Antimicrobial and Antiquorum-Sensing Studies. Part 3: Synthesis and Biological Evaluation of New Series of [1,3,4]Thiadiazoles and Fused [1,3,4]Thiadiazoles

Article abstract of DOI:10.1002/ardp.201400381

New series of [1,3,4]thiadiazoles and fused [1,3,4]thiadiazoles were synthesized. The newly synthesized compounds were screened for their antibacterial activity against Staphylococcus aureus, Bacillus cereus, and Escherichia coli. Compounds 3b and 10a displayed the highest activity against E. coli with MIC value of 78.125μg/mL. In addition, compound 10a exhibited the highest activity against B. cereus with MIC value of 156.25μg/mL. The antifungal activity of these compounds was also tested against Candida albicans and Aspergillus flavus 3375. Compounds 3b, 5a, 10a, and 12b showed the best activity against A. flavus 3375 with MIC value of 19.531μg/mL. The same compounds were examined for their antiquorum-sensing activity against Chromobacterium violaceum ATCC 12472, whereas compounds 3b, 5a, and 12b exhibited moderate activity. In vitro cytotoxicity testing of compounds 3b,c, 5a, 6a, 10a, and 12a,b against human normal lung fibroblast (W138) cell line was performed. The in vivo acute toxicity of the same compounds was also tested and the obtained results indicated that compound 10a is the least toxic analog. The same compounds were studied for their DNA-binding affinity and the obtained results showed that compounds 3b, 10a, and 12a,b have moderate DNA-binding affinity. New series of [1,3,4]thiadiazoles and fused [1,3,4]thiadiazoles were synthesized and screened for their antibacterial activity against Staphylococcus aureus, Bacillus cereus, and Escherichia coli, as well as their antifungal activity against Candida albicans and Aspergillus flavus 3375. The compounds were also examined for their antiquorum-sensing activity against Chromobacterium violaceum ATCC 12472. Compounds 3b and 10a showed interesting activity in all assays.

Full text of DOI:10.1002/ardp.201400381

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Arch. Pharm. Chem. Life Sci. 2015, 348, 283–297
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Full Paper
Antimicrobial and Antiquorum-Sensing Studies. Part 3: Synthesis
and Biological Evaluation of New Series of [1,3,4]Thiadiazoles and
Fused [1,3,4]Thiadiazoles
Nadia S. El-Gohary1 and Mona I. Shaaban2,3
1
Department of Medicinal Chemistry, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt
Department of Microbiology, Faculty of Pharmacy, Mansoura University, Mansoura, Egypt
Department of Microbiology, College of Pharmacy, Taibah University, Almadinah Almunawwarah,
2
3
Saudi Arabia
New series of [1,3,4]thiadiazoles and fused [1,3,4]thiadiazoles were synthesized. The newly synthesized
compounds were screened for their antibacterial activity against Staphylococcus aureus, Bacillus cereus,
and Escherichia coli. Compounds 3b and 10a displayed the highest activity against E. coli with MIC value of
78.125 mg/mL. In addition, compound 10a exhibited the highest activity against B. cereus with MIC value
of 156.25 mg/mL. The antifungal activity of these compounds was also tested against Candida albicans and
Aspergillus flavus 3375. Compounds 3b, 5a, 10a, and 12b showed the best activity against A. flavus 3375
with MIC value of 19.531 mg/mL. The same compounds were examined for their antiquorum-sensing
activity against Chromobacterium violaceum ATCC 12472, whereas compounds 3b, 5a, and 12b exhibited
moderate activity. In vitro cytotoxicity testing of compounds 3b,c, 5a, 6a, 10a, and 12a,b against human
normal lung fibroblast (W138) cell line was performed. The in vivo acute toxicity of the same compounds
was also tested and the obtained results indicated that compound 10a is the least toxic analog. The same
compounds were studied for their DNA-binding affinity and the obtained results showed that compounds
3b, 10a, and 12a,b have moderate DNA-binding affinity.
Keywords: [1,3,4]Thiadiazoles / Fused [1,3,4]thiadiazoles / Antimicrobial activity / Antiquorum-sensing
activity / Cytotoxic activity / In vivo acute toxicity / In silico studies
Received: October 14, 2014; Revised: February 15, 2015; Accepted: February 17, 2015
DOI 10.1002/ardp.201400381
Additional supporting information may be found in the online version of this article at the publisher's web-site.
:
quorum-sensing (QS). QS is a cell communication mechanism
through which signal molecules called autoinducers activate
Introduction
Antibacterial drugs are a group of the most effective
agents in medicine. However, continued emergence and
spread of multidrug resistant bacteria necessitated the
development of alternative treatment strategies, including
antipathogenic drugs [1, 2]. A promising approach is to
target cell-to-cell communications, commonly known as
specific receptors associated with transcription signals for
controlling various biochemical processes such as biofilm
formation [3–6], bioluminescence [7–10], virulence fac-
tors [11], and swarming [12, 13] which has been shown to
contribute to bacterial pathogenesis. In biofilm, bacteria do
not exist as independent entities, but as a coordinated and
metabolically integrated microbial community [14]. This
interaction provides enormous benefits to bacteria, includ-
ing increased diversity, increased metabolic efficiency, and
greater resistance to environment, antimicrobial agents, and
host defenses [14]. In view of the fact that QS is involved in
bacterial pathogenesis, recent research is focused on the
development of antipathogenic agents to control bacterial
diseases by inhibiting QS.
Correspondence: Dr. Nadia S. El-Gohary, Department of Medici-
nal Chemistry, Faculty of Pharmacy, Mansoura University,
Mansoura 35516, Egypt.
E-mail: dr.nadiaelgohary@yahoo.com
Fax: þ2 050 2247496
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carbamate analog 4. Compound 4 was previously prepared
starting from ethyl 5-mercapto-[1,3,4]thiadiazol-2-ylcarbamate
[47]. Reaction of 4 with the appropriate aniline in refluxing
glacial acetic acid produced 5-(3-(substituted phenyl)ureido)-
[1,3,4]thiadiazole-2-sulfonamide analogs 5a–c (Scheme 1).
Referring to Scheme 2, reaction of amine 1 with the
appropriate benzaldehyde in absolute ethanol in the
presence of glacial acetic acid afforded the azomethine
derivatives 6a–c. Reaction of 6a–c with thioglycolic acid in
refluxing dioxane yielded 5-(thiazolidin-3-yl)-[1,3,4]thiadi-
azole derivatives 7a–c.
Regarding Scheme 3, heating amine 1 with ethyl chloro-
acetate in glacial acetic acid gave the imidazo[2,1-b][1,3,4]-
thiadiazole derivative 8 [16]. Condensation of 8 with the
appropriate diazonium chloride 9a,b (obtained in situ by
diazotization of the appropriate aniline using a mixture of
sodium nitrite and hydrochloric acid in the presence of sodium
acetate) produced 5-(4-substituted phenyl)hydrazono-5H-
imidazo[2,1-b][1,3,4]thiadiazole derivatives 10a,b. Reaction
of 8 with the appropriate benzylidenemalononitrile 11a,b
[48] in absolute ethanol in the presence of piperidine as a
catalyst gave 5H-pyrano[30,20:5,6]imidazo[2,1-b][1,3,4]- thia-
diazole analogs 12a,b. Heating 8 with acetic anhydride
produced 5-acetyl-6-hydroxyimidazothiadiazole derivative 13.
In Scheme 4, cyclocondensation of 1 with the appropriate
benzylidenecyanoacetamide 14a–c [49] in absolute ethanol in
the presence of piperidine as a catalyst afforded thiadiazolo-
[3,2-a]pyrimidine derivatives 15a–c in 45–62% yields.
Literature survey revealed that [1,3,4]thiadiazolo[3,2-a]pyrimi-
dine nucleus is associated with diverse pharmacodynamic and
chemotherapeutic activities [15–26]. Moreover, many imidazo-
[2,1-b][1,3,4]thiadiazole derivatives are known to possess
interesting pharmacological properties, including antibacteri-
al [27–31], antifungal [32], and antimicrobial [16, 33–35]
activities. The arylideneamino-[1,3,4]thiadiazoles possess anti-
microbial activity [36–39]. In addition, thiazolidin-3-yl-[1,3,4]-
thiadiazoles were extensively investigated for their pharmaco-
logical uses, some of these compounds showed antimicrobial
activity [40–44]. Considering these published data and in
continuation to our previous work [15, 16] and in searching
for new compounds of potent antimicrobial and antiquorum-
sensing activities, we described herein the synthesis of new
series of [1,3,4]thiadiazoles and fused [1,3,4]thiadiazoles. The
synthesized compounds were evaluated for their antibacterial,
antifungal, antiquorum-sensing,andinvitrocytotoxicactivities
as well as in vivo acute toxicity and DNA-binding affinity.
