Antimicrobial and antiquorum-sensing studies. Part 2: Synthesis, antimicrobial, antiquorum-sensing and cytotoxic activities of new series of fused [1,3,4]thiadiazole and [1,3]benzothiazole derivatives

Article abstract of DOI:10.1007/s00044-013-0637-x

New series of [1,3,4]thiadiazolo[3,2-a]pyrimidines, [1,3,4]thiadiazolo[2,3- b]quinazolines, and pyrimido[2,1-b][1,3]benzothiazoles have been synthesized and characterized by analytical and spectrometrical methods (IR, MS, ¹H, and ¹³C NMR). Sixteen of the synthesized compounds; namely, 3a, b, 5a-f, 8a, b, 10, 11a-c, and 13a, b 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 selected microorganisms. The antifungal activity of these compounds were also tested against Candida albicans, Aspergillus fumigatus 293, and Aspergillus flavus 3375. Compound 11a showed potent antifungal activity against the three selected fungi; the rest of the tested compounds displayed either weaker activity or were completely inactive. The same compounds were examined for antiquorum-sensing activity against Chromobacterium violaceum ATCC 12472, where compounds 3a, 10, 11a, and 13a, b exhibited promising activity. The in vitro cytotoxic activity of these compounds was also studied by brine shrimp lethality bioassay, and results indicated that compounds 3a, 11a, and 13a have the highest cytotoxic activity.

Full text of DOI:10.1007/s00044-013-0637-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-013-0637-x
ORIGINAL RESEARCH
Antimicrobial and antiquorum-sensing studies. Part 2: synthesis,
antimicrobial, antiquorum-sensing and cytotoxic activities of new
series of fused [1,3,4]thiadiazole and [1,3]benzothiazole derivatives
•
N. S. El-Gohary M. I. Shaaban
Received: 11 March 2013 / Accepted: 20 May 2013
ꢀ Springer Science+Business Media New York 2013
Abstract New series of [1,3,4]thiadiazolo[3,2-a]pyrimi-
dines, [1,3,4]thiadiazolo[2,3-b]quinazolines, and pyrimi-
do[2,1-b][1,3]benzothiazoles have been synthesized and
characterized by analytical and spectrometrical methods (IR,
MS, 1H, and 13C NMR). Sixteen of the synthesized compounds;
namely, 3a,b, 5a–f, 8a, b, 10,11a–c, and13a, b were screened
for antibacterial activity against Escherichia coli, Staphylo-
coccus aureus, and Bacillus cereus. They were found to be
either moderately active, slightly active or inactive against the
selected microorganisms. The antifungal activity of these
compounds were also tested against Candida albicans, Asper-
gillus fumigatus 293, and Aspergillus flavus 3375. Compound
11a showed potent antifungal activity against the three selected
fungi; the rest of the tested compounds displayed either weaker
activity or were completely inactive. The same compounds
were examined for antiquorum-sensing activity against Chro-
mobacterium violaceum ATCC 12472, where compounds 3a,
10, 11a, and 13a, b exhibited promising activity. The in vitro
cytotoxic activity of these compounds was also studied by brine
shrimp lethality bioassay, and results indicated that compounds
3a, 11a, and 13a have the highest cytotoxic activity.
Introduction
The presence of multi-drug resistant pathogenic bacteria is
increasing worldwide. This resistance resulted from growth
of bacteria in specialized surface-attached communities,
called biofilms (Costerton et al., 1999). Bacteria in biofilms
were shown to be resistant to antibiotics (Olson et al., 2002).
Consequently, the development of novel chemotherapeutics
that inhibit bacterial pathogenesis is urgent (Hentzer and
Givskov, 2003). A promising approach is to target cell-to-
cell communications, commonly known as quorum-sensing
(QS). Quorum-sensing produce signal molecules known as
quorum-sensing autoinducers (QSAs) (Hastings and
Greenberg, 1999), such as N-acyl-homoserine lactones (acyl
HSLs) that allow intraspecies and interspecies communica-
tions. Once the threshold level of QSAs is reached, the au-
toinducers will bind to cognate receptors (Williams, 2007) to
form a complex, which will in turn regulate the expression of
target genes, particulary those responsible for virulence
(Chan et al., 2010). Thus, antiquorum-sensing treatment may
be a plausible way to attenuate bacterial virulence without
killing the pathogens, and this may reduce bacterial drug
resistance (Adonizio et al., 2006).
Keywords Thiadiazolopyrimidines ꢀ
Thiadiazoloquinazolines ꢀ Pyrimidobenzothiazoles ꢀ
Antimicrobial activity ꢀ Antiquorum-sensing activity ꢀ
Cytotoxic activity
Literature survey revealed that [1,3,4]thiadiazolo[3,2-
a]pyrimidine nucleus is associated with diverse pharma-
codynamic and chemotherapeutic activities (Coburn and
Glennon, 1973; Coburn et al., 1974; El-Ashmaway et al.,
2010; El-Gohary and Shaaban, 2013; El-Sayed et al., 2011;
Herrling, 1978; Maekawa and Mizumitsu, 1977; Mahran
et al., 1998; Suiko and Maekawa, 1977; Taher et al., 2012),
including antimicrobial (Coburn and Glennon, 1973; Co-
burn et al., 1974; El-Gohary and Shaaban, 2013; Mahran
et al., 1998). In addition, [1,3,4]thiadiazolo[2,3-b]quina-
zolines are known to possess interesting pharmacological
properties, including antitumor (El-Ashmaway et al., 2010)
N. S. El-Gohary (&)
Department of Medicinal Chemistry, Faculty of Pharmacy,
Mansoura University, Mansoura 35516, Egypt
e-mail: dr.nadiaelgohary@yahoo.com
M. I. Shaaban
Department of Microbiology, Faculty of Pharmacy,
Mansoura University, Mansoura 35516, Egypt
123

Med Chem Res
and CNS depressant activities (Alagarsamy et al., 2006).
