Synthesis, antimicrobial, antiquorum-sensing, antitumor and cytotoxic activities of new series of fused [1,3,4]thiadiazoles

Article abstract of DOI:10.1016/j.ejmech.2013.02.010

New series of [1,3,4]thiadiazolo[3,2-a]pyrimidines, benzo[h][1,3,4] thiadiazolo[2,3-b]quinazolines, benzothiadiazolotriazocine, and imidazo[2,1-b][1,3,4]thiadiazoles have been synthesized and characterized by analytical and spectrometrical methods (IR, MS, ¹H and ¹³C NMR). Twenty of the synthesized compounds were screened for antibacterial activity against Escherichia coli, Staphylococcus aureus and Bacillus cereus. They were found to be either moderately active, slightly active or inactive against the tested microorganisms. The antifungal activity of these compounds were also tested against Candida albicans, Aspergillus fumigatus 293 and Aspergillus flavus 3375. Compounds 3g, h showed potent antifungal activity against C. albicans. In addition, the same compounds were tested for antiquorum-sensing activity against Chromobacterium violacium ATCC 12472, where compounds 3b,c, 3f-h, 6b-d, 9, 10 and 12 demonstrated acceptable activity. Compounds 3d, 9 and 10 were screened for antitumor activity at National Cancer Institute, USA. The in vitro cytotoxic activity of eighteen of the synthesized compounds was studied by brine shrimp lethality bioassay, and results indicated that compounds 6c, 13, 3h, 6d and 3d have the highest cytotoxic activity.

Full text of DOI:10.1016/j.ejmech.2013.02.010

Accepted Manuscript
Synthesis, Antimicrobial, Antiquorum-Sensing, Antitumor and Cytotoxic Activities of
New Series of Fused [1,3,4]Thiadiazoles
Nadia S. El-Gohary, Mona I. Shaaban
PII:
S0223-5234(13)00096-2
10.1016/j.ejmech.2013.02.010
EJMECH 6000
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 October 2012
Revised Date: 4 February 2013
Accepted Date: 9 February 2013
Please cite this article as: N.S. El-Gohary, M.I. Shaaban, Synthesis, Antimicrobial, Antiquorum-Sensing,
Antitumor and Cytotoxic Activities of New Series of Fused [1,3,4]Thiadiazoles, European Journal of
Medicinal Chemistry (2013), doi: 10.1016/j.ejmech.2013.02.010.
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ACCEPTED MANUSCRIPT
Graphical Abstract
New series of fused [1,3,4]thiadiazoles have been synthesized and screened for
antimicrobial, antiquorum-sensing and antitumor activities. Compounds 3h, 9 and 15b
are the most active ones.
H C
3
CH3
N
O
N
N
S
CN
N
S
CN
N
N
N
N
H
2
NO
S
2 2
H NO S
2
OCH3
H NO S
2
2
N
O
S
N
O
OCH3
9
15b
3h
Strong activity against C. albicans
Strong activity against S. aureus
Inhibition zone =18 mm
Strong activity against A. Fumigatus
Inhibition zone =32 mm
Inhibition zone =16 mm

ACCEPTED MANUSCRIPT
Synthesis, Antimicrobial, Antiquorum-Sensing, Antitumor and
Cytotoxic Activities of New Series of Fused [1,3,4]Thiadiazoles
,
a
b
Nadia S. El-Gohary* , Mona I. Shaaban
a
Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura
5516, Egypt
3
b
Department of Microbiology, Faculty of Pharmacy, Mansoura University, Mansoura 35516,
Egypt
∗
Corresponding author: Tel.: +2 010 00326839, Fax: +2 050 2247496
E-mail address: dr.nadiaelgohary@yahoo.com (N. S. El-Gohary)
Abstract:
New
series
of
[1,3,4]thiadiazolo[3,2-a]pyrimidines,
benzo[h][1,3,4]thiadiazolo[2,3-b]quinazolines, benzothiadiazolotriazocine, and imidazo[2,1-
b][1,3,4]thiadiazoles have been synthesized and characterized by analytical and spectrometrical
1
13
methods (IR, MS, H and C NMR). Tweenty of the synthesized compounds were screened for
antibacterial activity against Escherichia coli, Staphylococcus aureus and Bacillus cereus. They
were found to be either moderately active, slightly active or inactive against the tested
microorganisms. The antifungal activity of these compounds were also tested against Candida
albicans, Asperigllus fumgatus 293 and Asperigllus flavus 3375. Compounds 3g,h showed
potent antifungal activity against Candida albicans. In addition, the same compounds were
tested for antiquorum-sensing activity against Chromobacterium violacium ATCC 12472,
where compounds 3b,c, 3f-h, 6b-d, 9, 10 and 12 demonstrated acceptable activity. Compounds
3
d, 9 and 10 were screened for antitumor activity at National Cancer Insitute, USA. The in
vitro cytotoxic activity of eighteen of the synthesized compounds was studied by brine shrimp
lethality bioassay, and results indicated that compounds 6c, 13, 3h, 6d and 3d have the highest
cytotoxic activity.
Keywords: Synthesis, Fused [1,3,4]thiadiazoles, Antimicrobial activity, Antiquorum-
sensing activity, Antitumor activity, Cytotoxic activity
1
. Introduction
The global rise of anti-microbial resistance, combined with the rapid rate of microbial
evolution, and the slower development of novel antibiotics, underscores the urgent need for
innovative therapeutics. Development of new antimicrobial or antipathogenic agents that act
upon new microbial targets is a necessity [1]. In view of the fact that quorum-sensing (QS) is
involved in microbial pathogenesis, research efforts have focused recently upon developing
antipathogenic agents to control bacterial diseases by inhibiting quorum sensing [2]. QS is a
term that describes an environmental sensing system that allows bacteria to monitor their own
population density which contributes significantly to the development and size of the biofilm.
Many gram negative bacteria use N-acyl-homoserine lactones (acyl HSLs) as quorum-sensing
signal molecules. The signal molecules aid in biofilm formation which in turn confer various
properties of pathogenicity including drug resistance [3]. Antiquorum-sensing agents are
-
1 -

ACCEPTED MANUSCRIPT
viewed as blockers of bacterial pathogenicity rather than as anti-microbials and therefore their
use is highly attractive, particularly with respect to the emergence of multi-antibiotic resistant
bacteria [4].
Also, cancer is one of the most insidious and feared diseases. It is a group of diseases
characterized by the uncontrolled growth of abnormal cells which have the ability to spread
throughout the body [5]. Therefore, development of novel chemotherapeutic agents is an
important and challenging task for the medicinal chemists. Hence, many research programs are
directed towards the design and synthesis of new drugs for their chemotherapeutic usage.
During recent years there have been intense investigations on fused thiadiazole systems.
Literature survey revealed that [1,3,4]thiadiazolo[3,2-a]pyrimidine nucleus is associated with
diverse pharmacodynamic and chemotherapeutic activities [6-15], including antimicrobial
[
9,10,14,15] and antitumor activities [6-9]. Moreover, many imidazo[2,1-b][1,3,4]thiadiazole
compounds are known to possess interesting pharmacological properties, including
antibacterial [16-20], antifungal [21], antimicrobial [22-24], antitumor activities [25,26] and
cyclooxygenase-2 inhibitors [27]. Considering these published data and our previous interests
on fused thiadiazole systems [7,8], and in searching for new compounds of potent
antimicrobial, antiquorum-sensing and antitumor activities, it seemed interesting to synthesize
new compounds incorporating these heterocyclic systems to explore their antimicrobial,
antiquorum-sensing and antitumor activities.
2
. Results and Discussion
2
.1. Chemistry
A general approach to synthesize the designed compounds is outlined in Schemes 1-3. 2-
Amino-1,3,4-thiadiazole-5-sulfonamide (1) was used as a key starting material in this study for
syntheses of other fused heterocycles. Amine 1 was synthesized via acid hydrolysis of 2-
acetamido-1,3,4-thiadiazole-5-sulfonamide [28]. Cyclocondensation of amine 1 with
benzylidene derivatives of diethyl malonate 2a-d [29] afforded ethyl 5,6-dihydro-7-oxo-5-
(substituted)phenyl-2-sulfamoyl-[1,3,4]thiadiazolo[3,2-a]pyrimidine-6-carboxylates
(3a-d)
(Scheme 1). When amine 1 was allowed to react with ethyl benzylidenecyanoacetates 2e-h [29]
in refluxing N,N-dimethylformamide, the two possible structures, 3e-h (as reported in literature
[
(
30-32] and 4e-h (as reported in literature [33]) could be proposed for the reaction product
Scheme 1). Interestingly, this reaction gave only one product, 3e-h as confirmed by spectral
data. The IR spectra of the products showed bands corresponding to nitrile group and no bands
1
for carbonyl ester group. In addition, H NMR spectra did not exhibit the triplet-quartet pattern
characteristic for the ethyl protons, thus structures 3e-h could be ruled out. Moreover, 5-amino-
7
-oxo-6-[4-(substituted)phenyldiazenyl]-5H,
7H-[1,3,4]thiadiazolo[3,2-a]pyrimidine-2-
sulfonamide derivatives 6a-d were obtained via reaction of amine 1 with ethyl 2-cyano-2-[4-
(substituted)phenyldiazenyl]acetates (5a-d) [34] in refluxing glacial acetic acid (Scheme 1).
Interaction of equimolar amounts of amine 1 with benzylidenetetralones 7a-d [35] in
refluxing propylene glycol gave the benzo[h][1,3,4]thiadiazolo[2,3-b]quinazoline derivatives
a-d in 45-55% yield (Scheme 2). When, compound 1 was allowed to react with ethyl 2-
ethoxymethylenecyano)acetate or 2-(ethoxymethylene)malononitrile in refluxing glacial acetic
8
(
acid, compounds 9 and 10, respectively, were obtained (Scheme 2). In addition, reaction of 1
with malononitrile in absolute ethanol and in the presence of catalytic amount of triethylamine
afforded 5-amino-7-imino-7H-[1,3,4]thiadiazolo[3,2-a]pyrimidine-2-sulfonamide (11) (Scheme
-
2 -