Results and discussion
Chemistry
The synthesis of the target compounds 3–15 is depicted
in Schemes 1–4. 5-Amino-[1,3,4]thiadiazole-2-sulfonamide (1)
was used as a key starting material in this study. Amine 1 was
synthesized via acid hydrolysis of N-(5-sulfamoyl-[1,3,4]thia-
diazol-2-yl)acetamide [45].
Biological screening
Heating amine 1 with ethyl N-(4-substituted phenyl)formimi-
dates 2a–d [46] in glacial acetic acid afforded 5-[N-((4-substituted
phenyl)aminomethylene)amino]-[1,3,4]thiadiazole-2-sulfon-
amide derivatives 3a–d in 60–70% yields. Moreover, refluxing 1
with ethyl chloroformate in glacial acetic acid yielded the ethyl
Antimicrobial and antiquorum-sensing screening
Compounds 3a–d, 4, 5a–c, 6a–c, 7a–c, 10a,b, 12a,b, and 15a–c
were screened for their in vitro antibacterial activity against
two species of Gram-positive bacteria (Staphylococcus aureus
N
R
R
N
N
N
N
C2H5O
_
2a d
Glacial acetic acid
H2NO2S
H2NO2S
NH2
N
H
3a d
N
S
S
1
_
R = a) Br; b) NO2; c) CH3; d) OCH3
ClCOOC2H5
Glacial acetic acid
N
N
N
N
H2N
Glacial acetic acid
R
COOC2H5
R
CONH
H2NO2S
H2NO2S
N
H
_
N
H
S
S
4
5a c
R = a) NO2; b) CH3; c) OCH3
Scheme 1. Synthesis of the target compounds 3a–d and 5a–c.
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Thiadiazoles and Fused Thiadiazoles as Antimicrobial Agents
Archiv der Pharmazie
R
N
N
N
N
OHC
H2NO2S
NH2
H2NO2S
N
6a c
S
1
R
S
Glacial acetic acid/ethanol
_
COOH
SH
Dioxane
O
N
N
H2NO2S
N
S
S
R
_
7a c
R = a) 4-Cl; b) 4-N(CH3)2; c) 3-OCH3-4-OH
Scheme 2. Synthesis of the target compounds 6a–c and 7a–c.
N
N
H2NO2S
NH2
S
1
Cl
COOC2H5
Glacial acetic acid
H
N
N
R
COCH3
N
9a,b
N Cl
R
N
S
N
N
S
N
N
8
N
Acetic anhydride
H2NO2S
H2NO2S
H2NO2S
S
Sodium acetate
Water/ethanol
O
N
N
O
OH
N
10a,b
13
R= a) NO2; b) CH3
NC
NC
R
11a,b
Piperidine/ethanol
R
CN
N
N
H2NO2S
S
N
O
NH2
12a,b
R = a) 4-Cl; b) 3,4-(OCH3)2
Scheme 3. Synthesis of the target compounds 10a,b, 12a,b, and 13.
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CONH2
CN
R
R
N
N
CONH2
N
S
_
N
14a c
H2NO2S
H2NO2S
NH2
S
1
Piperidine/ethanol
N
NH2
_
15a c
R = a) 4-Cl; b) 4-N(CH3)2; c) 3-OCH3-4-OH
Scheme 4. Synthesis of the target compounds 15a–c.
Also, these compounds were tested for their antifungal
activity against Candida albicans and Aspergillus flavus 3375
applying the agar diffusion method [52]. The antifungal
screening results were determined by measuring the average
diameter of the inhibition zones, expressed in millimeters.
Based on the results (Table 2), it is assumed that compounds
3b, 5a, 6a, 7a, 10a,b, and 12a,b have the highest activity
against A. flavus 3375, whereas compounds 3c, 4, and 6b,c
displayed moderate activity against the same microorganism.
Compounds 3b, 3d, 4, 5b,c, 7b, 10b, 12a,b, and 15b,c
exhibited weak activity against C. albicans. The results are
compared to fluconazole as a reference antifungal agent. The
minimal inhibitory concentrations (MICs, mg/mL) for the most
and Bacillus cereus) and one Gram-negative bacterium
(Escherichia coli). The antibacterial screening was carried out
applying the broth microdilution method using 96-multiwell
microtiter plates [50] and minimal inhibitory concentrations
(MICs, mg/mL) werecalculated forthe tested compounds. Based
on the results (Table 1), it could be inferred that compounds 3b
and 10a have the highest antibacterial activity against E. coli
with MIC value of 78.125 mg/mL. In addition, compound 10a
showed the highest activity against B. cereus with MIC value of
156.25 mg/mL. The results are compared to amoxicillin as a
reference antibacterial agent. The tested microorganisms were
resistant to amoxicillin according to the Clinical Laboratory
Standard Institute (CLSI 2012) [51].
Table 1. Antibacterial activity of the synthesized compounds.
Comp. no.
Minimal inhibitory concentration (MIC, mg/mL)a),b)
S. aureus
B. cereus
E. coli
3a
3b
3c
3d
4
5a
5b
2500
2500
312.5
2500
2500
2500
–
2500
2500
2500
2500
2500
625
–
2500
78.125
2500
–
1250
312.5
–
5c
6a
6b
–
–
–
–
–
–
–
–
–
6c
7a
7b
7c
–
–
–
2500
2500
2500
–
2500
2500
2500
156.25
–
2500
2500
625
78.125
–
10a
10b
12a
12b
15a
15b
15c
Amoxicillin
–
–
625
312.5
–
–
–
–
–
–
2500
2500
156.25
1250
2500
2500
1250
2500
78.125
Bold values represent the best MIC values.
a)Results are calculated after subtraction of DMSO activity.
b)Not active (–, MIC > 2500 mg/mL).
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Table 2. Antifungal and antiquorum-sensing activities of the synthesized compounds.
Comp. no.
Inhibition zone diameter (mm)a),b)
QS inhibition (mm)c)
C. albicans A. flavus
Ch. violaceum
3a
3b
3c
3d
4
5a
5b
5c
6a
6b
6c
7a
7b
7c
–
–
–
12
9
9
–
40
12
–
2
2
–
–
12
40
8
5
10
–
7
5
–
–
–
40
10
10
23
–
–
–
–
–
–
–
–
7
–
–
–
–
10a
10b
12a
12b
–
40
17
30
40
–
–
7
8
2
–
–
7
13
–
15a
15b
15c
Fluconazole
Catechin
9
2
17
nt
–
–
–
–
–
nt
4
nt
Bold values represent the best antifungal activity against A. flavus.
nt, not tested.
a)Results are calculated after subtraction of DMSO activity.
b)Not active (–, inhibition zone <2 mm); weak activity (2–9 mm); moderate activity (10–15 mm); strong activity (>15 mm).
c)QS inhibition (radius of pigment inhibition in mm) ¼ radius of growth and pigment inhibition (r2) ꢀ radius of bacterial growth
inhibition (r1).
active compounds (3b, 5a, 6a, 7a, 10a,b, and 12a,b) against
A. flavus 3375weredeterminedapplyingthebrothmicrodilution
method using 96-multiwell microtiter plates [50]. Results
indicated that compounds 3b, 5a, 10a, and 12b (with the lowest
MIC value of 19.531 mg/mL) have the highest activity (Table 3).
QS is not essential for the growth of bacteria, but it regulates
virulence in many bacterial pathogens; thus, being a promising
target for antivirulence therapy which may inhibit virulence
instead of cell growth and division. This means that there is a
little selective pressure for the evolution of resistance [53].
Based on these facts, the newly synthesized compounds were
screened for their antiquorum-sensing activity against Chro-
mobacterium violaceum ATCC 12472 [54]. The QS system of Ch.
