Synthesis, docking and evaluation of antioxidant and antimicrobial activities of novel 1,2,4-triazolo[3,4-b][1,3,4]thiadiazol-6-yl)selenopheno[2,3- d]pyrimidines

Article abstract of DOI:10.1016/j.ejmech.2014.01.006

A series of 1,2,4-(triazolo[3,4-b][1,3,4]thiadiazol-6-yl)selenopheno[2,3-d] pyrimidines (10a-j) were synthesized with various substituted anilines and benzoic acids. Structures of newly synthesized compounds were established by IR, ¹H & ¹³C NMR and LC-MS spectral data. The antioxidant activity of the synthesized compounds was evaluated by DPPH, NO and H ₂O₂ radical scavenging methods. The newly synthesized compounds were evaluated for their antimicrobial activity against Gram +ve and Gram -ve bacteria and antifungal activity by well diffusion method. Compounds 10d, 10h and 10i showed promising antioxidant, antibacterial as well as antifungal activity and these were found to be the most potent activity molecules when compared with that of standard drugs. Molecules docking studies have been performed on Staphylococcus aureus (SA) of Gram +ve bacteria.

Full text of DOI:10.1016/j.ejmech.2014.01.006

European Journal of Medicinal Chemistry 75 (2014) 195e202
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, docking and evaluation of antioxidant and antimicrobial
activities of novel 1,2,4-triazolo[3,4-b][1,3,4]thiadiazol-6-yl)
selenopheno[2,3-d]pyrimidinesq
,
b
b
Y. Kotaiah a, K. Nagaraju a, N. Harikrishna a, C. Venkata Rao a , L. Yamini , M. Vijjulatha
*
a Department of Chemistry, Sri Venkateswara University, Tirupati 517 502, Andhra Pradesh, India
b Molecular Modeling and Medicinal Chemistry Group, Department of Chemistry, Nizam College, Hyderabad 500001, Andhra Pradesh, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of 1,2,4-(triazolo[3,4-b][1,3,4]thiadiazol-6-yl)selenopheno[2,3-d]pyrimidines (10aej) were syn-
thesized with various substituted anilines and benzoic acids. Structures of newly synthesized compounds
were established by IR, 1H & 13C NMR and LCeMS spectral data. The antioxidant activity of the syn-
thesized compounds was evaluated by DPPH, NO and H2O2 radical scavenging methods. The newly
synthesized compounds were evaluated for their antimicrobial activity against Gram þve and Gram ꢀve
bacteria and antifungal activity by well diffusion method. Compounds 10d, 10h and 10i showed prom-
ising antioxidant, antibacterial as well as antifungal activity and these were found to be the most potent
activity molecules when compared with that of standard drugs. Molecules docking studies have been
performed on Staphylococcus aureus (SA) of Gram þve bacteria.
Received 19 May 2013
Received in revised form
2 January 2014
Accepted 3 January 2014
Available online 22 January 2014
Keywords:
Selenopheno[2,3-d]pyrimidines
Triazolothiadiazoles
Synthesis
Ó 2014 Elsevier Masson SAS. All rights reserved.
Antimicrobial activities
Antioxidant properties
Molecular docking
1. Introduction
nucleuses. Moreover, triazolo-thiadiazoles are a class of fused
heterocyclic compounds, which have attracted great interest in
In the past few decades, the incidence of microbial infection
has increased on frightening levels over the world as a result of
medicinal chemistry owing to their wide range of pharmaco-
logical activities including antitumor/antiproliferative/anticancer
activities [1e9].
antimicrobial resistance. Microbial infections are
a growing
problem in contemporary medicine and the use of antibiotics is
common across the world. Consequently, there is an urgent need
to widen new antimicrobial agents, which have a broad spectrum
of activity against the resistant micro-organisms. The current
literature is enriched with progressive findings about the syn-
thesis and pharmacological action of fused heterocycles. It is well
known that the N-bridged heterocycles consequent from 1,2,4-
triazoles find applications in the field of medicine, agriculture
and industry. There is existing therapeutically important medi-
cines Terconazole, Itraconazole, Fluconazole, Cefazoline, Rib-
avarin, Triazolam, Alprazolam, Etizolam and Furacylin are some
of the examples which contain anyone of these heterocyclic
In contrast, selenium has attracted great interest as an essential
element and certain diseases have been eradicated by dietary
supplementation of this element. Selenium is essential for cell
metabolism as a component of glutathione peroxidase and other
enzyme systems. Current interest lies in the prevention of certain
cancers by supplementation with selenium [10e12]. Furthermore,
Selenium is noted to regulate the expression and activity of sele-
noenzymes and thus provides protection from oxidative stress
induced cell damage, which otherwise would lead to neuropsy-
chiatric diseases and disorders like cerebrovascular disease, Alz-
heimer’s disease, Parkinson’s disease, obsessive compulsive
disorders, stroke and epilepsy [13,14]. It is noted that selenium
modulates endogenous antioxidant enzyme systems in quenching
reactive oxygen species in the neuronal cells [15e17]. Similarly,
organo selenium heterocycles have attracted much attention and
have been widely used in medicinal chemistry research. Synthesis
of selenophenes has been of considerable interest during the past
few decades as they exhibit better biological activity compared to
other heterocycles.
q
This is an open-access article distributed under the terms of the Creative
Commons Attribution-NonCommercial-No Derivative Works License, which per-
mits non-commercial use, distribution, and reproduction in any medium, provided
the original author and source are credited.
* Corresponding author. Tel.: þ91 9849605140; fax: þ91 877 2249532.
E-mail address: cvrsvu@gmail.com (C. Venkata Rao).
0223-5234/$ e see front matter Ó 2014 Elsevier Masson SAS. All rights reserved.
http://dx.doi.org/10.1016/j.ejmech.2014.01.006

196
Y. Kotaiah et al. / European Journal of Medicinal Chemistry 75 (2014) 195e202
Table 1
Prompted by these observations and continuation of our
Antimicrobial activity (MIC profiles) of the synthesized compounds (10aej).
