Design, synthesis, antimicrobial evaluation and molecular docking studies of some new thiophene, pyrazole and pyridone derivatives bearing sulfisoxazole moiety

Article abstract of DOI:10.1016/j.ejmech.2014.07.052

Development of new antimicrobial agents is a good solution to overcome drug-resistance problems. In this context, new functionalized thiophene, acrylamide, arylhydrazone, pyrazole and pyridone derivatives bearing sulfisoxazole moiety were designed, synthesized and evaluated for their in vitro antibacterial and antifungal activities. Among the synthesized compounds, thiophene 4d and 6-thioglucosylpyridone 17 displayed significant antibacterial activities against Escherichia coli (MIC, 0.007 μg/mL vs gentamycin 1.95 μg/mL) and Bacillis subtilis (MIC, 0.007 μg/mL vs ampicillin 0.24 μg/mL), respectively. Whereas, the pyrazole 6 showed the highest antifungal activity against Aspergillus fumigates (MIC, 0.03 μg/mL vs amphotericin B 0.12 μg/mL). In general, most of the synthesized compounds exhibited better antimicrobial activities than sulfisoxazole; this might be attributed to the synergistic effect of the sulfonamide and attached heterocyclic moieties as well as the increased lipophilic characters of the synthesized compounds. Molecular docking studies indicated that the synthesized compounds could occupy both p-amino benzoic acid (PABA) and pterin binding pockets of the dihydropteroate synthase (DHPS), suggesting that the target compounds could act by the inhibition of microbial DHPS enzyme. The results provide important information for the future design of more potent antimicrobial agents.

Full text of DOI:10.1016/j.ejmech.2014.07.052

European Journal of Medicinal Chemistry 84 (2014) 491e504
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis, antimicrobial evaluation and molecular docking
studies of some new thiophene, pyrazole and pyridone derivatives
bearing sulfisoxazole moiety
,
,
Tamer Nasr a *, Samir Bondock b c, Sameh Eid d
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Helwan University, 11795 Helwan, Cairo, Egypt
b Department of Chemistry, Faculty of Science, Mansoura University, ET-35516 Mansoura, Egypt
c Department of Chemistry, Faculty of Science, King Khalid University, 9004 Abha, Saudi Arabia
d BioMed X Innovation Center, Im Neuenheimer Feld 583, 69120 Heidelberg, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Development of new antimicrobial agents is a good solution to overcome drug-resistance problems. In
this context, new functionalized thiophene, acrylamide, arylhydrazone, pyrazole and pyridone de-
rivatives bearing sulfisoxazole moiety were designed, synthesized and evaluated for their in vitro anti-
bacterial and antifungal activities. Among the synthesized compounds, thiophene 4d and 6-
thioglucosylpyridone 17 displayed significant antibacterial activities against Escherichia coli (MIC,
Received 5 May 2014
Received in revised form
13 July 2014
Accepted 16 July 2014
Available online 16 July 2014
0.007
respectively. Whereas, the pyrazole 6 showed the highest antifungal activity against Aspergillus fumigates
(MIC, 0.03 g/mL vs amphotericin B 0.12 g/mL). In general, most of the synthesized compounds
mg/mL vs gentamycin 1.95 mg/mL) and Bacillis subtilis (MIC, 0.007 mg/mL vs ampicillin 0.24 mg/mL),
Keywords:
m
m
Antimicrobial agents
Molecular docking
Sulfonamide
Thiophene
exhibited better antimicrobial activities than sulfisoxazole; this might be attributed to the synergistic
effect of the sulfonamide and attached heterocyclic moieties as well as the increased lipophilic characters
of the synthesized compounds. Molecular docking studies indicated that the synthesized compounds
could occupy both p-amino benzoic acid (PABA) and pterin binding pockets of the dihydropteroate
synthase (DHPS), suggesting that the target compounds could act by the inhibition of microbial DHPS
enzyme. The results provide important information for the future design of more potent antimicrobial
agents.
Pyridone
© 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
antimicrobial pharmacophores are often combined together in one
molecule to get powerful synergistic effect [7].
Many drug-resistant human pathogenic microbes have been
observed in the past few decades [1]; the reasons are the misuse
and widespread use of antimicrobial agents as well as inaccurate
diagnosis [2]. Among these drug-resistant microbes are methicillin-
resistant Staphylococcus aureus, vancomycin-resistant Enterococci
and azole-resistant Candida species [3]. Treatment of these in-
fections is a major impediment especially in immunocompromised
patients [4]; to overcome this problem we need to search for new
powerful antimicrobial agents [5]. The discovery of completely new
antimicrobial pharmacophore and modifying the structure of a well
known antimicrobial agent are the main two strategies to accom-
plish this [6]. In the second strategy; two or more different
Sulfonamides have diverse biological activities including anti-
bacterial [8], carbonic anhydrase inhibitor [9], insulin release
inducer [10], antiviral [11], antifungal [12], anticancer [13], and
anti-inflammatory activities [14]. The antimicrobial sulfonamides
act as competitive inhibitors to PABA substrate for the DHPS
enzyme active site and thus inhibit the biosynthesis of dihydrofolic
acid [15]. DHPS facilitate the biosynthesis of the folate intermedi-
ate, 7,8-dihydropteroic acid, from PABA and dihydropterin-6-
hydroxymethyl pyrophosphate (DHPP). Despite the relative abun-
dance of DHPS crystal structures in the Protein Data Bank (PDB),
only two structures to-date were solved with a sulfa-related drug in
the active site of the enzyme. Yun et al., reported the crystal
structure of Bacillus anthracis dihydropteroate synthase (BaDHPS)
bound to sulfathiazole-6-hydroxymethyl-7,8-dihydropterin-pyro-
phosphate (STZ-DHPP) adduct. They showed that STZ-DHPP adduct
occupies both the PABA and pterin binding pockets of HDPS (Fig. I,
* Corresponding author.
E-mail address: tamerhefni@yahoo.com (T. Nasr).
http://dx.doi.org/10.1016/j.ejmech.2014.07.052
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

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T. Nasr et al. / European Journal of Medicinal Chemistry 84 (2014) 491e504
Fig. 1. Design of antimicrobial DHPS inhibitors.
supplementary data) [16]. The Co-administration of sulfonamides
and the dihydrofolate reductase inhibitor, trimethoprim, increases
the therapeutic efficacy [17].
chloroacetonitrile, was evaluated. Thus, treatment of an ethanolic
sodium ethoxide solution of compound with series of
3
a
substituted isothiocyanates, namely, methyl-, ethyl-, allyl-,
adamantly- and phenyl-isothiocyanates, at room temperature fol-
lowed by addition of chloroacetone or chloroacetonitrile afforded,
in each case, a single product, in moderate yields (55e65%).
Elemental analyses and spectral data (IR, MS, 1H and 13C NMR
spectra) confirmed the reaction products as 4-aminothiophene-3-
carboxamides 4aef. The one-pot formation of the functionalized
thiophenes 4aef starting from activated nitrile, isothiocyanates,
and haloalkanes is in the line of the work reported earlier by
Gewald et al. [24].
Literature survey revealed that thiophene, acrylamide, arylhy-
drazone, pyrazole and 2-pyridone derivatives are important scaf-
folds in pharmacologically active compounds. Regarding thiophene
ligands, their metal complexes possess antimicrobial activities [18].
For cyanothiophene scaffolds, they act as MurF ligase inhibitor
which prevents the bacterial peptidoglycan biosynthesis [19].
Interestingly; several cyanoacrylamide [20], arylhydrazone [21],
pyrazole [22], and 2-pyridone derivatives exhibit promising anti-
microbial activities [23].
Sulfisoxazole is an available sulfa drug acting as PABA compet-
itive inhibitor. Here we aimed to substitute its primary amino group
by flexible antimicrobial pharmacophores to occupy both the PABA
and pterin binding pockets for the DHPS enzyme (Fig. 1) to get new
potent antimicrobial agents.
Based on the above considerations and as extension of our
search for effective antimicrobial agents, we designed and syn-
thesized new thiophene, acrylamide, arylhydrazone, pyrazole and
pyridone compounds tagged with sulfisoxazole moiety. The syn-
thesized compounds were evaluated in vitro for their antimicrobial
activities against human pathogenic microbes. Molecular docking
and lipophilicity studies were used to explain the obtained bio-
logical data.
a,b-Unsaturated nitriles are versatile intermediates and were
utilized for the synthesis of functionalized aminopyrazoles. In this
context, the Knoevenagel condensation of compound 3 with aro-
matic and heteroaromatic aldehydes was investigated. Thus,
treatment of 3 with various types of aldehydes in refluxing ethanol
containing a catalytic amount of piperidine produced, in each case,
a single stereoisomer, identified as (E)-2-cyano-3-aryl-acrylamides
5aee (Scheme 2). The E-configuration of compound 5a, as repre-
sentative example, was assigned based on its 1H NMR spectrum
which displayed a downfield singlet signal at d 8.34 ppm due to the
olefinic (CH]) proton that agrees with the chemical shift of the
olefinic proton (8.36 ppm) of similar structure, (E)-N-(pyridin-2-
yl)-2-cyano-3-phenylprop-2-enamide, confirmed by X-ray anal-
ysis [25].
2. Results and discussion
Treatment of (E)-2-cyano-3-(4-methoxyphenyl)acrylamide 5b
with hydrazine hydrate in ethanol, under reflux, furnished the
functionalized pyrazole derivative 6. Formation of pyrazole 6 is
believed to proceed through the Michael addition of hydrazine
2.1. Chemistry
The synthetic strategies adopted for constructing the target
molecules are illustrated in Schemes 1e3. The starting material, 2-
cyano-N-(4-{[(3,4-dimethylisoxazole-5-yl)amino]sulfonyl}-
phenyl)acetamide (3), was prepared in a quantitative yield by
hydrate to a,b-unsaturated nitrile 5b and in situ intramolecular 1,5-
dipolar cyclization via the addition of amino group to a cyano
function to give the non-isolable dihydropyrazole which under-
went auto-oxidation to give the target pyrazole. The pyrazole for-
mation is in the line with our previous report [26].
cyanoacetylation of sulfisoxazole
1 with 1-cyanoacetyl-3,5-
dimethylpyrazole (2) in refluxing dry dioxan (Scheme 1). The
reactivity of a CH2 group of compound 3, with various types of
substituted isothiocyanates and each of chloroacetone and
Treatment of 3 with dimethylformamide-dimethylacetal (DMF-
DMA), in dry dioxan, at reflux temperature, afforded a yellow
product
identified
as
(E)-2-cyano-3-(N,N-dimethylamino)

T. Nasr et al. / European Journal of Medicinal Chemistry 84 (2014) 491e504
493
Scheme 1. Synthetic route to functionalized thiophenes tagged with sulfisoxazole.
Scheme 2. Synthetic route to functionalized pyrazoles bearing sulfisoxazole moiety.

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T. Nasr et al. / European Journal of Medicinal Chemistry 84 (2014) 491e504
Scheme 3. Synthesis of functionalized pyridones bearing sulfisoxazole moiety.
acrylamide 7. The E-configuration of enaminonitrile
7
was
d 5.72 ppm characteristic for the CH-proton of the CH-azo com-
confirmed according to the chemical shift of a methine proton of
similar E-enaminonitriles reported earlier by Yogavel et al. [27].
In addition, the reactivity of enaminonitrile 7 with nitrogen
binucleophile was investigated. Thus, when enaminonitrile 7 was
treated with hydrazine hydrate in refluxing ethanol, it afforded a
single product identified as 5-aminopyrazole 8. The IR spectrum of
compound 8 was free of absorption band corresponding to a nitrile
function and showed bands corresponding to amino group, in
addition to a strong absorption band corresponding to a conjugated
carbonyl group. Its mass spectrum revealed a molecular ion peak at
m/z 376 (Mþ) corresponding to a molecular formula C15H16N6O4S.
In addition, inspection of 1H NMR spectrum enabled establishing
structure 8 for this pyrazole derivative since the pyrazole H-3
pounds [28]. It is worth mentioning that the arylhydrazones 9aed
can exist in, two geometric structures, E- and Z-configuration. The
preference formation of E-configuration was elucidated from their
1H NMR spectra which displayed the hydrazone proton (C]NeNH)
signal at
hydrazone proton of Z-configuration has been reported to resonate
more downfield at
d
¼ 11.90e12.05 ppm while the chemical shift of the
d
¼ 13.83e13.92 ppm [29].
Alkylation of hydrazonyl nitrile 9d with chloroacetonitrile in
boiling ethanol containing a catalytic amount of triethylamine
afforded
a single product identified as 2-cyano-2-[(cyano-
methyl)(4-ethoxyphenyl)hydrazono]-N-(4-{[(3,4-
dimethylisoxazol-5-yl) am-ino]sulfonyl}phenyl)acetamide (10).
The base catalyzed Thorpe-Ziegler cyclization of compound 10 led
to the formation of 4-amino-3-cyano-1H-pyrazole-3-carboxamide
derivative 11.
appeared as a singlet at
d
8.23 ppm. We could not trace in the 1H
NMR spectrum any signals for the tautomeric 3-aminopyrazole as
this could reveal pyrazole-H5 as a doublet signal. Formation of
compound 8 is assumed to take place via a Michael type addition of
the amino group of the hydrazine to the activated double bond in
compound 7 to form the non-isolable intermediate which readily
undergoes intramolecular cyclization followed by loss of dime-
thylamine molecule to form the target compound (Scheme 2).
The behavior of compound 3 towards diazotized aromatic
amines as potential precursors for 4-aminopyrazoles was also
investigated. Thus, coupling of compound 3 with a series of diaz-
otized aromatic amines in pyridine furnished the arylhydrazones
9aed in excellent yields. The formation of arylhydrazones rather
than arylazo derivatives (CH-azo) was confirmed by IR spectra. In
addition, their 1H NMR spectra were free of a singlet peak at
It is well known that the reaction of N-hetaryl-2-
cyanoacetamides with either 1,3-dicarbonyl compounds or
a,b-
unsaturated nitriles represent a facile and efficient synthetic route
to attain functionalized pyridone derivatives [25,30]. In this
context, we investigated the reaction of compound 3 with each of
acetylacetone, arylidene malononitriles and arylidene cyanothioa-
cetamides (Scheme 3). Thus, reaction of equimolar amount of the
cyanoacetamide 3 and acetylacetone in boiling ethanol containing
a catalytic amount of piperidine afforded 2-pyridone 12. On the
other hand, treatment of 3 with, Michael acceptor, 2-cyano-3-(4-
methoxyphenyl)acrylonitrile, in ethanolic sodium ethoxide solu-
tion, under reflux, furnished the functionalized pyridone derivative
15. Compound 15 was assumed to be formed via an initial Michael-

