Synthesis and in vitro antimicrobial activity of novel N-(6-chlorobenzo[d] thiazol-2-yl) hydrazine carboxamide derivatives of benzothiazole class

Article abstract of DOI:10.3109/14756366.2010.508441

In this study, a series of novel 1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazole (6a-g) and 1,3,4-oxadiazole (7a-g, 8) were synthesized from N-(6-chlorobenzo[d] thiazol-2-yl) hydrazine carboxamide derivatives of benzothiazole class. Antimicrobial properties of the title compound derivatives were investigated against one Gram (+) bacteria (Staphylococcus aureus), three Gram (-) bacteria (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae) and five fungi (Candida albicans, Aspergillus niger, Aspergillus flavus, Monascus purpureus and Penicillium citrinum) using serial plate dilution method. The investigation of antibacterial and antifungal screening data revealed that all the tested compounds showed moderate to good inhibition at 12.5-100μg/mL in DMSO. It has been observed that triazolo-thiadiazole derivatives are found to be more active than 1,3,4-oxadiazole derivatives against all pathogenic bacterial and fungal strains.

Full text of DOI:10.3109/14756366.2010.508441

Journal of Enzyme Inhibition and Medicinal Chemistry, 2011; 26(3): 332–340
© 2011 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2010.508441
RESEARCH ARTICLE
Synthesis and in vitro antimicrobial activity of novel
N-(6-chlorobenzo[d]thiazol-2-yl) hydrazine carboxamide
derivatives of benzothiazole class
Sadaf J. Gilani, Suroor A. Khan, Nadeem Siddiqui, Suraj P. Verma, Pooja Mullick, and Ozair Alam
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Jamia Hamdard (Hamdard University), New Delhi
110062, India
Abstract
In this study, a series of novel 1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazole (6a–g) and 1,3,4-oxadiazole (7a–g, 8) were
synthesized from N-(6-chlorobenzo[d]thiazol-2-yl) hydrazine carboxamide derivatives of benzothiazole class.
Antimicrobial properties of the title compound derivatives were investigated against one Gram (+) bacteria
(Staphylococcus aureus), three Gram (−) bacteria (Escherichia coli, Pseudomonas aeruginosa, Klebsiella pneumoniae)
and five fungi (Candida albicans, Aspergillus niger, Aspergillus flavus, Monascus purpureus and Penicillium citrinum)
using serial plate dilution method. The investigation of antibacterial and antifungal screening data revealed that all
the tested compounds showed moderate to good inhibition at 12.5–100µg/mL in DMSO. It has been observed that
triazolo-thiadiazole derivatives are found to be more active than 1,3,4-oxadiazole derivatives against all pathogenic
bacterial and fungal strains.
Keywords: N-(6-chlorobenzo[d]thiazol-2-yl) hydrazine carboxamide, 1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazole,
1,3,4-oxadiazole, antimicrobial activity
Introduction
of hybrid molecules through the combination of different
Antimicrobialsreduceorcompletelyblockthegrowthand
multiplication of bacteria. is has made them unique
for the control of deadly infectious diseases caused by a
variety of pathogens. ey have transformed our ability
to treat infectious diseases such as pneumonia, menin-
gitis, tuberculosis, malaria and AIDS1. Although deaths
from bacterial and fungal infections have dropped in the
developed world, these are still major causes of death in
the developing world2.
According to WHO, each year 1.4 million children died
of gut infections and diarrhoea caused by gram-negative
bacteria like Pseudomonas, Salmonella, Shigellae and
gram positive rods like Corynebacterium diptheriae2.De-
cades of antibiotic use have resulted in the development
of widespread resistance to commonly prescribed anti-
bacterial agents3. erefore, there is a need to develop
new, potent, fast-acting antimicrobial drugs with low
toxicity. In the design of new compounds, development
pharmacophoresinonestructuremayleadtocompounds
with increased antimicrobial activity4.
Despite numerous attempts to develop new structural
prototype in the search for more effective antimicrobials,
benzothiazole still remain as one of the most versatile
class of compounds against microbes and therefore, are
useful substructures for further molecular exploration.
Benzothiazole’s literature is enriched with progressive
findings of the moiety in respect of anticonvulsant and
antimicrobial activities5. Benzothiazole and its deriva-
tives constitute the most versatile and valuable source
of antimicrobial compounds. ey appear to transcend
the chemotherapeutic boundaries of other antipara-
sitic drugs with a spectrum of activity that includes the
majority of fungi, bacteria, protozoa and helminthic
species.
