Aspergillusflavus and Macrophominaphaseolina than the unsubstituted parent
compound 6a. On the other hand, the 5-arylidene-3-aryl-4-thiazolidinones 6d,ewhich
bear the hetaryl ring at position-5 as istain and ninhydrin showed high efficacy
towards all the fungus strainsexcept the Aspergillusochraceousfungi compared with
positive antifungal control (Ridomil gold plus and Mancozeb) for Alternaria
10

solani.Also, compounds 6i,j revealed the high efficacy towards all the five fungus
strains compared with positive antifungal control due to bearing the hetaryl moiety
with the bromine atom as substituent in the phenyl ring.Moreover, the designed
compounds 5-benzylidene-3-aryl-4-thiazolidinones 9a,b which containing the
hydroxyl group in the phenyl ring at position-5 appeared the high activity against the
all types of fungus strain except the compound 9a showed low activity against the
Macrophominaphaseolina which unsubstituted phenyl. Additionally, compounds
10a,cexposed potent antifungal activity against all types of fungus strain which may
be attributed due to the presence of phenolic hydroxyl and arylazo groups in phenyl
ring. Compounds 10b and 10fshowed high activity against four type of fungus strain
and showed a lower activity against Macrophominaphaseolina. Consistent with the
previous results, compound 10d appeared to have a high activity against four types of
fungus and showed a low activity against Aspergillusflavous. Also,compound 10e
showed a high activity against the two types of fungus strain and low activity against
the three types of fungus.
2.2.5. Molecular docking study
Compound 4a binds into the active site of 1ZOY with minimum binding energy (∆G)
-8.8994 kcal/mol as shown in Table 2 and formed one hydrogen bond between
carbonyl function and the residue amino acid SerD50 with bond length 2.19 Å (Fig.
3). Also, compound 6i binds into the active site of 1ZOY with minimum binding
energy (∆G) -12.4351 kcal/mol (Table 2). The isatin ring in compound 6i formed one
hydrogen bond interaction between carbonyl group of 4-thiazolidinone and ArgD47
with bond length 1.99 Å. in addition to arene-arene interaction between ArgC46 and
ArgD47 with pyridine and isatin ring, respectively of compound 6i (Figure 5). In
addition, compound 6j appeared one hydrogen bond between carbonyl function and
ArgD47 with bond length 2.61 Å, beside to two arene-arene interaction between
phenyl ring with ArgD47 and pyridine ring with ArgC46 (Fig. 7). Moreover,
compound 9b binds into the active site of 1ZOY with minimum binding energy (∆G)
-11.3364 kcal/mol and at the active site of 1ZOY, shows onehydrogen bond
interaction between hydroxyl group in phenyl ring at position-5 and LysB239 with
bond length 2.04,whereas residue ArgD47 is involved in arene-arene interaction with
one phenyl ring (Figure 9). On the other hand, the 5-(3-hydroxy-4-[(4-
11

methoxyphenyl)-diazenyl)benzylid-ene]-2-imino-3-phenylthiazolidin-4-one10c binds
into the active site of 1ZOY with minimum binding energy (∆G) -14.6964 kcal/mol
and at the active site of 1ZOY, shows three hydrogen bond interaction between
hydroxyl group in phenyl ring at position-5 and SerC53 with bond length 1.68Å, the
other two hydrogen bonds interaction between methoxy group which present in the
aryl azo substituent and the residues two amino acids LysB239 and TrpD43 with bond
length 2.30 and 3.53Å, respectively. The last interaction between phenyl group of azo
function with ArgD47 and HisD216,respectively via arene-arene interaction (Fig.
11). Compound 10f shows two hydrogen bond between interactions methoxy group
and LysB239 with bond length 1.93Å and the other between nitrogen for imino group
and serC53 with bond length 1.64Å, also appeared two interactions as arene-arene
stabilized by phenyl ring and HisB216 and ArgD47 (Fig. 13). However, molecular
docking result showed a quite different orientation of compounds 6i, 9b, 10c and 10f
at the active site of 1ZOY due to substituent changing at position-5 of 4-
thiazolidinone ring. These results supported our binding and enzyme activity results,
that these compounds offers sufficient number of interactions to 1ZOY especially
compound number 10c, resultantly formed a stable complex. Also, these results are
also in close agreement with the results of antifungal activity as shown in Table 1.
Table 2: The protein-ligand interactions of compounds 4a, 6i, 6j, 9b, 10c and 10f
with active site pocket 1ZOY.
Length of hydrogen bonds
Amino acids involved
in the interaction
and other interaction
Compound
S
Kcal/mol
Å
-8.8994
SerD50
4a
6i
H-bond acceptor 2.02 Å
-12.4351
ArgD47
ArgD47
ArgC46
H-bond acceptor 1.99 Å
Arene-arene interaction
Arene-arene interaction
H-bond acceptor 2.61 Å
Arene-arene interaction
Arene-arene interaction
-13.3429
-11.3364
ArgD47
ArgD47
ArgC46
6j
LysB239
ArgD47
H-bond
acceptor
2.04
9b
ÅArene-arene interaction
12