T. Nasr et al. / European Journal of Medicinal Chemistry 84 (2014) 491e504
495
type adduct 13 followed by an intramolecular cyclization to the
final product 15 (Scheme 3).
against the following fungal strains; Aspergillus fumigatus (RCMB
02568), Syncephalastrum racemosum (RCMB 05922) and Geotricum
candidum (RCMB 05097). Agar-diffusion method was used for the
preliminary screening of the antibacterial and antifungal activities.
Ampicillin, gentamycin, amphotericin B and sulfisoxazole were
used as reference drugs. The results were recorded for each tested
compound as the average diameter of inhibition zones (IZ) of mi-
crobial growth around the disks in mm and were attributed to the
original tested concentration (5 mg/mL) as a preliminary test. The
minimum inhibitory concentration (MIC) measurements were
determined for compounds showed significant growth inhibition
zones (>10 mm) using twofold serial dilution method [31].
In a similar manner, compound 3 was reacted with a series of
arylidene cyanothioacetamides under the same experimental
condition to give the functionalized pyridonethiol derivatives
16aed. Elemental analysis and spectral data (IR, MS, 1H, and 13C
NMR) confirmed the structure of the latter products. Additionally,
the elucidation of structures 16aed was supported chemically
through its alternative synthesis from the reaction of acrylamides
5aed with cyanothioacetamide in refluxing ethanol and in the
presence of a catalytic amount of triethylamine.
Finally, coupling of the pyridonethiol 16b with, activated sugar,
20,30,40,60-tetra-O-acetyl-
a-D-glucopyranosyl bromide in refluxing
acetone containing a catalytic amount of anhydrous potassium
carbonate afforded the respective thioglycoside 17 in a moderate
yield (66%). The structure of thioglycoside 17 was established on
the basis of its elemental analysis and spectral data. The 1H NMR
spectrum of 17 showed the anomeric proton as a doublet at
2.2.1. Antibacterial activity
Preliminary antibacterial screening was carried out for most of
the newly synthesized compounds and the results were summa-
rized in Table 1.
Some of the thiophene derivatives 4aef (Scheme 1) displayed
significant antibacterial activities. It was observed that the 2-
allylamino- and 2-adamantylamino-thiophenes 4c and 4d were
the most potent among their series; they showed one to three folds
the activity of sulfisoxazole against both Gram-positive and Gram-
negative bacteria. Interestingly, the thiophene 4d was more potent
0
0
d
6.10 ppm with a coupling constant (J1 ,2 ¼ 9.8 Hz) corresponding
to a trans orientation of H-10 and H-20 protons indicating the
configuration (Scheme 3).
b-
2.2. Antimicrobial evaluation
than gentamycin against E. coli (MIC, 0.007
and equipotent to ampicillin against B. subtilis (MIC, 0.24
m
g/mL vs 1.95
m
m
g/mL)
g/mL)
The antimicrobial screening and minimal inhibitory concentra-
tions of the tested compounds were carried out at the Regional
Center for Mycology and Biotechnology, Al-Azhar University, Cairo,
Egypt. Twenty two of the newly synthesized target compounds
were evaluated for their in vitro antimicrobial activities against the
human pathogens Streptococcus pneumoniae (RCMB 010010), Ba-
cillis subtilis (RCMB 010067) and Staphylococcus epidermidis (RCMB
010024) as examples of Gram-positive bacteria and Escherichia coli
(RCMB 010052), Proteous vulgaris (RCMB 010085) and Klebsiella
pneumonia (RCMB 010093) as examples of Gram-negative bacteria.
They were also evaluated for their potential antifungal activities
(Table 1). Moreover, it was active against P. vulgaris taking advan-
tage over sulfisoxazole. On the other hand, the thiophene 4c was
one fold more potent than ampicillin against B. subtilis (MIC,
0.12 mg/mL vs 0.24 mg/mL).
Regarding the antibacterial activities of acrylamides, the 4-
chlorophenyl- and 3-pyridyl-derivatives 5c, 5e were the most
active among this series 5aee (Scheme 2), their MIC values were
(0.03e0.98
(0.06e3.9
amides 5c and 5e were eight folds more potent than ampicillin
mg/mL) against Gram-positive bacteria and
mg/mL) against Gram-negative bacteria (Table 1). Acryl-
Table 1
Antibacterial inhibition zone in mm standard deviation and minimal inhibitory concentrations (MIC, mg/ml, between brackets) of some new synthesized compounds.
Compounds
Gram-positive bacteria
Gram-negative bacteria
S. pneumoniae
B. subtilis
S. epidermidis
E. coli
P. vulgaris
K. pneumonia
4a
4c
4d
4f
5a
5c
5d
5e
6
7
8
9a
9c
9d
11
12
16.6 0.38 (16.63)
20.6 0.34 (0.98)
21.6 0.28 (0.98)
15.2 0.44 (62.5)
18.4 0.29 (7.81)
20.3 0.44 (0.98)
16.9 0.44 (15.63)
24.6 0.43 (0.06)
22.0 0.33 (0.49)
20.0 0.43 (1.95)
17.8 0.44 (7.81)
15.5 0.37 (62.5)
17.3 0.63 (15.63)
19.6 0.63 (1.95)
20.0 0.43 (1.95)
23.8 0.44 (0.12)
20.3 0.44 (0.98)
20.6 0.63 (0.98)
22.3 0.43 (0.49)
22.6 0.63 (0.24)
21.3 0.63 (0.98)
25.1 0.44 (0.06)
19.6 0.44 (1.95)
23.8 0.2 (0.12)
NT
19.1 0.41 (3.90)
23.7 0.25 (0.12)
22.9 0.24 (0.24)
17.4 0.25 (15.63)
20.3 0.41 (1.95)
25.6 0.63 (0.03)
17.4 0.25 (15.63)
26.2 0.58 (0.03)
20.4 0.36 (1.95)
21.4 0.72 (0.98)
18.0 0.67 (7.81)
18.4 0.53 (7.81)
21.2 0.44 (0.98)
20.0 0.30 (1.95)
19.6 0.53 (1.95)
25.3 0.58 (0.06)
22.2 0.67 (0.49)
22.4 0.44 (0.98)
24.4 0.53 (0.12)
23.6 0.67 (0.12)
22.6 0.58 (0.24)
29.8 0.58 (0.007)
20.8 0.63 (0.98)
32.4 0.3 (0.24)
NT
18.7 0.68 (7.81)
19.3 0.63 (3.90)
20.9 0.39 (0.98)
14.2 0.63 (125)
19.1 0.56 (3.90)
22.3 0.63 (0.49)
16.2 0.53 (31.25)
22.3 0.53 (0.49)
18.9 0.46 (3.90)
18.2 0.58 (7.81)
16.1 0.32 (31.25)
19.8 0.58 (1.95)
20.2 0.44 (1.95)
17.6 0.53 (7.81)
18.9 0.58 (3.90)
21.4 0.53 (0.98)
18.1 0.53 (7.81)
19.9 0.53 (1.95)
20.2 0.53 (0.49)
21.4 0.32 (0.98)
20.0 0.63 (1.95)
24.2 0.58 (0.12)
17.5 0.72 (7.81)
25.4 0.18 (0.06)
NT
17.1 0.37 (15.63)
18.3 0.58 (7.81)
29.9 0.51 (0.007)
11.2 0.33 (500)
20.6 0.37 (1.95)
22.1 0.25 (0.49)
14.9 0.44 (0.12)
23.7 0.63 (0.12)
17.8 0.36 (7.81)
16.3 0.33 (31.25)
13.0 0.46 (125)
13.5 0.43 (125)
15.9 0.37 (31.25)
15.9 0.46 (31.25)
16.4 0.58 (31.25)
20.9 0.72 (0.98)
16.2 0.46 (31.25)
20.6 0.37 (0.98)
20.6 0.25 (0.98)
21.6 0.46 (0.98)
19.2 0.58 (3.90)
23.2 0.19 (0.24)
15.2 0.25 (62.5)
NTb
18.2 0.50 (3.90)
16.9 0.31 (15.63)
18.9 0.63 (3.90)
20.3 0.34 (0.98)
13.6 0.63 (125)
21.9 0.30 (0.49)
23.9 0.61 (0.06)
15.0 0.58 (0.12)
24.2 0.72 (0.12)
17.1 0.58 (15.63)
17.1 0.58 (15.63)
14.6 0.46 (125)
19.1 0.44 (3.90)
20.2 0.58 (1.95)
16.3 0.58 (31.25)
25.1 0.44 (0.06)
21.4 0.63 (0.98)
22.3 0.53 (0.49)
21.4 0.58 (0.98)
23.2 0.44 (0.24)
22.6 0.46 (0.24)
21.0 0.58 (0.98)
25.8 0.72 (0.03)
18.2 0.58 (7.81)
NT
_a
17.4 0.45 (15.6)
_
18.0 0.48 (7.81)
19.2 0.63 (3.90)
_
19.3 0.58 (3.9)
19.6 0.50 (1.95)
_
11.0 0.46 (500)
_
_
_
18.2 0.44 (7.81)
19.5 0.53 (1.85)
17.2 0.58 (15.63)
18.6 0.44 (3.90)
19.3 0.53 (3.90)
18.3 0.46 (7.81)
17.6 0.72 (7.81)
15
16a
16b
16c
16d
17
Sulfisoxazole
Ampicillin
Gentamycin
21.2 0.58 (0.98)
_
NT
19.9 0.3 (1.95)
23.4 0.3 (0.24)
26.3 0.15 (0.03)
a
No activity.
NT, Not tested.
b