Moreover, pyrimido[2,1-b][1,3]benzothiazoles have also
been extensively investigated for their pharmacological
uses (Chaitanya et al., 2010; Deshmukh et al., 2011;
El-Sherbeny, 2000; Gupta et al., 2009; Sahu et al., 2012;
Shendarkar et al., 2011; Vartale et al., 2011), some of these
compounds showed antimicrobial activity (Sahu et al.,
2012; Shendarkar et al., 2011; Vartale et al., 2011). Con-
sidering these published data and in continuation to our
previous study (El-Ashmaway et al., 2010; El-Sayed et al.,
2011, El-Gohary and Shaaban, 2013) and in searching for
new compounds of potent antimicrobial activity, we are
reporting herein, the synthesis of new series of thiadiazo-
lopyimidine, thiadiazoloquinazoline and pyrimidobenzo-
thiazole derivatives and evaluation of their antibacterial,
antifungal, antiquorum-sensing, and cytotoxic activities.
sulfonamide (1) and 2-amino-6-methyl-[1,3]benzothiazole (9)
were used as key starting materials in this study for syntheses of
new fused heterocycles. Amine 1 was synthesized via acid
hydrolysis of 2-acetamido-[1,3,4]thiadiazole-5-sulfonamide
(Petrow et al., 1958). Cyclocondensation of 1 with cyanoace-
tanilide derivatives 2a, b (Deka et al., 2012) in refluxing glacial
acetic acid afforded 7-imino-5-(substituted phenyl)amino-7H-
[1,3,4]thiadiazolo[3,2-a]pyrimidine-2-sulfonamide derivatives
3a, b (Scheme 1). On the other hand, refluxing 1 with benzy-
lidenecyanoacetanilide derivatives 4a–f (Meskini et al., 2010)
in absolute ethanol and in the presence of catalytic amount of
piperidine gave 6-cyano-7-(substituted phenyl)-5-(substituted
phenyl)amino-7H-[1,3,4]thiadiazolo[3,2-a]pyrimidine-2-
sulfonamide derivatives 5a–f (Scheme 1). The 1H NMR
spectrum of compound 3b showed four significant singlets at d
5.20, 8.28, 10.03, and 11.65 ppm for (C6–H), (NH2), and
(2NH), respectively. The IR spectrum of compound 5b
showed absorption bands at 3,309, 3,193 cm-1 for (NH2 and
NH), in additionto the absorption band characteristic for cyano
group that appeared at 2,200 cm-1. 1H NMR spectrum of the
same compound displayed three singlets at d 2.08, 3.00, and
5.05 ppm assignableto (CH3), (NH), and(C7–H), respectively.
Reaction of dimedone 6 with the triethyl orthoformate or
triethyl orthoacetate in refluxing absolute ethanol, and in the
presence of catalytic amount of piperidine/glacial acetic acid
Results and discussion
Chemistry
A general approach to synthesize the designed compounds is
outlined in Schemes 1, 2, and 3. 2-Amino-[1,3,4]thiadiazole-5-
Scheme 1 Synthesis of
compounds 3a, b and 5a–f
R
H
O
N
HN
N
N
NC
R
N
S
2a,b
N
H2NO2S
NH2
S
1
H2NO2S
Glacial acetic acid
N
NH
H
N
3a,b
O
R= a) CH3; b) Br
CN
R
R1
4a-f
Piperidine/absolute ethanol
R
HN
CN
N
N
H2NO2S
S
N
R1
5a-f
R= CH3 for 5a-c; Br for 5d-f
R1= H for 5a,d; 4-Br for 5b,e; 3,4-(OCH3)2 for 5c,f
123

Med Chem Res
Scheme 2 Synthesis
of compounds 8a, b
O
6
RC(OC2H5)3
+
O
R
O
O
R
N
O
N
N
N
S
C2H5O
N
Glacial acetic acid
H2NO2S
H2NO2S
NH2
S
+
1
7a,b
8a,b
R = a) H; b) CH3
Scheme 3 Synthesis
of compounds 10, 11a–d, and
13a, b
COOCH3
C
C
COOCH3
COOCH3
H3C
N
N
H3C
NH2
CN
S
Methanol
S
O
N
9
10
R
Piperidine/
absolute ethanol
RNHNH2
CN
12a,b
Absolute ethanol
R
CONHNHR
H3C
CN
NH2
N
N
S
O
N
H3C
N
S
11a-d
13a,b
R= a) 3,4-(OCH3)2; b) 4-Cl
R= a) H; b) C6H5; c) 4-Cl-C6H4; d) 2,4-(NO2)2-C6H3
(3:2) afforded 2-(ethoxymethylene)-5,5-dimethylcyclohex-
ane-1,3-dione (7a) and 2-(1-ethoxyethylidene)-5,5-dimeth-
ylcyclohexane-1,3-dione (7b), respectively. 1H NMR
spectrum of 7b displayed the triplet–quartet pattern charac-
teristic for the ethyl protons at the expected regions, in
addition to three singlets at d 1.03, 2.23, and 2.56 ppm for
(2CH3), (C=C–CH3), and (2CH2), respectively. Cyclocon-
densation of aminothiadiazole 1 with 7a, b in refluxing
glacial acetic acid afforded the thiadiazolo[2,3-b]quinazo-
lines 8a, b in acceptable yields (Scheme 2). Mass spectrum
of 8a exhibited a molecular ion peak at m/z 312 which is in
8-methyl-2-oxopyrimido[2,1-b][1,3]benzothiazole-4-car-
boxylate (10) in 65 % yield (Scheme 3). The IR spectrum of
10 demonstrated the characteristic absorption bands for the
two carbonyl groups at 1,745 and 1,664 cm-1. In addition,
1H NMR spectrum showed three characteristic singlets at d
2.23, 3.96, and 7.88 ppm for (CH3), (OCH3), and (C3-H),
respectively.
New series of pyrimido[2,1-b][1,3]benzothiazole-4-car-
bohydrazide derivatives 11a–d were prepared through
reaction of compound 10 with hydrazine hydrate and
phenylhydrazine derivatives in refluxing absolute ethanol
(Scheme 3). The IR spectrum of 11b revealed the disap-
pearance of the carbonyl ester absorption band with con-
comitant appearance of the characteristic absorption bands
for (2NH) groups at 3,363 and 3,321 cm-1. The mass
spectrum of the same compound showed a molecular ion
peak at m/z 350, which is in agreement with its molecular
1
agreement with its molecular formula C11H12N4O3S2. H
NMR spectrum of the same compound showed three sig-
nificant singlets at d 6.73, 7.16, and 8.56 ppm for (C9-H),
(C5-H), and (NH2), respectively.
Treating of 2-aminobenzothiazole 9 with dimethyl acet-
ylenedicarboxylate in refluxing methanol yielded methyl
123

Med Chem Res
weight along with a base peak at m/z 210. 1H NMR
spectrum of 11d displayed two characteristic singlets d
9.98 and 11.34 ppm for (2NH).
1975). The antimicrobial screening results were determined
by measuring the average diameter of the inhibition zones,
expressedinmillimeters(mm). Basedontheresults(Table 1),
it could be inferred that compounds 5a, d, 10, 11a, and 13a
have moderate activity against S. aureus. The rest of the tested
compounds showed either weaker activity or were completely
inactive against the same microorganism. Compounds 10,
11a, b, and13a displayedinterestingactivityagainstB.cereus
compared to the reference drug, ampicillin.
Cyclocondensation of 9 with the appropriate benzylid-
enemalononitrile 12a, b (Bigi et al., 2000) in absolute
ethanol, and in the presence of catalytic amount of piper-
idine afforded the corresponding pyrimido[2,1-b][1,3]ben-
zothiazole-3-carbonitrile derivatives 13a, b (Scheme 3).