ACCEPTED MANUSCRIPT
2
). Moreover, the synthesis of benzo[g][1,3,4]thiadiazolo[3,2-a][1,3,5]triazocine-2-sulfonamide
derivative 12 was achieved through reaction of
1
with 2-[(thiophen-3-
yl)methyleneamino]benzoic acid in refluxing glacial acetic acid (Scheme 2).
The reaction of 2-aminothiadiazole 1 with 3-chloropropionyl chloride or ethyl chloroacetate
in refluxing glacial acetic acid afforded 6,7-dihydro-7-oxo-5H-[1,3,4]thiadiazolo[3,2-
a]pyrimidine-2-sulfonamide (13) and 5,6-dihydro-6-oxoimidazo[2,1-b][1,3,4]thiadiazole-2-
sulfonamide (14), respectively (Scheme 3). When imidazo[2,1-b][1,3,4]thiadiazole 14 was
heated under reflux with benzaldehyde derivatives in dioxane and in the presence of catalytic
amount of piperidine, 5-(substituted benzylidene)imidazo[2,1-b][1,3,4]thiadiazoles 15a,b were
obtained (Scheme 3).
2
.2. Antimicrobial and antiquorum-sensing screening
Tweenty of the synthesized compounds; namely, 3b-d, 3f-h, 6a-d, 8a,b, 9-14, and 15a,b
were screened for in vitro antimicrobial activity against two species of gram-positive bacteria,
Staphylococcus aureus & Bacillus cereus and one gram-negative bacteria, Escherichia coli
[
36]. Antifungal assay against Candida albicans, Aspergillus fumgitis 293 and Aspergillus
flavus 3372 was also performed [37]. The antimicrobial screening results were determined by
measuring the average diameter of the inhibition zones, expressed in millimeters (mm). As
shown in the results (Table 1), all tested compounds exhibited varying degree of inhibitory
effect on the growth of different tested microbial strains. Compound 3g showed promising
activity towards Bacillus cereus. Compounds 9 and 13 showed excellent antibacterial activity
against Staphyllococcus aureus, while compounds 3g and 6b showed moderate activity against
the same microorganism. Furthermore, compounds 6a and 13 demonstrated moderate activity
against Escherichia coli. The rest of the tested compounds showed either weaker activity or
were completely inactive against the selected microorganisms. Regarding the antifungal
activity, compounds 3g,h demonstrated strong antifungal activity against Candida albicans,
compounds 6c, 8a and 13 exhibited moderate activity towards the same microorganism. In
addition, compounds 15b showed interesting antifungal activity against Asperigallus fumigatus.
These observations may promote a further development of fused [1,3,4]thiadiazoles and may
lead to compounds with better pharmacological profile than the standard antibacterial and
antifungal drugs.
The same compounds were tested for antiquorum-sensing activity against Chromobacterium
violaceum ATCC 12472 [38]. The QS system of Chromobacterium violaceum was used for this
assay. QS in this wild type strain of bacteria produces violacein (a purple pigment) in response
to autoinducer molecules known as acyl HSLs [39,40]. Thus, drugs that inhibit acyl HSL-
mediated QS activity in Chromobacterium violaceum will prevent the production of this purple
pigment. Screening results for their ability to inhibit QS regulated violacein production against
Chromobacterium violaceum (based on measuring the radius of pigment inhibition in mm) are
presented in Table 1 and indicated that compounds 3b,c, 3f-h, 6b-d, 9, 10, and 12 have
antiquorum-sensing activity.
Structure-activity relationship (SAR) studies:
•
Concerning series 3a-h, compounds possessing an electron-donating substituent on the 5-
phenyl moiety produce enhanced antibacterial and antifungal activities over those substituted
with electron-withdrawing groups (compare 3c,d and 3b & 3g,h and 3f), on the other hand, the
antiquorum-sensing activity was decreased. In addition, the presence of 6-cyano group
-
3 -

ACCEPTED MANUSCRIPT
improved the antibacterial and antifungal activities over those analogues with 6-ethoxycarbonyl
group (compare 3g,h and 3c,d).
•
Regarding series 6a-d, the presence of para substituent regardless of its type on the phenyl
ring of the 6-phenyldiazenyl moiety increased the activity against Staphylococcus aureus, also
the antifungal and antiquorum-sensing activities were improved. On the other hand, the activity
against Bacillus cereus and Escherichia coli was decreased (compare 6b-d and 6a).
•
Taking into account the structure of imidazothiadiazoles 14 and 15a,b, it is assumed that the
presence of 5-benzylidene moiety substituted with electron-donating group improved the
antibacterial activity against Staphylococcus aureus and Bacillus cereus. Furthermore, it
enhanced the antifungal activity against Asperigallus fumigatus and Aspergillus flavus, but
decreased the activity against Candida albicans (compare 15b versus 14 and 15a).
2
.3. Preliminary in vitro antitumor screening
Out of the newly synthesized compounds, three derivatives 3d, 9 and 10 were selected by
the National Cancer Institute (NCI) in vitro disease-oriented human cells screening panel assay.
A single dose (10 µM) of the test compounds were used in the full NCI 60 cell lines panel
assay which includes nine tumor subpanels, namely; leukemia, non-small cell lung, colon,
CNS, melanoma, ovarian, renal, prostate, and breast cancer cells [41-44]. The data reported as
mean-graph of the percent growth of the treated cells, and presented as percentage growth
inhibition (GI%). The obtained results of the tested compounds 3d, 9, and 10 (Table 2); showed
distinctive potential pattern of selectivity, as well as broad-spectrum antitumor activity.
Concerning the activity towards individual cell lines; the three tested compounds showed
selective activity against leukemia (HL-60(TB)) cell line, non-small lung cancer (HOP-92) cell
line, melanoma (LOX IMVI) cell line and renal cancer (A498) cell line. With regard to broad-
spectrum antitumor activity; close examination of the data presented in table 2, revealed that
compound 3d is the most active member in this study, showing effectiveness towards numerous
cell lines belonging to different tumor subpanels.
2
.4. Cytotoxicity testing using brine shrimp lethality bioassay
Cytotoxicity via brine shrimp lethality test was studied in order to reveal new antimicrobial
and anticancer compounds [45]. Toxicity to brine shrimps has a good correlation with
antitumor, pesticidal [46] and anti-trypanosomal activities [47] in man. The brine shrimp larvae
(Artemia salina) responds similary to the corresponding mammalian systems [48] since the
DNA-dependant RNA polymerases of Artemia salina have been shown to be similar to the
mammalian type [49]. This test is not only used for predicting cytotoxicity, but also it is used to
predict antitumor, antibacterial and pesticidal activities [50]. Thus, it is possible to evaluate the
cytotoxicity of compounds using brine shrimp lethality bioassay rather than the more tedious in
vitro and in vivo antitumor assays.
Eighteen of the synthesized compounds; namely, 3b-d, 3f-h, 6a-d, 8a,b, 9, 10, 12, 13 and
1
5a,b were screened for cytotoxic activity against brine shrimp larvae (nauplii) adopting the
microplate assay, method of Meyer et al., [51] and using 5-flurouracil as a reference drug.
Cytotoxic activity of the tested compounds was determined by measuring the median lethal
concentration; LC50 (concentration that kills 50% of brine shrimp nauplii) expressed in µg/mL.
-
4 -

ACCEPTED MANUSCRIPT
LC90 (90% mortality) values were also determined to establish the therapeutic index. The results
were shown in table 3 and indicated that compounds 6c, 13, 3h, 6d and 3d have good cytotoxic
activity against brine shrimp nauplii in comparison with 5-flurouracil with LC50 values of
3
92.46, 398.72, 399.68, 403.87 and 404.85 ꢀg/mL, respectively, which represent high
cytotoxicity and hence may predict antitumor and antimicrobial potential of those compounds.
The rest of the tested compounds showed weaker activity, whereas compounds 3b, 15a, 3g and
3
f are the least cytotoxic analogues in this study with LC50 values of 508.13, 551.26, 598.08
and 659.63 ꢀg/mL, respectively.
Structure-activity relationship (SAR) studies:
•
Concerning series 3a-h, compounds possessing an electron-donating substituent on the 5-
phenyl moiety showed enhanced cytotoxic activity over those substituted with electron-
withdrawing groups (compare 3c,d and 3b & 3g,h and 3f). In addition, the presence of 4-
dimethylamino substituent on the 5-phenyl moiety improved the activity over that of the 3,4-
dimethoxy substituent (compare 3d and 3c & 3h and 3g).
•
Regarding series 6a-d, the presence of electron-donating substituent at the para position of
the phenyl ring of the 6-phenyldiazenyl moiety increased the cytotoxic activity against brine
shrimp larvae (compare 6c,d and 6a,b).
•
Taking into account the series of benzothiadiazoloquinazolines 8a-d, it is assumed that the
presence of 5-phenyl moiety substituted with electron-withdrawing group increased the
cytotoxic activity (compare 8b and 8c).
•
For the imidazothiadiazoles 15a,b, it is clear that the presence of 5-benzylidene moiety
substituted with electron-donating group enhanced the cytotoxic activity against brine shrimp
larvae (compare 15b and 15a).
3
. Conclusion
From the results of antimicrobial and cytotoxicity testing we can conclude that:
The unsubstituted thiadiazolopyrimidin-7-one nucleus enhances the antibacterial activity
•
against Staphylococcus aureus, antifungal activity against Candida albicans, and cytotoxic
activity against brine shrimp larvae (compound 13).
•
The thiadiazolopyrimidine nucleus possessing dimethylamino substituent at the para position
of the 5-phenyl moiety improved antifungal activity against Candida albicans as well as
cytotoxic activity against brine shrimp larvae (compound 3h). On the other hand, the presence
of chloro substituent at the para position of the 5-phenyl moiety decreased the antibacterial,
antifungal, and cytotoxic activities (compounds 3b and 3f).
•
The thiadiazolopyrimidine nucleus possessing methyl substituent at the para position of 6-
phenyldiazenyl moiety increased the antifungal activity against Candida albicans as well as
cytotoxic activity (compounds 6c).
•
The imidazothiadiazole nucleus possessing 5-benzylidene moiety substituted with electron-
donating group increased antibacterial activity against Bacillus cereus & Staphylococcus
aureus, antifungal activity against Aspergillus fumgitis 293 & Aspergillus flavus, and cytotoxic
-
5 -