496
T. Nasr et al. / European Journal of Medicinal Chemistry 84 (2014) 491e504
Table 2
Antifungal inhibition zone in mm standard deviation and minimal inhibitory concentrations (MIC,
m
g/ml, between brackets) of some new synthesized compounds.
Compounds
Fungi
Compounds Fungi
A. fumigatus
S. racemosum
G. candidum
A. fumigatus
S. racemosum
G. candidum
4a
4c
4d
4f
5a
5c
5d
5e
15.8 0.33 (62.5)
20.6 0.63 (0.98)
25.3 0.28 (0.06)
16.3 0.25 (31.25)
17.1 0.53 (15.63)
20.3 0.39 (0.98)
13.9 0.29 (125)
23.4 0.24 (0.49)
26.1 0.29 (0.03)
18.3 0.34 (7.81)
18.6 0.25 (7.81)
23.7 0.1 (0.12)
14.1 0.48 (62.5)
21.1 0.27 (0.98)
26.9 0.24 (0.007)
15.2 0.58 (62.5)
18.8 0.42 (7.81)
21.6 0.16 (0.98)
12.1 0.18 (500)
18.7 0.62 (3.90)
24.4 0.34 (0.12)
21.1 0.25 (0.98)
17.2 0.34 (15.63)
19.7 0.2 (7.81)
16.4 0.26 (31.25)
21.9 0.35 (0.49)
24.6 0.39 (0.12)
17.3 0.17 (15.63)
20.9 0.31 (0.98)
24.6 0.58 (0.12)
14.6 0.35 (62.5)
25.6 0.16 (0.03)
13.6 0.46 (125)
21.3 0.38 (0.98)
18.9 0.58 (7.81)
28.7 0.2 (0.007)
9a
9c
9d
11
12
15
16a
16b
16c
16d
17
16.5 0.36 (15.63)
16.3 0.44 (31.25)
19.3 0.44 (3.90)
23.0 0.34 (0.24)
22.9 0.44 (0.24)
20.6 0.25 (0.98)
20.4 0.34 (0.98)
21.6 0.58 (0.98)
22.0 0.36 (0.49)
21.2 0.58 (0.98)
22.3 0.25 (0.49)
17.3 0.53 (15.63)
18.6 0.58 (3.90)
18.6 0.36 (3.90)
24.6 0.52 (0.06)
20.3 0.58 (1.95)
19.7 0.34 (1.95)
16.3 0.52 (31.25)
20.2 0.25 (1.95)
20.6 0.44 (0.98)
19.2 0.63 (3.90)
20.3 0.25 (3.90)
13.2 0.58 (125)
17.8 0.38 (7.81)
19.8 0.25 (1.95)
21.4 0.58 (0.98)
21.9 0.53 (0.49)
23.1 0.37 (0.24)
20.9 0.58 (0.98)
22.4 0.31 (0.49)
22.8 0.38 (0.24)
24.3 0.58 (0.12)
6
7
8
22.0 0.37 (0.49)
25.8 0.58 (0.03)
16.8 0.58 (15.63)
Amphotericin B
Sulfisoxazole 15.1 0.39 (62.5)
against the growth of B. subtilis (MIC, 0.03
Moreover, 5e displayed doubled potency of ampicillin against
S. pyogenes (MIC, 0.06 g/mL vs 0.12 g/mL). Interestingly, the ac-
rylamides 5ce5e exhibited superior activities than gentamycin
against E. coli (MIC, 0.49 g/mL, 0.12 g/mL and 0.12 g/mL vs
1.95 g/mL; respectively). While, acrylamide 5a was equipotent to
m
g/mL vs 0.24
m
g/mL).
The 2-adamantylaminothiophene 4d (Scheme 1) showed more
potent antifungal activities than the 2-allylaminothiophene 4c. The
former was extremely stronger than amphotericin B against
m
m
S. racemosum (MIC, 0.007
double the potency of amphotericin B against A. fumigatus (MIC,
0.06 g/mL vs 0.12 g/mL).
mg/mL vs 7.81 mg/mL) and displayed
m
m
m
m
m
m
gentamycin against E. coli. Conversion of sulfisoxazole 1 to acryl-
amides 5a, 5c and 5e enhanced the antibacterial activity against
The acrylamides 5aee (Scheme 2) showed varied antifungal
activities. Acrylamide 5c was the most potent derivative; it had
eight folds the activity of amphotericin B against S. racemosum
P. vulgaris. The N,N-dimethyl-aminoacrylamide
7 was almost
equipotent to sulfisoxazole and less potent than ampicillin and
gentamycin. In general, the acrylamides 5aee were more potent
than the thiophenes 4aef against all tested bacteria.
In some cases, the arylhydrazone derivatives 9a, 9c and 9d
(Scheme 2) displayed better activities than sulfisoxazole and were
less potent than ampicillin and gentamycin.
(MIC, 0.98
and 5e showed moderate to weak antifungal activities. N,N-Dime-
thylaminoacrylamide exhibited eight folds the activity of
amphotericin B against S. racemosum (MIC, 0.98 g/mL vs 7.81 g/
mL); However, it was less potent than amphotericin B against
A. fumigatus and G. candidum.
mg/mL vs 7.81 mg/mL). While, the hetaryl acrylamides 5d
7
m
m
The pyrazole 6 (Scheme 2) displayed better antibacterial activ-
ities than sulfisoxasole against most of the tested bacteria. Simi-
larly, the pyrazole 11 was more potent than its corresponding
precursor 9d against the S. epidermidis, P. vulgaris and K. pneumonia.
On the other hand, transformation of the acrylamide 7 to pyrazole 8
reduced the antibacterial activities.
The arylhydrazone derivatives 9a, 9c and 9d showed good
antifungal activities. 4-Ethoxyphenylhydrazones 9c and 9d were
twofold more potent than amphotericin B against S. racemosum
(MIC, 3.9 mg/mL vs 7.81 mg/mL).
The pyrazole 6 showed promising antifungal activities, it was
much more active than amphotericin B against S. racemosum (MIC,
It was noticed that the 2-pyridones 12e17 (Scheme 3) were
more potent than sulfisoxazole against all screened bacteria
(Table 1). The 4,6-dimethylpyridone 12 was four folds stronger than
0.12
m
g/mL vs 7.81
m
g/mL) and four folds more active than
g/mL vs 0.12 g/
amphotericin B against A. fumigatus (MIC, 0.03
m
m
mL). Transformation of arylhydrazone 9d to pyrazole 11 increased
the antifungal activity. Pyrazole 11 exhibited much better anti-
fungal activity than amphotericin B against S. racemosum (MIC,
0.06 mg/mL vs 7.81 mg/mL). On the other hand, the pyrazole 8 was
less potent than its precursor acrylamide 7.
ampicillin against B. subtilis (MIC, 0.06 mg/mL vs 0.24 mg/mL) and
equipotent to ampicillin against S. pneumoniae (Table 1). Moreover,
it showed double the potency of gentamycin against E. coli. Inter-
estingly, the pyridonethiols 16b and 16c showed double potency of
ampicillin in inhibiting the growth of B. subtilis (MIC, 0.12
m
g/mL vs
The antifungal activities of the 2-pyridones 12, 15 and 16b were
four folds more than amphotericin B against S. racemosum (MIC,
0.24 g/mL) while compound 16d was equipotent to ampicillin
m
against the same bacteria. On the other hand, the pyridonethiols
16aec displayed double activity of gentamycin against E. coli. The
results displayed in Table 1 revealed that the pyridones 12 and
16aed had superior activity to the aminopyridone 15 against most
screened bacteria; and only pyridonethiols 16a and 16d were less
potent than the aminopyridone 15 against K. pneumonia. Trans-
formation of pyridonethiols 16d to thioglucoside 17 increased its
antibacterial activities to a great extent. The thioglucoside 17
1.95
eight folds the activity of amphotericin B against S. racemosum
(MIC, 0.98 g/mL vs 7.81 g/mL). Compound 12 showed more
antifungal activities than the pyridonethiols 16aed against
A. fumigatus; it was equipotent to the pyridonethiol 16b and
twofold less potent than the pyridonethiol 16c against G. candidum.
The thioglucoside 17 displayed double potency of amphotrecin B
mg/mL vs 7.81 mg/mL). Moreover, the pyridonethiol 16c showed
m
m
against S. racemosum (MIC, 3.9 mg/mL vs 7.81 mg/mL) and was more
showed excellent activity against B. subtilis (MIC, 0.007
0.24 g/mL for ampicillin) and double the potency of ampicillin
against S. pneumoniae (MIC, 0.06 g/mL vs 0.12 g/mL). Addition-
ally, the thioglucoside 17 showed eight folds the activity of genta-
mycin against E. coli (MIC, 0.24 g/mL vs 1.95 g/mL) and was
m
g/mL vs
potent than its pyridonethiol precursor 16b against A. fumigatus
and G. candidum; moreover, it possessed better antifungal activities
than the other 2-pyridones 12, 15, 16aed against G. candidum.
m
m
m
m
m
2.3. Molecular modeling
equipotent to gentamycin against K. pneumonia.
2.3.1. Validation of MOE docking methodology
2.2.2. Antifungal activity
The proposed docking algorithm was validated by self-docking
in the BaDHPS crystal structure (PDB code 3TYE) [16], by
removing the bound ligand, STZ-DHPP adduct, from the complex
then docking it back into the binding site. The top ranked pose
Sulfisoxazole showed weaker antifungal activities than
amphotericin B (Table 2) and some of the target compounds
showed promising antifungal activities.

T. Nasr et al. / European Journal of Medicinal Chemistry 84 (2014) 491e504
497
exhibited heavy-atom root-mean-square deviation (RMSD) value of
0.62 Å from the experimental crystal structure (Fig. 2). This result
indicates that Molecular Operating Environment (MOE) docking
can reliably predict docking poses for the studied compounds to
BaDHPS.
such as Met145 and Ile122. In addition to the characteristic network
of interactions at the PABA-pocket, the pyrazole ring in compounds
6 and 8 can form a hydrogen bond to the acidic side chain of Asp101
(Fig. 4). Similarly, compounds in the arylhydrazone series can form
a hydrogen bond to one of the polar residues Asp101 or Asn120 in
the pterin-binding pocket, e.g. compound 9a (Fig. 4).
Docking studies for the 2-pyridone derivatives in Scheme 3
showed that, unlike the derivatives of the other classes, they do
not have good poses with the BaDHPS enzyme. This could be
attributed to the direct link between the pyridone ring and the
sulfisoxazole phenyl ring, resulting in steric clash with binding site
residues. Nevertheless, Yun et al. reported significant flexibility in
the binding pocket of BaDHPS, as evidenced by the rearrangement
of loop 1 and loop 2 to form the PABA binding pocket [16]. There-
fore, bulky or rigid compounds that might not dock well into DHPS
binding pocket could in principle bind to a slightly altered confor-
mation of the enzyme, where pocket residues rearrange to
accommodate the larger substituents. In the present work, docking
studies were performed using a rigid representation of the enzyme
rather than the methods that take receptor flexibility into account,
e.g. induced-fit docking [34]. The reported DHPS binding pocket
conformational changes involve some loop rearrangements [16],
involving back-bone movements, which are more challenging to
account for in docking simulations. Future DHPS enzymatic assay
studies will be carried out to further confirm the target of the re-
ported compounds.
2.3.2. MOE docking results
In general, the top-ranked poses obtained from MOE docking
show that the all studied compounds can interact with BaDHPS in
a manner similar to that observed for the STZ-DHPP covalent
adduct in the solved crystal structure. In addition, the obtained
docking poses generally maintain most of the key interactions
observed in the aforementioned complex. In the thiophene de-
rivatives, for instance compound 4a, the benzene-sulfonamide
moiety occupies virtually the same position observed in the
STZ-DHPPeBaDHPS complex; thereby establishing the charac-
teristic hydrogen bond between its sulfonamide moiety and the
backbone NH group of Ser221 while its phenyl ring packs against
the side chains of Lys220 and Pro69 (Fig. 3). The predicted binding
mode of the benzene-sulfonamide moiety, occupying the p-amino
benzoic acid (PABA) pocket and preventing the key substrate from
binding, is common for all sulfa drugs and is the basis of their
inhibitory action on DHPS [32,33]. In addition to the PABA pocket,
the substituted thiophene moiety of compound 4a extends in to
the pterin-binding pocket establishing hydrogen bonds between
primary amino group and side chain of Lys220 and between the
acetyl oxygen and the side chain of Asn120. Meanwhile, the
thiophene ring itself forms a face-to-edge interaction with the
aromatic side chain of Phe189 (Fig. 3).
2.4. Structure activity relationship (SAR)
It was observed that most of the synthesized N-substituted
sulfisoxazole derivatives have superior antimicrobial activities than
sulfisoxazole itself. The synthesized compounds have higher lipo-
philic characters than sulfisoxazole (Table 3) and hence they have
more intracellular concentration due to their improved cell pene-
tration. Consequently, the synergistic effect of two different phar-
macophores (Fig. 5) and/or their ability to occupy both the PABA
and pterin binding pockets of DHPS enzyme (Figs. 3 and 4) could
account for the good results obtained.
Regarding the thiophene series 4aed (Scheme 1), the larger the
N-alkyl substituents the better antimicrobial activities were
noticed. This could be explained by the increased lipophilic char-
acters of these compounds by increasing the size of the alkyl group
(Table 3).
Predicted binding modes for the acrylamide derivatives show
essentially the same picture in the PABA-binding pocket, where the
sulfonamide group makes the key hydrogen bond to Ser221.
Additionally, the phenyl group in compound 5a makes face-to-edge
interaction with Phe189 (H-to-C distances ranging from 3.0 to
3.6 Å) and cation-p interaction with the positively-charged side
chain of Lys220 (Fig. 3). In the acrylamide series, however, the
terminal aryl substituent is mostly hydrophobic in nature. There-
fore, the interactions in the pterin-binding pocket are dominated by
van der Waals contacts to the hydrophobic side chains of residues
In case of the acrylamide 5cee and 7 (Scheme 2), the synergistic
effect of both the sulfonamide and acrylamide moieties could ac-
count for the good antimicrobial activities observed. Moreover, the
additional synergistic effect of the 3-pyridyl moiety in compound
5e resulted in excellent antimicrobial activities. Additionally, the
presence of the electron withdrawing chlorine atom in the acryl-
amide 5c and the 2-pyridone 16b increased the antimicrobial ac-
tivities of these compounds. On the other hand, the presence of
alkoxyphenyl substituent in pyrazoles 6 and 11 (Scheme 2) and the
replacement of the amino group by the thiol moiety at position 6 in
the 2-pyridone 16a (Scheme 3) increased the Clog P values (Table 3)
and hence their antimicrobial activities. Finally, the insertion of the
thioglucosyl moiety at position 6 in the 2-pyridone 17 significantly
increased the antimicrobial activities.
3. Conclusion
Fig. 2. Self-docking of STZ-DHPP adduct with BaDHPS active site. PDB code 3TYE, (STZ:
sulfathiazole, DHPP: 6-hydroxymethyl-7,8-dihydropterin-pyrophosphate); magenta
carbons: crystal structure pose, green carbons: top-ranked docking pose (red: oxygen,
blue: nitrogen, yellow: sulfur, white: hydrogen). The protein is shown as light gray
cartoons. Key residues in the binding site are shown as light gray sticks. (For inter-
pretation of the references to color in this figure legend, the reader is referred to the
web version of this article.)
New series of thiophene, acrylamide, pyrazole and pyridone
derivatives tagged with sulfisoxazole moieties were synthesized
successfully and evaluated for their in vitro antimicrobial activities.
Most of newly synthesized compounds were more potent than
sulfisoxazole. Moreover, some of the targets exhibited better