-14.6964
LysB239
TrpD43
SerC53
ArgD47
HisB216
10c
10f
H-bond acceptor2.30 Å
H-bond acceptor2.53 Å
H-bond acceptor 1.68 Å
Arene-arene interaction
Arene-arene interaction
H-bond acceptor 1.39Å
H-bond acceptor 1.64 Ao
Arene-arene interaction
Arene-arene interaction
-16.5038.1753 LysB239
SerC53
ArgD47
HisB216
Fig. 3. 2D ligand interaction of the compound 4a with the active site amino acids of
1ZOY.
13

Fig. 4. 3Ddocking of compound 4a with the active site pocket of the protein1ZOY.
Fig. 5. 2D ligand interaction of the compound 6i with the active site amino acids of
1ZOY.
14

Fig. 6. 3D docking of compound 6i with the active site pocket of the protein 1ZOY.
Fig. 7. 2D ligand interaction of the compound 6j with the active site amino acids of
1ZOY.
15

Fig. 8. 3D docking of compound 6j with the active site pocket of the protein 1ZOY.
Fig. 9. 2D ligand interaction of the compound 9b with the active site amino acids of
1ZOY.
16

Fig. 10. 3D docking of compound 9b with the active site pocket of the protein 1ZOY
.
Fig. 11. 2D ligand interaction of the compound 10c with the active site amino acids of
1ZOY.
17

Fig. 12. 3D docking of compound 10c with the active site pocket of the protein
Fig. 13. 2D ligand interaction of the compound 10f with the active site amino acids of
1ZOY.
18

Fig. 14. 3D docking of compound 10f with the active site pocket of the protein
Fig. 15. The active site pocket of the protein 1ZOY.
19

Fig. 15. The active site pocket of the protein1ZOY.
Conclusion
A series of 4-thiazolidinone derivatives incorporated pyridine moiety was synthesized
and evaluated for their antifungal activity. The antifungal activity of the prepared
compounds 6d, 6e, 6i, 6j, 9a, 9b and 10a-f showed high potent inhibitory activity
against Alternaria solani strain than Ridomil gold plus as the standard reference. One
of the promising findings which can glorify the domain of developing these 4-
thiazolidinones, was the interaction of 6d, 9a, 10b, 10c and 10f with Alternaria solani
DNA Plasmid, the electrophoresis pattern obtained manifested specific DNA
cleavage. Thus, DNA assessment accorded generous hint with supporting proof for
describing the mechanism of inhibitory action of these 4-thiazolidinones. That can
enhance the future researches of 4-thiazolidinone and their potential to act as artificial
nucleas mimics and metal-free nuclelytic agents. Qualitatively, theoretical molecular
docking study consulted the data obtained from in vitro antifungal activity.
3. Experimental section
All melting points are uncorrected and were taken on electro-thermal capillary
melting point apparatus (6100). IR spectra were recorded on a Perkin Elmer 1430
spectrophotometer. 1HNMR and 13CNMR spectroscopy were recorded in
deuterateddimethylsulfoxide at mercury 400 and 100 MHz on a Varian Gemini NMR
spectrophotometer usingtetramethylsilane as internal reference and the results are
expected as δ value, the main chemical warfare Laboratories, chemical warfare
department, ministry of defense. Mass spectra were taken on a Shimaduze GMCS-
20