ACCEPTED MANUSCRIPT
activity (compounds 15b). On the other hand, electron-withdrawing substituent on 5-
benzylidene moiety reduced the antibacterial and antifungal activities against the same
microorganisms, as well as cytotoxic activity (compounds 15a).
4
. Experimental
4
.1. Chemistry
o
All melting points ( C) were recorded on Fisher-Johns melting point apparatus and are
uncorrected. The infrared spectra were recorded in KBr disc using a Unicam SP 1000 IR
-1
spectrometer (v in cm ) at Faculty of Science, Mansoura University. Nuclear magnetic
1
13
resonance ( H and C NMR) spectra were obtained on 300 MHz FT-NMR spectrometer at
Faculty of Science, Cairo University. The chemical shifts are expressed in δ ppm using
tetramethylsilane (TMS) as internal reference and DMSO-d as solvent. Mass spectra were
6
recorded on JEOL JMS-600H spectrometer using electron impact technique at 70 eV at
Microanalytical Unit, Cairo University. Microanalyses (C, H, N) were performed at
Microanalytical Unit, Cairo University, and were in agreement with the proposed structures.
Reaction times were monitored using TLC plates, Silica gel 60 F254 precoated (E. Merck) and
the spots were visualized by UV. Chloroform: methanol (9:1) was adopted as elution solvent.
Compound 1 was prepared as reported [28], compounds 2a-h were prepared according to the
literature method [29], compounds 5a-d were prepared adopting the published method [34],
compounds 7a-d were prepared following the literature procedure [35].
4
.1.1. General procedure for the preparation of 6-(ethoxycarbonyl or cyano)-5,6-dihydro-7-
oxo-5-(substituted phenyl)-[1,3,4]thiadiazolo[3,2-a]pyrimidine-2-sulfonamides (3a-h)
A mixture of 2-amino-1,3,4-thiadiazole-5-sulfonamide (1) (0.45 g, 0.0025 mol) and the
appropriate benzylidene derivative of diethyl malonate 2a-d or ethyl cyanoacetate 2e-h (0.0025
mol) in DMF (10 mL) was heated under reflux for 18-20 hours. The reaction mixture was
cooled and poured onto ice-water (50 mL). The precipitated solid was collected by filtration,
dried and crystallized from ethanol/water (3:1).
4
.1.1.1.
Ethyl
5-(4-bromophenyl)-5,6-dihydro-7-oxo-2-sulfamoyl-[1,3,4]thiadiazolo[3,2-
a]pyrimidine-6-carboxylate (3a)
0
-1
Yield 45%, m.p. 197-199 C. IR spectrum (KBr, v, cm ): 3265, 3192 (NH
2
), 1750 (COOC
628 (C=O). MS m/z (%): 462 (0.22, M +1), 64 (100.00). Anal. Calcd for C14
461.31): C 36.45, H 2.84, N 12.15. Found: C 36.67, H 2.55, N 11.87.
.1.1.2. Ethyl 5-(4-chlorophenyl)-5,6-dihydro-7-oxo-2-sulfamoyl-[1,3,4]thiadiazolo[3,2-
a]pyrimidine-6-carboxylate (3b)
2 5
H ),
H13BrN O S
4 5 2
+
1
(
4
0
1
Yield 56%, m.p. 187-189 C. H NMR spectrum: (DMSO-d
6
, δ ppm): 1.30 (t, 3H, CH
2 3
CH ),
3
2
1
.86 (d, 1H, C H), 4.28-4.35 (q, 2H, CH CH ), 5.52 (d, 1H, C H), 7.68 (d, 2H, Ar-H), 8.06 (d,
6
-
2
3
5-
1
3
H, Ar-H), 8.41 (s, 2H, NH ). C NMR spectrum: (DMSO-d , δ ppm): 13.0, 53.2, 54.6, 61.1,
2
6
28.7 (2C), 130.1 (2C), 133.4, 135.2, 157.8, 161.1, 168.7, 169.2. MS m/z (%): 419 (10.10,
+
+
+
M +2), 418 (14.00, M +1), 417 (16.10, M ), 101 (100.00). Anal. Calcd for C14
416.86): C 40.34, H 3.14, N 13.44. Found: C 40.67, H 3.47, N 13.19.
4 5 2
H13ClN O S
(
-
6 -

ACCEPTED MANUSCRIPT
4
.1.1.3. Ethyl 5,6-dihydro-5-(3,4-dimethoxyphenyl)-7-oxo-2-sulfamoyl-[1,3,4]thiadiazolo[3,2-
a]pyrimidine-6-carboxylate (3c)
0
1
Yield 45%, m.p. 238-240 C. H NMR spectrum: (DMSO-d , δ ppm): 1.29 (t, 3H, CH CH ),
6
2
3
3
7
.80 (s, 3H, OCH ), 3.82 (s, 3H, OCH ), 4.26-4.34 (m, 3H, CH CH , C H), 5.45 (d, 1H, C H),
3 3 2 3 6- 5-
+
.17-7.78 (m, 3H, Ar-H), 8.31 (s, 2H, NH
(442.47): C 43.43, H 4.10, N 12.66. Found: C 43.75, H 4.38, N 12.37.
.1.1.4. Ethyl 5,6-dihydro-5-(4-dimethylaminophenyl)-7-oxo-2-sulfamoyl-[1,3,4]thiadiaz-
olo[3,2-a]pyrimidine-6-carboxylate (3d)
2
). MS m/z (%): 442 (20.98, M ), 151 (100.00). Anal.
18 4 7 2
Calcd for C16H N O S
4
0
-1
Yield 65%, m.p. 165-166 C. IR spectrum (KBr, v, cm ): 3183 (NH
2
), 1742 (COOC
), 3.08 (s, 6H, 2CH
), 5.47 (d, 1H, C5-H), 6.83 (d, 2H, Ar-H), 7.94 (d, 2H,
). Anal. Calcd for C16 (425.48): C 45.17, H 4.50, N 16.46.
Found: C 45.40, H 4.85, N 16.14.
.1.1.5. 5-(4-Bromophenyl)-6-cyano-5,6-dihydro-7-oxo-[1,3,4]thiadiazolo[3,2-a]pyrimidine-2-
sulfonamide (3e)
Yield 65%, m.p. 173-174 C. IR spectrum (KBr, v, cm ): 3430, 3327 (NH
2
H
5
), 1635
1
(C=O). H NMR spectrum: (DMSO-d
6
, δ ppm): 1.28 (t, 3H, CH
2
CH
3
3
), 3.82
(d, 1H, C6-H), 4.23-4.29 (q, 2H, CH CH
2
3
Ar-H), 8.01 (s, 2H, NH
2
19 5 5 2
H N O S
4
0
-1
2
), 2212 (C≡N), 1640
, δ ppm): 4.16 (d, 1H, C6-H), 5.06 (d, 1H, C5-H), 7.28-
). C NMR spectrum: (DMSO-d , δ ppm): 38.6, 46.2, 116.4, 122.3,
1
(C=O). H NMR spectrum: (DMSO-d
6
1
3
7
1
3
4
.76 (m, 6H, Ar-H, NH
2
6
28.9 (2C), 130.6 (2C), 139.8, 151.9, 162.3, 169.7. Anal. Calcd for C12
4.79, H 1.95, N 16.91. Found: C 34.45, H 2.23, N 16.69.
8 5 3 2
H BrN O S (414.26): C
.1.1.6. 5-(4-Chlorophenyl)-6-cyano-5,6-dihydro-7-oxo-[1,3,4]thiadiazolo[3,2-a]pyrimidine-2-
sulfonamide (3f)
Yield 70%, m.p. 179-180 C. IR spectrum (KBr, v, cm ): 3376, 3318 (NH ), 2220 (C≡N), 1637
0
-1
2
+
+
+
(
C=O). MS m/z (%): 371 (0.40, M +2), 370 (0.51, M +1), 369 (0.59, M ), 344 (100.00). Anal.
Calcd for C12 ClN (368.98): C 38.97, H 2.18, N 18.94. Found: C 39.25, H 1.93, N
H
8
5 3 2
O S
1
8.72.
4
.1.1.7.
6-Cyano-5,6-dihydro-5-(3,4-dimethoxyphenyl)-7-oxo-[1,3,4]thiadiazolo[3,2-a]-
pyrimidine-2-sulfonamide (3g)
0
-1
Yield 60%, m.p. 191-192 C. IR spectrum (KBr, v, cm ): 3376, 3318 (NH
2
), 2220 (C≡N), 1662
1
(
C=O). H NMR spectrum: (DMSO-d
6
, δ ppm): 3.84 (s, 3H, OCH
3
), 3.86 (s, 3H, OCH ), 4.31
3
1
3
(d, 1H, C6-H), 5.65 (d, 1H, C5-H), 7.02-7.81 (m, 3H, Ar-H), 9.20 (s, 2H, NH
2
). C NMR
spectrum: (DMSO-d , δ ppm): 38.6, 46.0, 55.5, 55.7, 111.6, 116.8, 118.7, 120.6, 136.5, 138.2,
6
+
1
48.4, 148.8, 160.7, 169.2. MS m/z (%): 395 (41.80, M ), 119 (100.00). Anal. Calcd for
(395.41): C 42.53, H 3.31, N 17.71. Found: C 42.36, H 3.57, N 17.98.
6-Cyano-5,6-dihydro-5-(4-dimethylaminophenyl)-7-oxo-[1,3,4]thiadiazolo[3,2-a]-
pyrimidine-2-sulfonamide (3h)
14 13 5 5 2
C H N O S
4
.1.1.8.
0
-1
Yield 75%, m.p. 177-178 C. IR spectrum (KBr, v, cm ): 3374 (NH
2
), 2210 (C≡N), 1662
), 2.97 (s, 3H, CH ), 4.21 (d,
H, C6-H), 5.09 (d, 1H, C5-H), 6.64-7.51 (m, 4H, Ar-H), 8.46 (s, 2H, NH ). Anal. Calcd for
(378.40): C 44.43, H 3.73, N 22.21. Found: C 44.67, H 4.11, N 21.96.
1
(C=O). H NMR spectrum: (DMSO-d
6
, δ ppm): 2.82 (s, 3H, CH
3
3
1
2
14 14 6 3 2
C H N O S
4
.1.2. General procedure for the preparation of 5-amino-7-oxo-6-[4-(substitu-
ted)phenyldiazenyl]-5H, 7H-[1,3,4]thiadiazolo[3,2-a]pyrimidine-2-sulfonamides (6a-d)
A mixture of amine 1 (0.45 g, 0.0025 mol) and the the appropriate ethyl 2-cyano-2-[4-
(substituted)phenyldiazenyl]acetate 5a-d (0.0025 mol) in glacial acetic acid (10 mL) was
heated under reflux for 14-16 hours. The reaction mixture was cooled and the precipitated solid
was collected by filtration, dried and crystallized from acetic acid/water (2:1).
-
7 -