The IR spectrum of 13a revealed the presence of two char-
acteristic absorption bands at 3,425 and 2,191 cm-1 for
(NH2) and (C:N) groups, respectively. 1H NMR spectrum
of the same compound showed two singlets at d 3.00 and
5.18 ppm assignable to (NH2) and (C4–H), respectively.
The mass spectrum of 13b showed a molecular ion peak at
m/z 353, which is in agreement with its molecular formula
C18H13ClN4S.
Regarding the antifungal activity, compound 11a dem-
onstrated promising antifungal activity against the three
selected fungi and it is proved to be the most active fun-
gicide in this study. The rest of the tested compounds were
found to be less active or completely inactive against the
selected fungi.
The same compounds were tested for antiquorum-sens-
ing activity against Chromobacterium violaceum ATCC
12472 (McClean et al., 1997). The QS system of C. vio-
laceum 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
(McClean et al., 2004; Cha et al., 2011). Thus, drugs that
inhibit acyl HSL-mediated QS activity in C. violaceum will
prevent production of this purple pigment. Screening
results for their ability to inhibit QS regulated violacein
Antimicrobial and antiquorum-sensing screening
Sixteen of the synthesized compounds; namely, 3a, b, 5a–f,
8a, b, 10, 11a–c, and 13a, b were screened for in vitro anti-
bacterial activity against Staphylococcus aureus, Bacillus
cereus, and Escherichia coli (Pearson et al., 1980). Also, they
were tested for antifungal activity against Candida albicans,
Aspergillus fumigatus 293, and Aspergillus flavus 3372 (Holt,
Table 1 Antimicrobial and antiquorum-sensing activities of compounds 3–13
Comp. no.
Inhibition zone diameter (mm)
E. coli
B. cereus
S. aureus
C. albicans
A. fumigatus
A. flavus
10
Ch. violaceum
3a
6
–
–
–
–
–
–
–
–
–
–
–
4
–
–
–
23
2
–
2
2
2
2
2
2
–
–
5
5
5
–
5
–
3.5
2
6
5
12
–
3b
–
4
–
–
–
–
–
–
–
–
–
7
18
–
–
9
–
3
–
–
–
–
–
–
–
–
–
18
–
–
9
–
5a
11
4
4
–
5b
4
–
5c
5
5
–
5d
9
3
–
5e
2
–
–
5f
7
–
–
8a
–
3
–
8b
–
4
–
10
9
6
9
11a
9
32
–
10
–
11b
4
11c
–
4
–
13a
11
4
15
–
12
6
13b
Ampicillin
Fluconazole
14
22
–
–
Sample concentration: 5 mg/mL, sample volume: 0.1 mL/well. Results are calculated after subtraction of DMSO activity. 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 Aspergillus
fumigatus, A. flavus Aspergillus flavus, Ch. violaceum Chromobacterium violaceum
123

Med Chem Res
production against C. violaceum (based on measuring the
radius of pigment inhibition in mm) are presented in
Table 1, and showed that compounds 3a, 10, 11a, and 13a,
b have antiquorum-sensing activity.
shrimp larvae (Artemia salina) responds similarly to the
corresponding mammalian systems (Solis et al., 1993)
since the DNA-dependent RNA polymerases of A. salina
have been shown to be similar to the mammalian type
(Birndorf et al., 1975). This test is not only used for pre-
dicting cytotoxicity, but also it is used to predict antitumor,
antibacterial and pesticidal activities (Sanchez et al., 1993).
Thus, it is possible to evaluate the cytotoxicity of com-
pounds using brine shrimp lethality bioassay, rather than
the more tedious in vitro and in vivo antitumor assays.
Sixteen of the synthesized compounds; namely, 3a, b,
5a–f, 8a, b, 10, 11a–c, and 13a, b were screened for
cytotoxic activity against brine shrimp larvae (nauplii)
adopting the microplate assay method (Mayer et al., 1982)
and using 5-flurouracil as a reference drug.
Structure–activity relationship (SAR) studies
•
For compounds 3a, b, the presence of 5-phenylamino
moiety substituted with electron-donating group
enhanced the antibacterial, antifungal and antiquorum-
sensing activities (compound 3a).
•
In the series of thiadiazolopyrimidines 5a–f, the
presence of 5-phenylamino moiety substituted with
electron-donating group (compounds 5a–c) improved
the antifungal activity against C. albicans, while those
substituted with electron-withdrawing group exhibited
either weaker activity (compound 5d) or were com-
pletely inactive (compounds 5e, f) against the same
microorganism. Regarding their antibacterial activity,
the presence of unsubstituted phenyl moiety at 7-posi-
tion increased the activity against S. aureus (com-
pounds 5a, d).
Cytotoxic activity of the tested compounds was deter-
mined by measuring the median lethal concentration; LC50
(concentration that kills 50 % of brine shrimp nauplii)
expressed in lg/mL. LC90 (concentration that kills 90 % of
brine shrimp nauplii) values were also determined to
establish the therapeutic index. The results were shown in
Table 2 and indicated that compounds 11a, 13a, and 3a
have good cytotoxic activity against brine shrimp nauplii in
comparison with 5-flurouracil with LC50 values of 404.85,
420.16, and 423.72 lg/mL, respectively, which represent
high cytotoxicity and hence may predict antimicrobial
potential of those compounds. The rest of the tested com-
pounds showed weaker activity, whereas compounds 3b,
5e, 5d, and 5f, are the least cytotoxic analogs in this study
with LC50 values of 714.28, 724.63, 781.25, and
806.45 lg/mL, respectively.
•
•
Concerning the thiadiazoloquinazolines 8a, b, the pres-
ence of methyl substituent at 5-position increased the
antifungal activity against C. albicans (compound 8b).
Taking into account the structure of pyrimido[2,1-
b][1,3]benzothiazole-4-carbohydrazides 11a–d, it is
assumed that the presence of free hydrazinocarbonyl
moiety at 4-position enhanced the activity against S.
aureus. In addition, the antifungal and antiquorum-
sensing activities were also increased (compound 11a).
However, the presence of unsubstituted phenylhydraz-
inocarbonyl moiety at 4-position resulted in improve-
ment of activity against E. coli, but decreased activity
against S. aureus. On the other hand, antifungal and
antiquorum-sensing activities were completely dimin-
ished (compound 11b). Furthermore, the presence of
electron-withdrawing substituent on the phenyl of
the 4-phenylhydrazinocarbonyl moiety abolished the
activity against all the tested microorganisms except
C. albicans (compound 11c).
Structure–activity relationship (SAR) studies
•
•
Concerning compounds 3a, b, the presence of 5-phe-
nylamino moiety substituted with electron-donating
group enhanced the cytotoxic activity (compound 3a).