GB1000 EX mass spectrophotometer at 70 ev in Micro analytical Center Laboratory,
Cairo University. Elemental analysis carried out at the Microanalyses Center of Cairo
University, Giza, Egypt and the results are within ±0.4% of the theoretical
values.Antifungal activityand gel electrophoresis assay were carried out at the
Agriculture Research Centre in Giza, Egypt.
4. Chemistry
General procedure for synthesis of 1-(6-methylpyridin-2-yl)-3-
arylthioureas(3a,b)
A mixture of 2-amino-6-picoline 1 (0.01 mol) and phenyl isothiocyanate derivatives
2a,b(0.01 mol) were stirred for 6 hours in methylene chloride (15 ml) at room
temperature. The precipitate so formed was collected, filtered off, and recrystallized
from ethanol.
1-(6-Methylpyridin-2-yl)-3-phenylthiourea (3a).
White crystals; yield; 70%; m.p. 186-188˚C; IR (KBr, cm-1): 3189 (NH), 3109 (NH),
1247 (C=S); 1H NMR (DMSO-d6):δ =2.21 (s, 3H, CH3), 6.86 (d, 1H, J= 7.2 Hz, Ar),
7.0 (d, 1H, J = 8 Hz, Ar), 7.23-7.78 (m, 6H, Ar), 10.68 (s, 1H, NH), 13.94 (s, 1H,
NH); Anal. Calcd for C13H13N3S (243.33): C, 64.17; H, 5.39; N, 17.27; S, 13.18.
Found: C, 64.0; H, 5.58; N, 17.04; S, 13.39%.
1-(4-Bromophenyl)-3-(6-methylpyridin-2-yl)thiourea (3b).
o
White crystals; yield 68.8%; m.p = 177-179 C; IR (KBr, cm-1): 3194 (NH), 3113
(NH), 1256 (C=S); 1H NMR (DMSO-d6): δ = 2.19 (s, 3H, CH3), 6.92 (d, 1H, J = 7.6
Hz, Ar), 7.04 (d, 2H, J = 8 Hz, Ar), 7.16-7.77 (m, 4H, Ar), 10.76 (s, 1H, NH), 14.16
(s, 1H, NH); MS: m/z (%) = 323 (M++1, 12.5), 322 (M+, 55.5), Anal. Calcd for
C13H12BrN3S (322.22): C, 48.46; H, 3.75; Br, 24.80; N, 13.04; S, 9.95. Found: C,
48.64; H, 3.91; N, 13.22; S, 9.78%.
General procedure of synthesis of 2-[(6-methylpyridin-2-yl)imino]-3-
phenylthiazolidin-4-one 4a,b
21

To a solution of each of 3a,b (0.01 mol), chloroacetic acid (0.01 mol) was added and
the mixture was heated under reflux in glacial acetic acid (15 ml) for 6 hours
containing anhydrous sodium acetate (0.01 mol), then the reaction mixture allowed to
cool. The solid so formed, filtered off, washed with ethanol and recrystallized from
ethanol-dioxane mixture.
2-[(6-Methylpyridin-2-yl)imino]-3-phenylthiazolidin-4-one (4a).
o
Yellow crystals; yield 87%; m.p = 260-262 C [lit. m.p = 262-264 oC] [39]. IR (KBr,
cm-1): 1722 (C=O); 1H NMR (DMSO-d6): δ =2.40 (s, 3H, CH3),4.0 (s, 2H, CH2), 6.67
(d, 1H,J = 7.6 Hz,Ar-H), 6.95 (d, 1H, J = 7.6 Hz, Ar), 7.32 (d, 2H, J = 7.6 Hz, Ar),
7.42-7.61 (m, 4H, Ar-H); MS: m/z (%) = 283 (M+, 99.9), 241 (95.3), 208 (50.8), 192
(97.0), 167 (17.3), 149 (85.7), 135 (35.6), 123 (37.8), 92 (86.2), 69 (99.9), Anal.
Calcd. for C15H13N3OS (283.35): C, 63.58; H, 4.62; N, 14.83; S,11.32.Found: C,
63.79; H, 4.80; N,15.03; S,11.65%
3-(4-Bromophenyl)-2-[(6-methylpyridin-2-yl)imino]thiazolidin-4-one (4b).Yellow
o
1
crystals; yield 76%; m.p = 250-252 C; IR (KBr, cm-1): 1717 (C=O); H NMR
(DMSO-d6): δ = 2.44 (s, 3H, CH3), 4.03 (s, 2H, CH2), 6.67 (d, 1H, J = 7.6 Hz, Ar),
6.69 (d, 1H, J = 7.8 Hz, Ar), 6.95 (d, 2H, J = 7.8 Hz, Ar ), 7.31-7.63 (m, 4H, Ar); 13C
NMR (DMSO-d6): δ = 23.85, 33.76, 117.70, 119.64, 122.01, 131.34, 132.42, 135.59,
139.05, 155.76, 157.45, 159.65, 172.50. MS: m/z (%) = 363 (M++1, 79.3), 362 (M+,
63.7), 321 (71.4), 288 (23.6), 261 (20.3), 240 (97.5), 192 (99.9), 180 (22.0), 135
(28.8), 120 (34.1), 92 (99.7), 65 (74.6), Anal. Calcd for C15H12BrN3OS (362.24): C,
49.73; H, 3.34; Br, 22.06; N, 11.60; S, 8.85. Found: C, 49.51; H, 3.49; N, 11.83; S,
8.66%.
General procedures for synthesis of compounds 6a-j
Method A
A mixture of aromatic aldehyde 5a-e(0.01 mol) and compounds 4a,b(0.01 mol) was
refluxed in glacial acetic acid (15 ml) for 7 hours in the presence of fused sodium
acetate (0.01mol). The precipitate obtained was filtered off, washed with water and
recrystallized from N,N-dimethylformamide (DMF).
22

