Synthesis and antimicrobial activity of styryl/pyrrolyl/pyrazolyl sulfonylmethyl-1,3,4-oxadiazolyl amines and styryl/pyrrolyl/pyrazolyl sulfonylmethyl-1,3,4-thiadiazolyl amines

Article abstract of DOI:10.1016/j.ejmech.2016.06.014

A new class of mono and bis heterocycles - styryl sulfonylmethyl-1,3,4-oxadiazolyl/1,3,4-thiadiazolyl amines, pyrrolyl sulfonylmethyl-1,3,4-oxadiazolyl/1,3,4-thiadiazolyl amines and pyrazolyl sulfonylmethyl-1,3,4-oxadiazolyl/1,3,4-thiadiazolyl amines were

Full text of DOI:10.1016/j.ejmech.2016.06.014

Accepted Manuscript
Synthesis and antimicrobial activity of styryl/pyrrolyl/pyrazolyl sulfonylmethyl-1,3,4-
oxadiazolyl amines and styryl/pyrrolyl/pyrazolyl sulfonylmethyl-1,3,4-thiadiazolyl
amines
G. Sravya, G. Yamini, V. Padmavathi, A. Padmaja
PII:
S0223-5234(16)30492-5
10.1016/j.ejmech.2016.06.014
EJMECH 8675
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 16 March 2016
Revised Date: 10 May 2016
Accepted Date: 10 June 2016
Please cite this article as: G. Sravya, G. Yamini, V. Padmavathi, A. Padmaja, Synthesis and
antimicrobial activity of styryl/pyrrolyl/pyrazolyl sulfonylmethyl-1,3,4-oxadiazolyl amines and styryl/
pyrrolyl/pyrazolyl sulfonylmethyl-1,3,4-thiadiazolyl amines, European Journal of Medicinal Chemistry
(2016), doi: 10.1016/j.ejmech.2016.06.014.
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
Synthesis and Antimicrobial Activity of Styryl / Pyrrolyl / Pyrazolyl Sulfonylmethyl-
1,3,4-Oxadiazolyl Amines and Styryl / Pyrrolyl / Pyrazolyl Sulfonylmethyl-1,3,4-
Thiadiazolyl Amines
Gundala Sravya, Gudi Yamini, Venkatapuram Padmavathi, Adivireddy Padmaja*
A new class of mono and bis heterocycles - styryl sulfonylmethyl-1,3,4-oxadiazolyl / 1,3,4-
thiadiazolyl amines, pyrrolyl sulfonylmethyl-1,3,4-oxadiazolyl / 1,3,4-thiadiazolyl amines and
pyrazolyl sulfonylmethyl-1,3,4-oxadiazolyl / 1,3,4-thiadiazolyl amines were prepared from
the synthetic intermediate Z-styrylsulfonylacetic acid adopting simple and well versed
synthetic methodologies and studied their antimicrobial activity. Amongst all the tested
compounds styryl thiadiazole 5c exhibited promising antimicrobial activity against P.
aeruginosa and P. chrysogenum.
N N
X
N N
X
N N
4
O
3
O
O
O
O
O
5
2
Ar
S
Ar
S
Ar
S
1
NH2
NH2
NH2
1
1
X
3
1
4
41
3
21
51
21
51
N
11
11
N
N
H
H
4 / 5
10 / 11
6 / 7
4 / 6 / 10 : X = O
5 / 7 / 11 : X = S

ACCEPTED MANUSCRIPT
Synthesis and antimicrobial activity of styryl / pyrrolyl / pyrazolyl
sulfonylmethyl-1,3,4-oxadiazolyl amines and styryl / pyrrolyl / pyrazolyl
sulfonylmethyl-1,3,4-thiadiazolyl amines
G. Sravya, G. Yamini, V. Padmavathi, A. Padmaja*
Department of Chemistry, Sri Venkateswara University, Tirupati 517 502, Andhra Pradesh, India.
E-mail: advireddyp@yahoo.co.in
ABSTRACT
A new class of mono and bis heterocycles - styryl sulfonylmethyl-1,3,4-oxadiazolyl / 1,3,4-
thiadiazolyl amines, pyrrolyl sulfonylmethyl-1,3,4-oxadiazolyl / 1,3,4-thiadiazolyl amines and
pyrazolyl sulfonylmethyl-1,3,4-oxadiazolyl / 1,3,4-thiadiazolyl amines were prepared from the
synthetic intermediate Z-styrylsulfonylacetic acid adopting simple and well versed synthetic
methodologies and studied their antimicrobial activity. Amongst all the tested compounds styryl
thiadiazole 5c exhibited promising antimicrobial activity against P. aeruginosa and P.
chrysogenum.
Keywords: Antimicrobial activity / Semicarbazide / Thiosemicarbazide / 1,3-Dipolar
cycloaddition
1. Introduction
Nitrogen containing heteroarenes have great utility in synthetic medicinal and material
chemistry. One such class of compounds are oxadiazoles, thiadiazoles, pyrroles and pyrazoles.
1,3,4-Oxadiazoles are associated with potent pharmacological activities due to the presence of
toxophoric –N=C–O linkage and display antimicrobial [1], antimycobacterial [2], antiviral [3] and
anticancer [4] activities. Further, they are very good bioisosteres of amides and esters, which can
contribute substantially in increasing pharmacological activity by participating in hydrogen
1

ACCEPTED MANUSCRIPT
bonding interactions with the receptors. Thiadiazole derivatives are known to possess anticancer
[5], antibacterial [6], antifungal [7], anti-inflammatory [8] and analgesic [9] activities. On the other
hand, pyrroles have been widely used as building blocks in total synthesis of many natural
products and exhibit biological activities such as powerful antibacterial, antiviral, anti-
inflammatory, antitumor, and antioxidant activities [10]. Pyrrole containing drugs Netropsin and
Distamycin are naturally anticancer antibiotics [11] (Fig. 1). Among the various synthetic methods,
the notable classical ones are Knorr [12] and Hantzsch [13] including multicomponent and metal-
catalyzed routes [14]. Literature survey indicates that pyrazole derivatives are well known for their
antibacterial [15], antifungal [16], antimicrobial [17], anticancer [18], analgesic and anti-inflammatory
[19, 20] activities. Pyrazole moiety makes the core structure of various drugs such as Difenamizole
[21], Celecoxib [22], Tepoxalin [23] etc (Fig. 1). The 1,3-dipolar cycloaddition methodology is
widely used for the syntheses of pyrazoles using diverse synthons such as nitrilimines and alkynes
[24] hydrazones and nitroolefins [25] and azomethine imines and alkynes [26]. In recent years
there is still a great demand for designing new compounds and broad spectrum antimicrobial
agents remains a challenge for medicinal chemistry researchers. Motivated by the aforesaid
findings and in continuation of our studies towards the development of a variety of biologically
potent heterocycles [27], herein we plan to synthesize a new class of mono and bis heterocycles –
styryl / pyrrolyl / pyrazolyl sulfonylmethyl-1,3,4-oxadiazolyl amines and styryl / pyrrolyl /
pyrazolyl sulfonylmethyl-1,3,4-thiadiazolyl amines and to study their antimicrobial activity.
2. Chemistry
A new class of mono and bis heterocycles- 5-Z-styrylsulfonylmethyl-1,3,4-oxadiazolyl /
thiadiazolyl amines, pyrrolyl sulfonylmethyl-1,3,4-oxadiazolyl / thiadiazolyl amines and pyrazolyl
sulfonylmethyl-1,3,4-oxadiazolyl / thiadiazolyl amines from the synthetic intermediate
2