498
T. Nasr et al. / European Journal of Medicinal Chemistry 84 (2014) 491e504
Fig. 3. Top-ranked docking poses of compound 4a (left) and compound 5a (right) showing key interactions with BaDHPS active site. Color codes as in Fig. 2. Hydrogen bonds are
shown as red dotted lines. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 4. Top-ranked docking poses of compound 6 (left) and compound 9a (right) showing key interactions with BaDHPS active site. Color codes as in Fig. 2. Hydrogen bonds are
shown as red dotted lines. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
antimicrobial activities than the reference drugs ampicillin, gen-
tamycin and amphotericin B. Docking simulations showed that the
studied compounds can be accommodated in the PABA pocket of
DHPS thereby inhibiting the enzyme in the same manner reported
for sulfa drugs. Additionally, the synergistic effect of combining
sulfonamide and biologically active heterocyclic rings in one
molecule could explain the targets' observed good results. It was
observed that the biological activities of the target compounds
increased as their CLog P values increased. Compounds 4d, 5e, 6
and 17 were the most active antimicrobial agents in this study.
4. Experimental
4.1. Chemistry
Melting points were determined on digital Gallen-Kamp MFB-
595 instrument using open capillary tubes and are uncorrected. IR
spectra were recorded on Schimadzu FTIR 440 spectrometer using
KBr pellets. Mass spectra were performed on Shimadzu Qp-2010
plus mass spectrometer at 70 eV. 1H NMR and 13C NMR spectra
were recorded on a Bruker model (500 MHz or 400 MHz) Ultra
Shield NMR spectrometer in DMSO-d6 using tetramethylsilane
(TMS) as an internal standard; chemical shifts are reported as dppm
units. The elemental analyses were done at the Microanalytical
Center, Cairo University, Cairo, Egypt. Sulfisoxazole 1 and (4-
methoxybenzylidene) malononitrile were purchased from sigma
Aldrich. 1-Cyanoacetyl-3,5-dimethylpyrazole 2 [35] and arylidene
cyanothioacetamides [36] were synthesized according to the re-
ported procedures.
Table 3
Calculated lipophilic characters of the target compounds.
Compounds
Clog Pa
Compounds
Clog Pa
Compounds
Clog Pa
Sulfisoxazole
1.02
1.66
2.10
2.27
3.68
3.97
3.86
2.98
2.87
5c
5d
5e
6
7
8
9a
9b
9c
3.59
2.08
1.90
2.17
1.20
0.53
3.09
3.41
2.99
9d
11
12
3.42
1.58
2.38
1.64
3.41
4.12
2.62
2.43
4.03
4a
4b
4c
4d
4e
4f
15
16a
16b
16c
16d
17
4.1.1. 2-Cyano-N-(4-{[(3,4-dimethylisoxazole-5-yl)amino]sulfonyl}
phenyl)acetamide (3)
To a hot solution of sulfisoxazole 1 (5.34 g, 0.02 mol) in dioxan
(60 mL) was added 1-cyanoacetyl-3,5-dimethylpyrazole 2 (3.26 g,
0.02 mol) and the reaction mixture was refluxed for 6 h. It was
5a
5b
a
Calculated by online program OSIRIS Property Explorer.

T. Nasr et al. / European Journal of Medicinal Chemistry 84 (2014) 491e504
499
Fig. 5. Factors improving the antimicrobial activities.
allowed to cool down to room temperature and the obtained solid
was filtered off, washed with dioxan and recrystallized from
ethanol/DMF to afford the target compound.
3.8), 463 (Mþ, 24.8), 433 (30.1), 175 (6.2), 169 (16.2), 133 (100); Anal.
Calcd. for C19H21N5O5S2 (463.53): C, 49.23; H, 4.57; N, 15.11%,
Found: C, 49.26; H, 4.54; N, 15.07%.
Colorless crystals, yield (82%), mp 241e242 ꢀC; IR (KBr) nmax
/
cmꢁ1: 3263 (NH), 3183 (NH), 3073 (CH-Ar), 2964 (CH-sp3), 2261
(CN), 1699 (CO); 1H NMR (500 MHz, DMSO-d6): dppm ¼ 1.63 (s, 3H,
CH3), 2.09 (s, 3H, CH3), 3.98 (s, 2H, CH2), 7.72e7.77 (m, 4H, CHAr),
10.73 (s, 1H, NHSO2), 10.97 (s, 1H, NHCO); 13C NMR (125 MHz,
DMSO-d6): dppm ¼ 5.8 (CH3), 10.2 (CH3), 27.0 (CH2), 105.1 (CN), 115.5
(Phenyl-C4), 119.0 (2C, Phenyl-C2 and Phenyl-C6), 128.0 (2C, Phenyl-
C3 and Phenyl-C5), 134.3 (Phenyl-C1), 142.5 (Isoxazole-C4), 155.4
(Isoxazole-C3), 161.3 (Isoxazole-C5), 161.9 (CO); MS m/z (%): 335
([MþH]þ, 22.8), 334 (Mþ, 24.7), 223 (100), 175 (30.2), 159 (84.8), 111
(78.8), 96 (16.2), 83 (13.7), 68 (78.2); Anal. Calcd. for C14H14N4O4S
(334.35): C, 50.29; H, 4.22; N, 16.76%, Found: C, 50.33; H, 4.17; N,
16.81%.
4.1.2.2. 5-Acetyl-4-amino-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]
sulfonyl}phenyl)-2-(ethylamino)thiophene-3-carboxamide
(4b).
Yellow powder, yield (51%), mp 140e141 ꢀC; IR (KBr) nmax/cmꢁ1
:
3422e3362 (NH2), 3310 (NH), 3244 (NH), 3170 (NH), 3049 (CH-Ar),
2978 (CH-sp3),1699 (CO),1646 (CO); 1H NMR (500 MHz, DMSO-d6):
dppm ¼ 1.23 (t, J ¼ 7.0 Hz, 3H, CH3),1.70 (s, 3H, CH3), 2.10 (s, 3H, CH3),
2.11 (s, 3H, COCH3), 3.24 (q, J ¼ 7.0 Hz, 2H, CH2N), 7.65 (broad s, 2H,
NH2), 7.71 (d, J ¼ 9.0 Hz, 2H, Phenyl-H2, Phenyl-H6), 7.81 (d,
J ¼ 9.0 Hz, 2H, Phenyl-H3, Phenyl-H5), 8.10 (s, 1H, NH), 9.90 (s, 1H,
NHSO2), 10.94 (s, 1H, CONH); 13C NMR (125 MHz, DMSO-d6):
dppm ¼ 5.9 (CH3), 10.3 (CH3), 13.9 (CH3), 27.9 (CH3CO), 41.5 (CH2N),
99.5, 104.9, 119.1, 119.9, 125.7, 127.4, 133.4 (9C, C6H4, Thiophene-C3,
Thiophene-C4, Thiophene-C5), 143.6 (Isoxazole-C4), 155.5 (Iso-
xazole-C3), 161.4 (Isoxazole-C5), 163.3 (CONH), 164.2 (Thiophene-
C2), 185.4 (CO); MS m/z (%): 478 ([MþH]þ, 45.0), 477 (Mþ, 45.0), 226
(32.5), 211 (32.5), 183 (2.5), 175 (19.7), 57 (100); Anal. Calcd. for
4.1.2. General procedure for the synthesis of functionalized
thiophene derivatives 4aef
An ethanolic solution of sodium ethoxide [prepared from so-
dium metal (0.23 g, 0.01 mol) in 10 mL absolute ethanol] was
added dropwise to a well stirred solution of compound 3 (3.34 g,
0.01 mol) in absolute ethanol (20 mL). After complete addition,
the reaction mixture was stirred at room temperature for 2 h.
Substituted isothiocyanate (0.01 mol) was added and the mixture
was heated on a water-bath at 70 ꢀC for 30 min then cooled to
25 ꢀC. Chloroacetone (0.93 g, 0.01 mol) or chloroacetonitrile
(0.76 g, 0.01 mol) was then added, and the temperature was raised
to 60 ꢀC for 30 min more, followed by addition of an ethanolic
solution of sodium ethoxide [prepared from sodium (0.12 g,
0.005 mol) in 5 mL absolute ethanol] and heating was continued
for additional 3 h. After cooling, water was added and the sepa-
rated product was filtered, dried and recrystallized from ethanol
to afford compounds 4aef.
C20H23N5O5S2 (477.56): C, 50.30; H, 4.85; N, 14.66%, Found: C,
50.28; H, 4.89; N, 14.67%.
4.1.2.3. 5-Acetyl-2-(allylamino)-4-amino-N-(4-{[(3,4-
dimethylisoxazol-5-yl)amino] sulfonyl}phenyl)thiophene-3-
carboxamide (4c). Brown powder, yield (49%), mp 155e156 ꢀC; IR
(KBr) nmax/cmꢁ1: 3417e3361 (NH2), 3314 (NH), 3280 (NH), 3193
(NH), 3096 (CH-Ar), 2983 (CH-sp3), 1697 (CO), 1650 (CO); 1H NMR
(500 MHz, DMSO-d6): dppm ¼ 1.71 (s, 3H, CH3), 2.09 (s, 3H, CH3),
2.52 (s, 3H, CH3CO), 3.87 (m, 2H, CH2N), 5.21 (dd, J ¼ 5.0 Hz, 12.0 Hz,
1H, CH2]), 5.28 (dd, J ¼ 5.0 Hz, 12.0 Hz, 1H, CH2]), 5.88 (m, 1H,
CH]), 7.62 (broad s, 2H, NH2), 7.71 (d, J ¼ 9.0 Hz, 2H, Phenyl-H2 and
Phenyl-H6), 7.81 (d, J ¼ 9.0 Hz, 2H, Phenyl-H3 and Phenyl-H5), 8.28
(s, 1H, NH), 9.93 (s, 1H, NHSO2), 10.94 (s, 1H, CONH); 13C NMR
(125 MHz, DMSO-d6): dppm ¼ 5.9 (CH3), 10.3 (CH3), 27.9 (CH3CO),
48.7 (CH2N),100.5, 104.9,105.0,117.0, 119.9, 127.4, 133.3, 133.4, 141.5
(11C, C6H4, eCH]CH2, Thiophene-C3, Thiophene-C4, Thiophene-
C5), 143.6 (Isoxazole-C4), 155.6 (Isoxazole-C3), 161.4 (Isoxazole-C5),
163.3 (CONH), 164.3 (Thiophene-C2), 185.6 (CO); MS m/z (%): 490
([MþH]þ, 9.9), 489 (Mþ, 7.9), 448 (5.9), 446 (3.6), 433 (6.3), 378
(4.4), 238 (5.7), 223 (8.3), 175 (2.3), 167 (100); Anal. Calcd. for
4.1.2.1. 5-Acetyl-4-amino-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]
sulfonyl}phenyl)-2-(methylamino)thiophene-3-carboxamide
(4a).
Brown powder, yield (58%), mp 156e157 ꢀC; IR (KBr) nmax/cmꢁ1
:
3412e3354 (NH2), 3314 (NH), 3247 (NH), 3173 (NH), 3047 (CH-Ar),
2987 (CH-sp3),1698 (CO),1650 (CO); 1H NMR (500 MHz, DMSO-d6):
dppm ¼ 1.63 (s, 3H, CH3), 2.09 (s, 3H, CH3), 2.30 (s, 3H, COCH3), 3.56
(s, 3H, NCH3), 7.70e7.85 (m, 6H, C6H4 and NH2), 8.17 (s, 1H, NH),
9.98 (s, 1H, NHSO2), 10.96 (s, 1H, CONH); MS m/z (%): 464 ([MþH]þ,
C21H23N5O5S2 (489.57): C, 51.52; H, 4.74; N, 14.31%, Found: C, 51.55;
H, 4.78; N, 14.35%.

