Green approach for the synthesis of thiophenyl pyrazoles and isoxazoles by adopting 1,3-dipolar cycloaddition methodology and their antimicrobial activity

Article abstract of DOI:10.1016/j.ejmech.2017.11.093

A variety of N-((1,3-diphenyl-5-aryl-1H-pyrazol-4-yl)sulfonyl)thiophene-2-carboxamides (7) and N-((5-aryl-3-phenylisoxazol-4-yl)sulfonyl)thiophene-2-carboxamides (8) were prepared from (E)-N-(arylethenesulfonyl)thiophene-2-carboxamides (4) adopting 1,3-di

Full text of DOI:10.1016/j.ejmech.2017.11.093

Accepted Manuscript
Green approach for the synthesis of thiophenyl pyrazoles and isoxazoles by adopting
1,3-dipolar cycloaddition methodology and their antimicrobial activity
D.V. Sowmya, G. Lakshmi Teja, A. Padmaja, V. Kamala Prasad, V. Padmavathi
PII:
S0223-5234(17)30996-0
10.1016/j.ejmech.2017.11.093
EJMECH 9968
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 1 September 2017
Revised Date: 20 November 2017
Accepted Date: 28 November 2017
Please cite this article as: D.V. Sowmya, G. Lakshmi Teja, A. Padmaja, V. Kamala Prasad, V.
Padmavathi, Green approach for the synthesis of thiophenyl pyrazoles and isoxazoles by adopting
1,3-dipolar cycloaddition methodology and their antimicrobial activity, European Journal of Medicinal
Chemistry (2018), doi: 10.1016/j.ejmech.2017.11.093.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
GRAPHICAL ABSTRACT
Green Approach for the Synthesis of Thiophenyl Pyrazoles And Isoxazoles By
Adopting 1,3-Dipolar Cycloaddition Methodology And Their Antimicrobial
Activity
•
•
Thiophenyl pyrazoles and isoxazoles were synthesized by 1,3-dipolar cycloaddition
of nitrile imines and nitrile oxides to N-(arylethenesulfonyl)thiophene-2-carboxamides
under green conditions followed by aromatization.
The presence of electronwithdrawing substituents on the aromatic ring increased the
activity.
•
•
4f, 7e, 7f, 8e, 8f; MIC = 6.25 µg/well against B.subtilis.
8e, 8f; MIC = 6.25 µg/well against A.niger.

ACCEPTED MANUSCRIPT
Green Approach for the Synthesis of Thiophenyl Pyrazoles And Isoxazoles By
Adopting 1,3-Dipolar Cycloaddition Methodology And Their Antimicrobial Activity
D. V. Sowmyaa, G. Lakshmi Tejaa, A. Padmajaa, V. Kamala Prasadb,
V. Padmavathia,*,
a
Department of Chemistry, Sri Venkateswara University, Tirupati 517 502, Andhra Pradesh,
India
b Denisco Chemicals Pvt. Ltd., Hyderabad 500 055, Telangana
ABSTRACT
A variety of N-((1,3-diphenyl-5-aryl-1H-pyrazol-4-yl)sulfonyl)thiophene-2-carboxamides
(7) and N-((5-aryl-3-phenylisoxazol-4-yl)sulfonyl)thiophene-2-carboxamides (8) were prepared
from (E)-N-(arylethenesulfonyl)thiophene-2-carboxamides (4) adopting 1,3-dipolar cycloaddition
of nitrile imines and nitrile oxides generated from araldehyde phenylhydrazones and araldoximes
in the presence of iodosobenzene and CTAB followed by oxidation with I2 in DMSO. The
compounds 4f, 7e, 7f, 8e and 8f showed potential antibacterial activity against B. subtilis
whereas 8e and 8f exhibited potential antifungal activity against A. niger.
Keywords: Pyrazole / Isoxazole / CTAB / 1,3-Dipolar cycloaddition / Antimicrobial activity
1. Introduction
Sulfonamides are useful as antimicrobial agents which are effective against bacterial
infections and possess enzyme inhibitory properties [1, 2]. In addition, sulfonamides constitute
an important class of pharmacological agents including antiviral [3], anti-inflammatory [4] and
anticancer [5, 6]. Several successful sulfonamide-based drugs have been introduced into the
market viz., Diovan HCT [7], and fluorine-containing Crestor and Celebrex [8] (Fig. 1). Amongst