ACCEPTED MANUSCRIPT
Z-styrylsulfonylacetic acid (3) (Schemes 1 and 2). The cyclocondensation of compound 3 with
semicarbazide / thiosemicarbazide in the presence of POCl3 produced 5-(2-arylethene-
sulfonylmethyl)-1,3,4-oxadiazol-2-amine (4) / 5-(2-arylethenesulfonylmethyl)-1,3,4-thiadiazol-2-
1
amine (5). The H NMR spectra of 4a and 5a displayed a singlet at δ 5.20 and 5.24 due to
methylene protons attached to C-5. Apart from this, a doublet was observed in these compounds at
6.84 and 6.93 ppm due to olefin proton HB and another doublet due to olefin proton, HA adjacent to
aryl group appeared at downfield region and merged with aromatic protons. The coupling constant
value J ≈ 9.3 Hz indicated that they possess cis geometry. Moreover, a broad singlet appeared at
4.25 and 4.30 ppm in 4a and 5a was assigned to NH2 which disappeared on deuteration.
The olefin moiety present in compounds 4 and 5 was utilized to develop five membered
heterocycles- pyrroles and pyrazoles. The 5-(4-aryl-1H-pyrrole-3-sulfonylmethyl)-1,3,4-oxadiazol-
2-amine (6) and 5-(4-aryl-1H-pyrrole-3-sulfonylmethyl)-1,3,4-thiadiazol-2-amine (7) were
prepared by the treatment of 4 and 5 with tosylmethyl isocyanide in the presence of sodium
1
hydride in a solvent mixture of DMSO and ether. The H NMR spectra of 6a and 7a exhibited a
singlet at δ 5.02 and 5.08 ppm due to methylene protons attached to C-5 of oxadiazole and
thiadiazole. In addition to these, a singlet was observed at 6.64 and 6.69 due to C5'-H of pyrrole
ring and another singlet due to C2'-H appeared at downfield region and merged with aromatic
protons. Moreover, in 6a and 7a two broad singlets appeared at 5.38, 8.70 and 5.43, 8.73 ppm were
attributed to NH2 and NH of pyrrole ring, respectively. The signals of highly acidic protons
disappeared when D2O was added. The cycloadditon of diazomethane to 4 and 5 in the presence of
Et3N in ether at -20 to -15ºC for 42-48 h gave 5-(4,5-dihydro-4-aryl-1H-pyrazol-3-sulfonyl-
methyl)-1,3,4-oxadiazol-2-amine (8) / 5-(4,5-dihydro-4-aryl-1H-pyrazol-3-sulfonylmethyl)-1,3,4-
1
thiadiazol-2-amine (9). In the H NMR spectra of 8a and 9a an AMX splitting pattern was
observed due to pyrazoline ring protons. Thus three double doublets present at δ 3.54, 4.21, 4.56 in
3

ACCEPTED MANUSCRIPT
8a and at 3.56, 4.29, 4.60 ppm in 9a were assigned to HX, HM and HA, respectively. The coupling
constant values JAM ≈ 12.2, JMX ≈ 10.4 and JAX ≈ 6.1 Hz indicated that HA, HM are cis, HA, HX are
trans while HM, HX are geminal. Further 8a and 9a displayed signals at δ 4.91, 4.95 (CH2), 5.51,
5.55 (NH2) and 10.08, 10.35 ppm (NH) respectively. The signals due to NH2 and NH disappeared
on deuteration. Aromatization of the compounds 8 and 9 with chloranil in xylene provided 5-(4-
phenyl-1H-pyrazol-3-sulfonylmethyl)-1,3,4-oxadiazol-2-amine (10) and 5-(4-phenyl-1H-pyrazol-
3-sulfonylmethyl)-1,3,4-thiadiazol-2-amine (11). The absence of AMX splitting pattern due to
pyrazoline ring protons in 10 and 11 indicated that aromatization took place. The structures of all
the compounds were further confirmed by IR, 13C NMR, mass spectra and elemental analyses.
O
O
N
N
O
O
O
O
i
ii
Ar
HA
S
Ar
Ar
S
iii
Ar
HA
S
NH2
OH
OH
X
HB
HB
1
2
3
4 / 5
Ar = a) Ph
(i) HSCH2CO2H / NaOH / MeOH
4 : X = O
5 : X = S
b) 4-Me.Ph
c) 4-Cl.Ph
(ii) H O2 /AcOH
2
(iii) NH NHCXNH2 / POCl3
2
Scheme 1. Synthesis of styrylsulfonylmethyl-1,3,4-oxadiazolylamines and styrylsulfonylmethyl-
1,3,4-thiadiazolylamines.
N
N
N
N
N N
4
HA
3
O
O
O
O
O
O
8 / 9
O
5
2
ii
Ar
S
i
Ar
S
1
X
Ar
HM
HX
S
NH2
NH2
NH2
41 31
21
X
X
51
N
11
N
N
H
H
4 / 5
6 / 7
iii
N
N
O
Ar
S
NH2
X
41 31
21
51
11
N
N
H
(i) TosMIC / NaH / DMSO / Et 2O
(ii) CH2N2 / Et3N / Et2O / -20 to -15oC
Ar = a) Ph
4 / 6 / 8 / 10 : X = O
5 / 7 / 9 / 11 : X = S
10 / 11
b) 4-Me.Ph
c) 4-Cl.Ph
(iii) Chloranil / Xylene
Scheme 2. Synthesis of pyrrolyl / pyrazolyl- sulfonylmethyl-1,3,4,-oxadiazolylamines and pyrrolyl
/ pyrazolyl- sulfonylmethyl-1,3,4,-thiadiazolylamines
4

ACCEPTED MANUSCRIPT
3. Biology
3.1. Antimicrobial activity
The compounds 4–11 were screened for antibacterial activity at four different
concentrations 12.5, 25, 50 and 100 ꢀg/well.
4. Results and discussion
4.1 Antibacterial activity
The results of antibacterial activity shown in Table 1 indicated that Gram-negative bacteria
were more susceptible towards the tested compounds than Gram-positive ones. It was observed
that mono heterocyclic compounds (4 and 5) displayed slightly higher activity than the respective
bis heterocyclic systems (6-11). This may be due to the presence of electron withdrawing styryl
moiety in mono heterocyclic derivatives. The compounds having thiadiazole moiety (5, 7, 9, 11)
were more active than those having oxadiazole moiety (4, 6, 8, 10). In fact, the compound 5c
showed excellent activity against P. aeruginosa when compared with the standard drug
Chloramphenicol. This may be due to the presence of more electronegative chlorine on the
aromatic ring. Among bis heterocyclic compounds aromatized derivatives (6, 7, 10 and 11) were
more effective. On the other hand the non-aromatized compounds (8 and 9) were inactive.
Moreover, it was observed that pyrazole containing bis heterocycles 10 and 11 displayed slightly
higher activity than the respective pyrrole containing bis heterocycles 6 and 7.
4.2. Antifungal activity
All the tested compounds inhibited the spore germination against tested fungi except
compound 8. In general, most of the compounds showed slightly higher antifungal activity towards
P. chrysogenum than A. niger. Amongst all the compounds 5c displayed greater inhibitory activity
particularly against P. chrysogenum when compared with the standard drug Ketoconazole
(Table 2). Moreover compounds 7c and 11c exhibited good activity. In fact compounds having
5

ACCEPTED MANUSCRIPT
pyrazole and pyrrole in combination with thiadiazole were displayed high inhibitory activity than
others.
4.3. MIC, MBC and MFC of compounds 5c, 7c and 11c
The minimum inhibitory concentration (MIC), minimum bactericidal concentration (MBC) and
minimum fungicidal concentration (MFC) values of the compounds tested 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 the MBC/MFC is not greater than four times the MIC. The compound 5c
exhibited low MIC values when compared with 7c and 11c. In addition MBC value in 5c was
2×MIC in case of P. aeruginosa and MFC value was 2×MIC in case of P. chrysogenum. However,
the other compounds showed bactericidal and fungicidal effects greater than 2×MIC. The
structure–antimicrobial activity relationship of the synthesized compounds revealed that mono
heterocyclic compounds with extended conjugation displayed greater activity than the
corresponding bis heterocycles. Amongst bis heterocyclic systems, the aromatized compounds
having thiadiazole moiety (7, 11) were more effective when compared with those having
oxadiazole unit (6, 10). The chloro substituted styryl-1,3,4-thiadiazole 5c exhibited excellent
antibacterial activity against P. aeruginosa with an inhibition zone of 34 mm at 100 ꢀg/well and
MIC and MBC of 6.25 and 12.5 ꢀg/ml, respectively. The compound 5c also displayed strong
antifungal activity against P. chrysogenum with an inhibition zone of 41 mm at 100 ꢀg/well and
MIC and MFC of 12.5 and 25 ꢀg/ml, respectively. Moreover, it was observed that the compounds
6