ACCEPTED MANUSCRIPT
4
.1.2.1. 5-Amino-7-oxo-6-phenyldiazenyl-5H, 7H-[1,3,4]thiadiazolo[3,2-a]pyrimidine-2-
sulfonamide (6a)
0
-1
Yield 75%, m.p. 237-238 C. IR spectrum (KBr, v, cm ): 3300, 3205 (2NH ), 1687 (C=O). MS
m/z (%): 351 (0.29, M ), 43 (100.00). Anal. Calcd for C H N O S (351.36): C 37.60, H 2.58,
2
+
1
1
9
7
3 2
N 27.90. Found: C 37.35, H 2.37, N 27.63.
.1.2.2. 5-Amino-6-[(4-bromophenyl)diazenyl]-7-oxo-5H,
a]pyrimidine-2-sulfonamide (6b)
4
7H-[1,3,4]thiadiazolo[3,2-
0
-1
1
Yield 60%, m.p. 238-240 C. IR spectrum (KBr, v, cm ): 3289, 3203 (2NH
NMR spectrum: (DMSO-d , δ ppm): 3.10 (s, 2H, C -NH ), 7.25-7.73 (m, 6H, Ar-H, SO
MS m/z (%): 431 (5.28, M +1), 91 (100.00). Anal. Calcd for C11 BrN (430.26): C
2
), 1689 (C=O). H
6
5
2
2 2
NH ).
+
H
8
7 3 2
O S
3
4
0.71, H 1.87, N 22.79. Found: C 30.43, H 1.59, N 23.12.
.1.2.3. 5-Amino-6-[(4-methylphenyl)diazenyl]-7-oxo-5H,
7H-[1,3,4]thiadiazolo[3,2-
a]pyrimidine-2-sulfonamide (6c)
0
1
Yield 65%, m.p. 175-177 C. H NMR spectrum: (DMSO-d , δ ppm): 2.18 (s, 3H, CH ), 3.19
6
3
+
(s, 2H, C
5
-NH
2
), 6.40-7.65 (m, 6H, Ar-H, SO
2 2
NH ). MS m/z (%): 365 (1.92, M ), 117 (100.00).
Anal. Calcd for C12
11 7 3 2
H N O S
(365.39): C 39.44, H 3.03, N 26.83. Found: C 39.77, H 2.85, N
2
6.56.
4
.1.2.4.
5-Amino-6-[(4-methoxyphenyl)diazenyl]-7-oxo-5H,
7H-[1,3,4]thiadiazolo[3,2-
a]pyrimidine-2-sulfonamide (6d)
0
1
Yield 70%, m.p. 200-202 C. H NMR spectrum: (DMSO-d
6
, δ ppm): 2.99 (s, 2H, C
5
-NH
). C NMR spectrum: (DMSO-d , δ
ppm): 55.1, 87.8, 113.7 (2C), 120.5 (2C), 132.4, 144.2, 154.9, 167.7, 168.8, 169.6. MS m/z
2
),
1
3
3
.81 (s, 3H, OCH
3
), 6.78-7.83 (m, 6H, Ar-H, SO
2
NH
2
6
+
(
%): 381 (0.16, M ), 60 (100.00). Anal. Calcd for C H N O S (381.39): C 37.79, H 2.91, N
12 11 7 4 2
2
5.71. Found: C 37.51, H 3.23, N 25.49.
4
5
.1.3. General procedure for the preparation of 6,7-dihydro-9-methoxy-5-(substituted phenyl)-
H-benzo[h][1,3,4]thiadiazolo[2,3-b]quinazoline-2-sulfonamides (8a-d)
A mixture of 2-amino-1,3,4-thiadiazole-5-sulfonamide 1 (0.45 g, 0.0025 mol) and the
appropriate benzylidenetetralone derivative 7a-d (0.0025 mol) in propylene glycol (10 mL) was
0
heated at 200-220 C for 10-12 hours. The mixture was cooled and diluted with water (50 mL)
with vigorous stirring. The separated solid was collected by filtration, washed with water, dried
and crystallized from ethanol/water (2:1).
4
.1.3.1.
5-(4-Bromophenyl)-6,7-dihydro-9-methoxy-5H-benzo[h][1,3,4]thiadiazolo[2,3-
b]quinazoline-2-sulfonamide (8a)
0
-1
1
Yield 50%, m.p. 226-227 C. IR spectrum (KBr, v, cm ): broad band at 3427 (NH
spectrum: (DMSO-d , δ ppm): 2.65-3.10 (m, 4H, 2CH ), 3.85 (s, 3H, OCH ), 4.18 (s, 1H, C5-
H), 7.46-7.70 (m, 9H, Ar-H, NH ). C NMR spectrum: (DMSO-d , δ ppm): 22.6, 28.5, 51.6,
5.8, 112.1, 112.3, 112.8, 117.9, 119.8, 126.5, 128.6 (2C), 130.7 (2C), 132.3, 139.2, 140.7,
2
). H NMR
6
2
3
1
3
2
6
5
1
+
+
+
56.4, 157.7, 161.3. MS m/z (%): 506 (0.09, M +1), 505 (0.01, M ), 504 (0.22, M -1), 77
(505.41): C 47.53, H 3.39, N 11.09. Found: C 47.75,
(100.00). Anal. Calcd for C20
H
17BrN
4
O
3
S
2
H 3.33, N 11.37.
4
.1.3.2.
5-(4-Chlorophenyl)-6,7-dihydro-9-methoxy-5H-benzo[h][1,3,4]thiadiazolo[2,3-b]
quinazoline-2-sulfonamide (8b)
0
1
Yield 55%, m.p. 211-212 C. H NMR spectrum: (DMSO-d
6
, δ ppm): 2.56-2.84 (m, 4H, 2CH
). MS m/z (%): 461 (4.29,
4 3 2
H17ClN O S (460.96): C 52.11, H
2
),
3
.83 (s, 3H, OCH ), 4.11 (s, 1H, C5-H), 7.19-7.49 (m, 9H, Ar-H, NH
M ), 460 (4.72, M -1), 59 (100.00). Anal. Calcd for C20
.72, N 12.15. Found: C 51.85, H 3.53, N 11.83.
3
2
+
+
3
-
8 -

ACCEPTED MANUSCRIPT
4
.1.3.3. 6,7-Dihydro-9-methoxy-5-(3,4-dimethoxyphenyl)-5H-benzo[h][1,3,4]thiadiazolo[2,3-
b]quinazoline-2-sulfonamide (8c)
0
1
Yield 45%, m.p. 145-146 C. H NMR spectrum: (DMSO-d , δ ppm): 2.72-3.11 (m, 4H, 2CH ),
6
2
3
4
5
4
.85-3.98 (m, 10H, 3OCH , C H), 7.26-7.70 (m, 6H, Ar-H), 8.06 (s, 2H, NH ). MS m/z (%):
3
5-
2
+
+
88 (9.67, M +2), 487 (16.67, M +1), 165 (100.00). Anal. Calcd for C22
4.31, H 4.56, N 11.51. Found: C 54.65, H 4.33, N 11.67.
H
22
N
4
O
5 2
S (486.56): C
.1.3.4.
6,7-Dihydro-9-methoxy-5-(4-dimethylaminophenyl)-5H-benzo[h][1,3,4]thiadiazo-
lo[2,3-b]quinazoline-2-sulfonamide (8d)
0
1
Yield 48%, m.p. 152-153 C. H NMR spectrum: (DMSO-d
.99 (s, 3H, CH ), 3.04 (s, 3H, CH ), 3.42 (s, 1H, C5-H), 3.88 (s, 3H, OCH
Ar-H), 7.82 (s, 2H, NH
6
, δ ppm): 2.65-2.92 (m, 4H, 2CH
2
),
2
3
3
3
), 6.60-7.53 (m, 7H,
1
3
2
). C NMR spectrum: (DMSO-d
6
, δ ppm): 22.7, 28.5, 40.6 (2C), 51.3,
5
1
5.6, 111.7, 111.9, 112.3, 112.8 (2C), 118.1, 125.8, 126.3 (2C), 131.5, 132.1, 138.8, 146.7,
53.4, 158.2, 160.6. MS m/z (%): 470 (0.24, M +1), 469 (0.39, M ), 101 (100.00). Anal. Calcd
+
+
for C H N O S (469.58): C 56.27, H 4.94, N 14.91. Found: C 56.55, H 4.67, N 14.73.
2
2
23
5
3 2
4
.1.4. Preparation of 6-cyano-5-oxo-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidine-2-sulfonamide (9)
A mixture of amine 1 (0.45 g, 0.0025 mol) and ethyl 2-(ethoxymethylene)cyanoacetate
(0.423 g, 0.0025 mol) in glacial acetic acid (10 mL) was heated under reflux for 16 hours. The
reaction mixture was cooled and the precipitated solid was collected by filtration, dried and
crystallized from acetic acid/water (3:1).
0
-1
2
Yield 72%, m.p. 280-282 C. IR spectrum (KBr, v, cm ): 3327, 3203 (NH ), 2220 (C≡N), 1680
1
13
(
C=O). H NMR spectrum: (DMSO-d
6
, δ ppm): 8.29 (s, 2H, NH
2
), 8.84 (s, 1H, C7-H).
C
NMR spectrum: (DMSO-d , δ ppm): 104.7, 116.2, 153.6, 157.9, 161.2, 168.7. MS m/z (%): 258
6
+
+
(
3.60, M +1), 257 (6.10, M ), 117 (100.00). Anal. Calcd for C H N O S (257.25): C 28.01, H
6 3 5 3 2
1
.18, N 27.22. Found: C 28.32, H 1.35, N 27.51.
4
.1.5. Preparation of 6-cyano-5-imino-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidine-2-sulfonamide
(
10)
A mixture of amine 1 (0.45 g, 0.0025 mol) and 2-(ethoxymethylene)malononitrile (0.305 g,
.0025 mol) in glacial acetic acid (10 mL) was heated under reflux for 20 hours. The reaction
0
mixture was cooled and the precipitated solid was collected by filtration, dried and crystallized
from ethanol.
0
-1
Yield 68%, m.p. 228-230 C. IR spectrum (KBr, v, cm ): 3429, 3327 (NH , NH), 2200 (C≡N).
2
1
H NMR spectrum: (DMSO-d
H). MS m/z (%): 257 (1.10, M +1), 256 (7.10, M ), 64 (100.00). Anal. Calcd for C
6
, δ ppm): 4.16 (s, 1H, NH), 7.25 (s, 2H, NH
2
), 8.13 (s, 1H, C7-
+
+
6
4 6 2 2
H N O S
(256.26): C 28.12, H 1.57, N 32.79. Found: C 27.93, H 1.73, N 32.98.
4
.1.6. Preparation of 5-amino-7-imino-7H-[1,3,4]thiadiazolo[3,2-a]pyrimidine-2-sulfonamide
(11)
A mixture of amine 1 (0.45 g, 0.0025 mol), malononitrile (0.165 g, 0.0025 mol) and
triethylamine (1 mL) in absolute ethanol (15 mL) was heated under reflux for 18 hours. The
reaction mixture was concentrated, cooled and the precipitated solid was collected by filtration,
dried and crystallized from ethanol/water (3:1).
0
-1
1
Yield 65%, m.p. >300 C. IR spectrum (KBr, v, cm ): 3407, 3332, 3245, 3175 (2NH
NMR spectrum: (DMSO-d , δ ppm): 4.10 (s, 2H, C -NH ), 7.29 (s, 1H, C6-H), 7.47 (s, 2H,
), 8.03 (s, 1H, NH). C NMR spectrum: (DMSO-d , δ ppm): 85.4, 157.2, 160.7, 163.5,
2
, NH). H
6
5
2
1
3
SO
68.1. MS m/z (%): 247 (0.12, M +1), 246 (0.14, M ), 64 (100.00). Anal. Calcd for
(246.27): C 24.39, H 2.46, N 34.13. Found: C 24.46, H 2.62, N 33.89.
2
NH
2
6
+
+
1
5 6 6 2 2
C H N O S
-
9 -