ACCEPTED MANUSCRIPT
five membered heterocycles, pyrazoles and isoxazoles are effective heteroaromatic constituents
because of their wide range of applications in medicinal and applied chemistry. Pyrazoles have a
significant influence on human health and agricultural productivity since they are often found in
the structures of a variety of leading drugs- Celebrex [8] and pesticides- Cyenopyrafen,
Fenpyroximate [9], Tebufenpyrad [10] (Fig. 1). Moreover, pyrazole is a versatile component
with diverse activities such as antimicrobial [11], anticonvulsant and antidepressant [12].
Isoxazole forms the basis for several drugs viz., Leflunomide, Valdecoxib, Sulfisoxazole,
Oxacillin and Cloxacillin (Fig. 1). Besides, Isoxazoles are known to exhibit antimicrobial [13],
antifungal [14] and analgesic [15] activities and also attracted chemists as non classical amide or
ester bioisosteres [16]. Amongst different methods for the synthesis of pyrazolines and
isoxazolines, 1,3-dipolar cycloaddition of an ylide onto an alkene in a 3+2 manner is a facile one
[17, 18]. Indeed, diazomethane, nitrile imines and nitrile oxides have been used extensively as
reactive intermediates [19, 20]. Although, these azoles have been intensely studied for over a
century, there is still a great demand for novel derivatives with enhanced biopotency to combat
various diseases and pathophysiological conditions. In fact for the last couple of years we have
been actively engaged towards the development of a variety of heteroaromatics with different
pharmacophores [21]. In continuation of our study we herein report the synthesis of
pyrazolyl/isoxazolyl sulfonyl thiophene carboxamides by adopting 1,3-dipolar cycloaddition
methodology and their antimicrobial activity.
2. Chemistry
The synthetic intermediates thiophenylamidosulfonylstyrenes were prepared as follows.
The reaction of styrene (1) with sulfuryl chloride resulted in styrylsulfonylchloride which on
ammonation with aqueous ammonia produced arylethenesulfonamide (2). The reaction of 2 with

ACCEPTED MANUSCRIPT
thiophene-2-carboxylic acid (3) in the presence of 1-ethyl-3-(3-dimethylaminopropyl)-
carbodiimide (EDCI), N,N-dimethylaminopyridine (DMAP) and triethylamine under
ultrasonication gave (E)-N-(arylethenesulfonyl)thiophene-2-carboxamide (4) (Scheme 1). The 1H
NMR spectrum of 4a displayed a doublet at δ 6.60 due to HB. However the signal corresponding
to HA merged with aromatic protons. The C4-H of thiophene exhibited a triplet at 6.96 while C3-
H and C5-H showed a multiplet in the region 8.31-8.43. Besides, a broad singlet appeared at
11.05 ppm was assigned to NH which disappeared on deuteration. The olefin moiety present in 4
was used to develop pyrazole and isoxazole rings. In our earlier communications we have
reported 1,3-dipolar cycloaddition of nitrile imines and nitrile oxides generated from araldehyde
phenylhydrazones and araldoximes in the presence of chloramine-T to activated olefins to
develop pyrazolines and isoxazolines [22]. We have also proved that chloramine-T also acts as a
reagent for aromatization [23]. As such when the reaction was carried out between 4a and
benzaldehyde phenylhydrazone in the presence of chloramine-T, instead of the expected N-
((1,3,5-triphenyl-4,5-dihydro-1H-pyrazol-4-yl)sulfonyl)thiophene-2-carboxamide (5a), cleavage
of 4a takes place at reflux temperature leading to thiophene-2-carboxamide (48%) along with a
gummy charred substance which could not be isolated (Scheme 2). Similar reaction of 4a with
benzaldoxime in the presence of chloramine-T resulted in thiophene-2-carboxamide (51%) and
dimerized product of nitrile oxide, furaxon (32%) (Scheme 2). The structure of thiophene-2-
1
13
carboxamide was confirmed by melting point, H and C NMR spectra [24]. In the literature it
was reported that iodosobenzene not only serves as a source of oxygen in various metal-
catalyzed oxidation reactions [25] but also oxidizes aldoximes to nitrile oxides. In fact, in the
presence of cationic surfactant cetyltrimethylammonium bromide (CTAB) the reaction
proceeded well [26]. However, there are no reports to generate nitrile imines from araldehyde

ACCEPTED MANUSCRIPT
phenylhydrazones in the presence of iodosobenzene and CTAB. We tried this green approach for
the synthesis of pyrazolines and isoxazolines. In fact, the reaction of 4 with benzaldehyde
phenylhydrazone in the presence of iodosobenzene and CTAB at room temperature led to N-((5-
aryl-1,3-diphenyl-4,5-dihydro-1H-pyrazol-4-yl)sulfonyl)thiophene-2-carboxamide (5). Similarly
N-((5-aryl-3-phenyl-4,5-dihydroisoxazol-4-yl)sulfonyl)thiophene-2-carboxamide
(6)
was
prepared by the treatment of 4 with araldoxime in the presence of iodosobenzene and CTAB
(Scheme 3). The mechanism of oxidation cum dehydration of benzaldehyde phenylhydrazones
and benzaldoximes to nitrile imines and nitrile oxides is depicted in scheme 4. The oxygen atom
of PhIO transferred to C=N of hydrazone/oxime by nucleophilic attack of PhIO followed by the
loss of PhI which resulted in 3-phenyl-1,2-oxaziridin-2-amine / ol. The N-O bond rupture of
unstable intermediate led to the formation of hydroxyl hydrazone / aldoxime by proton shift.
Removal of hydroxyl ion and deprotonation led to nitrile imines and nitrile oxides. The 1H NMR
spectra of 5a and 6a showed two doublets at δ 5.09, 5.05 and 5.43, 5.65 due to C4ʹ-H and C5ʹ-H
in addition to the signals of thiophene and aromatic ring protons. The oxidation of 5 and 6 with I2
in DMSO gave the aromatized products- N-((5-aryl-1,3-diphenyl-1H-pyrazol-4-
yl)sulfonyl)thiophene-2-carboxamide (7) and N-((5-aryl-3-phenylisoxazol-4-yl)sulfonyl)thiophene-
2-carboxamide (8) (Scheme 5). The iodine picks the hydride ion at 4th carbon of the heterocycle
followed by the loss of proton. The HI generated during the course of the reaction liberates I2 in
the presence of DMSO (Scheme 6). In the 1H NMR spectra of 7a and 8a the absence of doublets
corresponding to pyrazoline and isoxazoline ring protons confirmed that aromatization occurred.
However the multiplets appeared at δ 8.36-8.45 and 8.30-8.41 were attributed to C3-H & C5-H
and the signals appeared at 6.93, 6.91 & 10.61, 10.56 ppm were assigned to C4-H and NH. The
structures of the compounds were further established by IR, 13C NMR and mass.