Method B
A mixture of diarylthioureas3a,b (0.01 mol), aromatic aldehyde 5a-e(0.01 mol) and
chloroacetic acid (0.01 mol) was heated under reflux in glacial acetic acid (15 ml) for
5 hours in the presence of fused sodium acetate (0.01 mol). The precipitate obtained
was filtered off, washed with water and recrystallized from N,N-dimethylformamide
(DMF) to give 6a-j.
5-Benzylidene-2-[(6-methylpyridin-2-yl)imino]-3-phenyltiazolidin-4-one (6a.)
o
Yellow crystals; yield 60% (Method A); 66% (Method B); m.p = 274-276 C. IR
1
(KBr, cm-1): 1707 (C=O). H NMR(DMSO-d6): δ = 2.49 (s, 3H, CH3), 6.72 (d, 1H, J
= 7.6 Hz, Ar), 6.97 (d, 1H, J = 7.4 Hz, Ar), 7.31 (s, 1H, CH Ar), 7.37-7.66 (m, 11H,
Ar); Anal. Calcd for C22H17N3OS (371.45): C, 71.14; H, 4.61; N, 11.31; S, 8.63.
Found: C, 71.32; H, 4.80; N, 11.52; S, 8.86%.
5-(4-Fluorobenzylidene)-2-[(6-methylpyridin-2-yl)imino]-3-phenylthiazoli-din-4-
one (6b).
o
Yellow crystals; yield 70% (Method A); 77% (Method B); m.p = 281-283 C; IR
(KBr, cm-1) 1713 (C=O). 1H NMR (DMSO-d6) δ = 2.46 (s, 3H, CH3), 6.77 (d, 2H, J =
8 Hz, Ar), 7.02 (d, 1H, J = 7.6 Hz, Ar), 7.42 (s, 1H, CH,Ar ), 7.43-7.74 (m, 9H, Ar);
Anal. Calcd for C22H16FN3OS (389.45): C, 67.85; H, 4.14; F, 4.88; N, 10.79; S, 8.23.
Found: C, 67.64; H, 4.30; N, 10.60; S, 8.42%.
5-(2-Chloro-4-fluorobenzylidene)-2-[(6-methylpyridin-2-yl)imino]-3-phen-
ylthiazolidin-4-one (6c).
o
Yellow crystals; yield 76% (Method A); 83% (Method B); m.p = 308-310 C; IR
1
(KBr, cm-1): 1713 (C=O). H NMR (DMSO-d6) δ = 2.48 (s, 3H, CH3), 6.73 (d, 2H, J
= 8 Hz, Ar), 7.06 (d, 1H, J = 7.2 Hz, Ar), 7.43 (d,1H, J= 7.2 Hz,Ar), 7.51-7.62 (m,
6H, Ar), 7.69 (d, 2H, J = 8.4 Hz, Ar); Anal. Calcd for C22H15ClFN3OS (423.89): C,
62.34; H, 3.57; Cl, 8.36; F, 4.48; N, 9.91; S, 7.56. Found: C, 62.50; H, 3.73; N, 9.73;
S, 7.77%.
2-[(6-Methylpyridin-2-yl)imino]-5-(2-oxoindolin-3-ylidene)-3-phenylthiazo-lidin-
4-one(6d).
23