Minimum inhibitory concentration (MIC) (
g mlꢀ1 sample)
Antifungal activity
research [18,19] program on the synthesis of novel heterocyclic
compounds exhibiting biological activity, it was thought worth-
while to prepare a new type of hybrid that clubbed both of sele-
nopheno[2,3-d]pyrimidin as well as 1,2,4-triazolo[3,4-b]-1,3,4-
thiadiazole ring systems with a view to produce promising bio-
logically active agents.
m
Compound
Antibacterial activity
Gram þve bacteria Gram eve bacteria
S. aureus B. subtilis K. pneumoniae E. coli A. flavus C. albicans
In order to know the binding interactions of these molecules
docking studies were performed on Staphylococcus aureus (SA) of
Gram þve bacteria. Pathogenicity for majority of diseases caused by
SA is multifunctional. Hence, it is difficult to define precisely the
role of any specific enzyme in the inhibition. In this regard docking
was performed on four different proteins namely dehydrosqualene
synthase (DHSS), Thymidine kinase (TK), hydroxymethylpterin
pyrophosphokinase (HPPK) and dihydropteroate synthetase
(DHPS). Among the four different proteins considered for the study
DHSS of SA considered to be important and aimed for targeting the
virulence of SA. Due to high structural similarity with the human
DHSS which is involved in cholesterol synthesis pathway, choles-
terol lowering drugs found to have inhibitory effect on DHSS of
SA [20]. Drugs reported and marketed are of phosphosulfonates,
they considered as most potent and selective inhibitors of DHSS of
SA. According to the study of Kahlon et al. [21] amino acids involved
in the functionality of this enzyme are hydrophilic, polar and
charged residues. In order the molecules to be active towards DHSS
it must and should have the hydrophilic functional groups in its
structure. Due to less hydrophilic character of these molecules,
active sites of other proteins mentioned earlier have been
considered.
10a
10b
10c
10d
10e
10f
10g
10h
10i
50
25
>100
50
25
>100
25
25
6.25
>100
100
12.5
6.25
3.12
50
>100 50
50 50
12.5 6.25
6.25 3.12
>100 >100
>100
>100
6.25
1.56
>100
25
12.5
6.25
>100
50
6.25
6.25
3.12
100
3.12
>100
50
12.5
3.12
3.12
100
6.25
Nt
100
50
12.5 12.5
6.25 3.12
3.12 1.56
25
3.12
1.56
>100
Nt
3.12
e
10j
50
>100
Ciprofloxacin 6.25
Fluconazole Nt
Control
6.25
Nt
6.25 Nt
Nt
3.12
e
e
e
e
e
Nt e denotes not tested.
Lower MIC values indicate higher the antimicrobial activity.
ethoxymethyleneimino-3-methylselenophene-2-carboxylic acid
ethyl ester 2 with good yield. Further, compound 2 was converted
in to corresponding selenopheno[2,3-d]pyrimidines 3a and 3b by
treating with appropriate substituted anilines in the presence of
acetic acid gave the satisfactory yields. The ester hydrolysis of
compounds 3a and 3b with aqueous sodium hydroxide in methanol
yielded the corresponding substituted carboxylic acids 4a and 4b
with an excellent yield. Meanwhile, compounds 9aee was pre-
pared from the appropriate substituted benzoic acids by employing
well known methods available in the literature [25,26]. Finally, the
triazolo thiadiazolyl selenopheno[2,3-d]pyrimidines were pre-
pared from the reaction of compounds 3a & 3b with compounds
9aee in the presence of POCl3 afforded the compounds (10aej) in
good yields. The detailed synthesis and spectral data analysis were
described in the experimental section.
2. Results and discussion
2.1. Chemistry
In order to generate the desired compounds, eight steps syn-
thetic strategies are adopted are depicted in Scheme 1. The basic
compound
ethyl
5-amino-4-cyano-3-methylselenophene-2-
carboxylate 1 was synthesized via expedient procedure of ethyl
acetoacetate, malononitrile, Selenium metal powder in DMF as
solvent and catalytic amount of imidazole by the standard reaction
of Gewald method [22]. In the next step, compound 1 was refluxed
with triethyl orthoformate [23,24] afforded 4-cyano-5-
2.2. Antimicrobial activity
All the synthesized compounds (10aej) were screened for their
antimicrobial activity determined by well plate method [27,28]. The
R
R
HN
N
HN
N
CN
a
c
b
CN
N
N
N
EtOOC
HOOC
EtOOC
EtOOC
Se
Se
Se
NH2
Se
OEt
R
R1
1
2
3a and 3b
4a and 4b
3a, 4a: R = 4-Cl
3b, 4b: R = 3-Cl,-4-F
HN
N
h
N
S
N
N
N
S K
CONHNHC
N
Se
S
N
N
10a-j
SH
COOH
d
COOCH3
e
CONHNH2
f
N
g
NH2
R1
R1
R1
R1
R1
5a-e
6a-e
7a-e
8a-e
9a-e
Reagents and conditions: (a) HC(OEt)3, (10 eq.), reflux, 6 h, (b) AcOH, halo substituted anilines, 4 h, reflux; (c) NaOH, MeOH, RT,
16 h; (d) SOCl2, MeOH, reflux 2 h; (e) NH2NH2 H2O, reflux, 1 h; (f) CS2, KOH, reflux, 12 h; (g) NH2NH2 H2O, reflux, 12 h;
(h) POCl3, reflux, 5-6 h.
Compound 10a
10b
4-Cl
4-CH3 4-OCH3
10c
4-Cl
10d
4-Cl
4-OH
10e
4-Cl
4-NO2
10f
3-Cl,4-F 3-Cl,4-F 3-Cl,4-F 3-Cl,4-F 3-Cl,4-F
4-CH3 4-OCH3 4-OH 4-NO2
10g
10h
10i
10j
R
R1
4-Cl
H
H
Scheme 1. Synthesis of novel 1,2,4-(triazolo[3,4-b][1,3,4]thiadiazol-6-yl)selenopheno[2,3-d]pyrimidines.

Y. Kotaiah et al. / European Journal of Medicinal Chemistry 75 (2014) 195e202
197
potentiality of the synthesized compounds as antimicrobials was
appraised for their antimicrobial studies against various Gram þve
such as S. aureus and Bacillus subtilis and Gram eve such as
Escherichia coli and Klebsiella pneumoniae strains of human patho-
gens. The result obtained as MIC is presented in Table 1. It is more
attractive to speculate the observation that the result of the anti-
microbial activity of the various derivatives appeared to be related
the nature of substituents on the phenyl ring. It is evident from
Table 1 that four compounds viz. 10d, 10g, 10h and 10i were found
more potent with either less or equal MIC as compared to control
drug ciprofloxacin. DMSO was also taken in a control experiment
which showed no effect in the experiment. The compound 10i
containing 3-chloro-4-fluoro substituted aniline on selenopheno
[2,3-d]pyrimidine and hydroxyl substituted benzene ring on tri-
azolothiadiazol was found potent against all the tested organisms
synthesized compounds (10aej) to act as hydrogen donors or free
radical scavengers was tested by in vitro antioxidant assays
involving DPPH radical, NO radical, H2O2, and the results were
compared with that of standard antioxidants including natural
antioxidant Ascorbic acid and synthetic antioxidant BHT (Butylated
Hydroxy Toluene). All the synthesized compounds showed inter-
esting antioxidant activity compared to the standard (Table 2).