ACCEPTED MANUSCRIPT
3. Biology
3.1. Antimicrobial activity
The compounds 4-8 were screened for antimicrobial activity at three different
concentrations 50, 75 and 100 µg/well.
4. Results and discussion
4.1. Antibacterial activity
All the compounds were evaluated for antibacterial activity at three different
concentrations 50, 75 and 100 µg/well. The results revealed that some of the compounds
displayed high antibacterial activity towards Gram +ve bacteria than on Gram -ve bacteria.
However 5a, 5c, 5d, 6c and 6d were inactive. Amongst bis heterocycles, pyrazolylsulfonyl
thiophenecarboxamides (7) and isoxazolylsulfonyl thiophenecarboxamides (8) showed higher
activity
than
the
corresponding
dihydro
derivatives,
pyrazolinylsulfonyl
thiophenecarboxamides (5) and isoxazolinylsulfonyl thiophenecarboxamides (6). It was also
observed that (E)-N-(arylethenesulfonyl)thiophene-2-carboxamides (4) exhibited greater
activity than 5 and 6 but lower than 7 and 8. The structure-activity relationship revealed that
compounds with isoxazole unit 8 showed slightly higher activity than those having pyrazole
7. Further, the presence of electron withdrawing substituents on the aromatic ring increases
the activity. It was observed that with increasing electronegativity the activity increases. In
fact 7e, 7f, 8e and 8f exhibited excellent antibacterial activity on B. subtilis greater than the
standard drug Chloramphenicol at all tested concentrations (Table 1 & Fig. 2). The compound
4f displayed equal activity to the standard drug at 75 and 100 µg/well.
4.2. Antifungal activity
All the compounds inhibited the spore germination of the tested fungi except 5a, 5d,
6a, 6c and 6d. All the compounds displayed higher activity towards A. niger than on

ACCEPTED MANUSCRIPT
P. chrysogenum. The aromatized bis heterocycles 7 and 8 showed greater activity than (E)-N-
(arylethenesulfonyl)thiophene-2-carboxamides (4). Amongst 7 and 8 those with isoxazole unit
displayed higher activity. On the other hand, the dihydro derivatives 5 and 6 exhibited least
activity. The presence of electron withdrawing substituents on the aromatic ring increases the
activity. The compounds 8e and 8f displayed prominent antifungal activity greater than the
standard drug, Ketoconazole at all tested concentrations (Table 2 & Fig. 3).
4.3. MIC, MBC and MFC of compounds 4e, 4f, 8e and 8f
The compounds which showed greater antibacterial and antifungal activities are further
assayed for minimum inhibitory concentration (MIC), minimum bactericidal concentration
(MBC) and minimum fungicidal concentration (MFC) and the values are listed in Table 3. MIC
is the lowest concentration of an antimicrobial that will inhibit the visible growth of a
microorganism. (But it is not sure that the microorganisms are completely killed). The
MBC/MFC is the lowest concentration of antibiotic required to kill a particular bacterium/fungi.
The MBC/MFC involves an additional set of steps performed once the minimum inhibitory
concentration (MIC) is determined. The antimicrobials are usually regarded as
bactericidal/fungicidal if MBC/MFC is not greater than four times MIC [27]. MIC value of
compounds 4f, 7e, 7f, 8e and 8f against B. subtilis is equal to standard drug Chloramphenicol and
MBC value is 2×MIC. Moreover, the compounds 8e and 8f displayed low MIC value on A. niger
than 4e, 4f, 7e and 7f and MFC value is 2×MIC. This indicated that 4f, 7e, 7f, 8e and 8f are the
potential antibacterial agents against B. subtilis while the compounds 8e and 8f are potential
antifungal agents against A. niger. The structure - activity relationship of the compounds
revealed that aromatic bis heterocycles - pyrazolyl and isoxazolyl derivatives (7 & 8) displayed
prominent antibacterial and antifungal activities than the corresponding pyrazolinyl and