Several benzothiazole derivatives were also associ-
ated with antitumor6, antimicrobial7,8, anticonvulsant9,
Address for Correspondence: Sadaf J. Gilani, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Jamia Hamdard (Hamdard
University), New Delhi 110062, India. E-mail: gilanisadaf@gmail.com
(Received 18 February 2010; revised 02 June 2010; accepted 10 July 2010)
332

Synthesis and in vitro antimicrobial activity of novel N-(6-chlorobenzo[d]thiazol-2-yl) hydrazine carboxamide derivatives 333
and anti-inflammatory7,10 activities. In addition,
triazolo-thiadiazoles nucleus constitutes the active part
of several biologically active compounds, including
antibacterial11, antifungal12, antitumor13, anti-inflamma-
tory14, analgesic15 and so on. Moreover, 1,3,4-oxadiazole
were also reported to possess significant antimicrobial16,
anti-inflammatory17 and analgesic activities18.
A triazolo-thiadiazole system may be viewed as a
cyclic analogue of two very important components-thi-
osemicarbazide 19,20 and biguanide 21, which often display
diverse biological activities. Inspired by these observa-
tions a composite system was investigated, which com-
bine these two biolabile components in a ring together
to give a compact and planar structure, and screened for
their biological activities.
(−) bacteria (Escherichia coli, Pseudomonas aeruginosa,
Klebsiella pneumoniae) and five fungi (Candida albi-
cans, Aspergillus niger, Aspergillus flavus, Monascus
purpureus and Penicillium citrinum) using serial plate
dilution method.
Experimental
Chemistry
Chemicals were purchased from Merck Chemical Com-
pany, S.D. Fine (India) and Qualigens (India). Melting
points were determined in open capillary tubes in a
Hicon melting point apparatus and are uncorrected.
IR (KBr) spectra were recorded on a Nicolet, 5PC FTIR
1
spectrometer (νmax in cm−1) and H NMR spectra were
e prime objective for the current study is to develop
novel derivatives of benzothiazole moiety and finally
screen them against different microbial strains (bacteria
and fungi) at variable concentrations. e rationale for
the study includes the designing of the derivatives having
some common structural features that are important for
the compound to exhibit an antimicrobial activity that
includes the following:22–24
recorded in CDCl3/DM SO-d6 on a Bruker DRX-300
(300 MHz FT NMR) spectrometer using tetramethylsi-
lane (TMS) as internal reference. Chemical shift (δ) are
expressed in parts per million (ppm); coupling constants
(J) are reported in hertz and refer to apparent peak mul-
tiplicities, which may not necessarily be true coupling
constants. Mass spectra were recorded using Jeol SR-102
(FAB) mass spectrometer. e purity of various synthe-
sized compounds was checked by TLC and elemental
analysis. Spectral data (1H NMR, IR and mass) of the
synthesized compounds were in full agreement with the
proposed structures.
1. A lipohilic bicyclic aromatic ring system.
2. Another bulky lipophilic group (e.g. phenyl, tert-
butyl) as a side chain.
3. Two lipophilic domain linked by a spacer of appro-
priate length with polar centre at defined position,
for example, Naftifine, Butenafine, Terbinafine,
Debacarb, Penicillins and Cephalosporins.
General procedure for the synthesis of
6-chloro-1,3-benzothiazole-2-amines (1)
A mixture of aniline (0.01 mol) and potassium thiocya-
nate (0.01 mol) in glacial acetic acid (10%) was cooled
and stirred. To this solution bromine (0.01 mol) was
added dropwise at such a rate as to keep the tempera-
ture below 10°C throughout the addition. Stirring was
continued for an additional 3 h and the separated hydro-
chloride salt was filtered, washed with acetic acid and
dried. It was dissolved in hot water and neutralized with
aqueous ammonia solution (25%), filtered, washed with
water and dried, recrystallized with benzene to obtain
6-chloro-1,3-benzothiazole-2-amines.
In view of the above mentioned facts and in continuation
of our interest in the synthesis of heterocycles contain-
ing benzothiazole moiety, to identify new candidates
that may be of value in designing new, potent, selective
and less toxic antimicrobial agents, we report herein
the synthesis and antimicrobial evaluation of some
novel structure hybrids incorporating both the benzo-
thiazole moiety with either the triazolo-thiadiazole or
oxadiazole ring systems through different linkages. is
combination was suggested in an attempt to investi-
gate the influence of such hybridization and structure
variation on the anticipated biological activities, hoping
to add some synergistic biological significance to the
target molecules. e substitution pattern of triazolo-
thiadiazole and oxadiazole rings was carefully selected
so as to confer different electronic environment to the
molecules.