Orange crystals, yield 75% (Method A); 80% (Method B), m.p = 292-294oC; IR
(KBr, cm-1): 3431 (NH), 1690 (C=O); 1H NMR (DMSO-d6) δ = 2.43 (s, 3H, CH3),
6.82-7.09 (m, 3H, Ar), 7.32-7.65 (m, 6H, Ar), 8.40 (s, 1H, Ar), 8.93 (d, 2H, J = 8 Hz,
Ar), 11.56 (s, 1H, NH); Anal. Calcd for C23H16N4O2S (412.46): C, 66.98; H, 3.91; N,
13.58; S, 7.77. Found: C, 66.80; H, 3.73; N, 13.41; S, 7.56%.
2-[2-((5-Methylpyridin-2-yl)imino)-4-oxo-3-phenylthiazolidin-5-ylidene]-1H-
indene-1,3(2H)-dione (6e).
o
Dark redcrystals yield 72% (Method A); 79% (Method B);m.p>300 C.IR (KBr, cm-
1
1): 1685, 1689 (C=O). H NMR (DMSO-d6): δ = 2.49 (s, 3H, CH3), 6.82 (d, 2H, J =
7.8 Hz, Ar), 7.38-7.87 (m, 10H,Ar); Anal. Calcd for C24H15N3O3S (425.46): C, 67.75;
H, 3.55; N, 9.88; S, 7.54. Found: C, 67.55; H, 3.72; N, 9.63; S, 7.36%.
5-Benzylidene-3-(4-bromophenyl)-2-[(6-methylpyridin-2-yl)imino]-thiazoli-din-4-
one (6f).
o
Pale yellow crystals; yield 60% (Method A); 66% (Method B); m.p = 270-272 C. IR
(KBr, cm-1): 1710 (C=O). 1H NMR (DMSO-d6): δ = 2.47 (s, 3H, CH3), 6.70 (d, 2H, J
= 8 Hz, Ar), 6.95 (d, 1H, J = 7.2 Hz, Ar), 7.29 (s, 1H, CH, Ar), 7.35-7.64 (m, 9H, Ar);
Anal. Calcd for: C22H16BrN3OS (450.35): C, 58.67; H, 3.58; Br, 17.74; N, 9.33; S,
7.12. Found: C, 58.88; H, 3.79; N, 9.55; S, 7.26%.
3-(4-Bromophenyl)-5-(4-fluorobenzylidene)-2-[(6-methylpyridin-2-yl)-
mino]thiazolidin-4-one (6g).
o
Pale yellow crystals; yield 69% (Method A); 75% (Method B); m.p = 273-275 C; IR
(KBr, cm-1): 1716 (C=O). 1H NMR (DMSO-d6): δ = 2.45 (s, 3H, CH3), 6.76 (d, 2H, J
= 8 Hz, Ar), 7.01 (d, 1H, J = 7.6 Hz, Ar), 7.41 (s, 1H, CH, Ar), 7.42-7.73 (m, 8H, Ar);
Anal. Calcd for C22H15BrFN3OS (468.34): C, 56.42; H, 3.23; Br, 17.06; F, 4.06; N,
8.97; S, 6.85. Found: C, 56.61; H, 3.43; N, 8.76; S, 6.66%.
3-(4-Bromophenyl)-5-(2-chloro-4-fluorobenzylidene)-2-[(6-methyl-pyridin-2-
yl)imino]thiazolidin-4-one (6h).
o
Pale yellow crystals; yield 80% (Method A), 84% (Method B); m.p = 302-304 C; IR
(KBr, cm-1): 1717 (C=O). 1H NMR (DMSO-d6): δ = 2.41 (s, 3H, CH3), 6.81 (d, 2H, J
24

= 8 Hz, Ar), 7.0 (d, 1H, J = 7.2 Hz, Ar), 7.44 (d, 2H, J = 9.2 Hz, Ar), 7.49-7.57(m,
3H, Ar), 7.64 (s, 1H, CH), 7.73 (d, 2H, J = 8.4 Hz, Ar); m/z (%) = 503 (M+, 38.3),
505 (M++2, 11.5), 466 (98.8), 334 (73), 287 (43.7), 186 (69.4) 92 (65.3), 65
(27.8);Anal. Calcd for C22H14BrClFN3OS (502.79): C, 52.56; H, 2.81; Br, 15.89; Cl,
7.05; F, 3.78; N, 8.36; S, 6.38. Found: C, 52.75; H, 2.64; N, 8.52; S, 6.60%.
3-[4-Bromophenyl)-2-((5-methylpyridin-2-yl)imino]-5-(2-oxoindolin-3-ylid-
ene)thiazolidin-4-one (6i).
Orange crystals, yield 68% (Method A),76%(Method B), m.p = 286-288oC;IR (KBr,
cm-1): 3437 (NH), 1688 (C=O);1H NMR (DMSO-d6): δ = 2.47 (s, 3H, CH3), 6.90 (d,
2H, J = 8 Hz, Ar), 7.07-7.72 (m, 9H, Ar), 8.40 (s, 1H, Ar), 11.68 (s, 1H, NH); MS:
(m/z) = 491 (M+, 18%), Anal. Calcd for C23H15BrN4O2S (491.31): C, 56.22; H, 3.08;
N, 11.40; S, 6.53. Found: C, 56.42; H, 3.27; N, 11.59; S, 6.73 %.
2-[3-(4-Bromophenyl)-2-((5-methylpyridin-2-yl)imino]-4-oxothiazolidin-5-
ylidene)-1H-indene-1,3(2H)-dione (6j).Dark red, yield 76%, m.p = 290-292oC. IR
1
(KBr, cm-1): 1686 (C=O). H NMR (DMSO-d6): δ = 2.48 (s, 3H, CH3), 6.81(d, 1H, J
= 7.8 Hz, Ar), 7.67-7.79 (m, 10H, Ar), 8.87 (s, 1H, Ar); Anal. Calcd for
C24H14BrN3O3S (504.36): C, 57.15; H, 2.80; Br, 15.84; N, 8.33; S, 6.36. Found: C,
57.33; H, 3.0; N, 8.56; S, 6.58%.
General procedure for synthesis of compounds 9a,b
A mixture of each of 4a,b(0.01 mol) and 3-hydroxybenzaldehyde (0.01 mol) was
heated in glacial acetic acid (18 ml) in the presence of anhydrous sodium acetate (0.01
mol) for 7 hours then left to cool. The solid so formed filtered off, washed with water
and recrystallized from N,N-dimethylformamide (DMF).
5-(3-Hydroxybenzylidene)-2-[(6-methylpyridin-2-yl)imino]-3-phenylthia-zolidin-
4-one (9a).
o
Yellow crystals; yield 89%; m.p = 281-283 C; IR (KBr, cm-1): 3408 (OH), 1702
1
(C=O), 1590 (C=C). H NMR (DMSO-d6): δ = 2.52 (s, 3H, CH3), 6.76 (d, 2H, J = 8
Hz, Ar), 6.85 (d, 2H, J = 8 Hz, Ar), 7.01 (d, 1H, J = 7.6 Hz, Ar), 7.91 (s, 1H, CH, Ar),
6.99-7.64 (m, 7H, Ar), 9.82 (s, 1H, OH, exchangeable D2O); MS: m/z (%) = 387 (M+,
25