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 homoserine lactones
(HSLs) [55, 56]. Thus, drugs that inhibit acyl HSL-mediated QS
activity in Ch. violaceum would prevent production of this
purple pigment. Screening results for the ability of the newly
synthesized compounds to inhibit QS-regulated violacein
production against Ch. violaceum (based on measuring the
radius of pigment inhibition in mm) are presented in Table 2
and showed that compounds 3b, 5a, and 12b have moderate
antiquorum-sensing activity. The rest of the tested compounds
were found to be less active or completely inactive. The results
are compared to catechin as a positive control.
Structure–activity relationship (SAR) studies
In the series of 5-[N-((4-substituted phenyl)aminomethylene)-
amino]-[1,3,4]thiadiazoles 3a–d, the presence of 4-nitro-
phenyl substituent enhanced the antibacterial activity against
E. coli. In addition, the antifungal and antiquorum-sensing
activities were also increased (compound 3b), whereas the
presence of 4-methylphenyl substituent increased the anti-
bacterial activity against S. aureus (compound 3c). Referring
to series 5a–c, the presence of 3-(4-nitrophenyl)ureido moiety
at 5-position of the thiadiazole nucleus improved the
antibacterial and antiquorum-sensing activities as well as
antifungal activity against A. flavus 3375 (compound 5a). On
the other hand, the presence of 3-(4-methylphenyl)ureido
moiety at the same position increased the antifungal activity
against C. albicans (compound 5b). Regarding series 6a–c, the
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Table 3. MICs of compounds 3b, 5a, 6a, 7a, 10a,b, and
Table 5. In vivo acute toxicity of compounds 3b,c, 5a, 6a,
12a,b against A. flavus 3375.
10a, and 12a,b.
Comp. no.
Minimal inhibitory
concentration
Comp. no.
LD50 (mg/kg)
(MIC, mg/mL)a),b)
3b
3c
390
197
3b
5a
6a
7a
10a
10b
12a
12b
19.531
19.531
39.062
78.125
19.531
78.125
39.062
19.531
–
5a
6a
10a
12a
12b
Amoxicillin
Fluconazole
275
350
650
350
470
2125
1130
Bold value represents the best LD50
.
Fluconazole
Bold values represent the best MIC values.
a)Results are calculated after subtraction of DMSO activity.
b)Not active (–, MIC > 2500 mg/mL).
5-position of pyrano[30,20:5,6]imidazo[2,1-b][1,3,4]thiadiazole
nucleus improved the activity against B. cereus, A. flavus 3375,
and Ch. violaceum ATCC 12472 but decreased the activity
against C. albicans (compound 12b vs. 12a). Referring to
thiadiazolopyrimidines 15a–c, the presence of 4-dimethyl-
aminophenyl substituent at 5-position of thiadiazolo[3,2-a]-
pyrimidine nucleus increased the antibacterial activity against
B. cereus and E. coli as well as the antifungal activity against C.
albicans (compound 15b).
presence of 4-chlorobenzylideneamino substituent at 5-
position of thiadiazole nucleus enhanced the antifungal
activity against A. flavus 3375 (compound 6a). In the series of
compounds 7a–c, the presence of 4-chlorophenyl substituent
at 2-position of thiazolidine moiety improved the antifungal
activity against A. flavus 3375 (compound 7a). However, the
presence of 4-dimethylaminophenyl substituent at the same
position resulted in an increased antifungal activity against
C. albicans (compound 7b). In addition, the presence of
4-hydroxy-3-methoxyphenyl substituent at the same position
increased the antibacterial activity against E. coli (compound
7c). Taking into account the structure of 5-(4-substituted
In vitro cytotoxicity testing
The most active antibacterial and/or antifungal analogs, 3b,c,
5a, 6a, 10a, and 12a,b, were screened for in vitro cytotoxic
activity against human normal lung fibroblast (W138) cell line
at Vacsera, Agouza, Giza, Egypt, adopting MTT assay [57–59]
and using 5-fluorouracil as a reference cytotoxic agent. IC50
values expressed in mg/mL were calculated for each of the
tested compounds. Based on the results (Table 4), it could be
inferred that all tested compounds are safe against W138 cell
line at their MICs.
phenyl)hydrazonoimidazo[2,1-b][1,3,4]thiadiazoles
10a,b,
the presence of 4-nitrophenylhydrazono substituent at 5-
position of imidazothiadiazole nucleus enhanced the anti-
bacterial activity against B. cereus and E. coli as well as the
antifungal activity against A. flavus 3375 (compound 10a),
whereas the presence of 4-methylphenylhydrazono substitu-
ent at the same position increased the antifungal activity
against C. albicans (compound 10b). Concerning compounds
12a,b, the presence of 3,4-dimethoxyphenyl substituent at
In vivo acute toxicity testing (LD50
)
LD50 is a measurement used in toxicological studies to
determine the potential impact of toxic substances on
different types of test animals typically mice, rabbits, guinea
Table 6. DNA-binding affinity of compounds 3b,c, 5a, 6a,
10a, and 12a,b.
Table 4. In vitro cytotoxic activity of compounds 3b,c, 5a,
6a, 10a, and 12a,b against human normal lung fibroblast
(W138) cell line.
Comp. no.
DNA-binding affinity
3b
3c
5a
6a
10a
12a
12b
þþ
þ
Comp. no.
IC50 (mg/mL)
3b
3c
5a
6a
>5000
>5000
>5000
3906
>5000
>5000
>5000
351
þ
þ
þþ
þþ
þþ
þþþ
10a
12a
12b
5-Fluorouracil
Ethidium bromide
þþþ, strong binding affinity; þþ, moderate binding affinity;
þ, weak binding affinity.
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Thiadiazoles and Fused Thiadiazoles as Antimicrobial Agents
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pigs, or even larger animals such as dogs. It provides an
objective measure to compare and rank the toxicity of
substances [60]. The LD50 is defined as the amount of the
substance required to kill 50% of a given test population, it is
usually expressed as the amount of substance per kg of
body weight. The most active antibacterial and/or antifungal
analogs, 3b,c, 5a, 6a, 10a, and 12a,b, were screened for acute
toxicity in mice to study their potential risk of toxicity on living
organisms [61] and the dose of each compound (mg/kg)
required to kill 50% of animals (LD50) within 24 h was
calculated. Results indicated that compound 10a (one of the
least cytotoxic analogs against W138 cell line) has the lowest
acute toxicity (LD50¼ 650 mg/kg) (Table 5).
DNA-binding assay on TLC plates
It was demonstrated that when DNA was applied to TLC
plates, migration was observed when methanol/water (8:2)
was used as the elution solvent. However, when DNA was
mixed with compounds with which it interacts (e.g., ethidium
bromide), the complex was retained at the origin with
the same elution solvent. On the other hand, DNA inactive
compounds did not cause the DNA to be retained at the
origin [63]. Results from DNA-binding assay of compounds
3b,c, 5a, 6a, 10a, and 12a,b revealed that 3b, 10a, and 12a,b
have moderate binding affinity to DNA which was demon-
strated by migration of the complex for short distances. The
rest of the tested compounds showed weak affinity toward
DNA (Table 6).
DNA-binding assay
Molecular properties and drug-likeness
The mechanisms by which several known antimicrobial agents
produce their effect involve interaction with DNA. Based on
this fact, some short-term procedures were designed to be
applied for the discovery and evaluation of both naturally
occurring and synthetic compounds that function by this
mechanism. DNA-binding assay [62, 63] was applied for
determination of the interaction of small molecular weight
compounds with DNA.
A molecular property is a complex balance of various
structural features which determines whether a particular
molecule is similar to the known drugs or not. It generally
means “molecules which contain functional groups and/or
have physical properties consistent with most of the known
drugs.” Hydrophobicity, molecular size, flexibility, and
presence of various pharmacophoric features are the main
Table 7. Topological polar surface area, number of rotatable bonds, and calculated Lipinski's rule of five for the
synthesized compounds.
Comp. no.