Hence, to discover new and useful agents for treat-
ment of microbial diseases, we have replaced the
hydrazide group of N-(6-chlorobenzo[d]thiazol-2-yl)
hydrazine carboxamide with additional heterocycles,
which have been found to possess an interesting profile
of antimicrobial activity. e heterocycles reported here
are triazolo-thiadiazole and 1,3,4-oxadiazole. us, the
synthesized compounds were investigated against one
Gram (+) bacteria (Staphylococcus aureus), three Gram
1. IR (KBr) νmax (cm−1): 817 (C-Cl), 1570 (C = N), 3480 (NH);
1H NMR (300 MHz) (DMSO-d6) δ (ppm) 6.12 (2H, s, NH2),
6.61–6.64 (3H, J= 9 Hz, m, Ar-H).
General procedure for the synthesis of
1-(6-chloro-1,3-benzothiazol-2-yl) urea (2)
To the solution of sodium cyanate in minimum quantity
of water, glacial acetic acid (5 mL) was added. is solu-
tion was heated with 2-amino-6-chloro-benzothiazole
1 (0.01 mol) in alcohol till the contents of mixture be-
come turbid and volume remained half of the original
volume. e contents were added to ice cool water. e
solid obtained was filtered off and dried.
2. IR (KBr) νmax (cm−1): 830 (C-Cl), 1560 (C = N), 1628
(C = O), 3310 (NH); 1H NMR (DMSO-d6) (300 MHz) δ
(ppm) 6.34 (2H, s, NH2), 6.68–6.70 (3H, J= 6 Hz, m, Ar-H),
8.10 (1H, s, NHC = O).
© 2011 Informa UK, Ltd.

334 Sadaf J. Gilani et al.
on to crushed ice with stirring. e mixture was allowed
to stand overnight and the solid separated out was fil-
tered, treated with dilute sodium hydroxide solution and
washed thoroughly with cold water. e compound so
obtained was dried and recrystallized from ethanol.
(6a). IR (KBr) νmax (cm−1): 610 (C-S-C benzothiazole), 674
(C-S-C triazolo-thiadiazole), 837 (C-Cl ), 1269 (N-N= C
triazolo-thiadiazole), 1416 (C-N benzothiazole), 1518
General procedure for the synthesis of N-(6-chlorobenzo[d]
thiazol-2-yl)hydrazine carboxamide (3)
To the warm hydrazine hydrate solution of compound 2
in alcohol, conc. NaOH was added and refluxed for 6 h.
Reaction mixture was poured into crushed ice and solid
obtained was filtered off and dried. e solid collected
out was recrystallized from suitable solvent to get the
compound N-(6-chlorobenzo[d]thiazol-2-yl) hydrazine
carboxamide.
1
(C = C aromatic), 3084 (C-H aromatic), 3314 (N-H); H
(3). IR (KBr) νmax (cm−1): 657 (C-S-C), 817 (C-Cl), 1588
(C = N), 1660 (C = O), 3300 (NH); 1H NMR (DMSO-d6)
(300 MHz) δ (ppm) 7.28 (1H, s, NHNH2), 7.70–7.74 (3H,
J = 12 Hz, m, Ar-H), 9.12 (1H, s, NHC = O).
NMR (DMSO-d6) (300 MHz) δ (ppm): 7.41–7.44 (8H,
J = 9 Hz, m, Ar-H), 8.06 (1H, s, NH); 13C NMR δ (ppm):
118.3,121.2, 125.8, 128.7, 129.2, 129.8, 130.9, 132.3, 133.5,
143.3, 151.3, 157.2, 167.6, 174.5; Mass (m/z): 384 (M+).
Analysis for C16H9ClN6S2 (384.87); Calcd: C; 49.96, H; 2.40,
N; 21.88, S; 16.69. Found: C; 49.93, H; 2.36, N; 21.84, S;
16.66.