ACCEPTED MANUSCRIPT
isoxazolinyl derivatives (5 & 6). The presence of electron withdrawing substituents on the
aromatic ring increased the activity.
5. Conclusion
A variety of pyrazolinylsulfonyl thiophenecarboxamides (5) and isoxazolinylsulfonyl
thiophene carboxamides (6) were prepared by 1,3-dipolar cycloaddition of nitrile imines and
nitrile oxides generated from benzaldehyde phenylhydrazone and benzaldoxime in the presence
of iodosobenzene and CTAB to N-(arylethenesulfonyl)thiophene-2-carboxamides (4). The
dehydrogenation of pyrazoline and isoxazoline rings in 5 and 6 was effected with I2 in DMSO to
get pyrazolylsulfonyl thiophenecarboxamides (7) and isoxazolylsulfonyl thiophenecarboxamides
(8). All the compounds are tested for antimicrobial activity. The compounds 4, 7 and 8 displayed
good antibacterial activity whereas 8 exhibited good antifungal activity. The presence of electron
withdrawing groups on the aromatic ring increased the activity. The compounds 4f, 7e, 7f, 8e
and 8f showed prominent antibacterial activity against B. subtilis whereas 8e and 8f exhibited
prominent antifungal activity against A. niger.
6. Experimental
6.1. Chemistry
Melting points were determined in open capillaries on a Mel-Temp apparatus and are
uncorrected. The IR spectra were recorded on a Thermo Nicolet IR 200 FT-IR spectrometer as
1
KBr pellets and the wave numbers were given in cm-1. The H NMR spectra were recorded in
13
DMSO–d6 on a Jeol JNM λ–400 MHz. The C NMR spectra were recorded in DMSO‒d6 on a
Jeol JNM spectrometer operating at 100 MHz. Ultrasonication was performed in a Bandelin
Sonorex RK 102 H ultrasonic bath operating at the frequency of 35 kHz. All chemical shifts are
reported in δ (ppm) using TMS as an internal standard. High-Resolution Mass spectra were

ACCEPTED MANUSCRIPT
recorded on Micromass Q-TOF mass spectrometer using electrospray ionisation. The
microanalyses were performed on a Perkin–Elmer 240C elemental analyzer. The synthetic
intermediate 2 was prepared as per the literature procedure [28].
6.1.1. General procedure for the synthesis of synthesis of (E)-N-(arylethenesulfonyl)thiophene-
2-carboxamide (4a-f)
Arylethenesulfonamide (2) (0.50 mmol) was dissolved in dichloromethane (2 ml) and to
this triethylamine (0.71 mmol) was added dropwise. Then thiophene-2-carboxylic acid (3) (0.60
mmol) and DMAP (0.13 mmol) in dichloromethane (1 ml) was added followed by EDCI (0.60
mmol) and was sonicated at room temperature for a period of 45-50 min. The reaction mixture
was quenched with 1N HCl and the filtrate was extracted with EtOAc. The organic layer was
washed with water, brine solution and dried over an. Na2SO4. The solvent was concentrated in
vacuo and the solid was purified by column chromatography using hexane-ethyl acetate (3:1) as
eluent.
6.1.2. General procedure for the synthesis of N-((5-aryl-1,3-diphenyl-4,5-dihydro-1H-pyrazol-4-
yl)sulfonyl)thiophene-2-carboxamide / N-((5-aryl-3-phenyl-4,5-dihydroisoxazol-4-yl)sulfonyl)
thiophene-2-carboxamide (5a-f)/(6a-f)
A
mixture of benzadehyde phenylhydrazone
/
benzaldoxime (0.75 mmol),
(E)-N-(arylethenesulfonyl)thiophene-2-carboxamide (4) (1.50 mmol), water (8 ml) and CTAB
(0.25 mmol) were stirred at room temperature for 15 min. Then iodosobenzene (244 mg, 2.10
mmol) was added to the reaction mixture and stirring was continued at room temperature for 3-4
h. After completion of the reaction (monitored by TLC) it was extracted with ethyl acetate. The
organic portion was washed with brine solution, dried over an. sodium sulfate and the solvent

ACCEPTED MANUSCRIPT
was concentrated in vacuo. The resultant solid was purified by column chromatography using
hexane-ethyl acetate (3:1) as eluent.
6.1.3. General procedure for the synthesis of N-((1,3-diphenyl-5-aryl-1H-pyrazol-4-
yl)sulfonyl)thiophene-2-carboxamide / N-((5-aryl-3-phenylisoxazol-4-yl)sulfonyl)thiophene-2-
carboxamide (7a-f)/(8a-f)
The compounds 5 / 6 (0.2 mmol) were dissolved in dimethyl sulfoxide (5 ml) followed by the
addition of iodine (1 mg, 2.2 mol %). The reaction mixture was stirred and refluxed for 3-4 h.
The solution was poured onto crushed ice and the resultant compound was filtered, dried and
recrystallized from 2-propanol.
6.2. Biological assays
6.2.1. Compounds
The compounds 4-8 were dissolved in DMSO at three different concentrations of 50, 75
and 100 µg/well.
6.2.2. Cells
Bacterial strains Staphylococcus aureus, Bacillus subtilis (Gram-positive), Pseudomonas
aeruginosa, Klebsiella pneumonia (Gram-negative) and fungi Aspergillus niger, Penicillium
chrysogenum were obtained from Department of Microbiology, S.V. University, Tirupati, India.
6.2.3. Antimicrobial activity
The in vitro antimicrobial studies were carried out by agar well diffusion method against test
organisms [29, 30]. Nutrient broth (NB) plates were swabbed with 24 h old broth culture (100
µl) of test bacteria. Using the sterile cork borer, wells (6 mm) were made into each petriplate.
Various concentrations of DMSO dissolved compounds (50, 75, 100 µg/well) were added into