Molecular properties
OH–NH
interact
O–N
interact
No. of
violations
TPSA
Nrotb
miLogP
M. wt.
3a
3b
3c
3d
4
5a
5b
5c
6a
6b
6c
7a
7b
7c
10a
10b
12a
12b
13
15a
15b
15c
Amoxicillin
Fluconazole
110.340
156.164
110.340
119.574
124.281
172.898
127.074
136.308
98.313
101.551
127.775
106.258
109.496
135.720
177.650
131.826
149.413
167.881
127.663
159.472
162.710
188.934
132.957
81.664
4
5
4
5
4
4
3
4
3
4
4
3
4
4
4
3
2
4
2
3
4
4
4
5
1.853
1.002
1.492
1.100
ꢀ0.057
0.800
1.289
0.897
1.846
1.271
0.508
1.693
1.117
0.354
0.673
1.163
1.572
0.541
ꢀ0.278
0.278
ꢀ0.298
ꢀ1.061
ꢀ1.352
ꢀ0.118
3
3
3
3
3
4
4
4
2
2
3
2
2
3
3
3
4
4
3
6
6
7
5
1
7
8
7
8
8
11
8
9
6
7
8
7
8
9
12
9
9
11
362.234
328.335
297.365
313.364
252.277
344.334
313.364
329.363
302.768
311.392
314.348
376.872
385.496
388.452
369.344
338.374
408.852
434.459
262.272
386.846
395.470
398.426
365.411
306.200
0
0
0
0
0
1
0
0
0
0
0
0
0
0
1
0
0
1
0
1
1
2
0
0
8
9
10
11
8
7
TPSA, topological polar surface area; Nrotb, number of rotatable bonds; miLogP, the parameter of lipophilicity.
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physical properties that influence the behavior of molecules
in a living organism. Good bioavailability can be achieved
with an appropriate balance between solubility and parti-
tioning properties. Thus, all the newly synthesized com-
pounds were analyzed for the prediction of solubility and
Lipinski's rule of five [64] as well as other properties (Tables 7
and 8) for filtering compounds for subsequent synthesis and
antimicrobial screening.
of oral bioavailability of drug molecules [66–69]. TPSA is
calculated based on the methodology published by Ertl
et al. [69] as the surface areas that are occupied by oxygen and
nitrogen atoms and by hydrogen atoms attached to them.
Thus, it is closely related to the hydrogen bonding potential of
a compound [66–69]. TPSA has been shown to be a very good
descriptor characterizing drug absorption, including intesti-
nal absorption, bioavailability, and blood–brain barrier
penetration. Molecules with TPSA values around 140 Å2 or
more are expected to exhibit poor intestinal absorption [65].
Results shown in Table 7 indicated that all the synthesized
compounds (except 3b, 5a, 10a, 12a,b, and 15a–c) have
TPSA values <140 Å2; thus, they are expected to have
good intestinal absorption. Molecules with more than 10
rotatable bonds may have problems with bioavailability [65].
All compounds under investigation have two to five
rotatable bonds and they might not have problems with
bioavailability (Table 7). MiLogP is calculated adopting the
methodology developed by Molinspiration as a sum of
fragment-based contributions and correction factors (http://
www.molinspiration.com). It has been shown that for the
compound to have a reasonable probability of being well
absorbed, miLogP value must be in the range of ꢀ0.4 to
þ5 [65]. On this basis, all the tested compounds (except 15c)
were found to have miLogP values within the acceptable
Molinspiration calculations
As a part of our study, the compliance of compounds to the
Lipinski'sruleoffivewasevaluated[64];thissimple ruleisbased
on the observation that most biologically active drugs have
molecular weight of 500 or less, logP values not higher than 5,
hydrogen bond donor sites not higher than 5, and hydrogen
bond acceptor sites not higher than 10. In addition, topological
polar surface area (TPSA) and number of rotatable bonds have
been linked to drug bioavailability [65]. Molecular properties
(TPSA, nrotb, miLogP, OH–NH interaction, O–N interaction,
molecular weight, and number of violations from Lipinski's
rule) of the newly synthesized compounds were calculated
using molinspiration software and compared to the values of
the standard drugs, amoxicillin and fluconazole (Table 7).
Topological polar surface area (TPSA) and lipophilicity
(logP) values are two important properties for the prediction
Table 8. Toxicity risks, solubility, drug-likeness, and drug score for the synthesized compounds.
Comp. no.
Toxicity risks
Mutagenicity
Tumorigenicity
Irritation
Solubility
Drug-likeness
Drug score
3a
3b
3c
3d
4
5a
5b
5c
6a
6b
6c
7a
7b
7c
10a
10b
12a
12b
13
15a
15b
15c
Amoxicillin
Fluconazole
ꢀ3.86
ꢀ3.48
ꢀ3.37
ꢀ3.04
ꢀ2.25
ꢀ3.78
ꢀ3.67
ꢀ3.34
ꢀ3.57
ꢀ2.87
ꢀ2.56
ꢀ3.86
ꢀ3.16
ꢀ2.85
ꢀ3.78
ꢀ4.05
ꢀ3.94
ꢀ3.24
ꢀ0.24
ꢀ5.60
ꢀ4.90
ꢀ4.58
ꢀ1.27
ꢀ2.17
ꢀ0.64
ꢀ9.86
ꢀ0.05
0.09
ꢀ7.43
ꢀ9.68
2.19
2.37
3.92
2.08
3.50
0.32
0.26
0.39
0.32
0.28
0.25
0.48
0.49
0.50
0.30
0.43
0.47
0.30
0.51
0.45
0.77
0.66
0.71
0.96
0.40
0.31
0.71
0.91
0.87
5.85
4.09
5.52
ꢀ6.68
3.73
1.04
1.52
4.23
6.77
4.53
5.80
10.21
1.99
Bold values represent the best drug score values.
, low risk; , moderate risk; , high risk.
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criteria and are expected to have reasonable oral absorption
(Table 7). It is worth mentioning that all analyzed compounds
(except 15c) have one or zero violation of Lipinski's rule;
therefore, they are expected not to have problems with
bioavailability (Table 7). Molecules violating more than one
may have problems with bioavailability [70].
antifungal activity against A. flavus 3375 as well as moderate
DNA-bindingaffinity. Invitrocytotoxicitytestingindicatedthat
bothcompoundsaresafeagainsthumannormallungfibroblast
(W138) cell line at their MICs. Moreover, in vivo acute toxicity
testing revealed that compound 10a has the lowest acute
toxicity. These preliminary encouraging results of biological
screening of the newly synthesized compounds could offer an
excellent framework that may lead to the discovery of new
potent antimicrobial agents.
Osiris calculations
Toxicity risks (mutagenicity, tumorigenicity, and irritation)
and physicochemical properties (solubility, drug-likeness, and
drug score) of the synthesized compounds were calculated by
the methodology developed by Osiris [66]. The toxicity risk
predictor locates fragments within a molecule which indicate
a potential toxicity risk. Toxicity risk alerts indicate that the
drawn structure may be harmful concerning the risk category
specified. From the data presented in Table 8, it is obvious that
the synthesized compounds are expected to be non-muta-
genic, non-tumorigenic (except 6b,c, 7b, and 15b) and non-
irritating (except 3d). The aqueous solubility of a compound
significantly affects its absorption and distribution character-
istics. Typically, low solubility goes along with bad absorption
and therefore the general aim is to avoid poorly soluble
compounds. It is well established that more than 80% of the
drugs on the market have an estimated solubility values
greater than ꢀ4. As shown in Table 8, the synthesized
compounds (except 10b and 15a–c) exhibited solubility values
above ꢀ4 and they are expected to have good aqueous
solubility which significantly affects their absorption and
distribution characteristics.
Drug-likeness is defined as a complex balance of various
molecular properties and structural features which indicates
whether a particular molecule is similar to the known drugs or
not [71]. Osiris program was used for calculating the
fragment-based drug-likeness of the synthesized compounds,
a positive value indicates that the designed molecule contains
fragments which are frequently present in commercial drugs.
Results shown in Table 8 indicated that compounds 5b,c, 6a–c,
7a–c, 10b, 13, and 15a–c have drug-likeness values higher
than that of the standard drug, fluconazole.
Experimental
Chemistry
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
spectrometer (v in cmꢀ1) at Faculty of Science, Mansoura
University, Mansoura, Egypt. Nuclear magnetic resonance
(1H and 13C NMR) spectra were obtained on 300 MHz FT-NMR
spectrometer at Faculty of Science, Cairo University and on
500 MHz FT-NMR spectrometer at National Research Centre,
Dokki, Giza, Egypt. The chemical shifts are expressed in d ppm
using tetramethylsilane (TMS) as internal reference and
DMSO-d6 as solvent. Mass spectra were 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 pre-coated (E. Merck Co., Germany) and the
spots were visualized by UV. Chloroform/methanol (9:1) was
adopted as an elution solvent. Compound 1 was prepared
following the literature procedure [45]. Compounds 2a–d
were prepared adopting the reported procedure [46]. Com-
pound 8 was prepared according to the literature proce-
dure [16]. Compounds 11a,b were prepared following the
literature method [48]. Compounds 14a–c were prepared
according to the reported procedure [49].