ACCEPTED MANUSCRIPT
having chloro substituent on aromatic ring enhanced the activity when compared with methyl and
unsubstitued compounds.
5. Conclusion
In conclusion we have prepared a new class of mono and bis heterocycles-
styrylsulfonymethyl-1,3,4-oxadiazolyl / thiadiazolyl amines, pyrrolyl sulfonylmethyl-1,3,4-
oxadiazolyl / thiadiazolyl amines and pyrazolyl sulfonylmethyl-1,3,4-oxadiazolyl / thiadiazolyl
amines from the simple substrate Z-styrylsulfonylacetic acid adopting simple and well versed
synthetic methodologies. All the new compounds were tested for antimicrobial activity. In general
compounds having thiadiazole unit displayed higher antimicrobial activity than those with
oxadiazole moiety. The compound 5c having chloro substituent on the aromatic ring showed
promising antimicrobial activity against P. aeruginosa and P. chrysogenum.
6. Experimental
6.1. Chemistry
Melting points were determined in open capillaries on a Mel-Temp apparatus and are
uncorrected. The homogeneity of the compounds was checked by TLC (silica gel H, BDH,
hexane/ethyl acetate, 3:1). The IR spectra were recorded on a Thermo Nicolet IR 200 FT-IR
spectrometer as KBr pellets and the wave numbers were given in cm−1. The 1H NMR spectra were
recorded in CDCl3 / DMSO-d6 on a Jeol JNM λ-400 MHz spectrometer. The 13C NMR spectra
were recorded in CDCl3 / DMSO-d6 on a Jeol JNM spectrometer operating at λ-100 MHz. High-
resolution mass spectra were recorded on Micromass Q-TOF micromass spectrometer using
electrospray ionization. All chemical shifts were reported in δ (ppm) using TMS as an internal
standard. The elemental analyses were determined on a Perkin-Elmer 240C elemental analyzer.
The temperature was measured by flexible probe throughout the reaction. The starting compound
Z-styrylsulfonylacetic acid (3) was prepared as per the literature precedent [28].
7

ACCEPTED MANUSCRIPT
6.1.1. General procedure for the synthesis of 5-(2-arylethenesulfonylmethyl)-1,3,4-oxadiazol-2-
amine / 5-(2-arylethenesulfonylmethyl)-1,3,4-thiadiazol-2-amine (4a-c / 5a-c)
To an equimolar mixture (1 mmol) of semicarbazide / thiosemicarbazide and
Z-styrylsulfonylacetic acid (3a-c), POCl3 (7 ml) was added and heated under reflux for 4-6 h. The
excess POCl3 was removed under vacuum and the residue was poured onto crushed ice. The
resulting precipitate was filtered, washed with saturated sodium bicarbonate solution and then with
water. It was dried and recrystallized from ethanol.
6.1.1.1. 5-(2-Phenylethenesulfonylmethyl)-1,3,4-oxadiazol-2-amine (4a). White solid (0.117g,
67%); m.p. 109-111oC; IR (KBr): 1150, 1324 (SO2), 1571 (C=N), 1615 (C=C), 3337, 3445 (NH2)
cm–1; 1H NMR (DMSO-d6): δ 4.25 (bs, 2H, NH2), 5.20 (s, 2H, CH2), 6.84 (d, 1H, HB, J = 9.2 Hz),
7.15-7.23 (m, 6H, HA & Ar-H) ppm; 13C NMR (DMSO-d6): δ 50.5 (CH2), 132.0 (C-HB), 142.2
(C-HA), 154.2 (C-5), 159.6 (C-2), 127.1, 128.0, 129.9, 130.0 (aromatic carbons) ppm; HRMS
(m/z): 288.2774 [M+Na]; Anal. Calcd. for C11H11N3O3S: C, 49.87; H, 4.20; N, 15.88; Found: C,
49.80; H, 4.18; N, 15.84%.
6.1.1.2. 5-(4-Methylstyrylsulfonylmethyl)-1,3,4-oxadiazol-2-amine (4b). White solid (0.181g,
65%); m.p. 118-120oC; IR (KBr): 1148, 1320 (SO2), 1564 (C=N), 1610 (C=C), 3319, 3438 (NH2)
cm–1; 1H NMR (DMSO-d6): δ 2.26 (s, 3H, Ar-CH3), 4.21 (bs, 2H, NH2), 5.16 (s, 2H,CH2), 6.87 (d,
1H, HB, J = 8.5 Hz), 7.06-7.20 (m, 5H, HA & Ar-H) ppm; 13C NMR (DMSO-d6): δ 24.5 (Ar-CH3),
49.8 (CH2), 131.4 (C-HB), 141.7 (C-HA), 154.0 (C-5), 159.2 (C-2), 126.8, 127.6, 128.4, 129.7
(aromatic carbons) ppm; HRMS (m/z): 302.3032 [M+Na]; Anal. Calcd. for C12H13N3O3S: C,
51.69; H, 4.70; N, 15.21; Found: C, 51.60; H, 4.69; N, 15.04%.
6.1.1.3. 5-(4-Chlorostyrylsulfonylmethyl)-1,3,4-oxadiazol-2-amine (4c). White solid (0.206g,
69%); m.p. 127-129oC; IR (KBr): 1153, 1330 (SO2), 1579 (C=N), 1620 (C=C), 3342, 3452 (NH2)
cm–1; 1H NMR (DMSO-d6): δ 4.27 (bs, 2H, NH2), 5.22 (s, 2H, CH2), 6.99 (d, 1H, HB, J = 9.2 Hz),
8

ACCEPTED MANUSCRIPT
7.21-7.29 (m, 5H, HA & Ar-H) ppm; 13C NMR (DMSO-d6): δ 50.9 (CH2), 132.4 (C-HB), 142.6 (C-
HA), 154.8 (C-5), 160.2 (C-2), 127.6, 128.4, 130.5, 132.4 (aromatic carbons) ppm; HRMS (m/z):
322.7241 [M+Na]; Anal. Calcd. for C11H10ClN3O3S: C, 44.02; H, 3.35; N, 14.13; Found: C, 44.08;
H, 3.36; N, 14.02%.
6.1.1.4. 5-(2-Phenylethenesulfonylmethyl)-1,3,4-thiadiazol-2-amine (5a). White solid (0.211g,
75%); m.p. 122-124oC; IR (KBr): 1160, 1336 (SO2), 1580 (C=N), 1625 (C=C), 3339, 3455 (NH2)
cm–1; 1H NMR (DMSO-d6): δ 4.30 (bs, 2H, NH2), 5.24 (s, 2H, CH2), 6.93 (d, 1H, HB, J = 9.4 Hz),
7.17-7.42 (m, 6H, HA & Ar-H) ppm; 13C NMR (DMSO-d6): δ 51.2 (CH2), 133.2 (C-HB), 142.8 (C-
HA), 154.4 (C-5), 160.0 (C-2), 128.4, 129.2, 129.8. 130.6 (aromatic carbons) ppm; HRMS (m/z):
304.3431 [M+Na]; Anal. Calcd. for C11H11N3O2S2: C, 47.07; H, 3.97; N, 14.99; Found: C, 46.96;
H, 3.94; N, 14.93%.
6.1.1.5. 5-(4-Methylstyrylsulfonylmethyl)-1,3,4-thiadiazol-2-amine (5b). White solid (0.206g,
70%); m.p. 130-132oC; IR (KBr): 1161, 1325 (SO2), 1563 (C=N), 1622 (C=C), 3332, 3432 (NH2)
cm–1; 1H NMR (DMSO-d6): δ 2.28 (s, 3H, Ar-CH3), 4.28 (bs, 2H, NH2), 5.18 (s, 2H, CH2), 6.89 (d,
1H, HB, J = 8.8 Hz), 7.08-7.22 (m, 5H, HA & Ar-H) ppm; 13C NMR (DMSO-d6): δ 24.7 (Ar-CH3),
50.6 (CH2), 131.9 (C-HB), 142.5 (C-HA), 154.2 (C-5), 159.5 (C-2), 127.0, 127.9, 128.7, 130.2
(aromatic carbons) ppm; HRMS (m/z): 318.3711 [M+Na]; Anal. Calcd. for C12H13N3O2S2: C,
48.87; H, 4.46; N, 14.36; Found: C, 48.79; H, 4.44; N, 14.23%.
6.1.1.6. 5-(4-Chlorostyrylsulfonylmethyl)-1,3,4-thiadiazol-2-amine (5c). White solid (0.246, 78%);
m.p. 141-143oC; IR (KBr): 1150, 1324 (SO2), 1571 (C=N), 1615 (C=C), 3337, 3445 (NH2) cm–1;
1H NMR (DMSO-d6): δ 4.25 (bs, 2H NH2), 5.20 (s, 2H CH2), 6.90 (d, 1H HB, J = 8.9 Hz), 7.15-
13
7.23 (m, 6H HA & Ar-H) ppm; C NMR (DMSO-d6): δ 50.5 (CH2), 132.0 (C-HB), 142.2 (C-HA),
154.2 (C-5), 159.6 (C-2), 127.1, 128.0, 129.9, 130.0 (aromatic carbons) ppm; HRMS (m/z):
9