ACCEPTED MANUSCRIPT
4
.1.7. Preparation of 11-oxo-5-(thiophen-3-yl)-5H, 6H-benzo[g][1,3,4]thiadiazolo[3,2-a]-
[
1,3,5]triazocine-2-sulfonamide (12)
A mixture of amine 1 (0.45 g, 0.0025 mol) and 2-[(thiophen-3-yl)methyleneamino]benzoic
acid (0.578 g, 0.0025 mol) in glacial acetic acid (10 mL) was heated under reflux for 20 hours.
The reaction mixture was cooled and the precipitated solid was collected by filtration, dried and
crystallized from acetic acid.
0
-1
Yield 75%, m.p. 160-162 C. IR spectrum (KBr, v, cm ): 3445, 3387 (NH
2
), 3329 (NH), 1688
-H), 7.00-8.52 (m, 9H, Ar-H,
6
, δ ppm): 81.1, 115.1, 116.8, 117.4,
1
(
C=O). H NMR spectrum: (DMSO-d
6
, δ ppm): 2.93 (s, 1H, C
5
1
3
NH
2
), 11.20 (s, 1H, NH). C NMR spectrum: (DMSO-d
1
20.7, 127.9, 128.2, 129.9, 130.4, 138.2, 146.0, 153.2, 154.6, 168.9. MS m/z (%): 395 (0.02,
+
+
+
M +2), 394 (0.01, M +1), 393 (0.02, M ), 179 (100.00). Anal. Calcd for C14
11 5 3 3
H N O S
(393.46): C 42.74, H 2.82, N 17.80. Found: C 42.85, H 2.97, N 17.59.
4
.1.8.
Preparation
of
6,7-dihydro-7-oxo-5H-[1,3,4]thiadiazolo[3,2-a]pyrimidine-2-
sulfonamide (13)
A mixture of amine 1 (0.45 g, 0.0025 mole) and 3-chloropropionyl chloride (0.317 g, 0.0025
mol) in glacial acetic acid (10 mL) was heated under reflux for 15 hours. The solvent was
concentrated and the precipitated solid was collected by filtration, dried and crystallized from
ethanol.
0
-1
1
Yield 72%, 257-258 C. IR spectrum (KBr, v, cm ): 3139, 3200 (NH
spectrum: (CDCl , δ ppm): 2.84 (t, 2H, CH ), 3.01 (t, 2H, CH ), 8.20 (s, 2H, NH
spectrum: (DMSO-d , δ ppm): 32.8, 40.3, 161.1, 164.3, 169.2. MS m/z (%): 395 (0.02, M +2),
2
), 1652 (C=O). H NMR
1
3
3
2
2
2
). C NMR
+
6
+
+
+
3
94 (0.01, M +1), 393 (0.02, M ), 179 (100.00). MS m/z (%): 234 (0.05, M ), 85 (100.00).
Anal. Calcd for C (234.26): C 25.64, H 2.58, N 23.92. Found: C 25.43, H 2.75, N
5
6 4 3 2
H N O S
2
3.64.
4
.1.9. Preparation of 5,6-dihydro-6-oxoimidazo[2,1-b][1,3,4]thiadiazole-2-sulfonamide (14)
A mixture of amine 1 (0.45 g, 0.0025 mole) and ethyl chloroacetate (0.306 g, 0.0025 mol) in
glacial acetic acid (10 mL) was heated under reflux for 15 hours. The solvent was concentrated
and the precipitated solid was collected by filtration, dried and crystallized from ethanol.
0
-1
1
Yield 56%, m.p. 268-270 C. IR spectrum (KBr, v, cm ): 3303, 3186 (NH ), 1679 (C=O). H
2
1
3
NMR spectrum: (DMSO-d , δ ppm): 2.88 (s, 2H, CH ), 8.17 (s, 2H, NH ). C NMR spectrum:
6
2
2
+
+
(DMSO-d
6
, δ ppm): 44.7, 157.6, 162.1, 167.9. MS m/z (%): 221 (0.23, M +1), 220 (0.10, M ),
17 (100.00). Anal. Calcd for C (220.23): C 21.81, H 1.83, N 25.44. Found: C
1.95, H 2.11, N 25.36.
1
2
4 4 4 3 2
H N O S
4
.1.10. Preparation of 5,6-dihydro-6-oxo-5-(substituted benzylidene)imidazo[2,1-b]
[
1,3,4]thiadiazole-2-sulfonamide (15a,b)
A mixture of compound 14 (0.55 g, 0.0025 mole), the appropriate benzaldehyde (0.0025
mol) and a catalytic amount of piperidine (0.3 mL) in dioxane (10 mL) was heated under reflux
for 8-10 hours. The solvent was concentrated and the precipitated solid was collected by
filtration, dried and crystallized from ethanol.
-
10 -

ACCEPTED MANUSCRIPT
4
.1.10.1.
5-(4-Bromobenzylidene)-5,6-dihydro-6-oxoimidazo[2,1-b][1,3,4]thiadiazole-2-
sulfonamide (15a)
0
1
Yield 50%, m.p. 210-211 C. H NMR spectrum: (DMSO-d , δ ppm): 7.31-7.65 (m, 7H, Ar-H,
6
+
C=CH, NH ). MS m/z (%): 385 (0.15, M -2), 84 (100.00). Anal. Calcd for C H BrN O S
2
11
7
4
3
2
(387.23): C 34.12, H 1.82, N 14.47. Found: C 34.36, H 1.97, N 14.71.
4
.1.10.2. 5,6-Dihydro-5-(3,4-dimethoxybenzylidene)-6-oxoimidazo[2,1-b][1,3,4]thiadiazole-2-
sulfonamide (15b)
Yield 65%, m.p. 177-179 C. H NMR spectrum: (DMSO-d
.20 (m, 3H, Ar-H), 7.35 (s, 1H, C=CH), 7.95 (s, 2H, NH
ppm): 55.6, 55.7, 112.2, 114.6, 118.5, 127.4, 129.2, 148.3, 148.5, 151.8, 157.3, 162.5, 169.8. MS
0
1
6
, δ ppm): 3.76 (s, 6H, 2OCH
). C NMR spectrum: (DMSO-d , δ
3
), 6.64-
1
3
7
2
6
+
m/z (%): 368 (12.30, M ), 238 (100.00). Anal. Calcd for C13
.28, N 15.21. Found: C 42.51, H 3.43, N 14.93.
12 4 5 2
H N O S (368.39): C 42.38, H
3
4
.2. Biological Testing
.2.1. Antimicrobial and antiquorum-sensing activities
4
Some of the prepared compounds, 3b-d, 3f-h, 6a-d, 8a,b, 9-14 and 15a,b were screened for
antibacterial activity against Escherichia coli, Staphylococcus aureus and Bacillus cereus in
Luria-Bertani agar media) [36] and antifungal activity against Candida albicans in (Saboured's
(
agar), Asperigllus fumgatus 293 and Asperigllus flavus 3375 in (glucose minimal media) [37]. In
addition, their antipathogenic potential was checked by examining the antiquorum-sensing
activity against Chromobacterium violaceum ATCC 12472 in (Luria-Bertani agar media) [38].
Escherichia coli, Bacillus cereus, Staphylococcus aureus and Candida albicans were
obtained from the culture collection of the Department of Microbiology, Faculty of Pharmacy,
Mansoura University, Mansoura, Egypt. Filamentous fungi; Aspergillus fumigatus 293 and
Aspergillus flavus 3375 were kindly provided from Keller Lab, UW, USA. Chromobacterium
violaceum ATCC 12472 was kindly provided from Prof. Bob Mclean, Department of Biology,
Texas State University, USA. All bacterial strains were propagated in Luria-Bertani (LB) agar
media (Merck, Germany). Candida albicans was grown in Saboured's media (Merck, Germany)
and filamentous fungi were cultivated in glucose minimal media (Merck, Germany). Ampicillin
was obtained from EPICO company; Fluconazole was obtained from Pfizer company.
4
.2.1.1. Antibacterial screening
All the bacterial strains were propagated in Luria Bertani (LB) broth (1% peptone, 0.5%
yeast extract, 0.5% NaCl) and solidified with 1.5% agar. Melted Muller Hinton agar (50 mL) at
0
6
5
0 C were seeded with 50 µl of 1x10 CFU/mL of 18 hours culture of tested microorganisms.
The inoculated agar was mixed and poured into 15-cm-diameter plates to solidify. Wells were
made in agar using cork borer. Tested compounds were dissolved in DMSO in eppendorff tubes
for final concentration 5 mg/mL. Aliquots each of (100 µl) of each compound were applied into
the wells, DMSO was also included as a negative control, and ampicillin in a concentration of 5
mg/mL was used as a reference antibacterial agent. The compounds were allowed to diffuse for 2
0
0
hours at 4 C and incubated at 37 C for 24 hours [36]. Inhibition zones were measured using
Vernier caliper and the activity of the tested compounds was estimated in comparison to
ampicillin (Table 1). The inhibition zone diameter of DMSO was subtracted from the
antibacterial acrivity of tested compounds
-
11 -