The drug score combines drug-likeness, miLogP, solubility,
molecular weight, and toxicity risks in one handy value that
may be used to judge the compound's overall potential to
qualify for a drug [66]. A value of 0.5 or more makes
the compound a promising lead for future development of
safe and efficient drugs. The overall drug score values for the
synthesized compounds were calculated and compared to
that of the standard drugs, amoxicillin and fluconazole
(Table 8). Compounds 6a, 7c, 10b, 12a,b, 13, and 15c possess
good drug score values.
General procedure for the preparation of 5-[N-((4-
substituted phenyl)aminomethylene)amino]-[1,3,4]-
thiadiazole-2-sulfonamides 3a–d
A mixture of amine 1 (0.36 g, 0.002 mol) and the appropriate
ethyl N-(4-substituted phenyl)formimidate 2a–d (0.002 mol) in
glacial acetic acid (10 mL) was heated at reflux temperature
for 16–18 h. On cooling, the precipitated solid was collected
by filtration, dried, and crystallized from ethanol/water (2:1).
5-[N-((4-Bromophenyl)aminomethylene)amino]-[1,3,4]-
thiadiazole-2-sulfonamide (3a)
Yield 60%, m.p. 295–296°C. 1H NMR (DMSO-d6, d ppm): 7.45
Conclusion
(d, 2H, Ar-H), 7.56 (d, 2H, Ar-H), 8.28 (s, 1H, CH N), 10.03 (s,
ResultsofantimicrobialscreeningandDNA-bindingassayofthe
newly synthesized compounds showed that compounds 3b and
10a have the highest antibacterial activity against E. coli and
1H, NH), 11.67 (s, 2H, NH2). MS m/z (%): 364 (0.23, Mþþ2), 363
(1.02, Mþþ1), 362 (1.64, Mþ), 71 (100.00). Anal. calcd. for
C9H8BrN5O2S2 (362.23) C, H, N.
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5-[N-((4-Nitrophenyl)aminomethylene)amino]-[1,3,4]-
thiadiazole-2-sulfonamide (3b)
5-(3-(4-Methylphenyl)ureido)-[1,3,4]thiadiazole-2-
sulfonamide (5b)
Yield 70%, m.p. 240–241°C. 1H NMR (DMSO-d6, d ppm): 7.79
Yield 60%, m.p. 139–140°C. IR (KBr, v, cmꢀ1): 3454, 3434 (NH2),
(d, 2H, Ar-H), 8.16 (d, 2H, Ar-H), 8.42 (s, 1H, CH N), 10.49 (s,
3292, 3191 (2NH), 1661 (C O). 1H NMR (DMSO-d , d ppm): 2.24
6
1H, NH), 11.45 (s, 2H, NH2). 13C NMR (DMSO-d6, d ppm): 118.5,
124.8, 141.9, 145.4, 161.1, 164.3, 169.2. Anal. calcd. for
C9H8N6O4S2 (328.33) C, H, N.
(s, 3H, CH3), 7.08 (d, 2H, Ar-H), 7.44 (d, 2H, Ar-H), 8.35 (s, 2H,
NH2), 9.80 (s, 1H, NH), 11.62 (s, 1H, NH). 13C NMR spectrum:
(DMSO-d6, d ppm): 24.4, 119.4, 129.4, 132.3, 137.3, 152.6,
168.5, 170.2. MS m/z (%): 314 (19.79, Mþþ1), 313 (24.31, Mþ),
107 (100.00). Anal. calcd. for C10H11N5O3S2 (313.36) C, H, N.
5-[N-((4-Methylphenyl)aminomethylene)amino]-[1,3,4]-
thiadiazole-2-sulfonamide (3c)
Yield 65%, m.p. >300°C. IR (KBr, v, cmꢀ1): 3445, 3387 (NH2),
3329 (NH). 13C NMR (DMSO-d6, d ppm): 21.0, 121.8, 129.4,
133.4, 136.1, 158.1, 165.3, 168.2. MS m/z (%): 299 (0.23,
Mþþ2), 298 (0.44, Mþþ1), 297 (0.35, Mþ), 117 (100.00). Anal.
calcd. for C10H11N5O2S2 (297.36) C, H, N.
5-(3-(4-Methoxyphenyl)ureido)-[1,3,4]thiadiazole-2-
sulfonamide (5c)
Yield 45%, m.p. 134–135°C. IR (KBr, v, cmꢀ1): 3456 (NH2), 3264,
3167 (2NH), 1661 (C O). 1H NMR (DMSO-d6, d ppm): 3.78 (s,
ꢀꢀ
ꢀꢀ
3H, OCH3), 6.93 (d, 2H, Ar-H), 7.42 (s, 2H, NH2), 7.55 (d, 2H, Ar-
H), 8.38 (s, 1H, NH), 9.82 (s, 1H, NH). MS m/z (%): 331 (2.19,
Mþþ2), 330 (2.08, Mþþ1), 329 (2.39, Mþ), 108 (100.00). Anal.
calcd. for C10H11N5O4S2 (329.36) C, H, N.
5-[N-((4-Methoxyphenyl)aminomethylene)amino]-[1,3,4]-
thiadiazole-2-sulfonamide (3d)
Yield 63%, m.p. >300°C. 1H NMR (DMSO-d6, d ppm): 3.70 (s,
3H, OCH3), 6.84 (d, 2H, Ar-H), 7.46 (d, 2H, Ar-H), 8.10 (s, 1H,
General procedure for the preparation of 5-(substituted
benzylideneamino)-[1,3,4]thiadiazole-2-sulfonamides 6a–c
A mixture of amine 1 (0.9 g, 0.005 mol), the appropriate
benzaldehyde (0.005 mol) and glacial acetic acid (0.5 mL) in
ethanol (15 mL) was heated at reflux temperature for 10–14 h.
The reaction mixture was concentrated and the precipitated
solid was collected by filtration, dried, and crystallized from
ethanol.
CH N), 9.74 (s, 1H, NH), 11.62 (s, 2H, NH ). 13C NMR (DMSO-d6,
d ppm): 55.1, 113.7, 120.5, 132.5, 144.2, 154.9, 167.7, 169.6. MS
m/z (%): 313 (0.08, Mþ), 312 (0.21, Mþꢀ1), 180 (100.00). Anal.
calcd. for C10H11N5O3S2 (313.36) C, H, N.
ꢀꢀ
ꢀꢀ
2
General procedure for the preparation of ethyl
5-sulfamoyl-[1,3,4]thiadiazol-2-ylcarbamate (4) [47]
A mixture of amine 1 (0.9 g, 0.005 mol) and ethyl chlorofor-
mate (0.543 g, 0.005 mol) in glacial acetic acid (15 mL) was
heated at reflux temperature for 16 h. On cooling, the
precipitated solid was collected by filtration, dried, and
crystallized from ethanol/water (2:1).
5-(4-Chlorobenzylideneamino)-[1,3,4]thiadiazole-2-
sulfonamide (6a)
Yield 70%, m.p. 245–246°C. 1H NMR (DMSO-d6, d ppm): þ7.34–
ꢀꢀ
8.01 (m, 7H, Ar-H, CH N, NH ). MS m/z (%): 304 (6.42, M þ1),
ꢀꢀ
2
Yield 65%, m.p. 228–229°C [47]. IR (KBr, v, cmꢀ1): 3354,
303 (7.87, Mþ), 80 (100.00). Anal. calcd. for C9H7ClN4O2S2
(302.76) C, H, N.
3254 (NH ), 3144 (NH), 1732 (C O). 1H NMR (DMSO-d6,
ꢀꢀ
ꢀꢀ
2
d ppm): 1.20 (t, 3H, CH2CH3), 4.08–4.21 (q, 2H, CH2CH3), 7.42
(s, 2H, NH2), 9.80 (s, 1H, NH). 13C NMR (DMSO-d6, d ppm):
14.7, 62.2, 154.0, 157.8, 169.8. Anal. calcd. for C5H8N4O4S2
(252.27) C, H, N.