ACCEPTED MANUSCRIPT
338.7882 [M+Na]; Anal. Calcd. for C11H10ClN3O2S2: C, 41.89; H, 3.18; N, 13.43; Found: C,
41.84; H, 3.19; N, 13.3%.
6.1.2. General method for the preparation of 5-(4-aryl-1H-pyrrole-3-sulfonylmethyl)-1,3,4-
oxadiazol-2-amine / 5-(4-aryl-1H-pyrrole-3-sulfonylmethyl)-1,3,4-thiadiazol-2-amine (6a-c / 7a-c)
A mixture of 4a-c / 5a-c (0.5 mmol) and TosMIC (1 mmol) in Et2O-DMSO (2:1) was
added dropwise under stirring to a suspension of NaH (50 mg) in Et2O (10 ml) at room
temperature and stirring was continued for 4-6 h. Then water was added and the reaction mass was
extracted with Et2O. The ethereal layer was dried over anhydrous Na2SO4 and filtered. Evaporation
of the solvent in vacuo gave a solid which was purified by column chromatography using silica gel
(hexane-ethyl acetate; 4:1).
6.1.2.1. 5-(4-Phenyl-1H-pyrrole-3-sulfonylmethyl)-1,3,4-oxadiazol-2-amine (6a). White solid
(0.095g, 63%); m.p. 136-138oC; IR (KBr): 1136, 1319 (SO2), 1560 (C=N), 1624 (C=C), 3237
(NH), 3315, 3439 (NH2) cm–1; 1H NMR (DMSO-d6): δ 5.02 (s, 2H, CH2), 5.38 (bs, 2H, NH2), 6.64
13
(s, 1H, C5’-H), 7.17-7.78 (m, 6H, C2’-H & Ar-H), 8.70 (bs, 1H, NH) ppm; C NMR (DMSO-d6):
δ 56.1 (CH2), 104.1 (C-4'), 110.3 (C-3'), 117.6 (C-5'), 121.8 (C-2'), 154.5 (C-5), 156.4 (C-2),
127.4, 129.1, 132.5, 136.4 (aromatic carbons) ppm; HRMS (m/z): 327.3137 [M+Na]; Anal. Calcd.
For C13H12N4O3S: C, 51.41; H, 4.00; N, 18.59; Found: C, 51.31; H, 3.97; N, 18.41%.
6.1.2.2. 5-(4-(4-Methylphenyl)-1H-pyrrole-3-sulfonylmethyl)-1,3,4-oxadiazol-2-amine (6b). White
solid (0.105g, 66%); m.p. 144-146oC; IR (KBr): 1132, 1314 (SO2), 1557 (C=N), 1621 (C=C), 3233
1
(NH), 3310, 3428 (NH2) cm–1; H NMR (DMSO-d6): δ 2.24 (s, 3H, Ar-CH3), 4.98 (s, 2H, CH2),
5.29 (bs, 2H, NH2), 6.55 (s, 1H, C5'-H), 7.09-7.71 (m, 5H, C2'-H & Ar-H), 8.69 (bs, 1H, NH) ppm;
13C NMR (DMSO-d6): δ 23.4 (Ar-CH3), 55.9 (CH2), 103.7 (C-4'), 109.4 (C-3'), 116.6 (C-5'), 121.1
(C-2'), 154.6 (C-5), 155.8 (C-2), 126.4, 128.5, 130.2, 135.4 (aromatic carbons) ppm; HRMS (m/z):
10

ACCEPTED MANUSCRIPT
341.3412 [M+Na]; Anal. Calcd. for C14H14N4O3S: C, 52.89; H, 4.44; N, 17.75; Found: C, 52.82;
H, 4.43; N, 17.60%.
6.1.2.3. 5-(4-(4-Chlorophenyl)-1H-pyrrole-3-sulfonylmethyl)-1,3,4-oxadiazol-2-amine (6c). White
solid (0.120g, 71%); m.p. 160-162oC; IR (KBr): 1143, 1322 (SO2), 1566 (C=N), 1629 (C=C), 3242
(NH), 3326, 3466 (NH2) cm–1; 1H NMR (DMSO-d6): δ 5.06 (s, 2H, CH2), 5.42 (bs, 2H, NH2), 6.67
(s, 1H, C5'-H), 7.21-7.82 (m, 5H, C2'-H & Ar-H), 8.75 (bs, 1H, NH) ppm; 13C NMR (DMSO-d6): δ
56.4 (CH2), 104.4 (C-4'), 110.6 (C-3'), 118.3 (C-5'), 122.2 (C-2'), 155.9 (C-5), 156.9 (C-2), 128.2,
130.5, 134.2, 136.9 (aromatic carbons) ppm; HRMS (m/z): 361.7588 [M+Na]; Anal. Calcd. for
C13H11ClN4O3S: C, 46.18; H, 3.30; N, 16.73; Found: C, 46.09; H, 3.27; N, 16.54%.
6.1.2.4. 5-(4-Phenyl-1H-pyrrole-3-sulfonylmethyl)-1,3,4-thiadiazol-2-amine (7a). White solid
0.107g, 67%); m.p. 148-150oC; IR (KBr): 1142, 1324 (SO2), 1562 (C=N), 1623 (C=C), 3253
(NH), 3320, 3446 (NH2) cm–1; 1H NMR (DMSO-d6): δ 5.08 (s, 2H, CH2), 5.43 (bs, 2H, NH2), 6.69
(s, 1H, C5'-H), 7.28-7.84 (m, 6H, C2'-H & Ar-H), 8.73 (bs, 1H, NH) ppm; 13C NMR (DMSO-d6): δ
56.6 (CH2), 104.6 (C-4'), 110.9 (C-3'), 117.9 (C-5'), 122.2 (C-2'), 158.1 (C-5), 159.6 (C-2), 128.2,
130.4, 133.5, 136.1 (aromatic carbons) ppm; HRMS (m/z): 343.3792 [M+Na]; Anal. Calcd. for
C13H12N4O2S2: C; 48.73, H; 3.78, N; 17.49; Found: C, 48.73; H, 3.82; N, 17.70%.
6.1.2.5. 5-(4-(4-Methylphenyl)-1H-pyrrole-3-sulfonylmethyl)-1,3,4-thiadiazol-2-amine (7b). White
solid (0.121g, 73%); m.p. 156-158oC; IR (KBr): 1139, 1310 (SO2), 1559 (C=N), 1619 (C=C), 3250
1
(NH), 3,312, 3,432 (NH2) cm–1; H NMR (DMSO-d6): δ 2.28 (s, 3H, Ar-CH3), 5.01 (s, 2H, CH2),
5.32 (bs, 2H, NH2), 6.62 (s, 1H, C5'-H), 7.21-7.80 (m, 5H, C2'-H & Ar-H), 8.71 (bs, 1H, NH) ppm;
13C NMR (DMSO-d6): δ 23.8 (Ar-CH3), 56.6 (CH2), 104.1 (C-4'), 110.2 (C-3'), 116.9 (C-5'), 121.8
(C-2'), 157.9 (C-5), 159.1 (C-2), 127.1, 129.1, 131.2, 135.8 (aromatic carbons) ppm; HRMS (m/z):
357.4075 [M+Na]; Anal. Calcd. For C14H14N4O2S2: C, 50.36; H, 4.15; N, 16.92; Found: C, 50.28;
H, 4.22; N, 16.75%.
11