General procedure for the synthesis of potassium
dithiocarbazinate (4)
(6b). IR (KBr) νmax (cm−1): 616 (C-S-C benzothiazole), 688
(C-S-C triazolo-thiadiazole), 821 (C-Cl), 1274 (N-N = C
triazolo-thiadiazole), 1422 (C-N benzothiazole), 1524
(C = C aromatic), 3114 (C-H aromatic), 3318 (N-H);1H
NMR (DMSO-d6) (300 MHz) δ (ppm) 7.36–7.40 (7H, J = 12
Hz, m, Ar-H), 8.01 (1H, s, NH); 13C NMR δ (ppm): 118.3,
121.2, 125.8, 127.3, 128.9, 129.3, 129.8, 130.1, 132.2, 132.3,
136.9, 151.3, 157.2, 167.6, 174.1, 174.5; Mass (m/z): 419
(M+). Analysis for C16H8Cl2N6S2 (419.31); Calcd: C; 45.86,
H; 1.94, N; 20.07, S; 15.31. Found: C; 45.83, H; 1.92, N;
20.04, S; 15.29.
Potassium hydroxide (0.03 mol) was dissolved in abso-
lute ethanol (50 mL). e solution was cooled in ice bath
and acid hydrazide (3; 0.02 mol) was added with stirring.
To this carbon disulphide (0.025 mol) was added in small
portions with constant stirring. e reaction mixture was
agitated continuously for 12 h at room temperature. e
precipitated potassium dithiocarbazinate was collected
by filtration, washed with anhydrous ether (100 mL) and
dried in vacuum. e potassium salt thus obtained was in
quantitative yield and was used in the next step without
further purification.
(6c). IR (KBr) νmax (cm−1): 614 (C-S-C benzothiazole), 686
(C-S-C triazolo-thiadiazole), 822 (C-Cl), 1275 (N-N = C
triazolo-thiadiazole), 1424 (C-N benzothiazole), 1526
General procedure for the synthesis of 4-amino-5-
(6-chlorobenzo[d]thiazol-2-ylamino)-4H-1,2,4-
triazole-3-thiol (5)
1
(C = C aromatic), 3112 (C-H aromatic), 3317 (N-H); H
NMR (DMSO-d6) (300 MHz) δ (ppm) 7.40–7.44 (6H, J= 12
Hz, m, Ar-H), 8.03 (1H, s, NH); 13C NMR δ (ppm): 118.3,
121.2, 125.8, 127.4, 129.8, 130.3, 130.9, 132.3, 133.6, 135.0,
135.7, 151.3, 157.2, 167.6, 174.1, 174.5; Mass (m/z): 453
(M+). Analysis for C16H17Cl3N6S2 (453.76); Calcd: C; 42.37,
H; 1.58, N; 18.56, S; 14.15. Found: C; 42.35, H; 1.55, N;
18.52, S; 14.13.
(6d). IR (KBr) νmax (cm−1): 618 (C-S-C benzothiazole), 691
(C-S-C triazolo-thiadiazole), 829 (C-Cl), 1281 (N-N = C
triazolo-thiadiazole), 1427 (C-N benzothiazole), 1531
(C = C aromatic), 3119 (C-H aromatic),3319 (N-H);1H
NMR (DMSO-d6) (300 MHz) δ (ppm) 2.34(3H, S, CH3),
7.35–7.37 (6H, J= 6 Hz, m, Ar-H), 8.07 (1H, s, NH); 13C NMR
δ(ppm): 18.7,118.3, 121.2, 125.8, 126.2, 127.4, 128.6,129.5,
129.8, 132.3, 136.9, 137.2, 151.3, 157.2, 167.6, 174.1, 174.5;
Mass (m/z): 398 (M+). Analysis for C16H17Cl3N6S2 (398.89);
Calcd: C; 51.21, H; 2.82, N; 21.10, S;16.12. Found: C; 51.19,
H; 2.78, N; 21.07, S; 16.08.
Asuspensionofpotassiumdithiocarbazinate(4;0.02 mol)
in water (50 mL) and hydrazine hydrate (99%,0.04 mol)
was refluxed for 18–20 h with occasional shaking. e
colour of the reaction mixture changed to green, with
evolution of hydrogen sulphide gas. A homogenous reac-
tion mixture was obtained during the reaction process.
e reaction mixture was cooled to room temperature
and diluted with water (20 mL). On acidification with
acetic acid, the required triazole was precipitated out. It
was filtered, washed thoroughly with cold water, dried
and recrystallized from ethanol. Purity of the compound
was checked by TLC using silica gel-G coated plates by
using toluene: ethyl acetate:formic acid (T:E:F); (5:4:1) as
solvent system, and observed in UV light.