Among the synthesized compounds,10d,10h and 10i were found to
be the most potent antioxidant activity compared with that of the
standard (AA and BHT 12.27 ꢁ 0.86 and 16.53 ꢁ 1.74 respectively)
with the least values of IC50 11.02 ꢁ 0.27, 10.41 ꢁ 0.23 and
9.46 ꢁ 0.91
m
g mlꢀ1, inhibition concentration respectively. Table 2
gives an account for this antioxidant evolution and indicates that
compounds were capable with significant scavenging properties
towards DPPH. The reason would be the presence of electron
releasing hydroxy and methoxy groups on phenyl ring which is
linked with triazolothiadiazole along with halo (4-Cl and 3-Cl,4-F-
phenyl) substituted seleneopheno[2,3-d]pyrimidines. Further,
insertion of substituted phenyl on both sides to that of the moiety
endowed notable improvement in radical scavenging activity.
Almost, all the tested compounds (10aej) exhibited optimistic ef-
ficacy for scavenging DPPH free radical. The next promising anti-
oxidant activity was showed by compounds 10a, 10b, 10c, 10g and
10f were found to be moderate to good. This may be due to the
presence of unsubstituted and electron donating groups such as e
CH3 and eOCH3 on phenyl ring which linked to the tri-
azolothiadiazole. Compounds 10d and 10e which are having NO2
group on phenyl ring showed least activity compared to other
compounds. This may due to the presence of electron withdrawing
nature of nitro group would decrease the scavenging ability.
The IC50 value is the concentration of sample required to inhibit
50% of the NO radicals. Except 10d and 10e all the tested com-
pounds exhibited strong NO radical scavenging with low IC50
values. The greater NO radical scavenging activity of the tested
compounds was showed by compounds 10d, 10h and 10i showed
13.72 ꢁ 1.26,12.74 ꢁ 0.18 and 8.20 ꢁ 1.60 respectively. This may due
to the presence of the electron donating groups such as eCH3 and e
OCH3 on phenyl ring. Almost all the synthesized compounds have
capable to exhibit the NO radical scavenging activity with satis-
factory results. The synthesized compounds (10aej) having various
with MIC 3.12
6.25
g mlꢀ1. Further, the compounds 10h and 10d having eOCH3
and eOH respectively found equal and considerable antibacterial
activity against all the tested microorganisms at MIC 6.25
g mlꢀ1
The compound 10g found equal and moderate activity against
tested microorganisms at MIC in the range 6.25e12.5
g mlꢀ1 while
the standard showed 6.25
g mlꢀ1. Further, the compounds 10b,
and 10c were found moderate to good activity against all the tested
organisms at a MIC range 50 to 12.5
g mlꢀ1. The compounds 10a,
10e, 10f and 10j were found inactive with MIC 50
g mlꢀ1
100
g mlꢀ1 and >100 g mlꢀ1 against all strains. In general, control
drug ciprofloxacin showed MIC 6.25
g mlꢀ1 against all the tested
m
g mlꢀ1 while the standard Ciprofloxacin showed
m
m
.
m
m
m
m
,
m
m
m
microorganisms. It is obvious from the analysis of activity results
that electron withdrawing groups such as chloro and chloro-fluoro
on selenopheno[2,3-d]pyrimidine these have strong effects in
determining the antibacterial activity. This observation is sup-
ported by the highest activity shown by the compound 10i against
both the Gram þve and Gram eve bacterial strains. Thus, we hy-
pothesize that the presence of electron withdrawing groups such as
chloro and chloro-fluoro and triazolothiadiazol with electron
releasing groups such as OH, OCH3 and CH3 has significant effect on
antibacterial activity as compared to withdrawing NO2 group.
Further, these compounds 10aej were also screened for their
antifungal activity against two fungal strains such as Aspergillus
flavus and Candida albicans. Fluconazole was taken as a standard
drug throughout the experiment. Compound 10d was found to be
concentrations (5, 10, 25, 50 and 100 m
g mlꢀ1) were subjected to
H2O2 radical scavenging activity. The findings of present study
(Table 2) indicated that the most of the synthesized compounds
exhibited moderate to good radical scavenging ability. Biological
the equal and potent antifungal activity with MIC 3.12
1.56
g mlꢀ1 against A. flavus and C. albicans respectively, while
standard drug Fluconazole with MIC 3.12
g mlꢀ1. The reason might
m
g mlꢀ1 and
m
m
be the presence of electron releasing hydroxyl group present on the
triazolothiadiazol ring in the moiety. The compounds 10h and 10i
containing 3-chloro-4-fluoro substitution on selenopheno[2,3-d]
pyrimidine but only compound 10i showed potent activity with
Table 2
Antioxidant activities of the compounds in IC50 values.
MIC 1.56
with MIC 3.12
m
g mlꢀ1 whereas compound 10d showed similar activity
Compound
Scavenging activity (IC50 m )
g mlꢀ1
m
g mlꢀ1 against both the fungal strains. The com-
DPPH
NO
H2O2
pound 10c showed moderate activity with MIC 6.25 m
g mlꢀ1 against
10a
10b
10c
10d
10e
10f
10g
10h
10i
14.82 ꢁ 0.26
13.31 ꢁ 0.17
12.75 ꢁ 0.36
11.02 ꢁ 0.27
33.28 ꢁ 0.85
13.24 ꢁ 0.48
12.86 ꢁ 0.17
10.41 ꢁ 0.23
9.46 ꢁ 0.91
26.94 ꢁ 0.67
12.27 ꢁ 0.86
16.53 ꢁ 1.74
e
18.41 ꢁ 0.78
21.64 ꢁ 0.42
19.82 ꢁ 0.61
13.72 ꢁ 1.26
46.92 ꢁ 1.02
20.27 ꢁ 0.81
16.52 ꢁ 0.49
12.74 ꢁ 0.18
8.20 ꢁ 1.60
30.17 ꢁ 0.44
14.62 ꢁ 0.97
19.06 ꢁ 1.04
e
17.03 ꢁ 0.40
19.14 ꢁ 0.81
21.09 ꢁ 0.44
15.38 ꢁ 0.96
48.31 ꢁ 0.93
22.92 ꢁ 0.74
18.36 ꢁ 0.22
17.08 ꢁ 0.12
12.54 ꢁ 1.17
36.42 ꢁ 0.92
15.24 ꢁ 0.44
17.82 ꢁ 0.28
e
both the fungal strains.
2.3. Antioxidant screening (in vitro)
In the present study, the antioxidant activity of the synthesized
compounds was assessed in vitro by the 1,1-diphenyl-2-
picrylhydrazyl (DPPH) radical scavenging assay, nitric oxide (NO)
and hydrogen peroxide (H2O2) [29e33]. These methods were based
on measuring the continual absorbance decrease of the methanolic
solution of the DPPH at 517 nm, in the presence of antioxidant
compound. The DPPH has an odd electron so it can accept an
electron or hydrogen free radical. In the presence of antioxidant,
this odd electron becomes paired due to H transfer from antioxi-
dant and hence DPPH absorbance decreases. The ability of newly
10j
AAa
BHTb
Blank
(e) Showed no scavenging activity. Lower IC50 values indicate higher radical scav-
enging activity. Values were the means of three replicates ꢁ SD.
a
Ascorbic acid.