ACCEPTED MANUSCRIPT
6.1.2.6. 5-(4-(4-Chlorophenyl)-1H-pyrrole-3-sulfonylmethyl)-1,3,4-thiadiazol-2-amine (7c). White
solid (0.135g, 76%); m.p. 165-167oC; IR (KBr): 1151, 1326 (SO2), 1573 (C=N), 1630 (C=C), 3262
1
(NH), 3,331, 3,451 (NH2) (cm–1); H NMR (DMSO-d6): δ 5.10 (s, 2H, CH2), 5.48 (bs, 2H, NH2),
6.71 (s, 1H, C5'-H), 7.25-7.88 (m, 5H, C2'-H & Ar-H), 8.77 (bs, 1H, NH) ppm; 13C NMR (DMSO-
d6): δ 56.9 (CH2), 104.9 (C-4'), 110.9 (C-3'), 118.9 (C-5'), 122.9 (C-2'), 158.4 (C-5), 159.8 (C-2),
129.1, 131.2, 134.9, 136.8 (aromatic carbons) ppm; HRMS (m/z): 377.8248 [M+Na]; Anal. Calcd.
for C13H11ClN4O2S2: C, 44.10; H, 3.16; N, 16.01; Found: C, 44.00; H, 3.12; N, 15.79%.
6.1.3. General method for the preparation of 5-(4,5-dihydro-4-aryl-1H-pyrazol-3-sulfonylmethyl)-
1,3,4-oxadiazol-2-amine / 5-(4,5-dihydro-4-aryl-1H-pyrazol-3-sulfonylmethyl)-1,3,4-thiadiazol-2-
amine (8a-c / 9a-c)
To a well cooled solution of 4a-c / 5a-c (0.5 mmol) in dichloromethane (20 ml), an ice-cold
ethereal solution of diazomethane (40 ml, 0.4 M) and triethylamine (0.12 g) were added. The
reaction mixture was kept at -20ºC to -15ºC for 42-48 h. The solvent was removed under vacuum
and the resultant solid was purified by passing through a column of silica gel using hexane-ethyl
acetate (3:1) as an eluent.
6.1.3.1. 5-(4,5-Dihydro-4-phenyl-1H-pyrazol-3-sulfonylmethyl)-1,3,4-oxadiazol-2-amine (8a).
White solid (0.105g, 68%); m.p. 138-140oC; IR (KBr): 1135, 1321 (SO2), 1572 (C=N), 1624
1
(C=C), 3271 (NH), 3354, 3472 (NH2) cm–1; H NMR (DMSO-d6): δ 3.54 (dd, 1H, HX, JAX = 5.9
Hz, JMX = 10.4 Hz), 4.21 (dd, 1H, HM, JAM = 11.8 Hz), 4.56 (dd, 1H, HA), 4.91 (s, 2H, CH2), 5.51
(bs, 2H, NH2), 7.16-7.69 (m, 5H, Ar-H), 10.08 (bs, 1H, NH) ppm; 13C NMR (DMSO-d6): δ 47.1
(CH2), 52.8 (C-5'), 66.1 (C-4'), 152.3 (C-3'), 158.2 (C-5), 159.5 (C-2), 126.9, 127.6, 129.8, 131.9
(aromatic carbons) ppm; HRMS (m/z): 330.3172 [M+Na]; Anal. Calcd. for C12H13N5O3S: C,
44.10; H, 3.16; N, 16.01; Found: C, 44.00; H, 3.12; N, 15.79%.
12

ACCEPTED MANUSCRIPT
6.1.3.2. 5-(4-(4-Methylphenyl)-4,5-dihydro-1H-pyrazol-3-sulfonylmethyl)-1,3,4-oxadiazol-2-amine
(8b). White solid (0.105g, 65%); m.p. 151-153oC; IR (KBr): 1128, 1319 (SO2), 1584 (C=N), 1620
1
(C=C), 3267 (NH), 3345, 3463 (NH2) (cm–1); H NMR (DMSO-d6): δ 2.26 (s, 3H, Ar-CH3), 3.47
(dd, 1H, HX, JAX = 5.6 Hz, JMX = 10.1 Hz), 4.11 (dd, 1H, HM, JAM = 11.6 Hz), 4.49 (dd, 1H, HA),
4.81 (s, 2H, CH2), 5.49 (bs, 2H, NH2), 7.13-7.66 (m, 4H, Ar-H), 9.93 (bs, 1H, NH) ppm; 13C NMR
(DMSO-d6): δ 21.2 (Ar-CH3), 45.9 (CH2), 52.3 (C-5'), 64.9 (C-4'), 151.8 (C-3'), 157.8 (C-5), 158.6
(C-2), 126.1, 127.2, 128.5, 130.5 (aromatic carbons) ppm; HRMS (m/z): 344.3433 [M+Na]; Anal.
Calcd. for C13H15N5O3S: C, 44.10; H, 3.16; N, 16.01; Found: C, 44.00; H, 3.12; N, 15.79 %.
6.1.3.3. 5-(4-(4-Chlorophenyl)-4,5-dihydro-1H-pyrazol-3-sulfonylmethyl)-1,3,4-oxadiazol-2-amine
(8c). White solid (0.129g, 76%); m.p. 161-163oC; IR (KBr): 1140, 1327 (SO2), 1587 (C=N), 1627
(C=C), 3280 (NH), 3,350, 3,480 (NH2) cm–1; 1H NMR (DMSO-d6): δ 3.59 (dd, 1H, HX, JAX = 6.1
Hz, JMX = 10.7 Hz), 4.25 (dd, 1H, HM, JAM = 12.1 Hz), 4.59 (dd, 1H, HA), 4.99 (s, 2H CH2), 5.58
(bs, 2H, NH2), 7.19-7.75 (m, 4H, Ar-H), 10.14 (bs, 1H, NH) ppm; 13C NMR (DMSO-d6): δ 47.4
(CH2), 53.2 (C-5'), 66.4 (C-4'), 152.9 (C-3'), 158.7 (C-5), 159.6 (C-2), 128.4, 129.1, 130.8, 132.6
(aromatic carbons) ppm; HRMS (m/z): 364.7615 [M+Na]; Anal. Calcd. for C12H12ClN5O3S: C,
44.10; H, 3.16; N, 16.01; Found: C, 44.00; H, 3.12; N, 15.79%.
6.1.3.4. 5-(4,5-Dihydro-4-phenyl-1H-pyrazol-3-sulfonylmethyl)-1,3,4-thiadiazol-2-amine (9a).
White solid (0.105g, 65%); m.p. 142-144oC; IR (KBr): 1132, 1331 (SO2), 1584 (C=N), 1628
1
(C=C), 3256 (NH), 3366, 3481 (NH2) cm–1; H NMR (DMSO-d6): δ 3.56 (dd, 1H, HX, JAX = 6.4
Hz, JMX = 10.5 Hz), 4.29 (dd, 1H, HM, JAM = 12.6 Hz), 4.60 (dd, 1H, HA), 4.95 (s, 2H, CH2), 5.55
(bs, 2H, NH2), 7.18-7.75 (m, 5H, Ar-H), 10.35 (bs, 1H, NH) ppm; 13C NMR (DMSO-d6): δ 47.6
(CH2), 53.5 (C-5'), 66.5 (C-4'), 152.7 (C-3'), 158.1 (C-5), 159.6 (C-2), 127.7, 128.5, 130.2, 132.4
(aromatic carbons) ppm; HRMS (m/z): 346.3829 [M+Na]; Anal. Calcd. for C12H13N5O2S2: C,
44.10; H, 3.16; N, 16.01; Found: C, 44.00; H, 3.12; N, 15.79%.
13

ACCEPTED MANUSCRIPT
6.1.3.5. 5-(4-(4-Methylphenyl)-4,5-dihydro-1H-pyrazol-3-sulfonylmethyl)-1,3,4-thiadiazol-2-amine
(9b). White solid (0.118g, 70%); m.p. 155-157oC; IR (KBr): 1129, 1324 (SO2), 1581 (C=N), 1622
1
(C=C), 3250 (NH), 3355, 3475 (NH2) cm–1; H NMR (DMSO-d6): δ 2.28 (s, 3H, Ar-CH3), 3.52
(dd, 1H, HX, JAX = 6.1 Hz, JMX = 10.3 Hz), 4.14 (dd, 1H, HM, JAM=12.20 Hz), 4.52 (dd, 1H, HA),
4.87 (s, 2H, CH2), 5.46 (bs, 2H, NH2), 7.12-7.70 (m, 4H, Ar-H), 9.98 (bs, 1H, NH) ppm; 13C NMR
(DMSO-d6): δ 21.6 (Ar-CH3), 46.5 (CH2), 52.7 (C-5'), 65.4 (C-4'), 151.8 (C-3'), 157.5 (C-5), 158.8
(C-2), 126.4, 127.9, 129.5, 131.9 (aromatic carbons) ppm; HRMS (m/z): 360.4116 [M+Na]; Anal.
Calcd. for C13H15N5O2S2: C, 44.10; H, 3.16; N, 16.01 %. Found: C, 44.00; H, 3.12; N, 15.79%.
6.1.3.6. 5-(4-(4-Chlorophenyl)-4,5-dihydro-1H-pyrazol-3-sulfonylmethyl)-1,3,4-thiadiazol-2-amine
(9c). White solid (0.139g, 78%); m.p. 168-170oC; IR (KBr): 1157, 1335 (SO2), 1583 (C=N), 1630
1
(C=C), 3269 (NH), 3374, 3489 (NH2) cm–1; H NMR (DMSO-d6): δ 3.62 (dd, 1H, HX, JAX = 6.7
Hz, JMX = 10.9 Hz), 4.28 (dd, 1H, HM, JAM = 12.8 Hz), 4.64 (dd, 1H, HA), 5.03 (s, 2H, CH2), 5.60
(bs, 2H, NH2), 7.22-7.87 (m, 4H, Ar-H), 10.39 (bs, 1H, NH) ppm; 13C NMR (DMSO-d6): δ 47.9
(CH2), 53.9 (C-5'), 66.9 (C-4'), 153.1 (C-3'), 158.4 (C-5), 159.6 (C-2), 128.6, 129.8, 131.1, 133.4
(aromatic carbons) ppm; HRMS (m/z): 380.8274 [M+Na]; Anal. Calcd. for C12H12ClN5O2S2: C,
44.10; H, 3.16; N, 16.01; Found: C, 44.00; H, 3.12; N, 15.79%.
6.1.4. General method for the preparation of 5-(4-phenyl-1H-pyrazol-3-sulfonylmethyl)-1,3,4-
oxadiazol-2-amine / 5-(4-phenyl-1H-pyrazol-3-sulfonylmethyl)-1,3,4-thiadiazol-2-amine (10a-c /
11a-c)
A solution of 8a-c / 9a-c (1 mmol) and chloranil (1.4 mmol) in xylene (10 ml) was refluxed
for 16-18 h. Then the reaction mixture was treated with 5% NaOH solution. The organic layer was
separated and repeatedly washed with water. It was dried over anhydrous Na2SO4 and the solvent
was removed on a rotary evaporator. The resultant solid was recrystallized from 2-propanol.
14