Regarding series 5a–f, the presence of 5-phenylamino
moiety substituted with electron-donating group (com-
pounds 5a–c) improved cytotoxic activity compared
with those substituted with electron-withdrawing group
(compounds 5d–f).
•
•
Regarding compounds 13a, b, the presence of 4-phenyl
moiety substituted with electron-donating group
improved the antibacterial, antifungal and antiquorum-
sensing activities (compound 13a).
•
•
For thiadiazoloquinazolines 8a, b, the absence of
substituent at 5-position increased the cytotoxic activity
(compound 8a).
Taking into account the structure of pyrimido[2,1-
b][1,3]benzothiazole-4-carbohydrazides 11a–d, the
presence of free hydrazinocarbonyl moiety at 4-posi-
tion enhanced the cytotoxic activity (compound 11a).
For compounds 13a, b, it is clear that the presence of
4-phenyl moiety substituted with electron-donating
group improved the cytotoxic activity (compound 13a).
Cytotoxicity testing using brine shrimp lethality bioassay.
Cytotoxicity via brine shrimp lethality test was studied in
order to reveal new antimicrobial compounds (Harborne,
1998). Toxicity to brine shrimps has a good correlation
with antitumor, pesticidal (Mclaughlin, 1991) and antitry-
panosomal activities (Zani et al., 1995) in man. The brine
•
123

Med Chem Res
Table 2 Cytotoxic activity of compounds 3–13 using brine shrimp lethality bioassay
Comp. no.
Conc. (lg/mL)
LogC
Number of dead nauplii
% Mortality
LC50 (lg/mL)
LC90 (lg/mL)
3a
1,000
500
100
10
3
25
21
16
10
16
11
8
100.0
84.0
64.0
40.0
64.0
44.0
32.0
16.0
92.0
76.0
64.0
36.0
88.0
76.0
64.0
40.0
64.0
48.0
28.0
16.0
56.0
44.0
36.0
20.0
64.0
40.0
28.0
12.0
56.0
40.0
28.0
16.0
68.0
48.0
36.0
24.0
64.0
48.0
28.0
16.0
92.0
76.0
64.0
36.0
100.0
96.0
80.0
60.0
423.72
762.71
2.698
2
1
3b
5a
5b
5c
5d
5e
5f
1,000
500
100
10
3
714.28
462.96
476.19
694.44
781.25
724.63
806.45
657.89
694.44
462.96
404.85
1,285.71
833.33
2.698
2
1
4
1,000
500
100
10
3
23
19
16
9
2.698
2
1
1,000
500
100
10
3
22
19
16
10
16
12
7
857.14
2.698
2
1
1,000
500
100
10
3
1,250.00
1,406.25
1,304.34
1,451.61
1,184.21
1,250.00
833.33
2.698
2
1
4
1,000
500
100
10
3
14
11
9
2.698
2
1
5
1,000
500
100
10
3
16
10
7
2.698
2
1
3
1,000
500
100
10
3
14
10
7
2.698
2
1
4
8a
8b
10
11a
1,000
500
100
10
3
17
12
9
2.698
2
1
6
1,000
500
100
10
3
16
12
7
2.698
2
1
4
1,000
500
100
10
3
23
19
16
9
2.698
2
1
1,000
500
100
10
3
25
24
20
15
728.74
2.698
2
1
123

Med Chem Res
Table 2 continued
Comp. no.
Conc. (lg/mL)
LogC
Number of dead nauplii
% Mortality
LC50 (lg/mL)
LC90 (lg/mL)
11b
1,000
500
100
10
3
19
15
11
6
76.0
60.0
44.0
24.0
72.0
52.0
36.0
20.0
100.0
88.0
64.0
44.0
76.0
56.0
40.0
20.0
100.0
92.0
80.0
56.0
0.0
574.71
1,034.48
2.698
2
1
11c
1,000
500
100
10
3
18
13
9
625.00
420.16
588.23
409.83
–
1,125.00
756.30
1,058.82
737.70
–
2.698
2
1
5
13a
1,000
500
100
10
3
25
22
16
11
19
14
10
5
2.698
2
1
13b
1,000
500
100
10
3
2.698
2
1
5-Fu
1,000
500
100
10
3
25
23
20
14
0
2.698
2
1
–
Control
–
LC50 compound concentration required to kill 50 % of brine shrimp nauplii, LC90 compound concentration required to kill 90 % of brine shrimp
nauplii
Conclusion
spectrometer at Faculty of Science, Cairo University. The
chemical shifts are expressed in d ppm using tetramethyl-
silane (TMS) as internal reference and DMSO-d6 or CDCl3
as solvents. Mass spectra were recorded on JEOL JMS-
600H spectrometer using electron impact technique at
70 eV at Microanalytical Unit, Cairo University. Microa-
nalyses (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 9 was obtained
from Aldrich Chemical Co. (Milwaukee, WI). Compounds
2a, b were prepared adopting the method of (Deka et al.,
2012). Compounds 4a–f were prepared according to the
reported procedure (Meskini et al., 2010), while com-
pounds 12a, b were prepared following the literature
method (Bigi et al., 2000).
Concerning the antibacterial activity, all tested compounds
exhibited lower activity than ampicillin against E. coli. On
the other hand, compounds 10, 11a, b, and 13a were found to
be more active than ampicillin against B. cereus. Regarding
the antifungal activity, compound 11a was the most active
analog compared to fluconazole against the three selected
fungi. The rest of the tested compounds displayed either
weaker activity or no activity toward the selected microor-
ganisms. Compounds 3a, 10, 11a, and 13a, b demonstrated
antiquorum-sensing activity. In addition, compounds 3a,
11a, and 13a are the most cytotoxic analogs.
Experimental
Chemistry
All melting points (ꢁC) were recorded on Fisher-Johns
melting point apparatus and are uncorrected. The infrared
spectra were recorded in KBr disk using a Unicam SP 1000
IR spectrometer (v in cm-1) at Faculty of Science,
Mansoura University. Nuclear magnetic resonance (1H and
13C NMR) spectra were obtained on 300 MHz FT-NMR
General procedure for the preparation of 7-imino-5-
(substituted phenyl)amino-7H-[1,3,4]thiadiazolo[3,2-
a]pyrimidine-2-sulfonamides (3a, b)
A mixture of amine 1 (0.45 g, 0.0025 mol) and the
appropriate cyanoacetanilide 2a, b (0.0025 mol) in glacial
123

Med Chem Res
7-(4-Bromophenyl)-6-cyano-5-(4-methylphenyl)amino-7H-
[1,3,4]thiadiazolo[3,2-a]pyrimidine-2-sulfonamide
acetic acid (10 mL) was heated under reflux for 12 h. The
reaction mixture was cooled and the precipitated solid was
collected by filtration, dried, and crystallized from ethanol/
water (2:1).