5. IR (KBr) νmax (cm−1): 635 (C-S-C), 817 (C-Cl), 1528
(C = N), 2518 (SH), 3338 (NH2); 1H NMR (DMSO-d6)
(300 MHz) δ (ppm): 5.15(s, 2H, NH2), 7.28–7.32 (3H, J = 12
Hz, Ar-H), 9.29 (1H, s, NH),13.18 (1H, br s, SH).
(6e). IR (KBr) νmax (cm−1): 626 (C-S-C benzothiazole), 684
(C-S-C triazolo-thiadiazole), 817 (C-Cl), 1267 (N-N = C
triazolo-thiadiazole), 1436 (C-N benzothiazole), 1527
(C = C aromatic), 3106 (C-H aromatic), 3324 (N-H);1H
NMR (DMSO-d6) (300 MHz) δ (ppm) 2.51(3H, s,
OCOCH3), 7.24–8.28 (7H, J= 12 Hz, m, Ar-H),8.10 (1H, s,
NH); 13C NMR δ (ppm): 20.3,118.3, 121.2, 123.2, 125.8,
126.0, 127.9, 129.1, 129.4,129.8, 132.3, 151.1, 151.3, 157.2,
167.6, 169.0, 174.1, 174.5; Mass (m/z): 442 (M+). Analysis
General procedure for the synthesis of 6-chloro-N-
(6-substituted-[1,2,4]triazolo[3,4-b][1,3,4]thiadiazol-3-yl)
benzo[d] thiazol-2-amine (6a–g)
An equimolar mixture (0.10 mol) of 4-amino-5-(6-
chlorobenzo[d]thiazol-2-ylamino)-4H-1,2,4-triazole-3-
thiol (5) and aromatic acids in phosphorus oxychloride
(10 mL) was refluxed for 5 h. e reaction mixture was
cooled to room temperature and then gradually poured
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis and in vitro antimicrobial activity of novel N-(6-chlorobenzo[d]thiazol-2-yl) hydrazine carboxamide derivatives 335
for C18H11ClN6O2S2 (442.90); Calcd: C; 48.84, H; 2.52,
N; 18.99, S; 14.51. Found: C; 48.81, H; 2.50, N; 18.97, S;
14.48.
C15H7 Cl3N4OS (397.67): C; 45.32, H; 1.79, N; 14.11, S; 8.08.
Found: C; 45.30, H; 1.77, N; 14.09, S; 8.06.
(7d). IR (KBr) νmax (cm−1): 632 (C-S-C benzothiazole), 812
(C-Cl), 1467 (C-N benzothiazole), 1496 (C-O-C oxadiaz-
ole), 1514 (C = C aromatic), 3331 (N-H); 1H NMR (DMSO-
d6) (300 MHz) δ (ppm) 2.21(3H, s, CH3), 7.64–7.68 (7H,
J = 12 Hz, m, Ar-H), 8.12 (1H, s, NH); 13C NMR δ (ppm):
18.7, 118.3, 121.2, 125.8, 126.2, 127.4, 128.6, 129.5, 129.8,
132.3, 136.9, 137.2, 151.3, 164.5, 169.3, 174.5; Mass (m/z):
342 (M+).Analysis for C16H11 ClN4OS (342.80): C; 56.06, H;
3.23, N; 16.34, S; 9.35. Found: C; 56.10, H; 3.25, N; 16.36,
S; 9.37.
(6f). IR (KBr) νmax (cm−1): 626 (C-S-C benzothiazole), 692
(C-S-C triazolo-thiadiazole), 836 (C-Cl), 1274 (N-N = C
triazolo-thiadiazole), 1434 (C-N benzothiazole), 1532
1
(C = C aromatic), 3123 (C-H aromatic), 3318 (N-H); H
NMR (DMSO-d6) (300 MHz) δ (ppm) 7.29–7.31 (m, J = 6
Hz,8H, Ar-H), 8.10 (1H, s, NH); 13C NMR δ (ppm): 118.3,
121.2, 122.7, 124.6, 125.8, 129.7, 129.8, 132.3, 151.3,
155.2, 157.2, 167.6, 174.5; Mass (m/z): 400 (M+). Analysis
for C16H9ClN6OS2 (400.87); C; 47.97, H; 2.30, N; 20.98, S;