99.1%), 283 (99.9), 240 (84.6), 192 (97.0), 69 (60.9); Anal. Calcd for: C22H17N3O2S
(387.45): C, 68.20; H, 4.42; N, 10.85; S, 8.28. Found: C, 68.39; H, 4.60; N, 11.07; S,
8.53%.
4.4.2
3-(4-Bromophenyl)-5-(3-hydroxybenzylidene)-2-[(6-methylpyridin-2-yl)-
imino]thiazolidin-4-one (9b).Yellow crystals; yield 87%; m.p = 270-272 oC; IR (KBr,
1
cm-1): 3413 (OH), 1707 (C=O), 1596 (C=C); H NMR (DMSO-d6): δ = 2.51 (s, 3H,
CH3), 6.75 (d, 2H, J = 8 Hz, Ar), 6.84 (d, 2H, J = 8 Hz, Ar), 7.0 (d, 1H, J = 7.6 Hz,
Ar), 7.90-7.63 (m, 7H, Ar), 9.78 (s, 1H, OH, exchangeable D2O); Anal. Calcd for:
C22H16BrN3O2S (466.35): C, 56.66; H, 3.46; Br, 17.13; N, 9.01; S, 6.88 Found: C,
56.84; H, 3.67; N, 9.23; S, 7.07%.
General procedure for synthesis of compounds 10a-f
Method A
The appropriated amine (0.01mol) was dissolved in hydrochloric acid (0.03 mol) then
allowed to cool in ice bath. The prepared ice cold diazonium salts7a-c were added
dropwise to a solution of each of 9a,b(0.01 mol) and sodium hydroxide (0.01 mol) in
DMF (4 ml). The mixture stirred with cooling two additional hours then the mixture
poured into crashed ice (10 ml), the colored solid so formed, filtered off, washed with
water and recrystallized from ethanol-dioxane to give compounds 10a-f.
Method B
o
A solution of conc. hydrochloric acid (0.01mol) cooled at 0 C was added to a
solution of aniline (0.01mol) and aqueous solution of NaNO2 (0.01mol) dropwise at
0-5oC. The formed diazonium chloride was added on (0.01mol) of 3-
hydroxysalicyaldehyde in 5ml of 0.4g NaOH and 0.74g of Na2CO3. The reaction
o
stirred at 0 C for 2hr, the solid so formed was filtered off and recrystallized from
ethanol to give compounds 8a-c.A mixture of compounds 8a-c (0.01mol) and
4a,b(0.01) refluxed for 6 hr in glacial acetic acid (15 ml) in the presence of anhydrous
sodium acetate (0.01 mol), the mixture allowed to cool at room temperature then the
solid so formed and recrystallized from ethanol to produce 10a-f.
26