ACCEPTED MANUSCRIPT
the wells by using sterile pipettes. Simultaneously the standard antibiotics, Chloramphenicol
for antibacterial activity and Ketoconazole for antifungal activity (as positive control) were
tested against the pathogens. The samples were dissolved in DMSO which showed no zone of
o
inhibition acts as negative control. The plates were incubated at 37 C for 24 h for bacteria
o
and at 28 C for 48 h for fungi. After appropriate incubation, the diameter of zone of
inhibition of each well was measured. Duplicates were maintained and the average values
were calculated for eventual antibacterial activity.
Broth dilution test was used to determine minimum inhibitory concentration (MIC) of
the above mentioned samples [31, 32]. Freshly prepared nutrient broth was used as diluents.
The 24 h old culture of the test bacteria S. aureus, B. subtilis, P. aeruginosa, K. pneumoniae
and fungi A. niger, P. chrysogenum were diluted 100 folds in nutrient broth (100 µl bacterial
cultures in 10 ml NB). Increasing concentrations of the test samples (1.25, 2.5, 5, 10, 20, 40
µl of stock solution contains 6.25, 12.5, 25, 50, 100, 200 µg/well of the compounds) were
added to the test tubes containing the bacterial and fungal cultures. All the tubes were
o
o
incubated at 37 C for 24 h for bacteria and at 28 C for 48 h for fungi. The tubes were
examined for visible turbidity and using NB as control. Control without test samples and with
solvent was assayed simultaneously. The lowest concentration that inhibited visible growth of
the tested organisms was recorded as MIC. To determine the minimum bactericidal
concentration (MBC) [33] and minimum fungicidal concentration (MFC) [34] for each set of
test tubes in the MIC determination, a loopful of broth was collected from those tubes which
did not show any growth an d inoculated on sterile nutrient broth (for bacteria) and PDA (for
fungi) by streaking. Plates inoculated with bacteria and fungi were incubated at 37 oC for 24 h

ACCEPTED MANUSCRIPT
o
and at 28 C for 48 h, respectively. After incubation, the lowest concentration was noted as
MBC (for bacteria) or MFC (for fungi) at which no visible growth was observed.
Acknowledgement
One of the authors V. Padmavathi is grateful to Council of Scientific and Industrial
Research (CSIR) for financial assistance under major research project.
References
1.
2.
Y. Genc, R. Özkanca, Y. Bekdemir, Ann. Clin. Microbiol. Antimicrob. 7 (2008) 17-23.
M. Ceruso, S.D. Prete, Z. Alothman, C. Capasso, C.T. Supuran, Med. Chem. Lett.
5 (2014) 826-830.
3.
4.
O.I. El-Sabbagh, Arch. Pharm. 346 (2013) 733-742.
J. Lal, S.K. Gupta, D. Thavaselvam, D.D. Agarwal, Eur. J. Med. Chem. 64 (2013) 579-
588.
5.
6.
M. Basanagouda, K. Shivashankar, M.V. Kulkarni, V.P. Rasal, H. Patel, S.S. Mutha,
A.A. Mohite, Eur. J. Med. Chem. 45 (2010) 1151-1157.
A. Kamal, D. Dastagiri, M.J. Ramaiah, J.S. Reddy, E.V. Bharathi, M.K. Reddy, M.V.
P. Sagar, T.L. Reddy, S.N.C.V.L. Pushpavalli, M. Pal-Bhadra, Eur. J. Med. Chem. 46
(2011) 5817-5824.
7.
8.
9.
S.H. Hong, J.L. Wang, J. Tang, Am. J. Hypertens. 26 (2013) 879-887.
D.O. Hagan, J. Fluorine Chem. 131 (2010) 1071-1081.
M. Riga, A. Myridakis, D. Tsakireli, E. Morou, E .G. Stephanou, R. Nauen, T. Van
Leeuwen, V. Douris, J. Vontas, Insect Biochem. Mol. Biol. 65 (2015) 91-99.
D. Marcic, Exp. Appl. Acarol. 36 (2005) 177-185.
10.

ACCEPTED MANUSCRIPT
11.
12.
13.
A.M. Vijesh, A.M. Isloor, P. Shetty, S. Sundershan, H.K. Fun, Eur. J. Med. Chem. 62
(2013) 410-415.
M. Abdel-Aziz, G.E.A. Abuo-Rahma, A.A. Hassan, Eur. J. Med. Chem. 44 (2009)
3480-3487.
S.S. Basha, K. Divya, A. Padmaja, V. Padmavathi, Res. Chem. Intermed. 41 (2015)
10067-10083.
14.
15.
R. Shetty, S. Shafi, Y. Kuberan, J. Pharmcol. Res. 6 (2013) 897-901.
E. Rajanarendar, M.N. Reddy, S.R. Krishna, K.R. Murthy, Y.N. Reddy, M.V. Rajam,
Eur. J. Med. Chem. 55 (2012) 273-283.
16.
M. Koufaki, T. Fotopoulou, M. Kapetanou, G.A. Heropoulos, E.S. Gonos,
N. Chondrogianni, Eur. J. Med. Chem. 83 (2014) 508-515.
Y. Wang, C.I.R. Vera, Q. Lin, Org. Lett. 9 (2007) 4155-4158.
M. Rakesh, A. Ahmad, P. Gupta, K. Chand, M. Kumar, Jayendra, P. Rawat, N. Rasheed,
P. Gautam, Med. Chem. Res. 20 (2011) 139-145.
17.
18.
19.
20.
21.
22.
G. Sravya, G. Yamini, V. Padmavathi, A. Padmaja, Eur. J. Med. Chem. 122 (2016)
647-655.
G. Lavanya, V. Padmavathi, A. Padmaja, M. Balaji, Med. Chem. Res. 24 (2015) 3785-
3793.
T.B. Prakash, G.D. Reddy, A. Padmaja, V. Padmavathi, Eur. J. Med. Chem. 82 (2014)
347-354.
A. Padmaja, C. Rajasekhar, S. Durgamma, B.C. Venkatesh, V. Padmavathi, Med. Chem.
Res. 23 (2014) 1084-1098.