ACCEPTED MANUSCRIPT
.
4.2.1.2. Antifungal screening
Saboured's media (50 mL) inoculated with 50 µl of 1x10 CFU/mL of 24 hours culture of
6
Candida albicans. For filamentous fungi, glucose minimal media (50 mL) was inoculated with
6
5
0 µl of 1x10 CFU/mL of Aspergilli. Wells were made in agar using cork borer. The tested
compounds were dissolved in DMSO in eppendorff tubes for final concentration 5 mg/mL and
00 µl of test solution was applied in the wells. The standard antifungal drug (fluconazole) was
1
also added at the same concentration to each plate. In addition, DMSO (control solvent) was
0
added to each plate. Plates were incubated at 37 C for 48 hours for Candida albicans and
0
Aspergillus fumigatus and at 30 C for 48 hours for Aspergillus flavus [37]. Antifungal activity of
the tested compounds was determined by measuring the diameter of the inhibition zone (Table
1
). The inhibition zone diameter of DMSO was subtracted from the antifungal acrivity of tested
compounds.
4
.2.1.3. Antiquorum-sensing screening
Cultures were prepared by growing Chromobacterium violaceum ATCC 12472 in Luria
0
Bertani (LB) broth and incubated for 16-18 hours in an orbital incubator running at 28 C and
6
1
50 rpm. Cultures were then adjusted to 0.5 McFarland standard (Ca. 1x10 CFU/mL).
Chromobacterium violaceum was inoculated (100 µl/plate) in 50 mL LB agar and solidified.
Wells were made in LB agar media using cork borer. The tested compounds were dissolved in
DMSO in eppendorff tubes for final concentration 5 mg/mL and 100 µl of test solution was
applied in the wells. DMSO (control solvent) was added to each plate. Plates were incubated at
0
3
0 C for 48 hours to check the inhibition of pigment production around the well. Bacterial
growth inhibition would result in a clear halo around the disc, while a positive quorum sensing
inhibition is exhibited by a turbid halo harboring pigmentless bacterial cells of Chromobacterium
violaceum ATCC 12472 monitor strain [38]. Bacterial growth inhibition by the tested
1
compounds was measured as radius (r ) in mm, while both growth and pigment inhibition was
measured as radius (r ) in mm. The pigment inhibition (QS inhibition) was determined by
2
substracting bacterial growth inhibition (r ) from the total radius (r ), thus; QS inhibition= (r -r )
1
2
2 1
in mm (Table 1).
4
.2.2. Antitumor screening
Under sterile conditions, cell lines were grown in RPMI 1640 media (Gibco, NY, USA)
supplemented with 10% fetal bovine serum (Biocell, CA, USA). The concentrations of the
compounds ranging from 0.01 to 100 µM were prepared in phosphate buffer saline. Each
compound was initially solubilized in DMSO, however, each final dilution contained less than
1
9
% DMSO. Solutions of different concentrations (0.2 mL) were pipetted into separate well of
6-multiwell microtiter plate, duplicate wells were prepared for each individual dose. Cell
4
culture (1.8 mL) containing a cell population of 6x10 cells/mL was pippited into each well.
Controls containing only phosphate buffer saline and DMSO at identical dilutions, were also
prepared in the same manner. The cultures were incubated with the compounds for 48 hours in a
0
humidified incubator at 37 C and in atmosphere of 5% CO
2
. After 48 hours, cells in each well
were diluted 10 times with saline and counted by using a coulter counter [41-44]. The data
reported as mean-graph of the percent growth of the treated cells, and presented as percentage
growth inhibition (GI%) (Table 2).
-
12 -

ACCEPTED MANUSCRIPT
4
.2.3. Cytotoxicity testing using brine shrimp lethality bioassay
Artemia salina Leach (brine shrimp eggs) are readily available as fish food in pet shops.
Artificial "sea water" is prepared by dissolving sea salt in distilled water (40 g/liter)
supplemented with dried yeast (6 mg/liter). Brine shrimp eggs are hatched in artificial sea water
0
during 48 hours incubation in a warm room (22-29 C), providing large numbers of larvae
(nauplii). Brine shrimp larvae (nauplii) are collected with a Pasteur pipette after attracting the
organisms to one side of the vessel with a light source. Nauplii are separated from the eggs by
pipetting them 2-3 times in small beakers containing sea water. The test samples were made up
to 1 mg/mL in artificial sea water (water insoluble compounds are dissolved in 50 µl DMSO
prior to adding sea water). Serial dilutions (1000, 500, 100, 10 µg/mL) were made in the wells in
9
6-well microplates in triplicate in 100 µl sea water. A suspension of nauplii containing 30 brine
shrimp larvae in sea water (100 µl) is added to each well with the help of a Pasteur pipette and
0
the covered microwell plate incubated at 22–29 C for 24 hours. The plates are then examined
under a binocular microscope and the number of dead (non-mobile) nauplii in each well is
counted. Methanol (100 ꢀl) is then added to each well and after 15 minutes the total number of
shrimps in each well is counted [51]. Plotting of log of concentration (logC) versus % mortality for
all tested samples showed an approximate linear correlation and the values of LC50 and LC90 were
calculated by using Microsoft Excel XP (Table 3). A ِl l values were compared with the standard
cytotoxic agent, 5-flurouracil with LC50 and LC90 values of 409.16 and 736.49 ꢀg/mL,
respectively.
Acknowledgements
The author would like to express her sincere thanks to Dr. Mona I. Shaaban, Microbiology
Department, Faculty of Pharmacy, Mansoura University, Egypt, for carrying out the
antimicrobial and antiquorom-sensing screening. Thanks to Mr. Ahmed Abbas, a technician at
Pharmacognosy Department, Faculty of Pharmacy, Mansoura University, Egypt, for carrying out
the cytotoxicity testing. Thanks also to the staff members of the Department of Health and
Human Services, National Cancer Institute (NCI), Bethesda, Maryland, USA for performing the
antitumor testing of the newly synthesized compounds.
References
[
1] A. Coates, Y. Hu, R. Bax, C. Page, The future challenges facing the development of new
antimicrobial drugs. Nat. Rev. Drug Discov. 1 (2002) 895-910.
[
2] A. Adonizio, K. Kong, K. Mathee, Inhibition of quorum sensing-controlled virulence factor
production in Pseudomonas aeruginosa by south Florida plant extracts. Antimicrob. Agents.
Chemother. 52 (2008) 198-203.
[
3] D. Anbazhagan, M. Mansor, G. O. S. Yan, M. Y. Md Yusof, H. Hassan, S. D. Sekaran,
Detection of quorum sensing signal molecules and identification of an autoinducer synthase gene
among biofilm forming clinical isolates of Acinetobacter spp.
,
PLoS ONE 7(7) (2012), e36696.
.
doi:10.1371/journal.pone.0036696.
-
13 -

ACCEPTED MANUSCRIPT
[
4] A. L. Adonizio, K. Downum, B. C. Bennett, K. Mathee, Antiquorum sensing activity of
medicinal plants in southern Florida. J. Ethnopharmacol. 105 (2006) 427-435.
[
(
5] C. M. Bagi, Summary-Cancer cell metastasis session. J. Musculoskel. Neuron Interact. 2
2002) 579-580.
[
6] A. T. Taher, H. H. Georgey, H. I. El-Subbagh, Novel 1,3,4-heterodiazole analogues:
Synthesis and in vitro antitumor activity. Eur. J. Med. Chem. 47 (2012) 445-451.
[
7] N. S. El-Sayed, E. R. El-Bendary, S. M. El-Ashry, M. M. El-Kerdawy, Synthesis and
antitumor activity of new sulfonamide derivatives of thiadiazolo[3,2-a]pyrimidines. Eur. J. Med.
Chem. 46 (2011) 3714-3720.
[
8] M. B. El-Ashmaway, M. A. El-Sherbeny, N. S. El-Sayed, Synthesis, in vitro antitumor
activity and DNA-binding affinity of novel thiadiazolopyrimidine and thiadiazoloquinazoline
derivatives. Mans. J. Pharm. Sci. 26 (1) (2010) 60-68.
[
9] M. A. Mahran, M. A. El-Sherbeny, A. M. A. El-Obaid, F. A. Badria, Heterocyclic systems
containing pyrimidine nucleus as potential antimicrobial and antitumor agents. Alexandria J.
Pharm. Sci. 12 (1998) 39-44.
[
10] Y. Tokunaga, S. Ito, Y. Kojima, S. Maeno, N. Sawai, Y. Sasao, Preparation of 2-
phenylsulfinyl-5H-1,3,4-thiadiazolo[3,2-a]pyrimidin-5-one derivatives as agricultural and
horticultural fungicides. Jpn. Kokai Tokkyo Koho JP Patent, 1989, 01 83,084.
[
11] S. Herrling, 5-Carboxy-7H-1,3,4-thiadiazolo[3,2-a]pyrimidin-7-ones as immune enhancers.
Ger. Offen. Patent, 1978, 2, 712, 932.
[
12] M. Suiko, K. Maekawa, Synthesis and antitumor activity of 2-alkanesulfinyl (or
alkanesulfonyl)-7-methyl-5H-1,3,4-thiadiazolo[3,2-a]pyrimidin-5-ones. Agric. Biol. Chem. 41
(1977) 2047-2053.
[
13] K. Maekawa, M. Mizumitsu, Thiadiazolo[3,2-a]pyrimidinone derivatives. Jpn. Kokai
Patent, 1977, 77, 118, 494.
14] R. A. Coburn, R. A. Glennon, Z. F. Chmielewicz, Mesoionic purinone analogues. 7. In vitro
antibacterial activity of mesoionic 1,3,4-thiadiazolo[3,2-a]pyrimidine-5,7-diones. J. Med. Chem.
7 (1974) 1025-1027.
15] R. A. Coburn, R. A. Glennon, Mesoionic purinone analogues. VI. Synthesis and in vitro
[
1
[
antibacterial properties of mesoionic thiazolo[3,2-a]pyrimidine-5,7-diones and mesoionic 1,3,4-
thiadiazolo[3,2-a]pyrimidine-5,7-diones. J. Pharm. Sci. 62 (1973) 1785-1789.
[
16] K. F. M. Atta, O. O. M. Farahat, A. Z. A. Ahmed, M. G. Marei, Synthesis and antibacterial
activities of novel imidazo[2,1-b][1,3,4]thiadiazoles. Molecules. 16 (2011) 5496-5506.
[
17] A. Jakhar, J. K. Makrandi, Synthesis and antibacterial properties of some novel 2-
substituted-6-(4-methyl-6-substituted cinnoline-3-yl)imidazo[2,1-b][1,3,4]thiadiazoles. Indian J.
Chem. 49B (2010) 1547-1551.
[
18] G. Q. Hu, L. L. Hou, S. Q. Xie, W. L. Huang, H. B. Zhang, Synthesis and antibacterial
activity of imidazothiadiazoles and heterocyclic-amine Mannich-base hydrochloride. Yao Xue
Xue Bao. 34 (2008) 926-929.
[
19] A. K. Gadad, C. S. Mahajanshetti, S. Nimbalkar, A. Raichurkar, Synthesis and antibacterial
activity of some 5-guanylhydrazone/thiocyanato-6-arylimidazo[2,1-b][1,3,4]thiadiazole-2-
sulfonamide derivatives. Eur. J. Med. Chem. 35 (2000) 853-857.
-
14 -