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T. Nasr et al. / European Journal of Medicinal Chemistry 84 (2014) 491e504
0
0
4.1.2.4. 5-Acetyl-2-(adamantylamino)-4-amino-N-(4-{[(3,4-
dimethylisoxazol-5-yl)amino] sulfonyl}phenyl) thiophene-3-
carboxamide (4d). Yellow powder, yield (61%), mp 262e263 ꢀC;
IR (KBr) nmax/cmꢁ1: 3417e3370 (NH2), 3282 (NH), 3238 (NH), 3165
(NH), 3079 (CH-Ar), 2910 (CH-sp3), 1696 (CO), 1643 (CO); 1H NMR
(500 MHz, DMSO-d6): dppm ¼ 1.61e1.73 (m, 9H, 9 ꢂ Adamantyl-H),
Phenyl-H2 , Phenyl-H6 ), 7.91 (d, J ¼ 9.0 Hz, 2H, Phenyl-H2, Phenyl-
H6), 8.01 (d, J ¼ 9.0 Hz, 2H, Phenyl-H3, Phenyl-H5), 8.34 (s, 1H,
acrylamide-H3), 10.82 (s, 1H, NHSO2), 11.00 (s, 1H, CONH); 13C NMR
(125 MHz, DMSO-d6): dppm ¼ 5.9 (CH3), 10.3 (CH3), 105.1, 106.9,
115.9, 120.3, 127.8, 129.3, 130.1, 131.7, 132.6, 134.8, 142.6 (15C,
0
0
Acrylamide-C2, CN, C6H4, C6 H5 , Isoxazole-C4), 151.5 (Acrylamide-
C3), 155.5 (Isoxazole-C3), 161.1 (Isoxazole-C5), 161.4 (CO); MS m/z
(%): 423 ([MþH]þ, 0.14), 422 (Mþ, 0.24), 326 (1.2), 311 (2.4), 247
(3.4), 171 (1.3), 156 (100), 128 (67.4), 111 (2.1), 90 (5.4), 77 (47.4);
Anal. Calcd. for C21H18N4O4S (422.46): C, 59.70; H, 4.29; N, 13.26%,
Found: C, 59.72; H, 4.33; N, 13.31%.
1.92e2.17 (m, 6H, CH3,
3
ꢂ
Adamantyl-H), 2.25 (s, 3H,
3 ꢂ Adamantyl-H), 2.42 (s, 3H, CH3), 2.50 (s, 3H, CH3CO), 7.61
(broad s, 2H, NH2),7.68 (d, J ¼ 9.0 Hz, 2H, Phenyl-H2 and Phenyl-H6),
7.78 (d, J ¼ 9.0 Hz, 2H, Phenyl-H3 and Phenyl-H5), 8.07 (s, 1H, NH),
9.49 (s,1H, NHSO2),10.83 (s,1H, CONH); MS m/z (%): 583 (Mþ, 72.5),
582 (50.4), 568 (47.8), 408 (51.3), 332 (71.7), 294 (60.2), 231 (100),
175 (16.8), 150 (16.8), 135 (7.1), 96 (21.2); Anal. Calcd. for
4.1.3.2. 2-Cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}
phenyl)-3-(4-methoxylphenyl)-acrylamide (5b). Yellow powder,
yield (91%), mp 175e176 ꢀC; IR (KBr) nmax/cmꢁ1: 3317 (NH), 3205
(NH), 3043 (CH-Ar), 2977 (CH-sp3), 2211 (CN), 1671 (CO); 1H NMR
(500 MHz, DMSO-d6): dppm ¼ 1.63 (s, 3H, CH3), 2.03 (s, 3H, CH3),
C
28H33N5O5S2 (583.72): C, 57.61; H, 5.70; N,12.00%, Found: C, 57.64;
H, 5.75; N, 12.04%.
4.1.2.5. 5-Acetyl-4-amino-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]
sulfonyl}phenyl)-2-(phenylamino)thiophene-3-carboxamide
(4e).
0
0
Yellow powder, yield (58%), mp 188e189 ꢀC; IR (KBr) nmax/cmꢁ1
:
3.88 (s, 3H, OCH3), 7.18 (d, J ¼ 9.0 Hz, 2H, Phenyl-H3 , Phenyl-H5 ),
0
0
7.74 (d, J ¼ 9.0 Hz, 2H, Phenyl-H2 , Phenyl-H6 ), 7.81 (d, J ¼ 9.0 Hz,
2H, Phenyl-H2, Phenyl-H6), 8.04 (d, J ¼ 9.0 Hz, 2H, Phenyl-H3,
Phenyl-H5), 8.25 (s, 1H, Acrylamide-H3), 10.60 (s, 1H, NHSO2), 10.90
(s,1H, CONH); 13C NMR (100 MHz, DMSO-d6): dppm ¼ 6.6 (CH3), 10.8
(CH3), 56.1 (OCH3), 99.8, 103.7, 115.2, 117.1, 120.5, 124.7, 127.8, 133.2,
3418e3362 (NH2), 3287 (NH), 3241 (NH), 3166 (NH), 3062 (CH-Ar),
2928 (CH-sp3),1697 (CO),1647 (CO); 1H NMR (400 MHz, DMSO-d6):
dppm ¼ 1.74 (s, 3H, CH3), 2.17 (s, 3H, CH3), 2.57 (s, 3H, COCH3),
7.40e7.50 (m, 5H, Ar-H), 7.56 (s, 2H, NH2), 7.75 (d, J ¼ 9.0 Hz, 2H,
Phenyl-H2, Phenyl-H6), 7.90 (d, J ¼ 9.0 Hz, 2H, Phenyl-H3, Phenyl-
0
0
H5), 9.86 (s, 1H, NH), 10.30 (s, 1H, NHSO2), 10.99 (s, 1H, CONH); 13
C
138.5, 141.9 (14C, Acrylamide-C2, CN, C6H4, C6 H4 ), 151.3 (Acryl-
amide-C3), 160.4 (Isoxazole-C4), 161.0 (Isoxazole-C3), 161.8 (Iso-
xazole-C5), 163.3 (CO); MS m/z (%): 453 ([MþH]þ, 0.3), 452 (Mþ,
0.2), 356 (0.8), 294 (0.5), 277 (2.4), 201 (0.9), 186 (58.9), 175 (0.3),
158 (10.9), 111 (0.7), 107 (0.8), 84 (100); Anal. Calcd. for
NMR (100 MHz, DMSO-d6): dppm ¼ 6.4 (CH3), 10.8 (CH3), 28.5 (CH3),
105.5 (Thiophene-C3), 119.2, 120.4, 121.0, 124.6, 128.0, 129.6, 129.8,
134.2, 138.5, 141.2 (14C, C6H4, C6H5, Thiophene-C4, Thiophene-C5),
143.9 (Isoxazole-C4), 156.0 (Isoxazole-C3), 158.6 (Isoxazole-C5),
161.8 (CONH), 162.5 (Thiophene-C2), 187.0 (CO); Anal. Calcd. for
C
22H20N4O5S (452.48): C, 58.40; H, 4.46; N, 12.38%, Found: C, 58.42;
H, 4.43; N, 12.35%.
C
24H23N5O5S2 (525.60): C, 54.84; H, 4.41; N, 13.32%, Found: C,
54.80; H, 4.44; N, 13.35%.
4.1.3.3. 2-Cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}
phenyl)-3-(4-chlorophenyl)acrylamide (5c). Yellow powder, yield
(86%), mp 125e126 ꢀC; IR (KBr) nmax/cmꢁ1: 3335 (NH), 3229 (NH),
3067 (CH-Ar), 2993 (CH-sp3), 2227 (CN), 1686 (CO); 1H NMR
(400 MHz, DMSO-d6): dppm ¼ 1.70 (s, 3H, CH3), 2.14 (s, 3H, CH3),
7.60e8.00 (m, 8H, 2 ꢂ C6H4), 8.65 (s, 1H, Acrylamide-H3), 10.85 (s,
1H, NHSO2), 10.05 (s, 1H, CONH); 13C NMR (100 MHz, DMSO-d6):
dppm ¼ 6.4 (CH3), 10.8 (CH3), 105.0, 119.5, 120.0, 120.3, 128.3, 128.6,
130.8, 134.0, 135.6, 142.7, 148.2 (15C, Acrylamide-C2, CN, C6H4,
4.1.2.6. 4-Amino-5-cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]
sulfonyl}phenyl)-2-(phenylamino)thiophene-3-carboxamide
(4f).
Yellowish green powder, yield (52%), mp 141e142 ꢀC; IR (KBr) nmax
/
cmꢁ1: 3405e3348 (NH2), 3306 (NH), 3243 (NH), 3168 (NH), 3052
(CH-Ar), 2976 (CH-sp3), 2183 (CN), 1652 (CO); 1H NMR (400 MHz,
DMSO-d6): dppm ¼ 1.75 (s, 3H, CH3), 2.16 (s, 3H, CH3), 7.35e7.55 (m,
5H, Ar-H), 7.56 (s, 2H, NH2), 7.77 (d, J ¼ 9.0 Hz, 2H, Phenyl-H2,
Phenyl-H6), 7.93 (d, J ¼ 9.0 Hz, 2H, Phenyl-H3, Phenyl-H5), 9.83 (s,
1H, NH), 10.41 (s, 1H, NHSO2), 11.15 (s, 1H, CONH); 13C NMR
(100 MHz, DMSO-d6): dppm ¼ 6.8 (CH3), 10.9 (CH3), 98.1 (Thio-
phene-C5), 103.5 (Thiophene-C3), 111.5 (CN), 116.3, 118.4, 119.9,
120.5, 121.6, 129.1, 129.8, 130.0, 130.9 (13C, C6H4, C6H5, Thiophene-
C4), 143.4 (Isoxazole-C4), 156.6 (Isoxazole-C3), 159.5 (Isoxazole-C5),
161.8, 162.5 (2C, CONH, Thiophene-C2); MS m/z (%): 509 ([MþH]þ,
2.6), 508 (Mþ, 1.5), 493 (2.0), 294 (1.5), 257 (4.5), 111 (10.1), 80
(100); Anal. Calcd. for C23H20N6O4S2 (508.57): C, 54.32; H, 3.96; N,
16.52%, Found: C, 54.35; H, 4.01; N, 16.48%.
0
0
C6 H4 , Isoxazole-C4), 153.9 (Acrylamide-C3), 156.4 (Isoxazole-C3),
160.9 (Isoxazole-C5), 161.8 (CO); MS m/z (%): 456 (Mþ, 0.1), 345
(1.8), 281 (3.9), 266 (2.2), 190 (1.2), 173 (100), 124 (2.1), 111 (7.3), 96
(4.9); Anal. Calcd. for C21H17ClN4O4S (456.90): C, 55.20; H, 3.75; N,
12.26%, Found: C, 55.23; H, 3.78; N, 12.27%.
4.1.3.4. 2-Cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}
phenyl)-3-(2-furyl)-acrylamide (5d). Brown powder, yield (71%),
mp 231-231 ꢀC; IR (KBr) nmax/cmꢁ1: 3326 (NH), 3216 (NH), 3037
(CH-Ar), 2956 (CH-sp3), 2214 (CN), 1681 (CO); 1H NMR (500 MHz,
DMSO-d6): dppm ¼ 1.63 (s, 3H, CH3), 2.02 (s, 3H, CH3), 7.37 (t,
J ¼ 4.0 Hz, 1H, Furan-H4), 7.75 (d, J ¼ 9.0 Hz, 2H, Phenyl-H2, Phenyl-
H6), 7.80 (d, J ¼ 9.0 Hz, 2H, Phenyl-H3, Phenyl-H5), 7.96 (d,
J ¼ 4.0 Hz, 1H, Furan-H5), 8.17 (d, J ¼ 4.0 Hz, 1H, Furan-H3), 8.56 (s,
1H, acrylamide-H3), 10.57 (s, 1H, NHSO2), 10.90 (s, 1H, CONH); 13C
NMR (125 MHz, DMSO-d6): dppm ¼ 6.1 (CH3), 10.3 (CH3), 102.3
(Acrylamide-C2), 116.1, 119.5, 120.0, 127.3, 128.7, 135.6, 135.7, 137.9,
138.2 (11C, CN, C6H4, Furan-C2, Furan-C3, Furan-C4, Furan-C5), 141.2
(Isoxazole-C4), 144.3 (Acrylamide-C3), 156.3 (Isoxazole-C3), 160.4
(Isoxazole-C5), 160.7 (CO); MS m/z (%): 412 (Mþ, 2.8), 345 (0.3), 301
(19.5), 237 (14.9), 146 (100), 118 (12.5), 111 (6.9), 96 (2.3), 80 (3.5),
67 (1.9); Anal. Calcd. for C19H16N4O5S (412.42): C, 55.33; H, 3.91; N,
13.58%, Found: C, 55.37; H, 3.93; N, 13.55%.
4.1.3. General procedure for the synthesis of compounds 5aed
To a solution of compound 3 (3.34 g, 0.01 mol) and piperidine (5
drops) in absolute ethanol (30 mL), aryl/hetaryl aldehyde
(0.01 mol) was added. The reaction mixture was refluxed for 6 h,
then cooled to room temperature and poured onto (100 mL) ice/
water. The obtained solid was filtered off, washed with water and
recrystallized from ethanol/DMF to afford the target compounds.
4.1.3.1. 2-Cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}
phenyl)-3-phenyl-acrylamide (5a). Yellow powder, yield (88%), mp
220e221 ꢀC; IR (KBr) nmax/cmꢁ1: 3329 (NH), 3216 (NH), 3056 (CH-
Ar), 2987 (CH-sp3), 2219 (CN), 1683 (CO); 1H NMR (500 MHz,
DMSO-d6): dppm ¼ 1.66 (s, 3H, CH3), 2.10 (s, 3H, CH3), 7.60e7.65 (m,
0
0
0
3H, Phenyl-H3 , Phenyl-H4 , Phenyl-H5 ), 7.80 (d, J ¼ 9.0 Hz, 2H,