ACCEPTED MANUSCRIPT
23.
24.
25.
V. Padmavathi, R.P. Sumathi, N.C. Babu, D.B. Reddy, J. Chem. Research (S), (1999)
610-611.
S. Zhang, H. Xu, C. Lou, A.M. Senan, Z. Chen, G. Yin, Eur. J. Org. Chem. (2017)
1870-1875.
(a) W. Adam, F.G. Gelalcha, C.R. Saha-Moller, V.R. Stegmann, J. Org. Chem. 65 (2000)
1915-1918.
(b) E.M. McGarrigle, D.G. Gilheany, Chem. Rev. 105 (2005) 1563-1602.
N. Chatterjee, P. Pandit, S. Halder, A. Patra, D.K. Maiti, J. Org. Chem. 73 (2008) 7775-
7778.
26.
27.
28.
G.L. French, J. Antimicrobial Chem. 58 (2006) 1107−1117.
H. Harada, J. Azami, S. Watanuki, R.Tsuzuki, K. Sudoh, A. Fujimori, T. Tokunaga,
A. Tanaka, S. Tsukamoto, I. Yanagisawa, Chem. Pharm. Bull. 49 (2001) 1593-1603.
K.T. Chung, W.R. Thomasson, C.D. Wu-Yuan, J. Appl. Bacteriol. 69 (1990) 498-503.
C. Azoro, World J. Biotechnol. 3 (2002) 347-357.
29.
30.
31.
32.
33.
J. Bishnu, L. Sunil, S. Anuja, J. Sci. Eng. Technol. 5 (2009) 143-150.
D. Janovska, K. Kubikova, L. Kokoska, Czech. J. Food Sci. 21 (2003) 107-110.
Clinical and Laboratory Standards Institute (CLSI), Methods for Dilution Antimicrobial
Susceptibility Tests for Bacteria that Grow Aerobically, 7th edition; Approved standard,
CLSI document M7-A7, Wayne, PA (2006).
34.
Clinical and Laboratory Standards Institute (CLSI), Reference Method for Broth Dilution
Antifungal Susceptibility Testing of Yeasts, 2nd edition; Approved standard, CLSI
document M27-A2, Wayne,PA (2008).

ACCEPTED MANUSCRIPT
Scheme 1. Synthesis of arylethenesulfonyl thiophene-2-carboxamides.
NH2
S
O
O
O
S2
S
Ph
N
H
i
Ph-CH=N-NHPh
O
4a
O
Ph
S2
S
N
R
H
N
Ph
Ph
N
5a
Ph
Ph
i
Ph-CH=N-OH
NH2
4a
N
N
S
O
O
O
(i) Chloramine-T
Scheme 2. Reaction of 4a with benzaldehyde phenylhydrazone and benzaldoxime

ACCEPTED MANUSCRIPT
Scheme 3. Synthesis of pyrazolinylsulfonyl thiophenecarboxamides and isoxazolinylsulfonyl
thiophenecarboxamides.
H
Ph
H
Ph
H
Ph
H
PhI
X H
C N X H
O
C N X H
N
Ph C N X H
O
O
I
Ph
O
I
Ph
iv
i
ii
iii
H+
OH
Ph
-
OH
C N X H
HO
Ph C N X
Ph C N X H
vii
vi
v
X = N-Ph / O
Scheme 4. Mechanism for formation of nitrile imines and nitrile oxides.

ACCEPTED MANUSCRIPT
O
O
S2
Ar
S
N
R
R
2
3
5
5'
i
4'
3'
H
N1'
5
4
2'
N
Ph
Ph
7
O
O
S2
Ar
S
N
i
2
3
5
5'
4'
3'
6
H
O1'
4
2'
N
Ph
8
Ar
a) Ph
b) Ph
R
H
Cl
Ar
R
Cl
H
(i) DMSO / I2
d) 4-Me.Ph
e) 4-NO2.Ph
c) 4-Me.Ph H
f) 4-NO2.Ph Cl
Scheme 5. Synthesis of pyrazolylsulfonyl thiophenecarboxamides and isoxazolylsulfonyl
thiophenecarboxamides.
Scheme 6. Mechanism for oxidation of pyrazolinylsulfonyl thiophenecarboxamides and
isoxazolinylsulfonyl thiophenecarboxamides.