Butylated Hydroxy Toluene.
b

198
Y. Kotaiah et al. / European Journal of Medicinal Chemistry 75 (2014) 195e202
systems can produce hydrogen peroxide. The generation of H2O2 by
activated phagocytes is known to play an important part in the
killing of several bacterial and fungal strains. Additionally, H2O2 is
formed in vivo by a variety of enzymes, including superoxide dis-
mutase. H2O2 is in general measured as a powerful oxidizing agent.
It is known that H2O2 is toxic and induces cell death in vitro. It can
attack many cellular energy-producing systems. Thus, removing
hydrogen peroxide, as well as superoxide anion, is very important
for the protection of pharmaceuticals and food systems. The highest
H2O2 scavenging effect was detected in compounds 10i and 10d
(12.54 ꢁ 1.17, and 15.38 ꢁ 0.96) than the standard Ascorbic acid and
Butylated Hydroxy Toluene 15.24 ꢁ 0.44 and 17.82 ꢁ 0.28 respec-
tively. The reason would be the presence of electron releasing hy-
droxyl group present on the phenyl ring. Almost all the tested
compounds exhibited moderate to weak antioxidant activity in the
H2O2 radical scavenging activity.
3. Conclusions
In summary, we have synthesized a series of ten 1,2,4-(triazolo
[3,4-b][1,3,4]thiadiazol-6-yl)selenopheno[2,3-d]pyrimidine
de-
rivatives and evaluated for their in vitro antimicrobial and antiox-
idant activities were successfully achieved. Three compounds 10d,
10h and 10i showed excellent antibacterial activities against all the
tested strains with MIC value comparable to those of Ciprofloxacin
and Fluconazole. Specially, compound 10i displayed the strain-
specific to both bacterial and fungal strains (MIC 3.12
m
g mlꢀ1 and
1.56
m
g mlꢀ1 respectively). The radical scavenging activities of the
synthesized compounds also showed that the tree compounds such
as 10d,10h and 10i with potent IC50 values than that of the standard
drugs in all the three methods. The structure activity relationship
(SAR) studies showed that the electron releasing groups such as
hydroxyl, methoxy and methyl were essential to activities, and the
functional groups presented on phenyl ring plays important role in
the antibacterial selectivity of these compounds. Further, complete
structure activity relationship (SAR) and mechanistic approach
should be taken into account while considering designing and
screening of much more compounds. Due to multifunctional
factorization of SA virulence docking studies were performed on
DHSS, TK, HPPK and DHPS to know the binding interactions of these
molecules. More attention has been paid for DHSS among other
proteins here we discussed the docking interactions of these mol-
ecules with DHSS. Due to high hydrophobic character of these
molecules showing limited interactions with the active site of
DHSS. Compare to other molecules in the series 10i showed good
interactions with the active site amino acids of the four proteins.
This confirms that the hydrophilic character should be possessed by
the molecules for the inhibition of DHSS from SA. Hence, further
modification of these molecules with the incorporation of hydro-
philic substituents will further result in increasing the activity to-
wards DHSS. The development of the second generation analogs is
in progress. More research in this direction is under progress and
results will be published in due course.
2.4. Docking results
The analysis of docking score and hydrogen bond interactions
were performed for all the molecules (10ae10j) including the
standard ciproflaxin. Re-dock pose of the crystal structure ligands
showed RMSD values in range and further confirmed the docking
validation. Docking results were analyzed with different parame-
ters like dock score and hydrogen bond interactions. Combined
analysis of these two parameters for TK, HPPK and DHSS gave
comparable results in accordance with one another. This is not seen
in the case of DHSS where a high dock score and less hydrogen
bond interactions were observed compare to existing cholesterol
lowering drugs. This was analyzed and confirmed to be the struc-
tural disparity between the molecule and the active site of the
DHSS protein. As the molecules taken for the study were hydro-
phobic in nature could not participate in hydrogen bond in-
teractions with the active site residues which are hydrophilic in
character. Docking analysis of all the molecules in the set empha-
sized that molecule 10i in the series is showing good results
compared to other candidates of the set. This is due to the hydro-
philic (OH) substituent in the molecule, which makes the hydrogen
bond contribution during the calculation of dock score that is
missing in other molecules of the series. This OH group is partici-
pating in hydrogen bond interaction with His 18 and Arg 265 of
DHSS in molecule 10i (Fig. 1), but are not seen with the other
molecules due to lack of polar groups this further giving an evi-
dence for hydrophilic requirement in the molecule towards protein
active site. The docking results for all the four proteins were given
in Table 3 (Supplementary material). As per the dock score is con-
cerned these molecules showed good results towards DHSS
ranging ꢀ8.99 to ꢀ5.68 whereas the other proteins showing dock
score as ꢀ5.03 to ꢀ3.62, ꢀ5.90 to ꢀ3.59 and ꢀ4.52 to ꢀ2.84 for TK,
HPPK and DHPS respectively.
4. Experimental section
4.1. Chemistry
Melting points were determined in open capillaries on a Mel-
Temp apparatus and are uncorrected. All the reactions were
monitored by thin layer chromatography (TLC) on precoated silica
gel 60 F254 (mesh); spots were visualized with UV light. Merck
silica gel (60e120 mesh) was used for column chromatography. The
IR spectra were recorded on a PerkineElmer BX1 FTIR Spectro-
photometer as KBr pellets and the wave numbers were given in
cmꢀ1 1H NMR (400 MHz), and 13C NMR (100 MHz) spectra were
.
recorded on a Bruker AMX 400 MHz NMR spectrometer in CDCl3/
Table 3
Dock score of ten analogues (10ae10j) taken for the study.
Mol. name
DHSS
TK
DPHS
HPPK
MIC of SA
10a
10b
10c
10d
10e
10f
10g
10h
10i
ꢀ7.0514
ꢀ5.6861
ꢀ6.5804
ꢀ7.8957
ꢀ6.618
ꢀ3.9187
ꢀ3.4578
ꢀ3.7924
ꢀ4.108
ꢀ2.8396
ꢀ3.6544
ꢀ2.9203
ꢀ3.6576
ꢀ3.6719
ꢀ3.0626
ꢀ3.0722
ꢀ3.902
ꢀ4.3213
ꢀ3.5958
ꢀ3.8199
ꢀ4.5312
ꢀ4.0565
ꢀ3.9658
ꢀ3.8094
ꢀ3.8645
ꢀ4.6417
ꢀ4.2034
ꢀ5.9075
50
25
12.5
6.25
>100
50
6.25
6.25
3.12
100
6.25
ꢀ4.5428
ꢀ3.2665
ꢀ4.3323
ꢀ3.479
ꢀ6.5028
ꢀ6.8441
ꢀ6.0305
ꢀ8.9898
ꢀ8.4159
ꢀ8.4888
ꢀ4.2737
ꢀ3.5492
ꢀ5.0387
ꢀ4.0857
ꢀ4.3743
ꢀ4.521
10j
Ciproflaxin
Fig. 1. Docking results.