5-(3-Hydroxy-4-(phenyldiazenyl)benzylidene)-2-[(6-methylpyrid-in-2-yl)-imino]-
3-phenylthiazolidin-4-one (10a).
Scarlet red crystals; yield 82% (method A), 70% (method B); m.p = 192-194oC; IR
(KBr, cm-1): 3408 (OH), 1710 (C=O), 1661 (C=N). 1H NMR (DMSO-d6): δ = 2.43 (s,
3H, CH3), 6.79 (d, 2H, J = 8 Hz, Ar), 6.88 (d, 2H, J = 8 Hz, Ar), 7.03 (d, 1H, J = 7.2
Hz, Ar), 7.10-7.66 (m, 11H, Ar), 7.93 (s, 1H, CH, Ar), 9.83 (s, 1H, OH, exchangeable
D2O); Anal. Calcd for C28H21N5O2S (491.56) C, 68.41; H, 4.31; N, 14.25; S, 6.52.
Found: C, 68.60; H, 4.52; N, 14.49; S, 6.75%.
[(2-Chlorophenyl)diazenyl]-3-hydroxybenzylidene)-2-[(6-methy-lpyridin-2-
yl)imino]-3-phenylthiazolidin-4-one (10b).
Red crystals; yield 78% (method A),70% (method B);m.p = 219-221oC; IR (KBr, cm-
1
1): 3399 (OH), 1688 (C=O), 1653 (C=N). H NMR (DMSO-d6): δ = 2.42 (s, 3H,
CH3), 6.78 (d, 2H, J =8 Hz, Ar), 6.87 (d, 2H, J = 8 Hz, Ar), 7.02 (d, 1H, J = 7.6 Hz,
Ar), 7.09-7.65 (m, 10H, Ar), 7.91 (s, 1H, CH, Ar), 9.81 (s, 1H, OH, exchangeable
D2O); MS: m/z (%) = 526 (M+, 64.4%) Anal.Calcd for C28H20ClN5O2S (526.01): C,
63.93; H, 3.83; Cl, 6.74; N, 13.31; S, 6.10 Found: C, 63.71; H, 3.63; N, 13.08; S,
5.89%.
5-(3-Hydroxy-4-[(4-methoxyphenyl)diazenyl)benzylidene]-2-[(6-methyl-pyridin-
2-yl)imino]-3-phenylthiazolidin-4-one (10c).
o
Red crystals; yield 83% (method A),70% (method B); m.p = 234-236 C; IR (KBr,
1
cm-1): 3411 (OH), 1713 (C=O), 1665 (C=N). H NMR(DMSO-d6); δ = 2.46 (s, 3H,
CH3), 3.82 (s, 3H, OCH3), 6.82 (d, 2H, J =8 Hz, Ar), 6.90 (d, 2H, J = 8 Hz, Ar), 7.06
(d, 1H, J = 7.2 Hz, Ar), 7.13-7.69 (m, 9H, Ar), 7.95 (s, 1H, CH, Ar), 9.86 (s, 1H, OH,
exchangeable D2O);Anal. Calcd for C29H23N5O3S (521.59): C, 66.78; H, 4.44; N,
13.43; S, 6.15. Found: C, 66.94; H, 4.64; N, 13.62; S, 6.34%.
3-(4-Bromophenyl)-5-[3-hydroxy-4-(phenyldiazenyl)benzylidene]-2-[(6-methyl-
pyridin-2-yl)imino]thiazolidin-4-one (10d).
o
Scarlet red crystals; yield 69% (method A), 70% (method B); m.p = 182-184 C; IR
(KBr, cm-1): 3412 (OH), 1714 (C=O), 1666 (C=N); 1H NMR (DMSO-d6): δ = 2.43 (s,
27