5-[(4-Dimethylamino)benzylideneamino]-[1,3,4]-
thiadiazole-2-sulfonamide (6b)
Yield 60%, m.p. 260–261°C. 1H NMR (DMSO-d6, d ppm): 3.07 (s,
6H, N(CH3)2), 6.75 (d, 2H, Ar-H), 7.70 (d, 2H, Ar-H), 8þ.37 (s, 1H,
ꢀꢀ
General procedure for the preparation of 5-(3-(4-
substituted phenyl)ureido)-[1,3,4]thiadiazole-2-
sulfonamides 5a–c
CH N), 9.67 (s, 2H, NH ). MS m/z (%): 312 (8.35, M þ1), 311
ꢀꢀ
2
(9.22, Mþ), 117 (100.00). Anal. calcd. for C11H13N5O2S2 (311.38)
C, H, N.
A mixture of compound 4 (0.252 g, 0.001 mol) and the
appropriate aniline (0.001 mol) in glacial acetic acid (10 mL)
was heated at reflux temperature for 12–18 h. On cooling, the
precipitated solid was collected by filtration, dried, and
crystallized from ethanol/water (2:1).
5-(4-Hydroxy-3-methoxybenzylideneamino)-[1,3,4]-
thiadiazole-2-sulfonamide (6c)
Yield 65%, m.p. decomp. 260°C. 1H NMR (DMSO-d6, d ppm):
ꢀꢀ
3.86 (s, 3H, OCH ), 6.67–7.53 (m, 6H, Ar-H, CH N, NH ), 9.78 (s,
ꢀꢀ
3
2
1H, OH). MS m/z (%): 316 (10.47, Mþþ2), 315 (9.47, Mþþ1), 314
(12.96, Mþ), 117 (100.00). Anal. calcd. for C10H10N4O4S2
(314.34) C, H, N.
5-(3-(4-Nitrophenyl)ureido)-[1,3,4]thiadiazole-2-
sulfonamide (5a)
Yield 70%, m.p. 188–189°C. 1H NMR (DMSO-d6, d ppm): 7.83
(d, 2H, Ar-H), 8.21 (d, 2H, Ar-H), 9.30 (s, 2H, NH2), 10.60 (s, 1H,
NH), 11.50 (s, 1H, NH). 13C NMR (DMSO-d6, d ppm): 119.0,
125.4, 142.5, 145.9, 154.9, 169.0, 170.3. MS m/z (%): 345 (5.56,
Mþþ1), 344 (3.37, Mþ), 117 (100.00). Anal. calcd. for
C9H8N6O5S2 (344.33) C, H, N.
General procedure for the preparation of 5-[4-oxo-2-
(substituted phenyl)thiazolidin-3-yl]-[1,3,4]thiadiazole-2-
sulfonamides 7a–c
A mixture of the appropriate Schiff base 6a–c (0.002 mol) and
thioglycolic acid (0.184 g, 0.002 mol) in dioxane (10 mL) was
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5-[2-(4-Hydroxy-3-methoxyphenyl)-4-oxothiazolidin-3-yl]-
(2NH2), 2220 (C
ꢀN). 1H NMR (DMSO-d6, d ppm): 3.61 (s, 2H,
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Thiadiazoles and Fused Thiadiazoles as Antimicrobial Agents
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heated at reflux temperature for 16–18 h. The solvent was
evaporated under reduced pressure and the obtained residue
was triturated with 10% sodium bicarbonate solution. The
separated solid was filtered, washed with water, dried, and
crystallized from ethanol.
5-(4-Methylphenyl)hydrazono-6-oxo-5H-imidazo[2,1-b]-
[1,3,4]thiadiazole-2-sulfonamide (10b)
Yield 65%, m.p. 120–122°C. IR (KBr, v, cmꢀ1): 3472, 3357 (NH2),
1
ꢀꢀ
3176 (NH), 1650 (C O). H NMR (DMSO-d , d ppm): 2.20 (s, 3H,
ꢀꢀ
6
CH3), 5.26 (s, 1H, NH), 6.94–8.53 (m, 6H, Ar-H, NH2). MS m/z
(%): 339 (57.75, Mþþ1), 338 (47.89, Mþ), 91 (100.00). Anal.
calcd. for C11H10N6O3S2 (338.37) C, H, N.
5-[2-(4-Chlorophenyl)-4-oxothiazolidin-3-yl]-[1,3,4]-
thiadiazole-2-sulfonamide (7a)
Yield 50%, m.p. 216–218°C. IR (KBr, v, cmꢀ1): 3355, 3254 (NH2),
General procedure for the preparation of 7-amino-6-
cyano-5-(substituted phenyl)-5H-pyrano[30,20:5,6]-
imidazo[2,1-b][1,3,4]thiadiazole-2-sulfonamides 12a,b
A mixture of imidazothiadiazole 8 (0.44 g, 0.002 mol), the
appropriate benzylidenemalononitrile 11a,b (0.002 mol), and
piperidine (0.3 mL) in absolute ethanol (15 mL) was heated at
reflux temperature for 12–16 h. The mixture was concentrat-
ed, then poured onto ice-water. The precipitated solid was
collected by filtration, dried, and crystallized from ethanol/
water (2:1).
1681 (C O). 1H NMR (DMSO-d6, d ppm): 3.35 (s, 2H, C5–H of
ꢀꢀ
ꢀꢀ
thiazolidine), 6.60 (s, 1H, C2–H of thiazolidine), 6.91–7.58 (m,
4H, Ar-H), 8.85 (s, 2H, NH2). 13C NMR (DMSO-d6, d ppm): 34.8,
76.0, 126.0, 128.3, 135.9, 138.6, 166.1, 169.7, 177.1. MS m/z
(%): 378 (46.20, Mþþ1), 377 (34.18, Mþ), 97 (100.00). Anal.
calcd. for C11H9ClN4O3S3 (376.86) C, H, N.
5-[2-(4-Dimethylaminophenyl)-4-oxothiazolidin-3-yl]-
[1,3,4]thiadiazole-2-sulfonamide (7b)
Yield60%,m.p.>300°C.IR(KBr,v,þcmꢀ1):3211,3141(NH2),1667
þ
(C O). MS m/z (%): 387 (18.06, M þ2), 386 (27.43, M þ1), 116
7-Amino-5-(4-chlorophenyl)-6-cyano-5H-pyrano[30,20:5,6]-
imidazo[2,1-b][1,3,4]thiadiazole-2-sulfonamide (12a)
Yield 65%, m.p. 148–149°C. IR (KBr, v, cmꢀ1): 3343, 3211
ꢀ
ꢀ
(100.00). Anal. calcd. for C13H15N5O3S3 (385.48) C, H, N.
[1,3,4]thiadiazole-2-sulfonamide (7c)
NH2), 4.35 (s, 1H, C5–H), 7.55–8.07 (m, 6H, Ar-H, NH2). 13C NMR
(DMSO-d6, d ppm): 29.2, 60.0, 117.8, 122.3, 126.1, 130.8, 131.7,
138.2, 143.8, 151.9, 158.2, 171.3. MS m/z (%): 410 (20.30,
Mþþ1), 409 (42.86, Mþ), 408 (6.02, Mþꢀ1), 174 (100.00). Anal.
calcd. for C14H9ClN6O3S2 (408.84) C, H, N.
Yield 65%, m.p. >300°C. IR (KBr, v, cmꢀ1): 3457 (OH), 3208, 3140
1
ꢀꢀ
(NH ), 1653 (C O). H NMR (DMSO-d , d ppm): 3.38 (s, 2H, C –H
ꢀꢀ
2
6
5
of thiazolidine), 3.68 (s, 3H, OCH3), 5.29 (s, 2H, NH2), 6.33 (s, 1H,
C2–H of thiazolidine), 6.95–7.38 (m, 3H, Ar-H), 12.20 (s, 1H, OH).
13C NMR (DMSO-d6, d ppm): 34.1, 55.7, 74.0, 114.7, 117.7, 121.9,
129.0, 149.2, 151.1, 160.2, 163.7, 168.2. MS m/z (%): 390 (15.95,
Mþþ2), 389 (22.70, Mþþ1), 388 (22.70, Mþ), 53 (100.00). Anal.
calcd. for C12H12N4O5S3 (388.44) C, H, N.