(5b)
Yield 65 %, m.p. 162–163 ꢁC. IR spectrum (KBr, v,
1
cm-1): 3308, 3193 (NH2, NH), 2200 (C:N). H NMR
spectrum: (DMSO-d6, d ppm): 2.08 (s, 3H, CH3), 3.00 (s,
1H, NH), 5.05 (s, 1H, C7–H), 6.99–7.79 (m, 10H, Ar–H,
NH2). MS m/z (%): 505 (0.63, M??2), 504 (0.4, M??1),
503 (0.09, M?), 107 (100.00). Anal. Calcd. for
C19H15BrN6O2S2 (503.40): C 45.33, H 3.00, N 16.69.
Found: C 45.07, H 2.85, N 16.43.
7-Imino-5-(4-methylphenyl)amino-7H-[1,3,4]thiadiazolo
[3,2-a]pyrimidine-2-sulfonamide (3a) Yield 65 %, m.p.
1
187–188 ꢁC. H NMR spectrum: (DMSO-d6, d ppm): 2.23
(s, 3H, CH3), 5.31 (s, 1H, C6–H), 7.07 (d, 2H, Ar–H), 7.43
(d, 2H, Ar–H), 9.79 (s, 2H, NH2), 11.74 (s, 1H, NH), 12.14
(s, 1H, NH). 13C NMR spectrum: (DMSO-d6, d ppm): 21.3,
89.4, 118.6 (2C), 127.3, 128.9 (2C), 139.7, 157.2, 159.6,
161.8, 163.9. MS m/z (%): 336 (9.47, M?), 59 (100.00).
Anal. Calcd. for C12H12N6O2S2 (336.39): C 42.85, H 3.60,
N 24.98. Found: C 43.10, H 3.76, N 24.72.
6-Cyano-7-(3,4-dimethoxyphenyl)-5-(4-methylphenyl)amino-
7H-[1,3,4]thiadiazolo[3,2-a]pyrimidine-2-sulfonamide (5c)
Yield 60 %, m.p. 172–173 ꢁC. IR spectrum (KBr, v,
1
cm-1): 3338, 3192 (NH2, NH), 2200 (C:N). H NMR
spectrum: (DMSO-d6, d ppm): 2.09 (s, 3H, CH3), 3.04 (s,
1H, NH), 3.80 (s, 3H, OCH3), 3.85 (s, 3H, OCH3), 5.35 (s,
1H, C7–H), 6.78–7.77 (m, 9H, Ar–H, NH2). 13C NMR
spectrum: (DMSO-d6, d ppm): 21.7, 41.3, 55.6, 55.7, 69.7,
112.5, 113.6, 116.8, 119.1 (2C), 121.7, 127.5, 128.2 (2C),
131.1, 140.1, 145.7, 148.2, 157.6, 162.8, 165.3. MS m/
z (%): 483 (3.61, M?-1), 84 (100.00). Anal. Calcd. for
C21H20N6O4S2 (484.55): C 52.05, H 4.16, N 17.34. Found:
C 52.31, H 3.95, N 17.05.
5-(4-Bromophenyl)amino-7-imino-7H-[1,3,4]thiadiazolo
[3,2-a]pyrimidine-2-sulfonamide (3b) Yield 70 %, m.p.
1
268–270 ꢁC. H NMR spectrum: (DMSO-d6, d ppm): 5.20
(s, 1H, C6–H), 7.45 (d, 2H, Ar–H), 7.52 (d, 2H, Ar–H),
8.28 (s, 2H, NH2), 10.03 (s, 1H, NH), 11.65 (s, 1H, NH).
MS m/z (%): 400 (0.03, M?-1), 171 (100.00). Anal. Calcd.
for C11H9BrN6O2S2 (401.26): C 32.93, H 2.26, N 20.94.
Found: C 32.77, H 2.45, N 21.23.
5-(4-Bromophenyl)amino-6-cyano-7-phenyl-7H-[1,3,4]thi-
adiazolo[3,2-a]pyrimidine-2-sulfonamide
General procedure for the preparation of 6-cyano-7-
(substituted phenyl)-5-(substituted phenyl)amino-7H-
[1,3,4]thiadiazolo[3,2-a]pyrimidine-2-sulfonamides (5a–f)
(5d) Yield
65 %, m.p. 176–177 ꢁC. IR spectrum (KBr, v, cm-1):
1
3334, 3189 (NH2, NH), 2201 (C:N). H NMR spectrum:
(DMSO-d6, d ppm): 2.98 (s, 1H, NH), 5.34 (s, 1H, C7–H),
7.11–7.71 (m, 11H, Ar–H, NH2). Anal. Calcd for
C18H13BrN6O2S2 (489.37): C 44.18, H 2.68, N 17.17.
Found: C 44.39, H 2.48, N 16.93.
A mixture of amine 1 (0.45 g, 0.0025 mol), the appropriate
benzylidenecyanoacetanilide 4a–f (0.0025 mol) and a cat-
alytic amount of piperidine (0.3 mL) in absolute ethanol
(15 mL) was heated under reflux for 12–16 h. The solvent
was concentrated, then poured onto cold water (50 mL).
The precipitated solid was collected by filtration, dried, and
crystallized from ethanol/water (3:1).
7-(4-Bromophenyl)-5-(4-bromophenyl)amino-6-cyano-7H-
[1,3,4]thiadiazolo[3,2-a]pyrimidine-2-sulfonamide
(5e)
Yield 65 %, m.p. 166–167 ꢁC. IR spectrum (KBr, v,
1
cm-1): 3420 (broad band, NH2, NH), 2201 (C:N). H
6-Cyano-5-(4-methylphenyl)amino-7-phenyl-7H-[1,3,4]thi
adiazolo[3,2-a]pyrimidine-2-sulfonamide
NMR spectrum: (DMSO-d6, d ppm): 2.91 (s, 1H, NH), 5.32
(s, 1H, C7-H), 7.14–7.84 (m, 10H, Ar–H, NH2). MS m/z (%):
570 (0.05, M??2), 569 (0.11, M??1), 568 (0.29, M?),
64 (100.00). Anal. Calcd. for C18H12Br2N6O2S2 (568.26):
C 38.04, H 2.13, N 14.79. Found: C 37.87, H 2.35,
N 14.63.
(5a) Yield
60 %, m.p. 164–165 ꢁC. IR spectrum (KBr, v, cm-1):
1
3429, 3303 (NH2, NH), 2209 (C:N). H NMR spectrum:
(DMSO-d6, d ppm): 2.17 (s, 3H, CH3), 2.99 (s, 1H, NH),
5.30 (s, 1H, C7–H), 6.94–7.69 (m, 11H, Ar–H, NH2). 13C
NMR spectrum: (DMSO-d6, d ppm): 21.5, 40.9, 69.5,
117.1, 119.0 (2C), 125.8, 126.2, 127.6 (2C), 128.9 (2C),
129.5 (2C), 140.5, 141.9, 158.3, 161.9, 166.3. MS m/z (%):
424 (0.03, M?), 84 (100.00). Anal. Calcd. for
C19H16N6O2S2 (424.50): C 53.76, H 3.80, N 19.80. Found:
C 53.47, H 3.62, N 20.05.