ACCEPTED MANUSCRIPT
Table 1 The in vitro antibacterial activity of compounds 4-8.
Zone of inhibition(mm)
Compound
Gram-positive
Gram-negative
S. aureus
B. subtilis
P. aeruginosa
K. pneumoniae
50
µg/
well
75
µg/
well
100
µg/
50
µg/
well
75
µg/
well
100
µg/
50
µg/
well
75
µg/
well
100
µg/
50
µg/
well
75
µg/
well
100
µg/
well
well
well
well
12±1.3 16±1.2 17±2.1 18±1.5
16±2.5 19±2.1 21±1.7 22±1.4
21±2.3
24±2.6
15±1.3
19±1.9
32±2.3
34±1.5
-
23±1.8
27±1.2
18±2.4
22±2.1
36±1.7
38±2.6
-
7±1.6
9±2.5
14±2.0
7±1.6
11±2.7
19±1.2
21±1.5
-
10±2.1
17±1.9
9±1.3
12±1.7
21±2.5
24±2.2
-
15±1.6
19±2.3
12±2.5
14±2.2
26±1.8
27±1.4
-
18±1.3
22±2.1
13±1.8
15±1.2
30±2.3
29±1.6
-
19±2.5
25±1.4
16±2.6
17±1.3
31±1.9
33±1.2
-
4a
4b
4c
4d
4e
4f
12±2.2
-
11±2.3 12±1.9 13±2.1
-
10±1.6 13±1.4 14±2.5 17±1.7
19±2.2 22±1.5 25±1.3 30±2.6
21±2.4 24±2.1 26±1.6 31±1.2
-
16±1.4
19±1.9
-
-
-
-
-
-
-
-
-
-
8±2.3
-
-
-
-
5a
5b
5c
11±1.7
-
15±1.9
-
-
-
-
-
11±2.1
-
-
-
-
-

ACCEPTED MANUSCRIPT
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
9±2.3
12±1.6
-
-
-
5d
5e
5f
9±2.5
12±1.6
14±1.3
15±2.4
10±1.8
12±2.3
-
17±1.5
19±2.1
13±1.2
16±2.6
-
11±2.2
13±1.3
-
13±1.5
14±2.4
9±1.2
12±1.7
-
10±1.8 13±1.2
-
7±2.2
6a
6b
6c
6d
6e
6f
8±2.3
10±1.5
8±2.1
-
10±1.5
-
-
-
-
-
-
-
-
-
-
11±1.9 12±1.6 15±1.4 15±2.5 16±2.1 20±1.4
13±2.6 14±1.3 16±1.8 16±1.9 19±1.4 21±2.5
9±1.2
9±1.6
10±2.4
11±2.2
11±2.5
13±1.4
15±1.5 18±2.2 20±2.4 22±2.0 23±1.3 26±2.1
19±1.2 21±2.5 24±1.7 25±1.2 28±2.1 32±1.6
9±1.8 12±1.3 14±1.8 16±1.3 18±1.4 21±2.3
9±2.3
14±1.9
-
10±1.3
16±2.4
8±2.2
-
12±1.8
19±2.1
11±2.5
18±2.5
23±1.3
14±1.7
20±1.2
25±2.6
16±1.5
23±2.1
27±1.8
18±1.3
7a
7b
7c
-
12±1.7 15±2.1 16±1.3 18±2.5 20±2.6 24±2.1
23±2.4 26±2.6 27±1.7 33±1.2 36±2.2 40±1.4
24±2.1 28±1.3 29±2.5 35±2.4 38±2.2 41±1.7
17±1.4 21±2.4 23±1.7 24±2.1 26±1.4 29±2.3
13±1.3
24±1.8
26±1.5
15±2.1
15±1.4
29±1.2
30±1.9
21±2.5
17±2.1
32±2.5
33±2.3
24±1.6
20±1.6
34±2.2
37±1.8
26±2.3
7d
7e
7f
20±1.2
22±2.4
11±2.6
21±2.6
23±2.1
13±1.8
8a

ACCEPTED MANUSCRIPT
23±2.2 26±1.9 28±1.4 30±1.8 32±1.7 35±2.1
17±1.5
-
19±2.4
9±2.1
21±2.2
12±1.5
14±2.6
26±2.1
28±1.5
30±2.6
26±2.6
16±2.4
17±1.8
31±1.7
32±2.1
38±1.4
29±1.5
20±1.2
22±2.3
34±1.4
35±2.6
40±2.1
32±1.2
21±2.5
24±2.2
36±1.9
38±1.4
42±2.5
8b
8c
8d
8e
8f
12±2.5 14±1.2 16±2.3 18±2.1 20±2.6 23±1.3
13±1.7 17±2.5 19±1.7 21±1.2 23±2.2 25±2.5
25±1.4 28±1.6 30±2.2 34±1.5 38±2.4 42±1.9
27±2.3 30±2.1 32±1.8 37±1.3 39±1.6 44±2.3
8±1.2
21±1.4
23±2.1
25±1.9
10±1.6
23±2.5
25±1.2
27±2.2
Chloramp 30±2.5 33±1.8 35±2.4 32±2.3 34±2.6 38±1.7
henicol
-
-
-
-
-
-
-
-
-
-
-
-
Control
(DMSO)
(-) No activity. (±) Standard deviation