3H, CH3), 6.78 (d, 2H, J = 8 Hz, Ar), 6.87 (d, 2H, J = 8 Hz, Ar), 7.02 (d, 1H, J = 7.6
Hz, Ar), 7.10-7.67 (m, 10H, Ar), 7.93 (s, 1H, CH, Ar), 9.83 (s, 1H, OH, exchangeable
D2O); Anal. Calcd for C28H20BrN5O2S (570.46): C, 58.95; H, 3.53; Br, 14.01; N,
12.28; S, 5.62. Found: C, 58.77; H, 3.71; N, 12.49; S, 5.80%.
3-(4-Bromophenyl)-5-[4-(2-chlorophenyl)diazenyl]-3-hydroxy-benzylid-ene)-2-
[(6-methylpyridin-2-yl)imino]thiazolidin-4-one (10e).
o
Red crystals; yield 83% (method A),70% (method B); m.p = 207-209 C; IR (KBr,
1
cm-1): 3407 (OH), 1699 (C=O), 1659 (C=N); H NMR (DMSO-d6): δ = 2.41 (s, 3H,
CH3), 6.77 (d, 2H, J = 8 Hz, Ar), 6.86 (d, 2H, J = 8.4 Hz, Ar), 7.01 (d, 1H, J = 7.6 Hz,
Ar), 7.08-7.64 (m, 9H, Ar), 7.90 (s, 1H, CH, Ar), 9.79 (s, 1H, OH, exchangeable
D2O); Anal. Calcd for C28H19BrClN5O2S (604.90): C, 55.60; H, 3.17; Br, 13.21; Cl,
5.86; N, 11.58; S, 5.30. Found: C, 55.80; H, 3.37; N, 11.79; S, 5.52%.
3-(4-Bromophenyl)-5-(3-hydroxy-4-((4-methoxyphenyl)diazenyl)-benzylid-ene)-
2-((6-methylpyridin-2-yl)imino)thiazolidin-4-one (10f).
o
Red crystals; yield 71% (method A), 70% (method B); m.p = 215-217 C; IR (KBr,
1
cm-1): 3415 (OH), 1717 (C=O), 1670 (C=N). H NMR (DMSO-d6); δ = 2.44 (s, 3H,
CH3), 3.80 (s, 3H, OCH3), 6.81 (d, 2H, J =8 Hz, Ar), 6.89 (d, 2H, J = 8.4 Hz, Ar),
7.05 (d, 1H, J = 7.2 Hz, Ar), 7.12-7.68 (m, 8H, Ar), 7.94 (s, 1H, CH, Ar), 9.85 (s, 1H,
OH, exchangeable D2O); Anal. Calcd for C29H22BrN5O3S (600.49): C, 58.00; H, 3.69;
Br, 13.31; N, 11.66; S, 5.34. Found: C, 58.19; H, 3.90; N, 11.85; S, 5.50.
5. Antifungal activity
The tested organisms were sub-cultured for fungi. Ridomil gold plus and
Mancozeb were used as a positive control and DMSO solvent as a negative control.
The plates were done in duplicate and the average zone of inhibition was calculated.
The fungal cultures were incubated at (25-30 °C) for 6-8 days. Antimicrobial activity
was determined by measurement zone of inhibition. In-vitro antifungal screening of
compounds 4a,b, 6a-j, 9a,b and 10a-fagainstcultures of five fungal strains
(Aspergillus Niger ASP, Aspergillusflavus, Alternaria solani, Aspergillus ochraceus,
Macrophominaphaseolina) Ridomil gold plus and Mancozeb were used as a positive
control. DMSO solvent was used as a negative control to evaluate the potency of the
28

tested compounds under the same conditions (Table 1). Nuclease activity experiments
were accomplished by agarosegel electrophoresis. The illuminated gel was
photographed by Alpha Innotech Corporation Instrument. Cleavage experiments of
plasmid DNA(200 mg) by 6d, 9a and (100 μM) in (5mM Tris-HCl/50 mM NaCl),
buffer (pH 7.2)were carried out. The samples were incubated for 1 hat 37 °C. A
loading buffer containing 25% bromophenol blue, 0.25% xylene cyanol and 30%
glycerol was added and electrophoresis was carriedout at 60 V for one hour in Tris-
HCl buffer using 1% agarose gel containing 1.0 μg/mL ethidiumbromide (EB)26.
References
[1] J. H. Leiminger, H. Hausladen, Advances in plant physiological phenomena
research and application. Plant Dis. 96 (2012) 124-130.
[2] S. T. Horsefield, Y. Wang, K. Hedfalk, U. Johanson, M. Karlsson, E. Tajkhorshid,
R. Neutze, P. Kjellbom, Structural mechanism of plant aquaporngating. Nature, 439
(2006) 688-694.
[3] P. Taheri, S. Tarighi, Cytomolecular aspects of rice sheath blight caused by
Rhizoctoniasolani. Eur. J. of plant pathology, 126 (2011) 511-528.
[4] K. Eicken, N. Goetz, A. Harreus, E. Ammermann, G. Lorenz, H. Rang. Eur. Pat.
Appl. 545099 (1993).
[5] K. Fairchild, L. Miles, T. Miles, P. Wharton, Discovering novel alternaria
solani succinate dehydrogenase inhibitors by in silico modeling and virtual screening
strategies to combat early blight. Phytopathology 102 (2012) 35.
[6] G. Nagalakshmi, T. K. Maity, B. C. Maiti, Synthesis, characterization and
antiviral evaluation of some novel 2- [(substitutedphenyl/heteroaryl)imino]-3-phenyl-
1,3-thiazolidin-4-ones. Der Pharmacia Lett.,5 (2013) 177-188.
[7] D. Havrylyuk, L. Mosula, B. Zimenkovsky, O. Vasylenko, A. Gzella, R. Lesyk,
Synthesis and anticancer activity evaluation of 4-thiazolidinones containing
benzothiazole moiety. Eur. J. of Med. Chem.,4 (2010) 5012-5021.
29