5-(4-Bromophenyl)amino-6-cyano-7-(3,4-dimethoxyphenyl)-
7H-[1,3,4]thiadiazolo[3,2-a]pyrimidine-2-sulfonamide (5f)
Yield 72 %, m.p. 146–147 ꢁC. 1H NMR spectrum:
(DMSO-d6, d ppm): 3.01 (s, 1H, NH), 3.83 (s, 3H, OCH3),
123

Med Chem Res
3.85 (s, 3H, OCH3), 5.19 (s, 1H, C7–H), 6.49–7.78 (m, 9H,
Ar–H, NH2). 13C NMR spectrum: (DMSO-d6, d ppm):
41.6, 55.7, 55.9, 70.6, 114.6 (2C), 115.1, 116.4, 118.9,
120.7 (2C), 131.6 (2C), 133.2, 140.8, 146.2, 148.6, 157.3,
161.8, 165.5. MS m/z (%): 551 (0.01, M??2), 549 (0.01,
M?), 112 (100.00). Anal. Calcd. for C20H17BrN6O4S2
(549.42): C 43.72, H 3.12, N 15.30. Found: C 43.95, H
3.41, N 15.12.
C9–H), 7.16 (s, 1H, C5–H), 8.56 (s, 2H, NH2). MS m/z (%):
313 (0.08, M??1), 312 (0.07, M?), 64 (100.00). Anal.
Calcd. for C11H12N4O3S2 (312.37): C 42.30, H 3.87, N
17.94. Found: C 42.12, H 3.98, N 18.27.
7,8-Dihydro-5,8,8-trimethyl-6-oxo-[1,3,4]thiadiazolo[2,3-b]
quinazoline-2-sulfonamide (8b) Yield 72 %, m.p.
268–269 ꢁC. IR spectrum (KBr, v, cm-1): 3302, 3182
(NH2), 1679 (C=O). 1H NMR spectrum: (DMSO-d6,
d ppm): 1.75 (s, 6H, C8–2CH3), 2.05 (s, 3H, C5–CH3), 2.48
(s, 2H, CH2), 6.50 (s, 1H, C9–H), 7.89 (s, 2H, NH2). 13C
NMR spectrum: (DMSO-d6, d ppm): 14.6, 28.9 (2C), 31.8,
51.5, 112.8, 131.7, 133.4, 144.6, 157.4, 161.3, 169.9. MS
m/z (%): 328 (0.01, M??2), 327 (0.02, M??1), 326 (0.02,
M?), 180 (100.00). Anal. Calcd. for C12H14N4O3S2
(326.39): C 44.16, H 4.32, N 17.17. Found: C 44.37, H
4.57, N 16.99.
Preparation of 2-substituted 5,5-dimethylcyclohexane-1,3-
diones (7a, b)
Piperidine (0.9 mL) and glacial acetic acid (0.6 mL) were
added to a mixture of dimedone 6 (1.4 g, 0.01 mol), and
triethyl orthoformate or triethyl orthoacetate (0.015 mol) in
absolute ethanol (20 mL). The mixture was heated under
reflux for 48 h, then evaporated under reduced pressure.
The residue obtained (7a) was triturated with crushed ice,
filtered, dried, and crystallized from ethanol/water (2:1).
For compound 7b, evaporation of reaction mixture leaves
yellow oily product.
Preparation of methyl 8-methyl-2-oxopyrimido
[2,1-b][1,3]benzothiazole-4-carboxylate (10)
Dimethyl acetylenedicarboxylate (DMAD) (1.42 g, 0.01 mol)
was added dropwise to a stirred solution of 2-amino-6-methyl-
[1,3]benzothiazole (9) (1.64 g, 0.01 mol) in methanol
(20 mL). The reaction mixture was heated under reflux for
12 h. On cooling, the precipitated solid was collected by fil-
tration, dried, and crystallized from ethanol/water (2:1).
Yield 65 %, m.p. 193–194 ꢁC. IR spectrum (KBr, v,
cm-1): 1745 (COOCH3), 1664 (C=O). 1H NMR spectrum:
(DMSO-d6, d ppm): 2.23 (s, 3H, CH3), 3.96 (s, 3H, OCH3),
6.65–7.43 (m, 3H, Ar–H), 7.88 (s, 1H, C3–H). 13C NMR
spectrum: (DMSO-d6, d ppm): 23.9, 52.6, 103.3, 121.6,
124.1, 125.3, 127.8, 128.7, 138.5, 141.1, 157.5, 166.8,
169.7. MS m/z (%): 275 (12.03, M??1), 274 (72.00, M?),
216 (100.00). Anal. Calcd. for C13H10N2O3S (274.30): C
56.92, H 3.67, N 10.21. Found: C 56.74, H 3.49, N 9.98.
2-(Ethoxymethylene)-5,5-dimethylcyclohexane-1,3-dione (7a)
Yield 60 %, m.p. 140–142 ꢁC. 1H NMR spectrum: (DMSO-
d6, d ppm): 1.03–2.43 (m, 13H, 2CH3, CH2CH3, 2CH2),
4.06–4.12 (q, 2H, CH2CH3), 5.06 (s, 1H, CH=C). MS m/z (%):
198 (2.55, M??2), 197 (1.65, M??1), 196 (1.97, M?), 55
(100.00). Anal. Calcd. for C11H16O3 (196.24): C 67.32, H
8.22. Found: C 67.54, H 8.41.
2-(1-Ethoxyethylidene)-5,5-dimethylcyclohexane-1,3-dione
(7b) Yield 75 %, yellow oil. 1H NMR spectrum: (CDCl3,
d ppm): 1.03 (s, 6H, 2CH3), 1.32 (t, 3H, CH2CH3), 2.23
(s, 3H, CH3), 2.56 (s, 4H, 2CH2), 3.92–3.97 (q, 2H,
CH2CH3). MS m/z (%): 211 (1.92, M??1), 210 (1.61,
M?), 83 (100.00). Anal. Calcd. for C12H18O3 (210.27): C
68.54, H 8.63. Found: C 68.41, H 8.76.
Preparation of 8-methyl-2-oxo-N0-(un)substituted
pyrimido[2,1-b][1,3]benzothiazole-4-carbohydrazides
(11a–d)
Preparation of 7,8-dihydro-8,8-dimethyl-6-oxo-5-
unsubstituted/methyl-[1,3,4]thiadiazolo[2,3-b]
quinazoline2-sulfonamides (8a, b)
A mixture of compound 10 (0.274 g, 0.001 mol) and hydrazine
hydrate 98 % (0.5 g, 0.01 mol, excess) or aromatic hydrazine
(0.001 mol) in absolute ethanol (15 mL) was heated under
reflux for 16–18 h. The reaction mixture was concentrated and
the precipitated solid was collected by filtration, washed with
water, dried, and crystallized from dioxane.