Y. Kotaiah et al. / European Journal of Medicinal Chemistry 75 (2014) 195e202
199
DMSO-d6 solution using TMS as an internal standard. All chemical
shifts are reported in (ppm) using TMS as an internal standard.
AreH, J ¼ 8.0 Hz), 8.51 (s, 1H, CeH); 13C NMR (CDCl3, 100 MHz);
d
d 14.05, 15.28, 61.10, 114.73, 117.79, 120.61, 124.18, 133.73, 140.12,
The mass spectra were recorded on Agilent 1100 LC/MSD instru-
ment with method API-ES at 70 eV. The microanalyses were per-
154.35, 158.62, 161.03, 162.35 and 166.36; LCeMS (negative ion
mode): m/z 394 (M ꢀ H)ꢀ for C16H14ClN3O2Se.
formed on
a PerkineElmer 240C elemental analyzer. The
antioxidant property was carried out by using Shimadzu UV-2450
spectrophotometer.
4.2.2. Ethyl 4-(3-chloro-4-fluorophenylamino)-5-
methlyselenopheno[2,3-d]pyrimidine-6-carboxylate (3b)
Yield 68%; Pale-yellow solid m.p. ¼ 158e160 ꢂC; IR (KBr)
n
4.1.1. Synthesis of ethyl 5-amino-4-cyano-3-methylselenophene-2-
carboxylate (1)
(cmꢀ1): 3412, 2926, 1706, 1555; 1H NMR (CDCl3, 400 MHz)
d 1.40 (t,
3H, eCH2eCH3), 3.07 (s, 3H, selenopheneeCH3), 4.37 (q, 2H, eCH2e
CH3), 7.16 (t, 1H, AreH, J ¼ 8.0 Hz), 7.42 (m, 1H, AreH), 7.47 (s, 1H,
NeH), 7.81 (q, 1H, AreH, J ¼ 4.0 Hz), 8.52 (s, 1H, CeH); 13C NMR
The starting ethyl 5-amino-4-cyano-3-methylselenophene-2-
carboxylate 1 was prepared according to reported Gewald syn-
thetic procedure (Huang et al., 2011). A mixture of ethyl acetoace-
tate (3.0 mmol), dicyanomethane (3.3 mmol), selenium powder
(4.5 mmol), and imidazole (0.3 mmol) in DMF (3.0 ml) was stirred
at 60 ꢂC under nitrogen atmosphere for 16 h. After completion of
starting materials, the reaction mixture was cool to room temper-
ature, and then the unreacted selenium powder was filtered. The
filtrate was then poured onto ice cold water, and stirred for 15 min.
The solid obtained was collected by filtration and recrystallized
from ethanol. Yellowish solid Yield 53%; m.p. ¼ 212e214 ꢂC; IR
(CDCl3, 100 MHz):
d 14.25, 15.96, 60.09, 112.36, 115.82, 117.08,
118.49, 122.84, 141.06, 144.62, 146.54, 150.76, 154.38, 156.61, 160.44
and 170.18; LCeMS (negative ion mode): m/z 412 (M ꢀ H)ꢀ for
C16H13ClFN3O2Se.
4.3. General procedure for the synthesis of 4-(substituted
phenylamino)-5-methylselenopheno[2,3-d]pyrimidine-6-carboxylic
acid (4a and 4b)
(Chloroform)
NMR (CDCl3, 400 MHz)
selenopheneeCH3), 4.27 (q, 2H, eCH2eCH3), 5.50 (br s, 2H, NH2);
13C NMR (CDCl3, 100 MHz);
14.14, 14.59, 60.02, 88.65, 106.88,
n
(cmꢀ1): 3435 (NH2), 2206 (C^N), 1641 (C]O); 1H
1.41 (t, 3H, eCH2eCH3), 2.48 (s, 3H,
The compound (3a and 3b) was dissolved in MeOH/H2O (12 ml:
6 ml), and 15% v/v NaOH aq (2 ml) was added. Stirring was
continued for 16 h at rt, then CHCl3 was added. The aqueous layer
was acidified with 1 N HCl, stirred for 15 min, the product was
separated by vacuum filtration and washed with water, dried well
and recrystallized from chloroform and methanol to give com-
pounds (4a and 4b) in good yields.
d
d
114.90, 146.25, 161.24 and 166.57; LCeMS (negative ion mode): m/z
257 (M ꢀ H)ꢀ for C9H10N2O2Se.
4.1.2. Synthesis of 4-cyano-5-ethoxymethyleneimino-3-
methylselenophene-2-carboxylic acid ethyl ester (2)
A
mixture of 5-amino-4-cyano-3-methylselenophene-2-
4.3.1. 4-(4-Chlorophenylamino)-5-methylselenopheno[2,3-d]
carboxylicacid ethyl ester 1 (2.0 g, 7.72 mmol) and triethyl ortho-
formate (7.15 ml, 38.61 mmol) was refluxed for 16 h. After
completion of starting compound the reaction mixture was cooled,
the excess amount of triethyl orthoformate was concentrated and
the solid obtained was recrystallized from ethanol. Yield: 86%;
pyrimidine-6-carboxylic acid (4a)
Yield 92%; Off-white solid, m.p. ¼ 324e326 ꢂC; IR (KBr)
n
(cmꢀ1): 3446, 2926, 1705, 1539; 1H NMR (DMSO-d6, 400 MHz)
d
3.12 (s, 3H, selenopheneeCH3), 7.45 (d, 2H, AreH, J ¼ 8.0 Hz), 7.71
(d, 2H, AreH, J ¼ 12.0 Hz), 8.52 (s, 1H, NeH), 8.69 (s, 1H, CeH), 13.60
(br s, 1H, eCOOH); 13C NMR (DMSO-d6, 100 MHz):
16.62, 120.93,
m.p.: 106e108 ꢂC; IR (KBr)
1H NMR (CDCl3, 400 MHz)
CH3eester), 2.58 (s, 3H, CH3eselenophene), 4.31(q, 2H,
OCH2CH3), 4.47 (q, 2H, CH2eester), 7.90 (s, 1H, N]CH); 13C NMR
(CDCl3, 100 MHz); 14.28, 15.84, 16.25, 61.31, 62.12, 109.86, 114.23,
n
(cmꢀ1): 2958, 2894, 2210, 1690, 1558;
1.36 (t, 3H, eOCH2CH3), 1.43 (t, 3H,
d
d
124.00, 127.50, 127.72, 127.99, 137.41, 140.61, 153.39, 157.24,
164.77and 171.55; LCeMS (positive ion mode): m/z 368 (M þ H)þ
for C14H10ClN3O2Se.
e
d
115.67, 145.12, 153.51, 156.49 and 167.21; LCeMS (positive ion
mode): m/z 315 (M þ H)þ for C12H14N2O3Se.