ACCEPTED MANUSCRIPT
6.1.4.1 5-(4-Phenyl-1H-pyrazol-3-sulfonylmethyl)-1,3,4-oxadiazol-2-amine (10a). White solid
(0.195g, 64%); m.p. 133-135oC; IR (KBr): 1145, 1328 (SO2), 1579 (C=N), 1623 (C=C), 3282
(NH), 3329, 3455 (NH2) cm–1; 1H NMR (DMSO-d6): δ 5.06 (s, 2H, CH2), 5.56 (bs, 2H, NH2), 6.34
13
(bs, 1H, NH), 6.83-7.77 (m, 6H, C5'-H & Ar-H) ppm; C NMR (DMSO-d6): δ 54.1 (CH2), 133.2
(C-4'), 137.9 (C-5'), 147.5 (C-3'), 157.8 (C-5), 159.3 (C-2), 126.9, 127.3, 128.5, 135.7 (aromatic
carbons) ppm; HRMS (m/z): 328.3016 [M+Na]; Anal. Calcd. for C12H11N5O3S: C, 47.32; H, 3.66;
N, 23.14; Found: C, 47.21; H, 3.63; N, 22.94%.
6.1.4.2.
5-(4-(4-Methylphenyl)-1H-pyrazol-3-sulfonylmethyl)-1,3,4-oxadiazol-2-amine (10b).
White solid (0.220g, 69%); m.p. 140-142oC; IR (KBr): 1131, 1323 (SO2), 1585 (C=N), 1626
(C=C), 3275 (NH), 3318, 3443 (NH2) cm–1; 1H NMR (DMSO-d6): δ 2.30 (s, 3H, Ar-CH3), 5.02 (s,
2H, CH2), 5.51 (bs, 2H, NH2), 6.32 (bs, 1H, NH),6.81-7.72 (m, 5H, C5'-H & Ar-H) ppm; 13C NMR
(DMSO-d6): δ 21.5 (Ar-CH3), 53.9 (CH2), 131.80 (C-4'), 137.4 (C-5'), 146.7 (C-3'), 157.4 (C-5),
159.1 (C-2), 125.8, 126.6, 128.1, 134.2 (aromatic carbons) ppm; HRMS (m/z): 342.3276 [M+Na];
Anal. Calcd. for C13H13N5O3S: C, 48.84; H, 4.11; N, 22.01; Found: C, 48.89; H, 4.10; N, 21.93%.
6.1.4.3. 5-(4-(4-Chlorophenyl)-1H-pyrazol-3-sulfonylmethyl)-1,3,4-oxadiazol-2-amine (10c).
White solid (0.244g, 72%); m.p. 158-160oC; IR (KBr): 1140, 1334 (SO2), 1565 (C=N), 1612
1
(C=C), 3288 (NH), 3325, 3471 (NH2) cm–1; H NMR (DMSO-d6): δ 5.12 (s, 2H, CH2), 5.62 (bs,
2H, NH2), 6.38 (bs, 1H, NH), 6.90-7.81 (m, 5H, C5'-H & Ar-H) ppm; 13C NMR (DMSO-d6): δ
54.8 (CH2), 133.9 (C-4'), 138.2 (C-5'), 148.4 (C-3'), 158.2 (C-5), 159.6 (C-2), 127.1, 129.6, 130.5,
136.2 (aromatic carbons) ppm; HRMS (m/z): 362.7482 [M+Na]; Anal. Calcd. for C12H10ClN5O3S:
C, 42.51; H, 2.99; N, 20.76; Found: C, 42.42; H, 2.97; N, 20.61%.
6.1.4.4. 5-(4-Phenyl-1H-pyrazol-3-sulfonylmethyl)-1,3,4-thiadiazol-2-amine (11a). White solid
(0.215g, 67%); m.p. 146-148oC; IR (KBr): 1146, 1337 (SO2), 1563 (C=N), 1615 (C=C), 3267
(NH), 3338, 3465 (NH2) cm–1; 1H NMR (DMSO-d6): δ 5.10 (s, 2H, CH2), 5.57 (bs, 2H, NH2), 6.42
15

ACCEPTED MANUSCRIPT
13
(bs, 1H, NH), 6.95-7.86 (m, 6H, C5'-H & Ar-H) ppm; C NMR (DMSO-d6): δ 54.6 (CH2), 133.8
(C-4'), 138.4 (C-5'), 148.2 (C-3'), 158.1 (C-5), 159.7 (C-2), 127.3, 128.9, 129.7, 136.2 (aromatic
carbons) ppm; HRMS (m/z): 344.3685 [M+Na]; Anal. Calcd. for C12H11N5O2S2: C, 44.92; H, 3.43;
N, 21.92; Found: C, 44.85; H, 3.45; N, 21.79%.
6.1.4.5. 5-(4(4-Methylphenyl)-1H-pyrazol-3-sulfonylmethyl)-1,3,4-thiadiazol-2-amine (11b). White
solid (0.248g, 74%); m.p. 155-157oC; IR (KBr): 1134, 1332 (SO2), 1567 (C=N), 1600 (C=C), 3259
1
(NH), 3327, 3454 (NH2) cm–1; H NMR (DMSO-d6): δ 2.34 (s, 3H, Ar-CH3), 5.07 (s, 2H, CH2),
5.49 (bs, 2H, NH2), 6.40 (bs, 1H, NH), 6.91-7.75 (m, 5H, C5'-H & Ar-H) ppm; 13C NMR (DMSO-
d6): δ 21.9 (Ar-CH3), 54.1 (CH2), 133.1 (C-4'), 137.4 (C-5'), 147.9 (C-3'), 157.7 (C-5), 159.5 (C-2),
126.8, 127.9, 128.6, 135.2 (aromatic carbons) ppm; HRMS (m/z): 358.3931 [M+Na]; Anal. Calcd.
for C13H13N5O2S2: C, 46.66; H, 3.93; N, 21.07; Found: C, 46.55; H, 3.91; N, 20.88%.
6.1.4.6. 5-(4-(4-Chlorophenyl)-1H-pyrazol-3-sulfonylmethyl)-1,3,4-thiadiazol-2-amine (11c).
White solid (0.284g, 80%); m.p. 165-167oC; IR (KBr): 1150, 1340 (SO2), 1580 (C=N), 1618
1
(C=C), 3274 (NH), 3333, 3470 (NH2) cm–1; H NMR (DMSO-d6): δ 5.16 (s, 2H, CH2), 5.63 (bs,
2H, NH2), 6.44 (bs, 1H, NH), 7.04-7.87 (m, 5H, C5'-H & Ar-H) ppm; 13C NMR (DMSO-d6): δ
55.1 (CH2), 134.2 (C-4'), 139.5 (C-5'), 148.7 (C-3'), 158.4 (C-5), 159.9 (C-2), 128.2, 129.8, 130.5,
137.2 ppm (aromatic carbons) ppm; HRMS (m/z): 378.8129 [M+Na]; Anal. Calcd. for
C12H10ClN5O2S2: C, 40.57; H, 2.84; N, 19.81; Found: C, 40.51; H, 2.83; N, 19.68%.
6.2. Biological assays
6.2.1. Compounds
The compounds 4-11 were dissolved in DMSO at different concentrations of 12.5, 25, 50
and 100 µg/well.
16