A mixture of amine 1 (0.45 g, 0.0025 mol) and the appropriate
cyclohexane-1,3-dione derivative 7a, b (0.0025 mol) in glacial
acetic acid (10 mL) was heated under reflux for 24 h. The
reaction mixture was evaporated under reduced pressure and the
remaining products were crystallized from ethanol/water (3:1).
7,8-Dihydro-8,8-dimethyl-6-oxo-[1,3,4]thiadiazolo[2,3-b]
quinazoline-2-sulfonamide (8a) Yield 68 %, m.p.
274–275 ꢁC. IR spectrum (KBr, v, cm-1): 3320 (broad
band, NH2), 1674 (C=O). 1H NMR spectrum: (DMSO-d6, d
ppm): 2.05 (s, 6H, 2CH3), 2.62 (s, 2H, CH2), 6.73 (s, 1H,
8-Methyl-2-oxopyrimido[2,1-b][1,3]benzothiazole-4-car-
bohydrazide (11a) Yield 50 %, m.p. decomp. 210 ꢁC. IR
spectrum (KBr, v, cm-1): 3319 (broad band, NH2, NH),
1640 (2C=O). 1H NMR spectrum: (DMSO-d6, d ppm):
123

Med Chem Res
2-Amino-4-(3,4-dimethoxyphenyl)-8-methyl-4H-pyrimi
2.29 (s, 3H, CH3), 4.40 (s, 2H, NH2), 6.64–7.38 (m, 3H,
Ar–H), 7.47 (s, 1H, C3–H), 10.67 (s, 1H, NH). 13C NMR
spectrum: (DMSO-d6, d ppm): 23.9, 105.1, 121.3, 124.7
(2C), 127.5, 128.8, 138.7, 153.2, 157.6, 166.1, 170.4. Anal.
Calcd. for C12H10N4O2S (274.30): C 52.54, H 3.67, N
20.43. Found: C 52.83, H 3.92, N 20.21.
do[2,1-b][1,3]benzothiazole-3-carbonitrile
(13a) Yield
60 %, m.p. 164–165 ꢁC. IR spectrum (KBr, v, cm-1): 3425
(broad band, NH2), 2191 (C:N). 1H NMR spectrum:
(DMSO-d6, d ppm): 2.30 (s, 3H, CH3), 3.00 (s, 2H, NH2),
3.83 (s, 3H, OCH3), 3.87 (s, 3H, OCH3), 5.18 (s, 1H,
C4-H), 6.81-7.65 (m, 6H, Ar–H). 13C NMR spectrum:
(DMSO-d6, d ppm): 23.9, 38.8, 55.6, 55.7, 68.7, 112.6,
114.8 (2C), 116.3, 121.2, 126.5, 127.3 (2C), 127.9, 133.1,
137.9, 145.9, 148.1, 158.3, 163.1. MS m/z (%): 380 (0.14,
M??2), 379 (0.51, M??1), 378 (0.22, M?), 164 (100.00).
Anal. Calcd. for C20H18N4O2S (378.45): C 63.47, H 4.79,
N 14.80. Found: C 63.68, H 4.91, N 14.63.
8-Methyl-2-oxo-N0-phenylpyrimido[2,1-b][1,3]benzothiazole-
4-carbohydrazide (11b) Yield 60 %, m.p. 169–170 ꢁC. IR
spectrum (KBr, v, cm-1): 3363, 3321 (2NH), 1647 (2C=O).
1H NMR spectrum: (DMSO-d6, d ppm): 2.31 (s, 3H, CH3),
6.56-7.93 (m, 9H, Ar–H, C3–H), 9.82 (s, 1H, NH), 10.35
(s, 1H, NH). MS m/z (%): 351 (0.04, M??1), 350 (0.01, M?),
210 (100.00). Anal. Calcd. for C18H14N4O2S (350.39): C
61.70, H 4.03, N 15.99. Found: C 61.93, H 3.86, N 16.27.
2-Amino-4-(4-chlorophenyl)-8-methyl-4H-pyrimido[2,1-b]
[1,3]benzothiazole-3-carbonitrile (13b) Yield 65 %, m.p.
176–177 ꢁC. IR spectrum (KBr, v, cm-1): 3421, 3385
(NH2), 2189 (C:N). 1H NMR spectrum: (DMSO-d6,
d ppm): 2.29 (s, 3H, CH3), 3.00 (s, 2H, NH2), 4.45 (s, 1H,
C4–H), 6.98–7.65 (m, 7H, Ar–H). MS m/z (%): 355 (0.01,
M??2), 354 (0.09, M??1), 353 (0.11, M?), 164 (100.00).
Anal. Calcd. for C18H13ClN4S (352.84): C 61.27, H 3.71, N
15.88. Found: C 61.14, H 3.54, N 16.15.
N0-(4-Chlorophenyl)-8-methyl-2-oxopyrimido[2,1-b][1,3]
benzothiazole-4-carbohydrazide (11c) Yield 65 %, m.p.
156–157 ꢁC. IR spectrum (KBr, v, cm-1): 3354, 3320
1
(2NH), 1654 (2C=O). H NMR spectrum: (DMSO-d6, d
ppm): 2.23 (s, 3H, CH3), 6.63–7.88 (m, 8H, Ar–H, C3–H),
9.98 (s, 1H, NH), 11.34 (s, 1H, NH). MS m/z (%): 387 (0.09,
M??2), 385 (0.22, M?), 58 (100.00). Anal. Calcd. for
C18H13ClN4O2S (384.84): C 56.18, H 3.40, N 14.56. Found:
C 55.96, H 3.67, N 14.72.
Biological testing
Antimicrobial and antiquorum-sensing activities
8-Methyl-N0-(2,4-dinitrophenyl)-2-oxopyrimido[2,1-b][1,3]
benzothiazole-4-carbohydrazide (11d) Yield 50 %, m.p.
172–174 ꢁC. IR spectrum (KBr, v, cm-1): 3367, 3325
(2NH), 1656 (2C=O). 1H NMR spectrum: (DMSO-d6,
d ppm): 2.34 (s, 3H, CH3), 6.93–8.84 (m, 7H, Ar–H,
C3–H), 9.98 (s, 1H, NH), 11.34 (s, 1H, NH). 13C NMR
spectrum: (DMSO-d6, d ppm): 23.7, 106.5, 114.8 (2C),
121.7, 123.1, 124.3, 125.2, 127.6, 128.7, 129.1 (2C), 138.9,
146.9, 151.8, 157.3, 166.7, 169.9. MS m/z (%): 441 (1.36,
M??1), 440 (1.38, M?), 164 (100.00). Anal. Calcd. for
C18H12N6O6S (440.39): C 49.09, H 2.75, N 19.08. Found:
C 49.31, H 2.97, N 19.37.