7-Amino-6-cyano-5-(3,4-dimethoxyphenyl)-5H-
pyrano[30,20:5,6]imidazo[2,1-b][1,3,4]thiadiazole-2-
sulfonamide (12b)
Yield 60%, m.p. 142–143°C. IR (KBr, v, cmꢀ1): 3442 (2NH2),
1
ꢀꢀ
General procedure for the preparation of 6-oxo-5-(4-
substituted phenyl)hydrazono-5H-imidazo[2,1-b][1,3,4]-
thiadiazole-2-sulfonamides 10a,b
2213 (CꢀꢀN). H NMR: (DMSO-d6, d ppm): 2.99 (s, 2H, NH2), 3.82
ꢀꢀ
(s, 6H, 2OCH3), 4.34 (s, 1H, C5–H), 6.59–7.56 (m, 3H, Ar-H), 8.36
(s, 2H, NH2). MS m/z (%): 435 (46.99, Mþþ1), 434 (32.53, Mþ),
79 (100.00). Anal. calcd. for C16H14N6O5S2 (434.45) C, H, N.
A solution of sodium nitrite (0.138 g, 0.002 mol) in water
(5 mL) was added to a stirred ice-cooled solution of the
appropriate aniline (0.002 mol) in 5 N hydrochloric acid (5 mL).
After 10 min, the reaction mixture was rendered alkaline by
addition of a saturated solution of sodium acetate. The
obtained diazonium solution 9a,b was then added slowly to a
stirred cold slurry of imidazothiadiazole 8 (0.44 g, 0.002 mol),
sodium acetate (0.25 g, 0.003 mol) and water (3 mL) in ethanol
(10 mL). The reaction mixture was stirred at room tempera-
ture for 1 h and the solid obtained was collected by filtration,
dried and crystallized from ethanol.
Preparation of 5-acetyl-6-hydroxyimidazo[2,1-b][1,3,4]-
thiadiazole-2-sulfonamide (13)
A mixture of imidazothiadiazole 8 (0.44 g, 0.002 mol) and
acetic anhydride (10 mL) was heated at reflux temperature for
10 h. The mixture was evaporated under reduced pressure and
the obtained solid was crystallized from ethanol.
Yield 55%, m.p. 223–225°C. IR (KBr, v, cmꢀ1): 3448 (OH),
1
ꢀꢀ
3208, 3143 (NH ), 1657 (C O). H NMR (DMSO-d , d ppm): 2.45
ꢀꢀ
2
6
(s, 3H, CH3), 11.71 (s, 1H, OH), 12.20 (s, 2H, NH2). 13C NMR
(DMSO-d6, d ppm): 25.2, 143.9, 144.6, 166.7, 168.3, 169.8. MS
m/z (%): 263 (32.57, Mþþ1), 262 (35.43, Mþ), 80 (100.00). Anal.
calcd. for C6H6N4O4S2 (262.27) C, H, N.
5-(4-Nitrophenyl)hydrazono-6-oxo-5H-imidazo[2,1-b]-
[1,3,4]thiadiazole-2-sulfonamide (10a)
Yield 80%, m.p. 165–166°C. IR (KBr, v, cmꢀ1): 3387, 3303 (NH2),
1
ꢀꢀ
3195 (NH), 1639 (C O). H NMR (DMSO-d , d ppm): 6.57 (s, 2H,
General procedure for the preparation of 7-amino-5-
(substituted phenyl)-2-sulfamoyl-5H-[1,3,4]-
ꢀꢀ
6
NH2), 6.76 (s, 1H, NH), 7.31 (d, 2H, Ar-H), 8.21 (d, 2H, Ar-H). 13
C
NMR (DMSO-d6, d ppm): 118.9, 124.6, 142.6, 144.5, 155.2,
166.0, 169.7, 175.9. MS m/z (%): 370 (36.88, Mþþ1), 369 (39.01,
Mþ), 74 (100.00). Anal. calcd. for C10H7N7O5S2 (369.34) C, H, N.
thiadiazolo[3,2-a]pyrimidine-6-carboxamides 15a–c
A mixture of amine 1 (0.36 g, 0.002mol), the appropriate
benzylidenecyanoacetamide 14a–c (0.002 mol), and piperidine
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N. S. El-Goharya and M. I. Shaaban
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(0.3 mL) in absolute ethanol (15 mL) was heated at reflux
temperature for 18–24 h. The solvent was concentrated, then
poured onto ice-water. The precipitated solid was collected by
filtration, dried, and crystallized from ethanol/water (2:1).
Antibacterial screening (determination of minimal
inhibitory concentration [MIC, mg/mL])
Minimal inhibitory concentrations (MICs) were determined
by serial dilution technique using 96-multiwell microtiter
plates. The investigated compounds were dissolved in
DMSO 100% to prepare stock solutions of 5000 mg/mL.
Two-fold serial dilutions of the dissolved compounds were
performed in LB broth providing eight different concen-
trations (2500, 1250, 625, 312.5, 156.25, 78.125, 39.062, and
19.531 mg/mL). Overnight cultures of microorganisms were
diluted to 1 ꢁ 106 CFU/mL in LB broth, and 20 mL of the
diluted cultures were added to the test solutions (50 mL) in
the wells (one well per concentration per bacterial strain).
The plates were incubated at 37°C for 24 h [50]. MIC was
detected visually as the least concentration inhibiting
bacterial growth (no turbidity) and the activity of the
tested compounds was estimated in comparison to amoxi-
cillin (Table 1).
7-Amino-5-(4-chlorophenyl)-2-sulfamoyl-5H-[1,3,4]-
thiadiazolo[3,2-a]pyrimidine-6-carboxamide (15a)
Yield 62%, m.p. 173–175°C. 1H NMR (DMSO-d6, d ppm): 4.96
(s, 1H, C5–H), 5.09 (s, 2H, NH2), 7.76–8.02 (m, 8H, Ar-H,
2NH2). 13C NMR (DMSO-d6, d ppm): 67.8, 123.4, 125.3, 130.6,
134.9, 136.8, 163.9, 165.3, 167.5, 169.8. MS m/z (%): 387
(9.23, Mþ), 80 (100.00). Anal. calcd. for C12H11ClN6O3S2
(386.84) C, H, N.
7-Amino-5-(4-dimethylaminophenyl)-2-sulfamoyl-5H-
[1,3,4]thiadiazolo[3,2-a]pyrimidine-6-carboxamide (15b)
Yield 45%, m.p. 158–159°C. IR (KBr, v, cmꢀ1): 3444 (broad
þ
ꢀꢀ
band, 3NH ), 1631 (C O). MS m/z (%): 396 (37.37, M þ1), 395
ꢀꢀ
2
(24.21, Mþ), 113 (100.00). Anal. calcd. for C14H17N7O3S2
(395.46) C, H, N.
Antifungal screening (determination of inhibition zone
diameter [mm])
7-Amino-5-(4-hydroxy-3-methoxyphenyl)-2-sulfamoyl-5H-
Sabouraud's medium (50 mL) was inoculated with 50 mL of
1 ꢁ 106 CFU/mL of 24 h culture of C. albicans. Glucose minimal
medium (50 mL) was inoculated with 50 mL of 1 ꢁ 103 SFU/mL
of A. flavus 3375. Wells were made in agar using cork borer.
The tested compounds were dissolved in DMSO 100% in
Eppendorf tubes for final concentration 5 mg/mL, and 50 mL
of the test solution was applied into the wells. The standard
antifungal drug (fluconazole) was also added at the same
concentration and volume 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 at 30°C for
48 h for A. flavus 3375 [52]. The antifungal activity of the
tested compounds was determined by measuring the diame-
ter of the inhibition zones (Table 2). The inhibition zone
diameter of DMSO was subtracted from the antifungal
activity of the tested compounds.
[1,3,4]thiadiazolo[3,2-a]pyrimidine-6-carboxamide (15c)
Yield 45%, m.p. >300°C. IR (KBr, v, cmꢀ1): 3444 (broad band,
1
ꢀꢀ
3NH , OH), 1631 (C O). H NMR (DMSO-d , d ppm): 3.98 (s, 3H,
ꢀꢀ
2
6
OCH3), 5.36 (s, 1H, C5–H), 7.19–8.60 (m, 9H, Ar-H, 3NH2), 13.00
(s, 1H, OH). 13C NMR (DMSO-d6, d ppm): 57.0, 62.7, 99.5, 114.6,
115.4, 123.7, 136.5, 143.7, 150.0, 152.5, 156.9, 160.7, 169.9. MS
m/z (%): 399 (60.64, Mþþ1), 398 (74.47, Mþ), 304 (100.00).