T. Nasr et al. / European Journal of Medicinal Chemistry 84 (2014) 491e504
501
4.1.3.5. 2-Cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}
4.1.6. Synthesis of 5-amino-N-(4-{[(3,4-dimethylisoxazol-5-yl)
phenyl)-3-(3-pyridyl)acrylamide (5e). Yellow powder, yield (69%),
mp 158e159 ꢀC; IR (KBr) nmax/cmꢁ1: 3372 (NH), 3236 (NH), 3045
(CH-Ar), 2963 (CH-sp3), 2195 (CN), 1677 (CO); 1H NMR (500 MHz,
DMSO-d6): dppm ¼ 1.66 (s, 3H, CH3), 2.10 (s, 3H, CH3), 7.74 (t,
J ¼ 5.0 Hz, 1H, Pyridine-H5), 7.79 (d, J ¼ 9.0 Hz, 2H, Phenyl-H2,
Phenyl-H6), 7.94 (d, J ¼ 9.0 Hz, 2H, Phenyl-H3, Phenyl-H5), 8.51 (s,
1H, acrylamide-H3), 8.55 (m, 1H, Pyridine-H6), 8.81 (d, J ¼ 5.0 Hz,
1H, Pyridine-H4), 9.09 (s, 1H, Pyridine-H2), 10.90 (s, 1H, NHSO2),
11.05 (s, 1H, CONH); 13C NMR (125 MHz, DMSO-d6): dppm ¼ 5.8
(CH3), 10.3 (CH3), 105.2 (Acrylamide-C2), 109.5, 115.4, 120.3, 124.6,
127.8, 128.0, 134.9, 137.0, 142.5, 148.2, 150.3, 151.6 (14C, CN, C6H4,
C5H4N, Isoxazole-C4, Acrylamide-C3), 155.4 (Isoxazole-C3), 160.6
(Isoxazole-C5), 161.4 (CO); MS m/z (%): 423 (Mþ, 0.9), 345 (0.3), 312
(4.9), 248 (7.6), 175 (3.7), 175 (3.7), 172 (5.6), 157 (100), 129 (21.5),
111 (23.0), 96 (9.4), 91 (23.3), 78 (53.1); Anal. Calcd. for C20H17N5O4S
(423.45): C, 56.73; H, 4.05; N, 16.54%, Found: C, 56.70; H, 4.01; N,
16.49%.
amino]sulfonyl}phenyl)-1H-pyrazole-4-carboxamide (8)
A solution of compound 7 (0.389 g, 1 mmol) and hydrazine
hydrate (1 mL) in ethanol (30 mL) was refluxed for 4 h then cooled
to room temperature and poured onto (50 mL) ice/water. The ob-
tained solid was filtered off, washed with water and recrystallized
from ethanol to afford the target compound.
Yellow white powder, yield (56%), mp 173e174 ꢀC; IR (KBr) nmax
/
cmꢁ1: 3477 (NH), 3405e3319 (NH2), 3286 (NH), 3201 (NH), 3053
(CH-Ar), 2985 (CH-sp3), 1664 (CO); 1H NMR (500 MHz, DMSO-d6):
dppm ¼ 1.68 (s, 3H, CH3), 2.20 (s, 3H, CH3), 6.08 (s, 2H, NH2), 7.88 (d,
J ¼ 9.0 Hz, 2H, Phenyl-H2, Phenyl-H6), 7.96 (d, J ¼ 9.0 Hz, 2H,
Phenyl-H3, Phenyl-H5), 8.23 (s, 1H, Pyrazole-H3), 8.75 (s, 1H, NH),
9.83 (s, 1H, NHSO2), 10.98 (s, 1H, CONH); MS m/z (%): 376 (Mþ, 2.4),
294 (3.3), 266 (7.5), 175 (100), 111 (12.3), 110 (1.8), 96 (17.0); Anal.
Calcd. for C15H16N6O4S (376.39): C, 47.87; H, 4.28; N, 22.33%, Found:
C, 47.91; H, 4.32; N, 22.36%.
4.1.7. General procedure for the synthesis of compounds 9aed
To a cold solution of compound 3 (1 g, 3 mmol) in pyridine
(20 mL) was added the appropriate arene diazonium salts [pre-
pared by treating a cold solution of aromatic amines (3 mmol) in
6 M HCl (4.5 mL) with sodium nitrite solution (0.21 g, 3 mmol) in
cold water (9 mL)]. The addition was carried out portionwise with
stirring at 0e5 ꢀC over a period of 30 min. After the complete
addition, the reaction mixture was stirred for further 4 h, then kept
in ice-chest for 12 h, and finally diluted with water. The obtained
solid was filtered off, washed with water, air dried, and recrystal-
lized from ethanol/DMF (5:1) to afford the corresponding arylhy-
drazones 9aed.
4.1.4. Synthesis of 5-amino-N-(4-{[(3,4-dimethylisoxazol-5-yl)
amino]sulfonyl}phenyl)-3-(4-methoxyphenyl)-1H-pyrazole-4-
carboxamide (6)
A solution of compound 5b (0.452 g, 0.001 mol) and hydrazine
hydrate (1 mL) in ethanol (30 mL) was refluxed for 2 h then cooled
to room temperature and poured onto (100 mL) ice/water. The
obtained solid was filtered off, washed with water and recrystal-
lized from ethanol to afford the target compound.
Yellowish white powder, yield (67%), mp 161e162 ꢀC; IR (KBr)
nmax/cmꢁ1: 3481 (NH), 3399e3312 (NH2), 3286 (NH), 3208 (NH),
3046 (CH-Ar), 2983 (CH-sp3), 1657 (CO); 1H NMR (500 MHz, DMSO-
d6): dppm ¼ 1.65 (s, 3H, CH3), 2.1 (s, 3H, CH3), 3.78 (s, 3H, OCH3), 6.10
4.1.7.1. 2-Cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}
phenyl)-2-(phenyl-hydrazono)acetamide (9a). Brown powder, yield
(92%), mp 235e236 ꢀC; IR (KBr) nmax/cmꢁ1: 3421 (NH), 3376 (NH),
3247 (NH), 3067 (CH-Ar), 2977 (CH-sp3), 2215 (CN), 1676 (CO); 1H
NMR (400 MHz, DMSO-d6): dppm ¼ 1.65 (s, 3H, CH3), 2.10 (s, 3H,
CH3), 7.16 (m, 1H, Ar-H0), 7.40e7.44 (m, 2H, 2 ꢂ Ar-H0), 7.74e7.78 (m,
5H, NH, Phenyl-H2, Phenyl-H6, 2 ꢂ Ar-H0), 7.98 (d, J ¼ 9.0 Hz, 2H,
Phenyl-H3, Phenyl-H5), 10.29 (s, 1H, NH), 12.1 (s, 1H, NH); 13C NMR
(100 MHz, DMSO-d6): dppm ¼ 6.6 (CH3), 10.7 (CH3), 106.0 (C]N),
108.0 (CN), 112.5, 116.9, 121.0, 125.0, 128.1, 129.6, 135.0, 142.0 (12C,
0
0
(s, 2H, NH2), 7.23 (d, J ¼ 9.0 Hz, 2H, Phenyl-H3 , Phenyl-H5 ), 7.75 (d,
0
0
J ¼ 9.0 Hz, 2H, Phenyl-H2 , Phenyl-H6 ), 7.86 (d, J ¼ 9.0 Hz, 2H,
Phenyl-H2, Phenyl-H6), 7.93 (d, J ¼ 9.0 Hz, 2H, Phenyl-H3, Phenyl-
H5), 8.95 (s,1H, NH), 9.72 (s, 1H, NHSO2),10.93 (s, 1H, CONH); MS m/
z (%): 482 (Mþ, 1.9), 451 (2.3), 371 (2.2), 294 (1.9), 231 (2.2), 175
(0.9), 127 (100), 107 (11.2), 96 (0.9), 77 (74.7); Anal. Calcd. for
C
22H22N6O5S (482.51): C, 54.76; H, 4.60; N, 17.42%, Found: C, 54.73;
H, 4.62; N, 17.46%.
0
0
C6H4, C6 H5 ), 143.0 (Isoxazole-C4), 156.0 (Isoxazole-C3), 161.0 (Iso-
xazole-C5), 163.3 (CO); MS m/z (%): 438 (Mþ, 41.7), 412 (5.7), 361
(3.7), 346 (3.9), 327 (43.2), 263 (44.3), 251 (2.5), 187 (3.8), 172
(58.4), 111 (16.8), 96 (10.8), 92 (49.2), 80 (100); Anal. Calcd. for
4.1.5. Synthesis of 2-cyano-3-(N,N-dimethylamino)-N-(4-{[(3,4-
dimethylisoxazol-5-yl) amino]sulfonyl}phenyl)acrylamide (7)
To a solution of compound 3 (0.6 g, 1.8 mmol) in dry dioxan
(30 mL) was added dimethylformamide-dimethylacetal (0.28 mL,
2.2 mmol) and the mixture was refluxed for 6 h. Then, the solvent
was distilled off under reduced pressure and the residual viscous
liquid was taken in ether and the resulting solid was collected by
filtration, washed with ether, air dried, and finally recrystallized
from ethanol to afford compound 7.
C
20H18N6O4S (438.46): C, 54.79; H, 4.14; N, 19.17%, Found: C, 54.76;
H, 4.16; N, 17.14%.
4.1.7.2. 2-Cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}
phenyl)-2-[(4-methyl phenyl)hydrazono]acetamide (9b). Yellow
powder, yield (83%), mp 248e249 ꢀC; IR (KBr) nmax/cmꢁ1: 3416
(NH), 3369 (NH), 3241 (NH), 3063 (CH-Ar), 2921 (CH-sp3), 2215
(CN), 1668 (CO); 1H NMR (400 MHz, DMSO-d6): dppm ¼ 1.62 (s, 3H,
CH3), 2.10 (s, 3H, CH3), 2.31 (s, 3H, CH3), 7.20 (d, J ¼ 9.0 Hz, 2H,
Yellow powder, yield (71%), mp 158e159 ꢀC; IR (KBr) nmax
/
cmꢁ1: 3307 (NH), 3178 (NH), 3032 (CH-Ar), 2930 (CH-sp3), 2191
(CN), 1675 (CO); 1H NMR (500 MHz, DMSO-d6): dppm ¼ 1.80 (s,
3H, CH3), 2.17 (s, 3H, CH3), 3.25 (s, 3H, NCH3), 3.31 (s, 3H, NCH3),
7.64 (d, J ¼ 9.0 Hz, 2H, Phenyl-H2, Phenyl-H6), 7.86 (d, J ¼ 9.0 Hz,
2H, Phenyl-H3, Phenyl-H5), 7.88 (s, 1H, acrylamide-H3), 9.65 (s,
1H, NHSO2), 10.90 (s, 1H, CONH); 13C NMR (100 MHz, DMSO-d6):
dppm ¼ 6.56 (CH3), 10.4 (CH3), 36.7 (NCH3), 38.2 (NCH3), 108.6
(Acrylamide-C2), 118.8 (CN), 119.5, 119.7, 128.4, 129.3, 144.6, 156.9,
158.4 (9C, C6H4, Acrylamide-C3, Isoxazole-C4, Isoxazole-C3), 161.7
(Isoxazole-C5), 164.4 (CO); MS m/z (%): 389 (Mþ, 1.7), 294 (1.5),
278 (16.8), 214 (12.2), 138 (2.9), 123 (100), 111 (2.1), 96 (7.8), 95
(9.5), 57 (2.4); Anal. Calcd. for C17H19N5O4S (389.43): C, 52.43; H,
4.92; N, 17.98%, Found: C, 52.48; H, 4.96; N, 17.95%.
0
0
0
Phenyl-H3 , phenyl-H5 ), 7.59 (d, J ¼ 9.0 Hz, 2H, Phenyl-H2 , phenyl-
0
H6 ), 7.71 (d, J ¼ 9.0 Hz, 2H, Phenyl-H2, Phenyl-H6), 7.94 (d,
J ¼ 9.0 Hz, 2H, Phenyl-H3, Phenyl-H5), 10.24 (s, 1H, NH), 11.15 (s, 1H,
NH), 11.95 (s, 1H, NH); 13C NMR (100 MHz, DMSO-d6): dppm ¼ 6.3
(CH3), 10.7 (CH3), 20.9 (CH3), 105.6 (C]N), 107.0 (CN), 112.0, 116.9,
121.0, 128.1, 130.0, 134.3, 134.7, 140.0 (12C, 2 ꢂ C6H4), 143.0 (Iso-
xazole-C4), 156.0 (Isoxazole-C3), 160.7 (Isoxazole-C5), 161.8 (CO);
MS m/z (%): 452 (Mþ, 4.1), 437 (6.3), 341 (8.5), 332 (1.0), 186 (1.5),
158 (7.5), 112 (100); Anal. Calcd. for C21H20N6O4S (452.49): C, 55.74;
H, 4.46; N, 18.57%, Found: C, 55.71; H, 4.42; N, 18.58%.