4.3.2. 4-(3-Chloro-4-fluorophenylamino)-5-methylselenenopheno
[2,3-d]pyrimidine-6-carboxylic acid (4b)
Yield 87%; White solid m.p. ¼ 315e317 ꢂC; IR (KBr)
n
(cmꢀ1):
d 3.04 (s, 3H,
4.2. General procedure for the synthesis of ethyl 4-(substituted
phenylamino)-5-methylselenopheno[2,3-d]pyrimidine-6-
carboxylate (3a and 3b)
3423, 2924, 1693, 1568; 1H NMR (DMSO-d6, 400 MHz)
selenopheneeCH3), 7.43 (t, 1H, AreH, J ¼ 8.0 Hz), 7.64 (t, 1H, AreH,
J ¼ 4.0 Hz), 7.92 (d, 1H, AreH, J ¼ 4.0 Hz), 8.52 (s, 1H, NeH), 8.70 (s,
1H, CeH), 13.56 (br s, 1H, eCOOH); 13C NMR (DMSO-d6, 100 MHz):
A
mixture
of
4-cyano-5-ethoxymethyleneimino-3-
(1.6
d
14.07, 113.61, 117.84, 123.57, 127.78, 138.52, 142.51, 146.34, 151.24,
methylselenophene-2-carboxylic acid ethyl ester
2
g
154.70, 156.66, 163.71, 166.81 and 169.23; LCeMS (positive ion
mode): m/z 386 (M þ H)þ for C14H9ClFN3O2Se.
5.09 mmol) was dissolved in AcOH to this added (6.11 mmol) of
appropriate halo substituted anilines and refluxed for 4 h. After
completion of the starting compounds, then the total reaction
mixture was cooled to room temperature for 2 h, poured into ice
cold water and stirred for 15 min. The product was separated by
filtration and washed with water, dried well and recrystallized from
chloroform and n-hexane to give compounds (3a and 3b) in good
yields.
4.4. General procedure for the synthesis of compounds 10aej
To a mixture of compound (4a and 4b) (0.01 mol) and
appropriate substituted phenyl triazoles (0.01 mol), phosphorus
oxychloride (10 ml) was added and the reaction contents were
refluxed for 5e6 h. After completion of the starting compounds,
then the total reaction mixture was cooled to room temperature
for 2 h, poured into ice cold water, basified with 10% NaOH and
stirred for 15 min. The product was separated by filtration and
washed with ice cold water, dried well and recrystallized from
chloroform and methanol to give compounds (10aej) in good
yields.
4.2.1. Ethyl 4-(4-chlorophenylamino)-5-methylselenoenopheno
[2,3-d]pyrimidine-6-carboxylate (3a)
Yield 86%; White crystalline solid m.p. ¼ 166e168 ꢂC; IR (KBr)
n
(cmꢀ1): 3421, 2926, 1710, 1565; 1H NMR (CDCl3, 400 MHz)
d 1.40 (t,
3H, eCH2eCH3), 3.08 (s, 3H, selenopheneeCH3), 4.37 (q, 2H, eCH2e
CH3), 7.37 (d, 2H, AreH, J ¼ 12.0 Hz), 7.50 (s, 1H, NeH), 7.59 (d, 2H,

200
Y. Kotaiah et al. / European Journal of Medicinal Chemistry 75 (2014) 195e202
4.4.1. N-(4-Chlorophenyl)-5-methyl-6-(3-phenyl-[1,2,4]triazolo
[3,4-b][1,3,4]thiadiazol-6-yl)selenopheno[2,3-d]pyrimidin-4-amine
(10a)
4.4.6. N-(3-Chloro-4-fluorophenyl)-5-methyl-6-(3-phenyl-[1,2,4]
triazolo[3,4-b][1,3,4]thiadiazol-6-yl)selenopheno[2,3-d]pyrimidin-
4-amine (10f)
Yield 71%; Pale yellow solid, m.p. ¼ 246e248 ꢂC; IR (KBr)
n
(cme
2.96 (s,
Yield 74%; Off-White solid, m.p. ¼ 228e230 ꢂC; IR (KBr)
n
1): 3438, 2937, 1605, 1538; 1H NMR (DMSO-d6, 400 MHz)
d
(cmꢀ1): 3442, 2923, 1607, 1547; 1H NMR (DMSO-d6, 400 MHz)
3H, selenopheneeCH3), 7.46 (d, 2H, AreH, J ¼ 8.0 Hz), 7.57e7.68 (m,
d 3.00 (s, 3H, selenopheneeCH3), 6.58e6.90 (m, 5H, Phenyl), 7.45 (t,
5H, Phenyl), 8.26 (d, 2H, AreH, J ¼ 8.0 Hz), 8.49 (s,1H, NeH), 8.91 (s,
1H, AreH, J ¼ 8.0 Hz), 7.63 (dd, 1H, AreH, J ¼ 4.0 Hz), 7.89 (dd, 1H,
1H, CeH), 13C NMR (DMSO-d6, 100 MHz):
d 18.05, 120.84, 124.30,
AreH, J ¼ 8.0 Hz), 8.28 (s, 1H, NeH), 8.51 (s, 1H, CeH), 13C NMR
125.46, 125.81, 128.39, 129.18, 129.79, 130.38, 137.76, 137.89, 140.58,
147.16, 151.95, 154.09, 157.15, 161.42, and 166.20; LCeMS (positive
ion mode): m/z 524 (M þ H)þ for C22H14ClN7SSe.
(DMSO-d6, 100 MHz): d 18.13, 115.83, 116.05, 118.17, 120.91, 121.28,
123.87, 124.08, 127.11, 129.70, 132.22, 136.39, 137.13, 140.08, 143.84,
147.48, 153.60, 155.52, 157.71 and 162.04; LCeMS (positive ion
mode): m/z 542 (M þ H)þ for C22H13ClFN7SSe.