ACCEPTED MANUSCRIPT
6.2.2. Cells
Bacterial strains Staphylococcus aureus, Bacillus subtilis (Gram-positive), Pseudomonas
aeruginosa, Klebsiella pneumoniae (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 study was 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. The
compounds were dissolved in DMSO of 5 mg/ml and from this 2.5, 5, 10 and 20 µl (12.5, 25, 50,
100 µg/well) were added into 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 inhibition acts as negative control. The plates were incubated at 37oC for
24 h for bacteria and at 28oC 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 antimicrobial 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 and K. pneumoniae and the test
fungi A. niger & P. chrysogenum were diluted 100 folds in nutrient broth (100 µl bacterial cultures
in 10 ml NB). The stock solution of the synthesized compounds was prepared in dimethyl
sulfoxide (DMSO) by dissolving 5 mg of the compound in 1 ml of DMSO. Increasing
concentrations of the test samples (1.25, 2.5, 5, 10, 20, 40 µl of stock solution contains 6.25, 12.5,
17

ACCEPTED MANUSCRIPT
25, 50, 100, 200 µg of the compounds) were added to the test tubes containing the bacterial and
fungal cultures. All the tubes were incubated at 37oC for 24 h for bacteria and at 28oC 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 and inoculated on sterile
nutrient broth (for bacteria) and PDA (for fungi) by streaking. Plates inoculated with bacteria and
fungi were incubated at 37oC for 24 h and at 28oC 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
The authors are grateful to University Grants Commission (UGC), New Delhi for sanction
of UGC-BSR fellowship.
References
[1]
[2]
[3]
[4]
B. Chandrakantha, S. Prakash, N. Vijesh, I. Nishitha, M. A. Isloor, Eur. J. Med. Chem.
45 (2010) 1206–1210.
G.V.S. Kumar, Y. Rajendraprasad, B.P. Mallikarjuna, S.M. Chandrashekar, C. Kistayya,
Eur. J. Med. Chem. 45 (2010) 2063–2074.
A.A. El-Emam, O.A. Al-Deeb, M. Al-Omara, J. Lehmannb, Bioorg. Med. Chem. 12
(2004) 5107–5113.
S. Bajaj, V. Asati, J. Singh, P.P. Roy, Eur. J. Med. Chem. 97 (2015) 124–141.
18

ACCEPTED MANUSCRIPT
[5]
[6]
[7]
[8]
[9]
M.N. Noolvi, H.M. Patel, N. Singh, A.K. Gadad, S.S. Cameotra, A. Badiger,
Eur. J. Med. Chem. 46 (2011) 4411–4418.
A.K. Gadad, C.S. Mahajanshetti, S. Nimbalkar, A. Raichurkar, Eur. J. Med. Chem.
35 (2000) 853–857.
I.A.M. Khazi, C.S. Mahajanshetti, A.K. Gadad, A.D. Tarnalli, C.M. Sultanpur, Arzneim.
Forsch./Drug. Res. 46 (1996) 949–952.
S.D. Joshi, H.M. Vagdevi, V.P. Vaidya, G.S. Gadaginamath, Eur. J. Med. Chem.
43 (2008) 1989–1996.
Z.A. Kaplancikli, G. Turan-Zitouni, A. Ozdemir, G. Revial, Eur. J. Med. Chem. 43 (2008)
155–159.
[10] (a) A. Furstner, Angew. Chem., Int. Ed. 42 (2003) 3528–3603; (b) S. S. Gholap, Eur. J. Med.
Chem. 110 (2016) 13-31.
[11] D.E. Wemmer, Biopolymers 52 (1999) 197–211.
[12] J.M. Manley, M.J. Kalman, B.G. Conway, C.C. Ball, J.L. Havens, R. Vaidyanathan
J. Org. Chem. 68 (2003) 6447–6450.
[13] Y.F. Wang, K.K. Toh, S. Chiba, K. Narasaka, Org. Lett. 10 (2008) 5019–5022.
[14] Y. Nishibayashi, M. Yoshikawa, Y. Inada, M.D. Milton, M. Hidai, S. Uemura, Angew.
Chem. Int. Ed. 42 (2003) 2681–2684.
[15]
A. Tanitame, Y. Oyamada, K. Ofuji, M. Fujimoto, N. Iwai, Y. Hiyama, K. Suzuki, H. Ito,
H. Terauchi, M. Kawasaki, K. Nagai, M. Wachi, J. Yamagishi, J. Med. Chem. 47
(2004) 3693-3696.
[16]
R.V. Ragavan, V. Vijay kumar, N. Suchetha kumari, Eur. J. Med. Chem. 45 (2010) 1173-
1180.
19

ACCEPTED MANUSCRIPT
[17] A. Tanitame, Y. Oyamada, K. Ofuji, H. Terauchi, M. Kawasaki, M. Wachi, J.I. Yamagishi,
Bioorg. Med. Chem. Lett. 15 (2005) 4299-4303.
[18]
[19]
S. Fletcher, E.P. Keaney, C.G. Cummings, M.A. Blaskovich, M.A. Hast, M.P. Glenn,
S.Y. Chang, C.J. Bucher, R.J. Floyd, W.P. Katt, M.H. Gelb, W.C. Van Voorhis, L.S.
Beese, S.M. Sebti, A.D. Hamilton, J. Med. Chem. 53 (2010) 6867-6888.
G. Ouyang, X.J. Cai, Z. Chen, B.A. Song, P.S. Bhadury, S. Yang, L.H. Jin, W. Xue, D.Y.
Hu, S. Zeng, J. Agric. Food. Chem. 56 (2008) 10160-10167.
[20] A. Hall, A.S. Billinton, H. Brown, N.M. Clayton, A. Chowdhury , G.M.P. Giblin, P.
Goldsmith, T.G. Hayhow , D.N. Hurst, I.R. Kilford, A. Naylor, B. Passingham, L.
Winyard, Bioorg. Med. Chem. Lett. 18 (2008) 3392-3399.
[21]
[22]
Kameyama T, Nabeshima T, Neuropharmacology 17 (1978) 249–256.
T.D. Penning, J.J. Talley, S.R. Bertenshaw, J.S. Carter, P.W. Collins, J. Med. Chem. 40
(1997) 1347–1365.
[23]
[24]
[25]
[26]
[27]
D.C. Argentieri, D.M. Ritchie, M.P. Ferro, T. Kirchner, M.P. Wachter, J.
Pharmacol. Exp. Ther. 271 (1994) 1399–1408.
P. Conti, A. Pinto, L. Tamborini, V. Rizzo, C. D. Micheli, Tetrahedron 63 (2007) 5554–
5560.
G. Palazzino, L. Cecchi, F. Melani, V. Colotta, G. Filacchioni, C. Martini, A. Lucacchini,
J. Med. Chem. 30 (1987) 1737–1742.
K. I. Washizuka, K. Nagai, S. Minakata, I. Ryu, M. Komatsu, Tetrahedron Lett.
40 (1999) 8849–8853.
(a) A. Padmaja, C. Rajasekhar, S. Durgamma, B. C. Venkatesh, V. Padmavathi,
Med. Chem. Res. 23 (2014) 1084–1098; (b) A. Padmaja, T. Payani, G. Dinneswara
Reddy, V. Padmavathi, Eur. J. Med. Chem. 44 (2009) 4557–4566.
20

ACCEPTED MANUSCRIPT
[28]
(a) M.V. Ramana Reddy, S. Reddy, Synthesis. (1984) 322–323; (b) M.V. Ramana Reddy,
S. Reddy, D. Bhaskar Reddy, V. Padmavathi, Synth. Commun. 19 (1989) 1101–1107.
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]
D. Janovska, K. Kubikova, L. Kokoska, J. Food Sci. 21 (2003) 107–110.
J. Bishnu, L. Sunil, S. Anuja, J. Sci. Eng. Technol. 5 (2009) 143–150.
National Committee for Clinical Laboratory Standards Methods for Dilution
Antimicrobial Susceptibility Tests for Bacteria that Grow Aerobically, 3rd edition;
Approved standard, NCCLS publication M7-A3; Villanova, PA (1993).
National Committee for Clinical Laboratory Standards, Reference Method for Broth
Dilution Antifungal Susceptibility Testing of Yeasts, Proposed standard, NCCLS
Document M27-P; Villanova, PA (1992).
[34]
21