502
T. Nasr et al. / European Journal of Medicinal Chemistry 84 (2014) 491e504
4.1.7.3. 2-Cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}
phenyl)-2-[(4-methoxyphenyl)hydrazono]acetamide (9c).
onto (100 mL) ice/water and neutralized by HCl. The obtained solid
was filtered off, washed with water and recrystallized from ethanol
to afford the target compound.
Yellow powder, yield (88%), mp 244e245 ꢀC; IR (KBr) nmax/cmꢁ1
:
3419 (NH), 3364 (NH), 3231 (NH), 3065 (CH-Ar), 2929 (CH-sp3),
2218 (CN), 1667 (CO); 1H NMR (400 MHz, DMSO-d6): dppm ¼ 1.65 (s,
3H, CH3), 2.10 (s, 3H, CH3), 3.78 (s, 3H, OCH3), 6.99 (d, J ¼ 9.0 Hz, 2H,
Brown powder, yield (72%), mp 163e164 ꢀC; IR (KBr) nmax/cmꢁ1
:
3387e3326 (NH2), 3278 (NH), 3204 (NH), 3034 (CH-Ar), 2987 (CH-
sp3), 2219 (CN), 1671 (CO); 1H NMR (400 MHz, DMSO-d6):
dppm ¼ 1.35 (t, J ¼ 6.8 Hz, 3H, CH3),1.68 (s, 3H, CH3), 2.15 (s, 3H, CH3),
4.11 (q, J ¼ 6.8 Hz, 2H, OCH2), 6.20 (s, 2H, NH2), 6.88 (d, J ¼ 8.8 Hz,
0
0
0
Phenyl-H3 , phenyl-H5 ), 7.69 (d, J ¼ 9.0 Hz, 2H, Phenyl-H2 , phenyl-
0
H6 ), 7.74 (d, J ¼ 9.0 Hz, 2H, Phenyl-H2, Phenyl-H6), 7.97 (m, 3H, NH,
Phenyl-H3, Phenyl-H5), 10.21 (s, 1H, NH), 12.05 (s, 1H, NH); 13C NMR
(100 MHz, DMSO-d6): dppm ¼ 6.3 (CH3), 10.7 (CH3), 55.8 (OCH3),
105.0 (C]N), 106.0 (CN), 111.5, 114.8, 118.4, 120.9, 128.1, 136.0, 137.0,
2H, Phenyl-H3 , phenyl-H5 ), 7.54 (d, J ¼ 8.8 Hz, 2H, Phenyl-H2 ,
0
0
0
0
phenyl-H6 ), 7.78 (d, J ¼ 8.8 Hz, 2H, Phenyl-H2, Phenyl-H6), 7.93 (d,
J ¼ 8.8 Hz, 2H, Phenyl-H3, Phenyl-H5), 8.32 (s, 1H, NHSO2), 10.92 (s,
1H, NHCO); MS m/z (%): 522 ([MþH]þ, 3.3), 521 (Mþ, 8.0), 505 (3.0),
294 (1.0), 270 (3.0), 251 (2.5), 175 (2.0), 121 (74), 80 (100); Anal.
Calcd. for C24H23N7O5S (521.55): C, 55.27; H, 4.44; N, 18.80%, Found:
C, 55.25; H, 4.39; N, 18.76%.
0
0
143.0, 155.0 (13C, C6H4, C6 H4 , Isoxazole-C4), 156.0 (Isoxazole-C3),
161.8 (Isoxazole-C5), 162.0 (CO); MS m/z (%): 468 (Mþ, 0.8), 437
(3.5), 357 (8.6), 346 (3.6), 293 (1.0), 266 (2.1), 217 (0.7), 174 (0.7), 112
(100), 111 (7.5), 96 (21.0); Anal. Calcd. for C21H20N6O5S (468.49): C,
53.84; H, 4.30; N, 17.94%, Found: C, 53.80; H, 4.32; N, 17.99%.
4.1.10. Synthesis of 4-(3-cyano-4,6-dimethyl-2-oxopyridin-1(2H)-
yl)-N-(3,4-dimethyl-isoxazol-5-yl)benzenesulfonamide (12)
To a mixture of compound 3 (1.67 g, 0.005 mol) and acetylace-
tone (0.51 mL, 0.005 mol) in absolute ethanol (30 mL) was added
triethylamine (0.5 mL). The reaction mixture was refluxed for 9 h
then cooled down to room temperature, poured onto (100 mL) ice/
water and the medium was neutralized by dilute HCl. The obtained
solid was filtered off, washed with water and recrystallized from
ethanol to afford the target compound.
4.1.7.4. 2-Cyano-N-(4-{[(3,4-dimethylisoxazol-5-yl)amino]sulfonyl}
phenyl)-2-[(4-ethoxy-phenyl)hydrazono]acetamide
(9d).
Orange powder, yield (83%), mp 215e216 ꢀC; IR (KBr) nmax/cmꢁ1
:
3418 (NH), 3368 (NH), 3233 (NH), 3059 (CH-Ar), 2955 (CH-sp3),
2219 (CN), 1657 (CO); 1H NMR (400 MHz, DMSO-d6): dppm ¼ 1.36 (t,
J ¼ 6.8 Hz, 3H, CH3), 1.69 (s, 3H, CH3), 2.14 (s, 3H, CH3), 4.08 (q,
0
J ¼ 6.8 Hz, 2H, OCH2), 6.98 (d, J ¼ 8.8 Hz, 2H, Phenyl-H3 , phenyl-
0
0
0
H5 ), 7.73 (d, J ¼ 8.8 Hz, 2H, Phenyl-H2 , phenyl-H6 ), 7.80 (d,
J ¼ 8.8 Hz, 2H, Phenyl-H2, Phenyl-H6), 8.01 (d, J ¼ 8.8 Hz, 2H,
Phenyl-H3, Phenyl-H5), 10.26 (s, 1H, NH), 10.90 (s, 1H, NH), 11.90 (s,
1H, NH),; 13C NMR (100 MHz, DMSO-d6): dppm ¼ 6.3 (CH3), 10.8
(CH3), 15.1 (CH3), 63.7 (OCH2), 105.0 (C]N), 106.0 (CN), 112.5, 115.3,
White powder, yield (77%), mp 182e183 ꢀC; IR (KBr) nmax/cmꢁ1
:
3200 (NH), 3068 (CH-Ar), 2985 (CH-sp3), 2218 (CN), 1655 (CO); 1H
NMR (400 MHz, DMSO-d6): dppm ¼ 1.60 (s, 3H, CH3), 1.97 (s, 3H,
CH3), 1.99 (s, 3H, CH3), 2.33 (s, 3H, CH3), 6.47 (s, 1H, Pyridone-H5),
7.44 (d, J ¼ 8.8 Hz, 2H, Phenyl-H2, Phenyl-H6), 7.89 (d, J ¼ 8.8 Hz, 2H,
Phenyl-H3, Phenyl-H5), 8.90 (s, 1H, NHSO2); 13C NMR (100 MHz,
DMSO-d6): dppm ¼ 6.7 (CH3), 10.9 (CH3), 21.1 (CH3), 21.8 (CH3), 97.2
(Pyridone-C5), 100.4 (Pyridone-C3), 109.5 (CN), 116.2 (Phenyl-C4),
128.0 (2C, Phenyl-C2, Phenyl-C6), 128.8 (2C, Phenyl-C3, Phenyl-C5),
139.8 (Phenyl-C1), 145.9 (Isoxazole-C4), 152.2, 160.3, 160.6, 160.8
(4C, Pyridone-C4, Pyridone-C6, Isoxazole-C3, Isoxazole-C3), 162.2
(CO); MS m/z (%): 399 ([MþH]þ, 1.2), 398 (Mþ, 3.3), 383 (2.0), 302
(100), 287 (0.98), 251 (1.4), 223 (21.8), 175 (0.4), 147 (0.3), 111 (8.0);
Anal. Calcd. for C19H18N4O4S (398.44): C, 57.27; H, 4.55; N, 14.06%,
Found: C, 57.31; H, 4.56; N, 14.09%.
0
0
115.5, 118.4, 120.8, 128.1, 133.5, 135.8 (12C, C6H4, C6 H4 ), 143.0
(Isoxazole-C4), 156.4 (Isoxazole-C3), 161.0 (Isoxazole-C5), 162.0
(CO); MS m/z (%): 482 (Mþ, 0.7), 307 (4.0), 251 (1.5), 231 (2.1), 216
(1.0), 188 (6.7), 175 (4.8), 150 (1.6), 136 (26.8), 121 (26.7), 111 (5.5),
108 (100), 96 (2.8); Anal. Calcd. for C22H22N6O5S (482.51): C, 54.76;
H, 4.60; N, 17.42%, Found: C, 54.80; H, 4.62; N, 17.39%.
4.1.8. Synthesis of 2-cyano-2-[(cyanomethyl)4-(ethylphenyl)
hydrazono]-N-(4-{[(3,4-dimethyl-isoxazol-5-yl)amino]sulfonyl}
phenyl)acetamide (10)
To a solution of compound 9d (1 g, 2.07 mmol) and chlor-
oacetonitrile (0.13 mL, 2.07 mmol) in ethanol (30 mL), triethyl-
amine (0.5 mL) was added. The reaction mixture was refluxed for
6 h then cooled to room temperature and poured onto (100 mL) ice/
water and neutralized by HCl. The obtained solid was filtered off,
washed with water and recrystallized from ethanol to afford the
target compound.
4.1.11. Synthesis of 4-[6-amino-3,5-dicyano-4-(4-methoxyphenyl)-
2-oxopyridin-1(2H)-yl]-N-(3,4-dimethylisoxazol-5-yl)
benzenesulfonamide (15)
To a solution of compound 3 (1.67 g, 0.005 mol) and (4-
methoxybenzylidene)malononitrile (0.92 g, 0.005 mol) in abso-
lute ethanol (30 mL), sodium ethoxide [prepared by dissolving
sodium metal (115 mg, 0.005 mol) in absolute ethanol (5 mL)] was
added. The reaction mixture was refluxed for 5 h then allowed to
cool to room temperature. Then, it was poured onto (100 mL) ice/
water and the medium was neutralized by dilute HCl. The obtained
solid was filtered off, washed with water and recrystallized from
ethanol to afford the target compound.
Brown powder, yield (68%), mp 194e195 ꢀC; IR (KBr) nmax/cmꢁ1
:
3379 (NH), 3235 (NH), 3025 (CH-Ar), 2980 (CH-sp3), 2259 (CN), 2211
(CN), 1661 (CO); 1H NMR (400 MHz, DMSO-d6): dppm ¼ 1.34 (t,
J ¼ 6.8 Hz, 3H, CH3), 1.67 (s, 3H, CH3), 2.05 (s, 3H, CH3), 4.01 (q,
J ¼ 6.8 Hz, 2H, OCH2), 4.65 (s, 2H, CH2CN), 6.93 (d, J ¼ 8.8 Hz, 2H,
0
0
0
Phenyl-H3 , phenyl-H5 ), 7.73 (d, J ¼ 8.8 Hz, 2H, Phenyl-H2 , phenyl-
0
H6 ), 7.77 (d, J ¼ 8.8 Hz, 2H, Phenyl-H2, Phenyl-H6), 7.91 (d, J ¼ 8.8 Hz,
2H, Phenyl-H3, Phenyl-H5), 10.38 (s, 1H, NHSO2), 11.51 (s, 1H, NHCO);
MS m/z (%): 521 (Mþ, 8.3), 481 (2.0), 294 (3.0), 266 (1.0),189 (1.0),175
(1.5), 121 (26.0), 108 (100); Anal. Calcd. for C24H23N7O5S (521.55): C,
55.27; H, 4.44; N, 18.80%, Found: C, 55.31; H, 4.47; N, 18.78%.
Yellow powder, yield (71%), mp 131e132 ꢀC; IR (KBr) nmax/cmꢁ1
:
3401e3345 (NH2), 3217 (NH), 3057 (CH-Ar), 2985 (CH-sp3), 2217
(CN, broad), 1650 (CO); 1H NMR (400 MHz, DMSO-d6): dppm ¼ 1.63
(s, 3H, CH3), 2.08 (s, 3H, CH3), 3.74 (s, 3H, OCH3), 6.61 (s, 2H, NH2),
0
0
6.91 (d, J ¼ 8.8 Hz, 2H, Phenyl-H3 , phenyl-H5 ), 7.14 (d, J ¼ 8.8 Hz,
0
0
4.1.9. Synthesis of 4-amino-5-cyano-N-(4-{[(3,4-dimethylisoxazol-
5-yl)amino]sulfonyl} phenyl)-1-(4-ethoxyphenyl)-1H-pyrazole-3-
carboxamide (11)
To a solution of compound 10 (1 g, 1.91 mmol) in absolute
ethanol (30 mL), sodium ethoxide [prepared by dissolving sodium
metal (44 mg, 1.91 mmol) in absolute ethanol (10 mL)] was added.
The reaction mixture was cooled to room temperature and poured
2H, Phenyl-H2 , phenyl-H6 ), 7.51 (d, J ¼ 8.8 Hz, 2H, Phenyl-H2,
Phenyl-H6), 7.75 (d, J ¼ 8.8 Hz, 2H, Phenyl-H3, Phenyl-H5), 10.51 (s,
1H, NHSO2); 13C NMR (100 MHz, DMSO-d6): dppm ¼ 6.5 (CH3), 10.7
(CH3), 55.8 (OCH3), 105.0, 113.1, 114.5, 114.6 (4C, Pyridone-C3, Pyr-
idone-C2, 2 ꢂ CN), 122.3, 125.1, 127.6, 128.0, 128.5, 129.1, 130.6, 131.7
(12C, 2 ꢂ C6H4), 154.0, 157.5 (2C, Isoxazole-C4, Pyridone-C6), 161.0,
161.5 (2C, Isoxazole-C3, Isoxazole-C5), 162.0 (CO), 168.0 (Pyridone-