ACCEPTED MANUSCRIPT
Table 2
The in vitro antifungal activity of compounds 4-8.
Zone of inhibition(mm)
Compound
A. niger
75 µg/well 100 µg/well
P. chrysogenum
50 µg/well
50 µg/well
75 µg/well
100 µg/well
11±2.1
17±1.9
10±1.3
12±2.6
27±1.8
28±1.5
-
4a
4b
4c
4d
4e
4f
10±1.7
13±1.2
17±1.4
8±2.3
11±1.3
25±1.9
29±2.2
-
15±2.3
21±1.6
12±1.8
14±1.4
28±2.3
31±1.5
-
-
8±1.5
15±2.1
10±1.2
13±2.4
-
-
-
9±2.5
-
10±2.2
23±1.4
22±2.1
24±1.4
27±1.2
24±1.3
26±2.5
5a
5b
5c
5d
-
-
-
-
-
-
-
-
-
8±1.9
11±2.5
8±1.7
-
13±2.1
10±1.3
-
9±2.2
-
-
-
-

ACCEPTED MANUSCRIPT
5e
5f
-
12±1.2
14±1.4
-
15±1.7
17±2.3
-
-
9±2.1
10±1.8
-
12±2.4
14±1.6
-
10±1.5
-
-
6a
6b
6c
6d
6e
6f
-
-
9±2.5
-
12±1.2
-
-
-
7±2.2
-
-
-
-
-
-
-
-
-
-
-
10±1.5
12±1.7
16±2.4
21±1.9
15±2.2
19±1.5
28±2.4
31±1.8
20±1.9
13±2.6
15±2.1
20±1.6
25±2.5
17±1.2
23±1.4
31±2.1
34±1.3
23±1.7
-
8±2.4
9±1.9
14±2.6
15±1.4
8±1.6
10±1.2
24±2.2
25±2.4
16±1.5
10±1.5
12±1.3
16±2.1
17±2.4
10±2.3
13±1.8
27±1.2
29±1.5
19±1.7
9±2.3
15±1.8
18±1.3
12±1.5
16±2.6
25±1.2
29±2.1
18±1.4
-
7a
7b
7c
7d
7e
7f
12±2.2
13±1.7
-
-
22±2.5
24±1.6
14±2.3
8a

ACCEPTED MANUSCRIPT
8b
8c
8d
8e
21±2.3
13±1.2
18±2.4
33±2.6
35±1.7
24±1.5
15±2.4
21±1.7
35±2.1
37±1.5
28±2.3
18±2.1
26±1.5
38±1.3
40±2.4
17±2.6
10±1.8
14±1.7
25±2.4
27±2.1
19±1.8
12±2.6
16±2.2
28±2.0
29±1.5
22±1.3
16±1.5
18±2.4
30±1.2
33±2.6
8f
Ketoconazole
Control (DMSO)
31±1.9
33±2.5
36±2.1
35±2.3
36±1.9
38±1.7
-
-
-
-
-
-
(-) No activity. (±) Standard deviation.

ACCEPTED MANUSCRIPT
Table 3 MIC, MBC and MFC of compounds 4e, 4f, 7e, 7f, 8e and 8f.
Minimum inhibitory concentration
MIC (MBC/MFC) g/well
µ
Compound
S. aureus
100 (>200)
50 (200)
50 (200)
50 (200)
50 (200)
50 (200)
12.5
B. subtilis P. aeruginosa K. pneumoniae A. niger
P. chrysogenum
100 (>200)
100 (>200)
100 (>200)
50 (200)
4e
12.5 (50)
6.25 (12.5)
6.25 (12.5)
6.25 (12.5)
6.25 (12.5)
6.25 (12.5)
6.25
25 (100)
25 (100)
25 (100)
12.5 (50)
12.5 (50)
12.5 (50)
6.25
50 (200)
25 (100)
25 (100)
25 (100)
25 (100)
25 (100)
6.25
25 (100)
25 (100)
25 (100)
12.5 (50)
6.25 (12.5)
6.25 (12.5)
—
4f
7e
7f
50 (200)
8e
8f
50 (200)
Chloramphenicol
—
Ketoconazole
—
—
—
—
6.25
12.5
(-) No activity. (±) Standard deviation.

ACCEPTED MANUSCRIPT
O O
S
OH OH
O
O O
S
O O
S
H2N
N
OH
H2N
NH
N
N
N
S
O
N
CF3
Cl
N
O
H
F
Crestor
Diovan HCT
Celebrex
O
Cl
O
H
O
O
O
N
N
N
N
N
N
N
O
N
C
N
O
Tebufenpyrad
Cyenopyrafen
Fenpyroximate
O O
S
CF3
O O
O
NH2
N
O
S
N
N
H
H
N
N
O
NH2
O
Leflunomide
Sulfisoxazole
Valdecoxib
O
N
O
N
Cl
H
H
H
N
H
N
N
S
S
O
O
N
O
O
OH
OH
O
O
Oxacillin
Cloxacillin
Fig. 1. Drugs containing sulfonamide, pyrazole and isoxazole units.

ACCEPTED MANUSCRIPT
Fig. 2. The in vitro antibacterial activity of compounds 4-8.
Fig. 3. The in vitro antifungal activity of compounds 4-8.

ACCEPTED MANUSCRIPT
Highlights:
•
•
•
•
•
A variety of pyrazolyl / isoxazolyl thiophene carboxamides were prepared.
Green conditions were used for addition of 1,3-dipolar reagents and aromatization.
Electron withdrawing substituents on aromatic ring increased antimicrobial activity.
4f, 7e, 7f, 8e and 8f showed potential antibacterial activity against B. subtilis.
8e and 8f exhibited prominent antifungal activity against A. niger.