Anal. calcd. for C13H14N6O5S2 (398.42) C, H, N.
Biological testing
Antimicrobial and antiquorum-sensing activities
Compounds 3a–d, 4, 5a–c, 6a–c, 7a–c, 10a,b, 12a,b, and 15a–c
were screened for their antibacterial activity against S. aureus,
B. cereus, and E. coli in Luria–Bertani (LB) agar medium [50].
The same compounds were also screened for their antifungal
activity against C. albicans in Sabouraud's agar medium and A.
flavus 3375 in glucose minimal medium [50, 52]. In addition,
their antipathogenic potential was checked by examining the
antiquorum-sensing activity against Ch. violaceum ATCC
12472 in LB agar medium [54].
Antifungal screening (determination of minimal
inhibitory concentration [MIC, mg/mL])
Minimal inhibitory concentrations (MICs) were determined
by serial dilution technique using 96-multiwell microtiter
plates. The investigated compounds, 3b, 5a, 6a, 7a, 10a,b,
and 12a,b, were dissolved in DMSO 100% to prepare stock
solutions of 5000 mg/mL. Two-fold serial dilutions of the
dissolved compounds were performed in glucose minimal
medium providing eight different concentrations (2500,
1250, 625, 312.5, 156.25, 78.125, 39.062, and 19.531 mg/mL).
A. flavus 3375 was diluted to 1 ꢁ 103 SFU/mL in glucose
minimal medium, and 20 mL of the diluted culture was added
to the test solutions (50 mL) in the wells (one well per
concentration). The plates were incubated at 30°C for
48 h [50]. MIC was detected visually as the least concentra-
tion inhibiting fungal growth (no turbidity) and the activity
of the tested compounds was estimated in comparison to
fluconazole (Table 3).
S. aureus, B. cereus, E. coli, and C. albicans were obtained
from the culture collection of the Department of Microbiolo-
gy, Faculty of Pharmacy, Mansoura University. A. flavus 3375
was kindly provided by Keller Lab (University of Washington,
Madison, WI, USA). Ch. violaceum ATCC 12472 was kindly
provided by Prof. Bob Mclean (Department of Biology, Texas
State University, San Marcos, TX, USA). All bacterial strains
were propagated in LB agar medium (Merck, Germany). C.
albicans was grown in Sabouraud's medium (Merck, Germany)
and A. flavus 3375 was cultivated in glucose minimal medium
(Merck, Germany). Amoxicillin was obtained from EPICO Co.
(Egypt). Fluconazole was obtained from Pfizer Co. (Egypt).
Catechin and DMSO 100% were obtained from Sigma–Aldrich
Co. (USA).
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Thiadiazoles and Fused Thiadiazoles as Antimicrobial Agents
Archiv der Pharmazie
Antiquorum-sensing screening
equation:
The culture was prepared by growing Ch. violaceum ATCC
12472 in LB broth and incubated for 16–18 h in an orbital
incubator running at 28°C and 150 rpm. The culture was
then adjusted to 0.5 McFarland standard (ca. 1 ꢁ 106 CFU/mL).
Ch. violaceum (50 mL) was inoculated into LB agar (50 mL),
poured into plates, and solidified. Wells were made in LB
agar medium using cork borer. The tested compounds were
dissolved in DMSO 100% in Eppendorf tubes for final
concentration of 5 mg/mL, and 50 mL of the test solution
was applied into the wells. The positive control (catechin) was
also added at the same concentration and volume to each
plate. In addition, DMSO (control solvent) was added to each
plate. Plates were incubated at 30°C for 48 h to check the
inhibition of pigment production around the wells. 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
Ch. violaceum ATCC 12472 monitor strain [54]. Bacterial
growth inhibition by the tested compounds was measured as
radius (r1) in mm, while both growth and pigment inhibition
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 ¼ (r2 ꢀ r1) in mm (Table 2).
Atreated cells ꢀ Ablank
% Cell viability ¼
ꢁ 100
Auntreated cells ꢀ Ablank
The relation between surviving fraction and drug concen-
tration is plotted to get the survival curve for W138 lung
normal cell line. The GraphPad Prism software (http://www.
graphpad.com) was used for calculation of the concentration
required for 50% inhibition of cell viability (IC50) of each of
the tested compounds (Table 4).
In vivo acute toxicity testing
Compounds 3b,c, 5a, 6a, 10a, and 12a,b were studied for
potential acute toxicity in mice [61]. Adult male Swiss albino
mice (weighing 20–25 g) were obtained from animal house,
Department of Pharmacology, Faculty of Pharmacy, Man-
soura University. Animals were housed in microlon boxes in a
controlled environment (temperature 25 ꢂ 2°C) with a regular
12 h light/12 h dark and were allowed free access to standard
laboratory food and water.
The solutions of different concentrations of the investigat-
ed compounds were prepared in normal saline (0.9% NaCl).
Each compound was initially solubilized in DMSO; however,
each final dilution contained less than 1% DMSO. Animals
were divided into six groups each consisting of six mice. One
of these groups served as control and received normal saline.
The other groups were subjected to acute intraperitoneal
toxicity study using different doses (1000, 500, 250, 100, and
50 mg/kg) of the tested compounds. All the animals had free
access to food and water after drug administration and they
were observed for toxic symptoms, signs of poisoning, and
mortality continuously after dosing. Finally, the number of
survivors in each group was noted after 24 h [61]. The
arithmetical method of Karber was used for the determina-
tion of LD50 values (mg/kg) of the tested compounds [72]. The
results are means of two experiments (Table 5).
In vitro cytotoxicity testing
Compounds 3b,c, 5a, 6a, 10a, and 12a,b were screened for
in vitro cytotoxic activity against human normal lung
fibroblast (W138) cell line adopting MTT assay [57–59] and
using 5-fluorouracil as a reference cytotoxic agent. The cell
line was obtained from ATCC via Holding company for
biological products and vaccines (VACSERA), Agouza, Giza,
Egypt. Cells were cultured in RPMI-1640 medium with 10%
fetal bovine serum, penicillin (100 U/mL), and streptomycin
(100 mg/mL) at 37°C in an atmosphere of 5% CO2 were
added. The reagents, RPMI-1640 medium, MTT, DMSO 100%,
and 5-fluorouracil, were obtained from Sigma–Aldrich Co.
(USA).
W138 cells were placed in a 96-multiwell microtiter plate
(104 cells/well) for 24 h at 37°C and in an atmosphere of 5%
CO2 before treatment with the compounds to allow attach-
ment of the cells to the wall of the plate. The tested
compounds were dissolved in DMSO and diluted with
phosphate buffer solution (PBS) to obtain six different
concentrations (5000, 2500, 1000, 500, 250, and 100 mg/mL).
Test compounds of different concentrations were added to
each well and cells were incubated with the compounds for
48 h at 37°C in an atmosphere of 5% CO2. Triplicate wells were
prepared for each individual dose. The treated cells were
washed with PBS and 100 mL of 3-(4,5-dimethylthiazol-2-yl)-
2,5-diphenyltetrazolium bromide solution (MTT) (5 mg/mL
MTT stock in PBS diluted to 1 mg/mL with 10% RPMI-1640
medium) was added. The absorbance was measured at 570 nm
using a microplate reader (EXL 800) [57–59]. The relative
percentage cell viability was calculated from the following
DNA-binding assay on TLC plates
DNA was obtained by extraction of genomic DNA from
human sperm cells. TLC plates, Silica gel 60 F254 pre-coated,
were obtained from E. Merck Co. (Germany).
TLC plates were pre-developed with methanol/water (8:2).
The tested compounds, 3b,c, 5a, 6a, 10a, and 12a,b, were then
applied (5 mg/mL in methanol) at the origin, followed by the
addition of DNA (1 mg/mL in methanol/water mixture) at the
same positions at the origin. The plates were then developed
with the same solvent system and the position of DNA was
determined by spraying with anisaldehyde reagent [62].
The reagent yields a blue color spot with DNA, and the
intensity of the color was proportional to the quantity of DNA
added to the plate. Ethidium bromide was used as a positive
control.
Thanks to Mr. Ahmed Abbas, a technician at Faculty of
Pharmacy, Mansoura University, Egypt, for performing the in
vivo acute toxicity testing and DNA-binding assay.
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N. S. El-Goharya and M. I. Shaaban
Archiv der Pharmazie
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