T. Nasr et al. / European Journal of Medicinal Chemistry 84 (2014) 491e504
503
C4); MS m/z (%): 517 ([MþH]þ, 1.9), 516 (Mþ, 1.9), 490 (2.5), 405
(1.9), 96 (48.9), 57 (100); Anal. Calcd. for C22H15N5O5S2 (493.52): C,
53.54; H, 3.06; N, 14.19%, Found: C, 53.59; H, 3.02; N, 14.23%.
(3.0), 265 (6.7), 251 (4.1),111 (1.4), 96 (4.3), 80 (100); Anal. Calcd. for
C
25H20N6O5S (516.53): C, 58.13; H, 3.90; N, 16.27%, Found: C, 58.17;
H, 3.93; N, 16.30%.
4.1.12.6. Synthesis of 4-[3,5-dicyano-4-(3-pyridyl)-6-mercapto-2-
oxopyridin-1(2H)-yl]-N-(3,4-dimethylisoxazol-5-yl)benzenesulfona-
mide (16d). Yellow powder, yield (57%), mp 146e147 ꢀC; IR (KBr)
nmax/cmꢁ1: 3216 (NH), 3068 (CH-Ar), 2971 (CHe sp3), 2219 (CN),
1655 (CO); 1H NMR (400 MHz, DMSO-d6): dppm ¼ 1.62 (s, 3H, CH3),
2.08 (s, 3H, CH3), 7.45e8.10 (m, 6H, C6H4, Pyridine-H5 and Pyridine-
H6), 8.50e8.80 (m, 2H, Pyridine-H2, Pyridine-H4), 10.55 (s, 1H,
NHSO2), 11.98 (s, 1H, SH); MS m/z (%): 505 ([MþH]þ, 8.8), 504 (Mþ,
6.4), 408 (8.0), 251 (11.1),175 (1.1), 80 (100), 78 (3.4); Anal. Calcd. for
4.1.12. General procedure for the synthesis of pyridone derivatives
16aed
4.1.12.1. Method (A). To
a solution of compound 3 (1.0 g,
2.99 mmol) and arylidene cyanothioacetamides (2.99 mmol) in
absolute ethanol (30 mL), sodium ethoxide [prepared by dissolving
sodium metal (69 mg, 2.99 mmol) in absolute ethanol (5 mL)] was
added. The reaction mixture was refluxed for 4 h then allowed to
cool down to room temperature. Then, poured onto (100 mL) ice/
water and the medium was neutralized by dilute HCl. The obtained
solid was filtered off, washed with water and recrystallized from
ethanol to afford the target compounds.
C23H16N6O4S2 (504.54): C, 54.75H, 3.20; N, 16.66%, Found: C, 54.78;
H, 3.22; N, 16.63%.
4.1.13. Synthesis of 4-[3,5-dicyano-6-(20,30,40,60-tetra-O-acetyl-
b-D-
glucopyranosylthio)-4-(4-chlorophenyl)-2-oxopyridin-1(2H)-yl]-N-
4.1.12.2. Method (B). To a solution of cyanothioacetamide (1 g,
0.01 mol) and the corresponding arylidene derivatives 5bee
(0.01 mol) in absolute ethanol (30 mL), triethylamine (0.5 mL) was
added. The reaction mixture was refluxed for 9 h then allowed to
cool to room temperature. The reaction mixture was poured onto
(100 mL) ice/water and the medium was neutralized by HCl. The
obtained solid was filtered off, washed with water and recrystal-
lized from ethanol to afford the target compound.
(3,4-dimethylisoxazol-5-yl)benzenesulfonamide (17)
A solution of 2,3,4,6-tetra-O-acetyl-a-D-glucopyranosyl bro-
mide (0.41 g, 0.001 mol) in dry acetone (10 mL) was added to a
mixture of compound 16b (0.54 g, 0.001 mol) and potassium car-
bonate (0.3 g) in dry acetone (20 mL). The reaction mixture was
refluxed for 18 h, cooled down to room temperature and filtered.
The filtrate was evaporated under vacuum. The obtained residue
was triturated with distilled water (10 mL), filtered, air dried and
purified by column chromatography using 60 N silica gel and
hexane/ethylacetate (10:1).
4.1.12.3. Synthesis of 4-[3,5-dicyano-6-mercapto-4-(4-
methoxyphenyl)-2-oxopyridin-1(2H)-yl]-N-(3,4-dimethylisoxazol-5-
yl)benzenesulfonamide (16a). Yellow powder, yield (77%), mp
120e121 ꢀC; IR (KBr) nmax/cmꢁ1: 3217 (NH), 3052 (CH-Ar), 2971 (CH-
sp3), 2212 (CN),1651 (CO); 1H NMR (500 MHz, DMSO-d6): dppm ¼ 1.62
(s, 3H, CH3), 2.08 (s, 3H, CH3), 3.82 (s, 3H, OCH3), 6.91 (d, J ¼ 9.0 Hz, 2H,
Yellow powder, yield (66%), mp 130e131 ꢀC; IR (KBr) nmax/cmꢁ1
:
3203 (NH), 3069 (CH-Ar), 2939 (CH-sp3), 2207 (CN),1752 (CO),1650
(CO); 1H NMR (400 MHz, DMSO-d6): dppm ¼ 1.95 (s, 3H, CH3),
1.99e2.01 (4s, 12H, 4 ꢂ CH3CO), 2.09 (s, 3H, CH3), 3.90e4.30 (m, 3H,
0
0 0
Glucose-H6b ,Glucose-H5 ), 4.90e5.20 (m, 2H,
Glucose-H6a
,
0
0
0
0
0
0
Phenyl-H3 , Phenyl-H5 ), 7.04 (d, J ¼ 9.0 Hz, 2H, Phenyl-H2 , Phenyl-
Glucose-H4 , Glucose-H3 ), 5.40 (m, 1H, Glucose-H2 ), 6.10 (d,
H6 ), 7.71e7.72 (m, 4H, C6H4),10.50(s,1H, NHSO2),11.91(s,1H, SH);13C
J ¼ 9.80 Hz, 1H, Glucose-H1 ), 7.40e7.70 (m, 8H, 2 ꢂ C6H4), 10.30 (s,
0
0
NMR (125 MHz, DMSO-d6): dppm ¼ 5.7 (CH3),10.2 (CH3), 55.0 (OCH3),
104.2, 109.5, 110.5, 112.6, 113.5, 113.9, 124.5, 128.0, 128.6, 129.5, 129.9,
139.5, 141.0 (17C, 2 ꢂ CN, Pyridone-C3, Pyridone-C5, Pyridone-C6,
2 ꢂ C6H4), 153.2, 156.2, 160.5 (3C, Isoxazole-C3, Isoxazole-C4, Iso-
xazole-C5),161.1 (CO),167.0 (Pyridone-C4); MS m/z (%): 533 (Mþ, 6.3),
507 (0.9), 426 (5.6), 422 (1.3), 282 (0.7), 251 (2.4),111 (24.7), 96 (29.3),
57 (100); Anal. Calcd. for C25H19N5O5S2 (533.58): C, 56.27; H, 3.59; N,
13.13%, Found: C, 56.31; H, 3.62; N, 13.15%.
1H, NHSO2); MS m/z (%): 870 ([Mþ2]þ, 10.7), 869 ([MþH]þ, 21.8),
832 (13.3), 824 (10.3), 808 (11.2), 251 (6.5),175 (7.1),153 (100); Anal.
Calcd. for C38H34ClN5O13S2 (868): C, 52.56H, 3.95; N, 8.07%, Found:
C, 52.61; H, 3.99; N, 8.03%.
4.2. Antimicrobial evaluation
4.2.1. Antimicrobial screening
The disks of Whatman filter paper were prepared with standard
size (6.0 mm diameter) and kept into 1.0 Oz screw capped wide
mouthed containers for sterilization. These bottles are kept into hot
air oven at a temperature of 150 ꢀC. Then, the standard sterilized
filter paper disks impregnated with a solution of the test compound
4.1.12.4. Synthesis of 4-[3,5-dicyano-6-mercapto-4-(4-
chlorophenyl)-2-oxopyridin-1(2H)-yl]-N-(3,4-dimethylisoxazol-5-yl)
benzenesulfonamide (16b). Yellow powder, yield (67%), mp
180e181 ꢀC; IR (KBr) nmax/cmꢁ1: 3217 (NH), 3050 (CH-Ar), 2974
(CHe sp3), 2206 (CN), 1656 (CO); 1H NMR (400 MHz, DMSO-d6):
in DMF (100 mL, 5 mg/mL) were placed on nutrient agar plate seeded
0
0
dppm ¼ 1.63 (s, 3H, CH3), 2.08 (s, 3H, CH3), 7.44e7.48 (m, 4H, C6 H4 ),
7.56 (d, J ¼ 9.0 Hz, 2H, Phenyl-H2, Phenyl-H6), 7.82 (d, J ¼ 9.0 Hz, 2H,
Phenyl-H3, Phenyl-H5), 10.49 (s, 1H, NHSO2), 11.85 (s, 1H, SH); MS
m/z (%): 540 ([Mþ2]þ, 1.3), 538 (Mþ, 4.1), 440 (3.7), 426 (7.4), 422
(1.3), 251 (2.4), 111 (25.6), 96 (2.2), 80 (100); Anal. Calcd. for
with the appropriate test organism in triplicates. Standard concen-
trations of 106 CFU/mL (Colony Forming Units/mL) and 104 CFU/mL
were used for antibacterial and antifungal assay, respectively. Pyrex
glass Petri dishes (9 cm in diameter) were used and two disks of
filter paper were inoculated in each plate. The utilized test organ-
isms were S. pneumoniae, B. Subtilis and S. epidermidis as examples of
Gram-positive bacteria and E. coli, P. vulgaris and K. pneumonia as
examples of Gram-negative bacteria. They were also evaluated for
their in vitro antifungal potential against A. fumigatus, S. racemosum
and G. candidum fungal strains. Ampicillin and gentamycin were
used as standard antibacterial agents; while amphotericin B was
used as standard antifungal agent. The starting antimicrobial agent
sulfisoxazole was also used as reference drug. DMF alone was used
as control at the same above-mentioned concentration and due this
there was no visible change in bacterial growth. The plates were
incubated at 37 ꢀC for 24 h for bacteria and for 48 h at 25 ꢀC for fungi.
The mean zone of inhibition measured in mm standard deviation
C
24H16ClN5O4S2 (538.04): C, 53.58; H, 3.00; N, 13.02%, Found: C,
53.61; H, 3.02; N, 13.05%.
4.1.12.5. Synthesis of 4-[3,5-dicyano-4-(2-furyl)-6-mercapto-2-
oxopyridin-1(2H)-yl]-N-(3,4-dimethyl-isoxazol-5-yl)benzenesulfona-
mide (16c). Yellow powder, yield (72%), mp 132e133 ꢀC; IR (KBr)
nmax/cmꢁ1: 3207 (NH), 3059 (CH-Ar), 2973 (CHe sp3), 2211 (CN),
1654 (CO); 1H NMR (400 MHz, DMSO-d6): dppm ¼ 1.62 (s, 3H, CH3),
2.08 (s, 3H, CH3), 6.70 (t, J ¼ 5.8 Hz,1H, Furan-H4), 7.20 (d, J ¼ 5.8 Hz,
1H, Furan-H3), 7.70e7.85 (m, 5H, C6H4 and Furan-H5), 10.59 (s, 1H,
NHSO2), 11.90 (s, 1H, SH); MS m/z (%): 494 ([MþH]þ, 0.3), 493 (Mþ,
0.3), 478 (0.2), 467 (0.3), 460 (0.3), 381 (0.4), 251 (1.9), 175 (1.6), 111

504
T. Nasr et al. / European Journal of Medicinal Chemistry 84 (2014) 491e504
beyond well diameter (6 mm) produced on a range of environ-
mental and clinically pathogenic microorganisms. Compounds that
showed growth inhibition zones (>10 mm) using the twofold serial
dilution technique, were further evaluated for their minimal
inhibitory concentrations (MICs).
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The microdilution susceptibility test in MüllereHinton Broth
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centrations 1000
standard method broth (Difco) to prepare serial twofold dilutions in
the range of 500e0.007 g/mL. 10 mL of the broth containing about
mg/mL. Each stock solution was diluted with
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106 CFU/mL of test bacteria or 104 CFU/mL of the test fungus was
added to each well of 96-well microtiter plate. The sealed micro-
plates were incubated at 37 ꢀC for 24 h for antibacterial activity and
at 25 ꢀC for 48 h for antifungal activity in a humid chamber. At the
end of the incubation period, the minimal inhibitory concentrations
(MIC) values were recorded as the lowest concentrations of the
substance that had no visible turbidity. Control experiments with
DMF and uninoculated media were run parallel to the test com-
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Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.07.052.