ACCEPTED MANUSCRIPT
Table 1. The in vitro antibacterial activity of compounds 4−11.
Zone of inhibition (mm)
Gram-positive bacteria
Gram-negative bacteria
P. aeruginosa K. pneumoniae
50 100 12.5 25 50 100 12.5 25 50 100
µg/ µg/ µg/ µg/ µg/ µg/ µg/ µg/ µg/ µg/ µg/ µg/ µg/ µg/ µg/ µg/
well well well well well well well well well well well well well well well well
S. aureus
B. subtilis
Compound
12.5 25 50 100 12.5 25
11±2 13±1 15±3 18±2 12±3 13±1 14±1 16±2 14±3 16±3 18±2 20±2
12±1 14±3 8±2 9±3 11±2 14±1 12±2 14±1 16±3 18±3
15±2 17±3 19±2 21±3
11±3 13±2 15±3 17±2
16±1 18±1 20±2 22±1
27±2 29±3 31±1 33±3
23±1 25±2 27±2 29±1
32±2 34±3 36±2 38±2
4a
4b
4c
-
-
12±3 14±1 16±3 20±1 13±2 14±2 15±3 17±3 15±1 17±2 19±1 21±2
21±1 22±3 23±2 25±2 27±1 28±1 29±1 31±2 23±3 25±3 27±2 29±1
19±3 20±2 21±3 23±1 25±3 26±1 27±2 29±3 21±2 23±2 25±3 27±2
22±2 23±1 24±2 26±3 28±2 29±2 30±1 32±3 24±3 26±3 28±2 34±3
5a
5b
5c
-
-
-
-
-
-
11±1
10±3
-
-
-
-
-
-
-
-
12±1 08±1 10±2 12±1 14±2
10±2 10±2 11±1
-
-
-
-
11±3 13±3
12±1
6a
-
-
-
6b
6c
07±1 08±1 09±1 12±2
11±3 13±1 09±3 11±3 13±1 15±3
08±1 10±2 12±2 14±2
26±2 27±1 29±2 31±1
20±3 22±3 24±3 26±3
28±2 30±2 32±2 34±2
17±3 19±3 22±2 24±1 20±1 21±3 22±1 24±3 19±1 21±2 23±2 25±1
15±2 17±2 19±2 22±3 18±2 19±1 20±2 22±2 17±2 19±1 21±3 23±3
17±1 19±1 21±3 23±1 21±3 22±3 23±3 25±3 20±3 22±3 24±1 26±1
7a
7b
7c
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
8a
8b
8c
-
-
-
-
9a
9b
9c
10±1 14±3 07±1 09±2 11±3 15±2 11±3 13±3 15±1 17±2
12±2 06±2 07±1 09±2 11±1 09±1 10±1 12±2 14±2
10±3 12±3 14±3 16±2
12±2 14±1
10a
10b
10c
11a
11b
11c
-
-
-
08±1 10±2 12±1 15±1 08±3 09±3 12±1 13±3 12±2 14±3 16±3 18±3
20±3 21±1 22±3 24±3 25±1 26±2 27±3 29±1 22±1 23±2 25±2 27±1
16±1 17±3 18±1 20±2 21±2 22±1 23±1 25±2 20±3 22±3 24±1 26±2
21±3 22±1 23±3 25±2 26±3 27±3 28±3 30±3 24±2 26±1 28±3 31±3
30±1 32±2 35±2 37±1 32±3 34±2 36±1 40±1 25±2 27±2 29±2 32±1
11±1 13±3 15±1 17±3
26±2 28±2 30±3 32±1
22±2 24±1 26±3 28±3
30±3 32±2 34±2 36±3
38±2 40±2 42±1 44±2
Chloram-
phenicol
Control
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
(DMSO)
(-) No activity, (±) Standard deviation.

ACCEPTED MANUSCRIPT
Table 2. The in vitro antifungal activity of compounds 4−11.
Zone of inhibition (mm)
Compound
A. niger
P. chrysogenum
100 µg/
12.5 µg/ 25 µg/ 50 µg/ 100 µg/ 12.5 µg/ 25 µg/ 50 µg/
well
well
well
well
well
well
well
well
4a
09±2
11±3
10±1
15±2
27±1
24±3
29±2
08±1
-
13±3
11±2
17±1
29±3
26±2
31±1
10±2
07±2
09±2
16±3
13±2
19±1
-
15±3
13±2
19±1
31±3
28±2
32±3
12±1
09±2
14±3
18±1
15±2
21±3
-
11±3
10±2
15±1
29±3
25±2
34±1
11±1
08±2
12±2
25±1
22±3
29±2
-
13±3
12±2
16±1
34±2
27±3
35±2
12±3
10±1
14±3
27±2
24±1
32±3
-
15±2
14±3
18±2
36±3
29±2
37±1
14±3
12±1
16±2
29±2
26±3
34±2
-
17±3
16±2
20±1
35±2
31±3
41±2
16±1
14±2
18±3
31±2
28±1
37±3
-
08±3
4b
13±3
4c
25±2
5a
22±1
5b
27±3
5c
-
6a
-
6b
-
10±3
14±1
11±2
17±3
-
6c
12±2
7a
09±3
7b
15±2
7c
-
8a
-
-
-
-
-
-
-
-
8b
8c
-
-
-
-
-
-
-
-
-
-
07±1
4±3
09±2
08±1
11±2
13±1
10±3
13±3
29±2
27±3
30±2
37±1
-
-
-
-
12±2
11±1
13±2
16±2
14±3
17±2
36±1
34±3
38±2
39±2
-
9a
-
-
-
-
-
9b
9c
-
-
10±2
11±1
10±2
11±2
27±1
25±3
28±1
34±1
-
-
-
10±2
14±3
11±1
15±2
34±1
32±2
35±3
37±2
-
-
-
09±2
08±1
09±2
25±2
23±3
26±1
31±2
-
-
12±2
-
10a
-
10b
-
12±1
30±2
28±3
31±2
34±1
-
13±3
32±2
30±1
33±3
36±1
-
10c
23±3
21±2
24±1
29±3
-
11a
11b
11c
Ketoconazole
Control
(DMSO)
(-) No activity, (±) Standard deviation.

ACCEPTED MANUSCRIPT
Table 3. MIC, MBC and MFC of compounds 5c, 7c and 11c.
Minimum inhibitory concentration
Compound
MIC (MBC / MFC) µg/ml
S. aureus B. subtilis P. aeruginosa K.pneumoniae A. niger P. chrysogenum
25(>100)
25(100)
6.25(12.5)
25(100)
12.5(50)
6.25
12.5(50)
25(>100)
25(>100)
12.5
12.5(50)
25(100)
25(100)
-
12.5(25)
50(>200)
12.5(50)
-
5c
7c
50(>200) 50(>200)
50(200)
25(100)
11c
Chloramphenicol
Ketoconazole
(-) No activity
6.25
-
6.25
-
-
-
6.25
12.5

ACCEPTED MANUSCRIPT
Figure 1. Drugs containing pyrrole and pyrazole moieties.
OH
F
F
N
N
O
F
N
O
N
N
H
N
CH3
N
N
H
N
O
Cl
O
S
O
NH2
Tepoxalin
Celecoxib
Difenamizole
O
NH
H
NH
N
NH2
HN
N
H
N
H3C
O
H
O
N
CH3
N
O
CH3
NH2
Distamycin
H
HN
N
N
H
H
N
O
N
H
N
NH2
O
CH3
N
O
NH
CH3
Netropsin

ACCEPTED MANUSCRIPT
Figure 2. The in vitro antibacterial activity of compounds 4−11.
Figure 3. The in vitro antifungal activity of compounds 4−11.

ACCEPTED MANUSCRIPT
Research Highlights
ꢀ We have synthesized Pyrrolyl Sulfonylmethyl-1,3,4-Oxadiazolyl / 1,3,4-Thiadiazolyl
Amines and Pyrazolyl Sulfonylmethyl-1,3,4-Oxadiazolyl / 1,3,4-Thiadiazolyl Amines
ꢀ Mono heterocyclic compounds (4, 5) displayed slightly higher antimicrobial activity than the
respective bis heterocyclic systems (6-11).
ꢀ
In all the tested compounds 5-(4-Chlorostyrylsulfonylmethyl)-1,3,4-thiadiazol-2-amine showed
greater antimicrobial activity.