ACCEPTED MANUSCRIPT
[
20] A. K. Gadad, I. M. Khazi, C. S. Mahajanshetti, Synthesis of 2-alkyl-6-arylimidazo[2,1-
b][1,3,4]thiadiazoles for antibacterial activity. Indian J. Heterocycl. Chem. 2 (1992) 125-128.
21] C. S. Andotra, T. C. Langer, A. Kotha, Synthesis and antifungal activity of some substituted
,3,4-thiadiazolo[3,2-a]-s-triazin-5-phenyl-7-thiones and imidazo[2,1-b][1,3,4]-thiadiazol-5-
ones. J. Indian Chem. Soc. 74 (1997) 125-127.
22] A. Kaur, R. Kumar, U. Kalidhar, Synthesis, spectral studies and biological activity of some
novel biphenyl imidazo[2,1-b][1,3,4]thiadiazole derivatives. Res. J. Pharm. Biol. Chem. Sci. 3
[
1
[
(2012) 1084-1096.
[
23] S. G. Alegaon, K. R. Alagawadi, Synthesis, characterization and antimicrobial activity
evaluation of new imidazo[2,1-b][1,3,4]thiadiazole derivatives. Eur. J. Chem. 2 (2011) 94-99.
24] J. Mohan, A. Rathee, Condensed bridgehead nitrogen heterocyclic systems: Facile synthesis
and antimicrobial activity of imidazo[2,1-b][1,3,4]thiadiazoles. Indian J. Heterocycl. Chem. 17
[
(2007) 101-102.
[
25] M. N. Noolvi, H. M. Patel, N. Singh, A. K. Gadad, S. S. Cameotra, A. Badiger, Synthesis
and anticancer evaluation of novel 2-cyclopropylimidazo[2,1-b][1,3,4]thiadiazole derivatives.
Eur. J. Med. Chem. 46 (2011) 4411-4418.
[
26] T. Nalan, A. Gursouy, Synthesis and anticancer evaluation of some new hydrazone
derivatives of 2,6-dimethylimidazo[2,1-b][1,3,4]thiadiazole-5-carbohydrazide. Eur. J. Med.
Chem. 38 (2003) 781-786.
[
27] A. K. Gadad, M. B. Palkar, K. Anand, M. N. Noolvi, T. S. Boreddyc, J. Wagwadec,
Synthesis and biological evaluation of 2-trifluoromethyl/sulfonamido-5,6-diaryl substituted
imidazo[2,1-b][1,3,4]thiadiazoles: A novel class of cyclooxygenase-2 inhibitors. Bioorg. Med.
Chem. 16 (2008) 276–283.
[
28] V. Petrow, O. Stephenson, A. J. Thomas, A. M. Wild, Preparation and hydrolysis of some
derivatives of 1,3,4-thiadiazole. J. Chem. Soc. (1958) 1508-1512.
[
29] I. Meskini, L. Toupet, M. Daoudi, A. Kerbal, B. Bennani, P. H. Dixneuf, Z. H. Chohan, A.
C. L. Leite, T. B. Hadda, An efficient protocol for accessing ß-amino dicarbonyl compounds
through aza-Michael reaction. J. Braz. Chem. Soc. 21 (2010) S1-S18.
[
30] S. A. Abdel-Mohsen, Heterocycles derived from 5-(2-aminothiazol-4-yl)-8-hydroxyl-
quinoline: Synthesis and antimicrobial activity. J. Chin. Chem. Soc. 50 (2003) 1085-1092.
[
31] A. A. Hassanien, A. E. Amrb, S. A. S. Ghozlan, Utility of (p-
sulfonamidophenyl)azomalononitrile, and ethyl acetoacetate in synthesis of fused azole and
azines derivatives II. J. Chin. Chem. Soc. 47 (2000) 1273-1278.
[
32] J. Quiroga, M. Alvarado, B. Insuasty, R. Mereno, Synthesis of 5-cyanopyrazolo[3,4-
b]pyridines in the reaction of 5-amino-3-methyl-1-phenylpyrazole with arylidene derivatives of
malononitrile and ethyl cyanoacetate. J. Heterocyclic Chem. 36 (1999) 1311-1316.
[
33] D. K. Sain, B. Thadhaney, A. Joshi, N. Hussain, G. L. Talesara, Synthesis, characterization
and biological evaluation of some heterocyclic compounds containing ethoxyphthalimide moiety
via key intermediate 6-chloro-1,3-benzothiazole-2-amine. Indian J. Chem. 49B (2010) 818-825.
[
34] K. M. Al Zaydi, R. M. Borik, Microwave assisted condensation reactions of 2-aryl-
hydrazonopropanals with nucleophilic reagents and dimethyl acetylenedicarboxylate.
Molecules. 12 (2007) 2061-2079.
-
15 -

ACCEPTED MANUSCRIPT
[
35] S. W. Yee, C. Simons, Synthesis and CYP24 inhibitory activity of 2-substituted-3,4-
dihydro-2H-naphthalen-1-one (tetralone) derivatives. Bioorg. Med. Chem. Lett. 14 (2004) 5651-
654.
5
[
36] R. D. Pearson, R. T. Steigbigel, H. T. Davis, S. W. Chapmann, Method for reliable
determination of minimal lethal antibiotic concentrations. Antimicrob. Agents Chemother. 18
(
[
[
1980) 699-708.
37] R. J. Holt, Laboratory tests of antifungal drugs. J. Clin. Pathol. 28 (1975) 767–774.
38] K. H. McClean, M. K. Winson, L. Fish, A. Taylor, S. R. Chhabra, M. Camara, M. Daykin,
J. H. Lamb, S. Swift, B. W. Bycroft, G. S. Stewart, P. Williams, Quorum sensing and
Chromobacterium violaceum: exploitation of violacein production and inhibition for the
detection of N-acyl homoserine lactones. Microbiology. 143 (1997) 3703-3711.
[
39] R. McClean, L. S. Pierson, C. Fuqua, A simple screening protocol for the identification of
quorum signal antagonists. J. Microbiol. Methods. 58 (2004) 351-360.
[
40] W. Cha, D. A. Vattem, V. Maitin, M. B. Barnes, R. J. Mclean, Bioassays of quorum sensing
compounds using Agrobacterium tumefaciens and Chromobacterium violaceum. Methods Mol.
Biol. 692 (2011) 3-19.
[
41] P. Skahan, R. Storeng, D. Scudiero, A. Monks, J. McMahon, D. Vistica, J. R. Warren, H.
Bokesch, S. Kenney, M. R. Boyd, New colorimetric cytotoxic assay for anticancer drug
screening. J. Natl. Cancer Inst. 82 (1990) 1107-1112.
[
42] M. R. Grever, S. A. Schepartz, B. A. Chabner, The National Cancer Institute: Cancer drug
discovery and development program. Semin. Oncol. 19 (1992) 622-638.
[
43] A. Monks, D. Scudiero, P. Skehan, Feasibility of a high-flux anticancer drug screen using a
diverse panel of cultured human tumor cell lines. J. Natl. Cancer Inst. 83 (1991) 757-766.
[
44] M. R. Boyd, K. D. Paull, Some practical considerations and applications of the national
cancer institute in vitro anticancer drug discovery screen. Drug Dev. Res. 34 (1995) 91-109.
rd
[
45] J. B. Harborne, Phytochemical Methods. 3 Edn., Chapman and Hall, London, ISBN: 0-
4
12-57260-5, (1998) 1-302.
[
46] J. L. Mclaughlin, Crown-gall Tumors in Potato Discs and Brine Shrimp Lethality: Two
Simple Bioassays for Higher Plant Screening and Fractionation. Methods in Plant Biochemistry,
Hostettmann, K. (Ed.), Academic Press, London, (1991).
[
47] C. L. Zani, P. G. Chaves, R. Queiroz, N. M. Mendes, A. B. De Oliveira, J. E. Cardoso, M.
G. Anjos, T. S. M. Grandi. Brine shrimp lethality assay as prescreening system for anti-
Trypanosoma cruzi. Phytomedicine 2 (1995) 47-50.
[
48] P. N. Solis, C. W. Wright, M. M. Anderson, M. P. Gupta, J. D. Phillipson, A microwell
cytotoxicity assay using Artemia salina. Planta Med. 59 (1993) 250-252.
[
49] H. C. Birndorf, J. D'Alossio, J. C. Bagshaw, DNA-dependant RNA-polymerases from
Artemia embryos. Characterization of polymerases I and II from nauplius larvae. Dev. Biol. 45
(1975) 34-43.
[
50] C. Sanchez, M. Gupta, M. Vasquez, de Noriega, G. Montenegro, Bioassay with Artemia to
predict antibacterial and pharmacologic activity. Rev. Med. Panama 18 (1993) 62-69.
-
16 -