4.4.2. N-(4-Chlorophenyl)-5-methyl-6-(3-p-tolyl-[1,2,4]triazolo
[3,4-b][1,3,4]thiadiazol-6-yl)selenopheno[2,3-d]pyrimidin-4-amine
(10b)
4.4.7. N-(3-Chloro-4-fluorophenyl)-5-methyl-6-(3-p-tolyl-[1,2,4]
triazolo[3,4-b][1,3,4]thiadiazol-6-yl)selenopheno[2,3-d]pyrimidin-
4-amine (10g)
Yield 68%; Off-white solid, m.p. ¼ 240e242 ꢂC; IR (KBr)
n
Yield 82%; Brown solid, m.p. ¼ 272e274 ꢂC; IR (KBr)
n
(cmꢀ1):
d 2.41 (s, 3H,
(cmꢀ1): 3437, 2922, 1603, 1543; 1H NMR (DMSO-d6, 400 MHz)
3412, 2936, 1608, 1546; 1H NMR (DMSO-d6, 400 MHz)
d
2.41 (s, 3H, PhenyleCH3), 2.98 (s, 3H, selenopheneeCH3), 7.39 (d,
PhenyleCH3), 3.00 (s, 3H, selenopheneeCH3), 7.39 (t, 1H, AreH,
J ¼ 8.0 Hz), 7.42 (dd, 1H, AreH, J ¼ 4.0 Hz), 7.53 (dd, 1H, AreH,
J ¼ 8.0 Hz), 7.86 (d, 2H, AreH, J ¼ 8.0 Hz), 8.15 (d, 2H, AreH,
J ¼ 8.0 Hz), 8.40 (s, 1H, NeH), 9.18 (s, 1H, CeH), 13C NMR (DMSO-d6,
2H, AreH, J ¼ 8.0 Hz), 7.46 (d, 2H, AreH, J ¼ 8.0 Hz), 7.68 (d, 2H, Are
H, J ¼ 8.0 Hz), 8.16 (d, 2H, AreH, J ¼ 8.0 Hz), 8.41 (s, 1H, NeH), 8.50
(s, 1H, CeH), 13C NMR (DMSO-d6, 100 MHz):
d 18.23, 20.96, 117.81,
123.04, 125.77, 126.60, 127.38, 129.52, 129.92, 131.43, 137.07, 137.46,
141.26, 148.50, 152.03, 153.14, 156.84, 162.10, and 167.48; LCeMS
(positive ion mode): m/z 538 (M þ H)þ for C23H16ClN7SSe.
100 MHz):
d 18.14, 21.01, 116.36, 116.57, 119.30, 120.17, 121.40,
123.34, 124.46, 125.70, 129.68, 129.97, 131.14, 137.10, 138.87, 141.22,
145.66, 147.87, 152.28, 154.13, 158.76 and 161.48; LCeMS (positive
ion mode): m/z 556 (M þ H)þ for C23H15ClFN7SSe.
4.4.3. N-(4-Chlorophenyl)-6-(3-(4-methoxyphenyl)-[1,2,4]triazolo
[3,4-b][1,3,4]thiadiazol-6-yl)-5-methylselenopheno[2,3-d]
pyrimidin-4-amine (10c)
4.4.8. N-(3-Chloro-4-fluorophenyl)-6-(3-(4-methoxyphenyl)-
[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-6-yl)-5-methylselenopheno
[2,3-d]pyrimidin-4-amine (10h)
Yield 74%; Off-white solid, m.p. ¼ 262e264 ꢂC; IR (KBr)
n
(cmꢀ1):
2.90 (s, 3H,
3448, 2928, 1657, 1543; 1H NMR (DMSO-d6, 400 MHz)
d
Yield 76%; Pale yellow solid, m.p. ¼ 262e264 ꢂC; IR (KBr)
n
selenopheneeCH3), 3.79 (s, 3H, PhenyleOCH3), 7.13 (d, 2H, AreH,
J ¼ 8.0 Hz), 7.36 (d, 2H, AreH, J ¼ 8.0 Hz), 7.59 (d, 2H, AreH,
J ¼ 8.0 Hz), 8.11 (d, 2H, AreH, J ¼ 8.0 Hz), 8.41 (s, 1H, NeH), 8.91 (s,
(cmꢀ1): 3446, 3150, 2836,1610,1547; 1H NMR (DMSO-d6, 400 MHz)
d
2.98 (s, 3H, selenopheneeCH3), 3.86 (s, 3H, PhenyleOCH3), 7.21 (d,
2H, AreH, J ¼ 8.0 Hz), 7.46 (t,1H, AreH, J ¼ 8.0 Hz), 7.61 (dd,1H, Are
H, J ¼ 4.0 Hz), 7.90 (dd, 1H, AreH, J ¼ 8.0 Hz), 8.21 (d, 2H, AreH,
J ¼ 8.0 Hz), 8.52 (s, 1H, NeH), 8.91 (s, 1H, CeH), 13C NMR (DMSO-d6,
1H, CeH), 13C NMR (DMSO-d6, 100 MHz):
d 17.68, 56.80, 116.53,
121.39, 125.12, 125.98, 128.31, 129.14, 130.44, 131.68, 136.24, 138.72,
140.80, 149.02, 151.37, 154.40, 156.81, 162.94, and 168.22; LCeMS
(positive ion mode): m/z 554 (M þ H)þ for C23H16ClN7OSSe.
100 MHz):
d 18.06, 52.98, 113.84, 117.40, 121.72, 123.61, 128.17,
119.20, 120.83, 121.62, 122.80, 124.07, 125.22, 129.41, 130.18, 131.77,
137.21, 138.29, 140.85, 144.38, 147.20, 153.12, 156.24, 157.68 and
162.07; LCeMS (positive ion mode): m/z 572 (M þ H)þ for
4.4.4. 4-(6-(4-(4-Chlorophenylamino)-5-methylselenopheno[2,3-d]
pyrimidin-6-yl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-3-yl)phenol
(10d)
C23H15ClFN7OSSe.
4.4.9. 4-(6-(4-(3-Chloro-4-fluorophenylamino)-5-
methylselenopheno[2,3-d]pyrimidin-6-yl)-[1,2,4]triazolo[3,4-b]
[1,3,4]thiadiazol-3-yl)phenol (10i)
Yield 62%; White solid, m.p. ¼ 242e244 ꢂC; IR (KBr)
n
(cmꢀ1):
2.99
3512, 3425, 2922, 1604, 1566; 1H NMR (DMSO-d6, 400 MHz)
d
(s, 3H, selenopheneeCH3), 6.24 (s, 1H, PhenyleOH), 7.44 (d, 2H, Are
H, J ¼ 8.0 Hz), 7.70 (d, 2H, AreH, J ¼ 8.0 Hz), 8.22 (d, 2H, AreH,
J ¼ 8.0 Hz), 8.48 (d, 2H, AreH, J ¼ 8.0 Hz), 8.51 (s, 1H, NeH), 9.17 (s,
Yield 69%; White solid, m.p. ¼ 225e227 ꢂC; IR (KBr)
n
(cmꢀ1):
2.99
3394, 3158, 2948, 1609, 1570; 1H NMR (DMSO-d6, 400 MHz)
d
(s, 3H, selenopheneeCH3), 5.99 (br s, 1H, PhenyleOH), 7.22 (t, 1H,
AreH, J ¼ 8.0 Hz), 7.44 (d, 2H, AreH, J ¼ 8.0 Hz), 7.62 (dd, 1H, AreH,
J ¼ 4.0 Hz), 7.92 (dd, 1H, AreH, J ¼ 8.0 Hz), 8.24 (d, 2H, AreH,
J ¼ 8.0 Hz), 8.52 (s, 1H, NeH), 9.12 (s, 1H, CeH), 13C NMR (DMSO-d6,
1H, CeH), 13C NMR (DMSO-d6, 100 MHz):
d 18.13, 120.76, 124.45,
127.39, 127.87, 128.36, 129.67, 131.43, 132.05, 137.76, 138.06, 140.26,
148.84, 153.14, 153.57, 157.63, 161.08, and 167.40; LCeMS (positive
ion mode): m/z 539 (M þ H)þ for C22H14ClN7OSSe.