Sixteen of the synthesized compounds; namely, 3a, b, 5a–f,
8a, b, 10, 11a–c, and 13a, b were screened for antibacterial
activity against E. coli, S. aureus, and B. cereus in (Luria–
Bertani agar media) (Pearson et al., 1980) and antifungal
activity against C. albicans in (Saboured’s agar), A. fu-
migatus 293, and A. flavus 3375 in (glucose minimal
media) (Holt, 1975). In addition, their antipathogenic
potential was checked by examining the antiquorum-sens-
ing activity against C. violaceum ATCC 12472 in (Luria–
Bertani agar media) (McClean et al., 1997).
Escherichia coli, B. cereus, S. aureus, and C. albicans
were obtained from the culture collection of the Depart-
ment of Microbiology, Faculty of Pharmacy, Mansoura
University, Mansoura, Egypt. Filamentous fungi; A. fu-
migatus 293 and A. 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 fila-
mentous fungi were cultivated in glucose minimal media
(Merck, Germany). Ampicillin was obtained from EPICO
company; Fluconazole was obtained from Pfizer company.
Preparation of 2-amino-8-methyl-4-(substituted)phenyl-
4H-pyrimido[2,1-b][1,3]benzothiazole-3-carbonitriles
(13a, b)
A mixture of amine 9 (0.41 g, 0.0025 mol), the appropriate
benzylidenemalononitrile 12a, b (0.0025 mol) and a cata-
lytic amount of piperidine (0.3 mL) in absolute ethanol
(15 mL) was heated under reflux for 12–16 h. The solvent
was concentrated, then poured onto cold water (50 mL).
The precipitated solid was collected by filtration, dried, and
crystallized from ethanol/water (2:1).
123

Med Chem Res
Method
borer. The tested compounds were dissolved in DMSO in
eppendorff tubes for final concentration 5 mg/mL and
100 lL of test solution was applied into the wells. DMSO
(control solvent) was added to each plate. Plates were
incubated at 30 ꢁC for 48 h to check the inhibition of pig-
ment production around the wells. Bacterial growth inhibi-
tion would result in a clear halo around the disk, while a
positive quorum sensing inhibition is exhibited by a turbid
halo harboring pigmentless bacterial cells of C. violaceum
ATCC 12472 monitor strain (McClean et al., 1997). Bacte-
rial growth inhibition by the tested compounds was measured
as radius (r1) in mm, while both growth and pigment inhi-
bition was measured as radius (r2) in mm. The pigment
inhibition (QS inhibition) was determined by subtracting
bacterial growth inhibition (r1) from the total radius (r2);
thus, QS inhibition = (r2 - r1) in mm (Table 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 50 ꢁC were seeded with 50 lL of
1 9 106 CFU/mL of 18 h culture of tested microorgan-
isms. 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 lL 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 h at 4 ꢁC and incubated at 37 ꢁC for
24 h (Pearson et al., 1980). Inhibition zones were measured
using Vernier caliper and the activity of the tested com-
pounds was estimated in comparison to ampicillin (Table 1).
The inhibition zone diameter of DMSO was subtracted from
the antibacterial activity of tested compounds.
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/L)
supplemented with dried yeast (6 mg/L). Brine shrimp
eggs are hatched in artificial sea water during 48 h incu-
bation in a warm room (22–29 ꢁC), providing large num-
bers of larvae (nauplii). Brine shrimp larvae (nauplii) were
collected with a Pasteur pipette after attracting the organ-
isms to one side of the vessel with a light source. Nauplii
were separated from the eggs by pipetting them 2–3 times
in small beakers containing sea water. The test compounds
were made up to 1 mg/mL in artificial sea water (water
insoluble compounds were dissolved in 50 lL DMSO prior
to adding sea water). Serial dilutions (1000, 500, 100,
10 lg/mL) were made in the wells in 96-multiwell
microtiter plate in triplicate in 100 lL sea water. A sus-
pension of nauplii containing 25 brine shrimp larvae in
100 lL sea water was added to each well with the help of a
Pasteur pipette and the covered microwell plate was incu-
bated at 22–29 ꢁC for 24 h. The plate was then examined
under a binocular microscope and the number of dead
(non-mobile) nauplii in each well was counted. Methanol
(100 lL) was then added to each well, and after 15 min the
total number of shrimps in each well was counted (Mayer
et al., 1982). Plotting of log concentration (logC) versus %
mortality for all tested compounds showed an approximate
linear correlation, and the values of LC50 and LC90 were
calculated by using Microsoft Excel XP (Table 2). All
values were compared with the standard cytotoxic agent,
5-flurouracil with LC50 and LC90 values of 409.83 and
737.70 lg/mL, respectively.
Antifungal screening
Saboured’s media (50 mL) was inoculated with 50 lL of
1 9 106 CFU/mL of 24 h culture of C. albicans. For fila-
mentous fungi, glucose minimal media (50 mL) was inoc-
ulated with 50 lL of 1 9 106 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 100 lL of test solution was
applied into the wells. The standard antifungal drug (fluco-
nazole) was also added at the same concentration to each
plate. In addition, DMSO (control solvent) was added to
each plate. Plates were incubated at 37 ꢁC for 48 h for
C. albicans and A. fumigatus and at 30 ꢁC for 48 h for A.
flavus (Holt, 1975). Antifungal activity of the tested com-
pounds was determined by measuring the diameter of the
inhibition zone (Table 1). The inhibition zone diameter of
DMSO was subtracted from the antifungal activity of tested
compounds.
Antiquorum-sensing screening
Cultures were prepared by growing C. violaceum ATCC
12472 in Luria–Bertani (LB) broth and incubated for
16–18 h in an orbital incubator running at 28 ꢁC and
150 rpm. Cultures were then adjusted to 0.5 McFarland
standard (Ca. 1 9 106 CFU/mL). Chromobacterium viola-
ceum was inoculated (100 lL/plate) in 50 mL LB agar and
solidified. Wells were made in LB agar media using cork
Acknowledgments The author would like to express her sincere
thanks to Dr. Mona I. Shaaban, Microbiology Department, Faculty of
123

Med Chem Res
Pharmacy, Mansoura University, Egypt, for carrying out the antimi-
crobial and antiquorum-sensing screening. The author would also like
to thank Mr. Ahmed Abbas, a technician at Pharmacognosy Depart-
ment, Faculty of Pharmacy, Mansoura University, Egypt, for carrying
out the cytotoxicity testing.
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Hastings JW, Greenberg EP (1999) Quorum sensing: the explanation
of a curious phenomenon reveals a common characteristic of
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