ACCEPTED MANUSCRIPT
[
51] B. Mayer, N. Ferrigni, J. Putnam, L. Jacobsen, D. Nichols, J. Mclaughlin, Brine shrimp: a
convenient general bioassay for active plant constituents. Planta Med. 45 (1982) 31-34.
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ACCEPTED MANUSCRIPT
R
N
N
S
CN
H NO S
2
2
N
O
3e-h
R = e) 4-Br; f) 4-Cl;
R
CN
g) 3,4-(OCH ) ; h) 4-N(CH )
3
2
3 2
COOC H
2
5
2e-h
DMF
R
N
R
COOC H
2
5
R
COOC H
2 5
N N
2
a-d
N
S
2
COOC H5
H NO S
NH2
H NO S
2 2
2
2
S
1
DMF
N
O
N
S
COOC
H
2 5
N
3a-d
H NO S
2
2
R = a) 4-Br; b) 4-Cl; c) 3,4-(OCH3)2;
N
NH2
d) 4-N(CH3)2
4e-h
NC
C H OOC
N
N
R
2
5
5
a-d
Glacial acetic acid
NH
N
NH2
N
S
N
O
N
R
N
S
N
O
N
R
N
N
H NO S
H NO S
2 2
2
2
N
6a-d
R = a) H; b) Br; c) CH ; d) OCH
3
3
Scheme 1. Synthesis of compounds 3a-h and 6a-d
-
18 -

ACCEPTED MANUSCRIPT
O
N
S
CN
N
H2NO2S
N
9
CN
C2H5O
COOC2H5
Glacial acetic acid
O
R
NH
N
CN
CN
R
C H O
N
S
CN
2
5
N
N
OCH3
N
S
N
N
7
a-d
H NO S
2 2
H2NO2S
H2NO2S
NH2
S
N
Propylene glycol
Glacial acetic acid
1
10
8a-d
OCH3
O
HO
R = a) 4-Br; b) 4-Cl; c) 3,4-(OCH ) ;
3
2
CN
CN
HC
S
N
d) 4-N(CH3)2
/
ine
l
o
m
n
yla
ute
a
h
t
h
t
e
e
i
r
T
a
l
o
s
b
O
NH2
N
H2NO2S
N
N
N
N
S
N
1
S
H2NO2S
NH
NH
1
S
12
Scheme 2. Synthesis of compounds 8a-d and 9-12
-
19 -

ACCEPTED MANUSCRIPT
Cl
N
S
N
N
S
COCl
N
N
N
H2NO2S
Cl
COOC H5
2 2
H NO S
2
H2NO2S
NH2
S
N
O
N
O
Glacial acetic acid
1
3
1
Glacial acetic acid
14
R
CHO
Piperidine/
dioxane
R
N
S
N
H2NO2S
N
O
1
5a,b
R = a) 4-Br; b) 3,4-(OCH3)2
Scheme 3. Synthesis of compounds 13, 14 and 15a,b
-
20 -

ACCEPTED MANUSCRIPT
a,b,c
Table (1): Antimicrobial and antiquorum-sensing activities of compounds 3-15
Inhibition zone diameter (mm)
Comp.
No
E. coli
B. cereus
S. aureus
C. albicans
A. fumigatus
A. flavus
Ch. violaceum
3
3
b
c
-
-
-
-
-
-
10
-
4
5
-
4
7
5
-
9
6
7
5
3
3
-
3
8
5
-
10
7
7
9
8
8
-
-
4
10
8
24
32
4
6
12
6
14
-
-
-
-
-
-
-
-
-
-
-
11
8
-
11
8
6
3d
4
6
8
8
-
4
6
-
3
f
3g
3h
6a
6b
-
12
12
11
-
6
c
5
5
-
6d
8a
8b
6
-
-
-
-
-
-
9
5
-
-
-
11
-
2
-
-
-
4
-
18
-
-
-
16
-
4
-
-
-
12
5
-
-
-
-
8
-
-
-
-
-
10
-
10
11
-
6
-
1
1
1
1
1
0
1
2
3
4
-
1
5a
-
-
23
2
4
3.5
2
4
14
8
4
-
16
-
7
-
-
15b
Ampicillin
Fluconazole
22
-
-
a
Sample concentration: 5 mg/mL, Sample volume 100 µl /well
Results are calculated after substraction of DMSO activity
b
c
Not active (-, inhibition zone<2 mm); weak activity (2-8 mm); moderate activity (9-15 mm); strong activity
> 15 mm)
(
E. coli: Escherichia coli; B. cereus: Bacillus cereus; S. aureus: Staphylococcus aureus; C. albicans: Candida
albicans; A. fumigatus: Asperigllus fumigatus; A. flavus: Asperigllus flavus; Ch. violaceum: Chromobacterium
violaceum
-
21 -

ACCEPTED MANUSCRIPT
Table (2): Percentage growth inhibition (GI %) of in vitro subpanel tumor cell lines at 10 µM
concentration of compounds 3d, 9 and 10.
a
%
Growth Inhibition (GI %)
Subpanel tumor cell lines
3d
9
10
Leukemia
-
-
-
CCRF-CEM
HL-60(TB)
K-562
MOLT-4
RPMI-8226
SR
2
1.0
-
18.4
29.3
-
-
-
-
-
-
-
-
-
-
1
0.3
Non-Small Lung Cancer
A549/ATCC
EKVX
HOP-62
HOP-92
-
-
-
1
2.2
-
-
-
-
10.7
5
1.2
-
12.3
20.3
-
-
-
-
-
-
-
-
NCI-H226
NCI-H23
-
-
NCI-H460
NCI-H522
Colon Cancer
COLO 205
HCC-2998
HCT-116
HCT-15
HT29
KM 12
SW-620
1
9.3
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
CNS Cancer
SF-268
SF-295
SF-539
SNB-19
-
-
-
-
-
-
-
-
-
-
-
-
-
1
0.1
10.3
SNB-75
Melanoma
LOX IMVI
MALME-3M
M14
MDA-MB-435
SK-MEL-2
SK-MEL-28
SK-MEL-5
UACC-257
UACC-62
Ovarian cancer
IGORV1
OVCAR-3
OVCAR-4
OVCAR-5
OVCAR-8
NCI/ADR-RES
SK-OV-3
Renal cancer
1
1.7
4.9
-
10.7
15.1
1
28.0
-
-
-
-
-
-
-
-
-
21.3
-
-
-
-
-
-
-
-
-
13.7
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
7
86-0
16.5
34.2
14.9
A498
-
22 -

ACCEPTED MANUSCRIPT
Table (2): Continued
-
-
-
ACHN
CAKI-1
RXF 393
SN12C
TK-10
UO-31
Prostate cancer
2
1.1
-
-
11.5
-
-
-
-
-
-
-
-
-
1
1
4.7
10.5
1.4
-
-
-
-
-
PC-3
DU-145
Breast cancer
MCF-7
MDA-MB-231/ATCC
HS 578T
BT-549
T-47D
1
3
1
0.2
4.6
8.5
-
-
-
-
12.0
12.8
-
-
-
-
-
-
-
-
-
MDA-MB-468
a
-
, GI <10%
-
23 -

ACCEPTED MANUSCRIPT
Table (3): Cytotoxic activity of compounds 3-10, 12, 13 and 15 using brine shrimp lethality
a
bioassay
Number
LogC of Dead
Nauplii
Comp.
No.
Conc.
(µg/mL)
%
LC50
LC90
Mortality (µg/mL) (µg/mL)
1
000
3
2.698
2
25
21
16
83.3
70.0
5
00
00
3b
508.13
442.47
404.85
659.63
598.08
399.68
419.46
459.55
392.46
403.87
438.21
914.63
796.46
728.74
1187.33
1076.55
719.42
755.03
827.20
706.43
726.97
788.78
1
53.3
1
000
0
1
3
11
28
36.6
93.3
1
5
00
00
2.698
2
25
21
83.3
70.0
3
c
1
10
1
3
15
30
50.0
100.0
1
000
5
00
00
2.698
2
28
25
93.3
83.3
3d
1
10
1
3
20
20
66.6
66.6
1
000
5
00
00
2.698
2
15
11
50.0
36.6
3
f
1
10
1
3
7
21
23.3
70.0
1
000
5
00
00
2.698
2
18
15
60.0
50.0
3
g
1
10
1
3
11
30
36.6
100.0
1
000
5
00
00
2.698
2
29
26
96.6
86.6
3h
1
10
1
3
21
29
70.0
96.6
1
000
5
00
00
2.698
2
27
24
90.0
80.0
6
a
1
10
1
3
20
27
66.6
90.0
1
000
5
00
00
2.698
2
24
20
80.0
66.6
6b
1
10
1
3
14
30
46.6
100.0
1
000
5
00
00
2.698
2
30
29
100.0
96.6
6
c
1
10
1
3
27
30
90.0
100.0
1
000
5
00
00
2.698
2
28
26
93.3
86.6
6
d
b
1
10
1
3
22
29
73.3
96.6
1
000
5
00
00
2.698
24
20
15
80.0
66.6
50.0
8
1
2
1
10
-
24 -

ACCEPTED MANUSCRIPT
Table (3): Continued
1
000
3
2.698
2
28
23
19
93.3
76.6
63.3
5
00
00
8
c
455.37
432.15
419.11
477.09
398.72
551.26
425.17
819.67
777.87
754.40
858.77
717.70
992.28
765.30
1
1
000
0
1
3
14
29
46.6
96.6
1
5
00
00
2.698
2
25
21
83.3
70.0
9
1
10
1
3
15
29
50.0
96.6
1
000
5
00
00
2.698
2
27
24
90.0
80.0
1
1
1
0
2
3
1
10
1
3
22
26
73.3
86.6
1
000
5
00
00
2.698
2
23
20
76.6
66.6
1
10
1
3
14
30
46.6
100.0
1
000
5
00
00
2.698
2
29
27
96.6
90.0
1
10
1
3
21
24
70.0
80.0
1
000
5
00
00
2.698
2
18
12
60.0
40.0
15a
1
10
1
3
7
29
23.3
96.6
1
000
5
00
00
2.698
2
26
23
86.6
76.6
15b
1
1
000
0
1
3
19
30
63.3
100.0
1
5
00
00
2.698
2
27
25
90.0
83.3
5
-Fu
409.16
-
736.49
-
1
1
0
-
1
-
19
0
63.3
0.0
Control
a
LC50, compound concentration required to kill 50% of brine shrimp nauplii.
LC , compound concentration required to kill 90% of brine shrimp nauplii.
9
0
-
25 -

ACCEPTED MANUSCRIPT
Research Highlights
New series of fused [1,3,4]thiadiazole systems have been synthesizedꢀ
Tweenty of the compounds were tested for antimicrobial and antiquorum-sensing
activityꢀCompounds 3d, 9, 10 were screened for antitumor activity at National
Cancer Institute

ACCEPTED MANUSCRIPT
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