100 MHz):
d 18.13, 116.40, 116.62, 118.76, 120.73, 121.08, 123.48,
124.64, 127.31, 135.93, 137.90, 142.68, 146.94, 149.27, 153.06, 153.70,
155.00,157.04 161.22 and 166.52; LCeMS (positive ion mode): m/z
558 (M þ H)þ for C22H13ClFN7OSSe.
4.4.5. N-(4-Chlorophenyl)-5-methyl-6-(3-(4-nitrophenyl)-[1,2,4]
triazolo[3,4-b][1,3,4]thiadiazol-6-yl)selenopheno[2,3-d]pyrimidin-
4-amine (10e)
Yield 68%; Yellow solid, m.p. ¼ 226e228 ꢂC; IR (KBr)
n
(cmꢀ1):
d 2.97 (s, 3H,
4.4.10. N-(3-Chloro-4-fluorophenyl)-5-methyl-6-(3-(4-
nitrophenyl)-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-6-yl)
selenopheno[2,3-d]pyrimidin-4-amine (10j)
3436, 2912, 1607, 1547; 1H NMR (DMSO-d6, 400 MHz)
selenopheneeCH3), 6.74 (d, 2H, AreH, J ¼ 8.0 Hz) 7.45 (d, 2H, AreH,
J ¼ 8.0 Hz), 7.68 (d, 2H, AreH, J ¼ 8.0 Hz), 7.93 (d, 2H, AreH,
J ¼ 8.0 Hz), 8.50 (s, 1H, NeH), 9.08 (s, 1H, CeH), 13C NMR (DMSO-d6,
Yield 61%; Yellow solid, m.p. ¼ 236e238 ꢂC; IR (KBr)
n
(cmꢀ1):
d 2.93 (s, 3H,
3206, 2957, 1602, 1576; 1H NMR (DMSO-d6, 400 MHz)
100 MHz):
d
18.04, 112.19, 113.58, 120.89, 124.21, 124.45, 127.08,
selenopheneꢀCH3), 7.26 (t, 1H, AreH, J ¼ 8.0 Hz), 7.38 (d, 2H, AreH,
J ¼ 8.0 Hz), 7.58 (dd, 1H, AreH, J ¼ 4.0 Hz), 8.03 (dd, 1H, AreH,
J ¼ 8.0 Hz), 8.16 (d, 2H, AreH, J ¼ 8.0 Hz), 8.79 (s, 1H, NeH), 9.01 (s,
127.62, 128.32, 137.58, 137.80, 139.27, 141.92, 147.17, 150.93, 153.97,
156.99 and 172.09; LCeMS (positive ion mode): m/z 569 (M þ H)þ
for C22H13ClN8O2SSe.
1H, CeH), 13C NMR (DMSO-d6, 100 MHz):
d 18.24, 115.84, 116.40,

Y. Kotaiah et al. / European Journal of Medicinal Chemistry 75 (2014) 195e202
201
119.29, 121.35, 122.82, 123.04, 126.27, 129.60, 134.88, 138.92, 141.84,
146.29, 148.90, 152.74, 153.81, 156.22, 157.41163.07 and 167.08; LCe
MS (positive ion mode): m/z 587 (M þ H)þ for C22H12ClFN8O2SSe.
Qikprop. Qikprop helps in analyzing the pharmacokinetics and
pharmacodynamics of the ligand by accessing the drug like prop-
erties. Before docking the screened ligands into the protein active
site, the protein was prepared by deleting the substrate cofactor as
well as the crystallographically observed water molecules and then
the active site of the protein was defined for generating the grid.
Receptor van der Waals scaling for the non polar atoms was set to
0.9 [41] which makes the protein site “roomier” by moving back the
surface of non polar regions of the protein and ligand. This kind of
adjustments emulate to some extent the effect of breathing motion
to the protein site, it is a kind of giving breathing to the receptor,
this approach softens the active site region of the receptor making
it flexible [42]. The screened ligands were docked into the prepared
grid, for which “standard precision mode” of docking was selected.
The dock pose of each ligand docked into protein was analyzed for
their hydrogen bond interactions with the receptor. The analysis of
dock poses of all the molecules showed hydrogen bond interaction
with the active site residues mentioned in the literature.
4.5. Biological essay
4.5.1. Antimicrobial activity
Microdilution broth susceptibility assay was used for the anti-
bacterial evaluation of the compounds [27], whereas antifungal
susceptibility of the fungus yeasts was examined according to
NCCLS reference method for broth dilution antifungal susceptibility
testing of yeasts [28]. Ciprofloxacin was used as standard antibac-
terial agent whereas flucanazol was used as antifungal agent and
both are prepared as described in the related references.
4.5.2. DPPH radical scavenging activity
The hydrogen atom or electron donation ability of the com-
pounds was measured from the bleaching of the purple colored
methanol solution of 1,1-diphenyl-1-picrylhydrazyl (DPPH). The
spectrophotometric assay uses the stable radical DPPH as a reagent.
1 ml of various concentrations of the test compounds (5, 10, 25, 50
Acknowledgments
and 100 m
g mlꢀ1) in methanol was added to 4 ml of 0.004% (w/v)
Authors are (Y. Kotaiah) is gratefully acknowledge to UGCeBSR
(RFSMS) for financial assistance.
methanol solution of DPPH. After a 30 min incubation period at
room temperature, the absorbance was read against blank at
517 nm. The percent of inhibition (I%) of free radical production
from DPPH was calculated by the following equation.
Appendix A. Supplementary data
% of scavenging ¼ ½ðA control ꢀ A sampleÞ=A blankꢃ ꢄ 100
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.01.006.
(1)
where A control is the absorbance of the control reaction (con-
taining all reagents except the test compound) and A sample is the
absorbance of the test compound. Tests were carried at in triplicate.
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