16.04. Found: C; 47.94, H; 2.26, N; 20.96, S; 16.00.
(6g). IR (KBr) νmax (cm−1): 621 (C-S-C benzothiazole), 689
(C-S-C triazolo-thiadiazole), 830 (C-Cl), 1280 (N-N = C
triazolo-thiadiazole), 1369 (NO2), 1429 (C-N benzothi-
azole), 1527 (C = C aromatic), 3118 (C-H aromatic), 3320
(N-H); 1H NMR (DMSO-d6) (300 MHz) δ (ppm) 7.39–7.41
(7H, J = 6 Hz, m, Ar-H), 8.12 (1H, s, NH); 13C NMR δ (ppm):
118.3, 121.2, 124.4, 125.8, 128.4,129.8, 132.3, 139.6, 143.3,
147.9, 151.3, 157.2, 167.6, 174.1, 174.5; Mass (m/z): 429
(M+). Analysis for C16H17Cl3N6S2 (429.86): C; 48.84, H;
2.52, N; 18.99, S; 14.51. Found: C; 48.81, H; 2.50, N; 18.97,
S; 14.48.
(7e). IR (KBr) νmax (cm−1): 638 (C-S-C benzothiazole), 824
(C-Cl), 1468 (C-N benzothiazole), 1489 (C-O-C oxadiaz-
ole), 1517 (C = C aromatic), 3336 (N-H). 1H NMR (DMSO-
d6) (300 MHz) δ (ppm) 2.59 (3H, s, OCOCH3), 7.24–7.28
(7H, J= 12 Hz, m, Ar-H),8.16 (1H, s, NH); 13C NMR δ(ppm):
20.3, 117.8, 118.3, 121.2, 123.2, 125.8, 126.0, 129.1, 129.8,
132.3, 137.1, 151.1, 151.3, 164.5, 169.0, 169.3, 174.5; Mass
(m/z): 386 (M+). Analysis for C17H11 ClN4O3S (386.81): C;
52.79, H; 2.87, N; 14.48, S; 8.29. Found: C; 52.81, H; 2.88,
N; 14.51, S; 8.32.
(7f). IR (KBr) νmax (cm−1): 634 (C-S-C benzothiazole), 824
(C-Cl), 1464 (C-N benzothiazole), 1492 (C-O-C oxadiaz-
ole), 1518 (C = C aromatic), 3326 (N-H). 1H NMR (DMSO-
d6) (300 MHz) δ (ppm) 7.25–7.28 (8H, J = 9 Hz, m, Ar-H),
8.14 (1H, s, NH); 13C NMR δ (ppm): 118.3, 121.2, 122.7,
124.6, 125.8, 129.7, 129.8, 132.3, 151.3, 155.2, 158.7, 169.3,
174.5; Mass (m/z): 344 (M+).Analysis for C15H9ClN4O2S
(344.78): C; 52.25, H; 2.63, N; 16.25, S; 9.30. Found: C;
52.22, H; 2.61, N; 16.23, S; 9.27.
General procedure for the synthesis of N-(6-Chlorobenzo[d]
thiazol-2-yl)-5-phenyl-1,3,4-oxadiazol-2-amine (7a)
Compound 3 (0.001 mol) and appropriate aromatic acid
(0.001 mol) was dissolved in phosphorus oxychloride
and refluxed for 20 h. e reaction mixture was slowly
poured over crushed ice and kept overnight. e solid
thus separated out was filtered, washed with water, dried
and recrystallized from ethanol.
(7g). IR (KBr) νmax (cm−1): 637 (C-S-C benzothiazole), 828
(C-Cl), 1378 (NO2), 1468 (C-N benzothiazole), 1496 (C-
(7a). IR (KBr) νmax (cm−1): 622 (C-S-C benzothiazole),
1454 (C-N benzothiazole), 1488 (C-O-C oxadiazole),
O-C oxadiazole), 1521 (C = C aromatic), 3336 (N-H). H
1
NMR (DMSO-d6) (300 MHz) δ (ppm) 7.21–7.24 (7H, J = 9
Hz, m, Ar-H), 8.16 (1H, s, NH); 13C NMR δ (ppm): 118.3,
121.2, 122.8, 123.9, 125.8, 127.0, 129.8, 130.1, 132.3, 133.6,
148.4, 151.3, 164.5, 169.3, 174.5; Mass (m/z): 373 (M+).
Analysis for C15H8ClN5O3S (373.77); C; 48.20, H; 2.16, N;
18.74, S; 8.58. Found: C; 48.23, H; 2.18, N; 18.76, S; 8.60.
1
1518 (C = C aromatic), 3318 (N-H),. H NMR (DMSO-d6)
(300 MHz) δ (ppm) 7.24–7.28 (8H, J = 12 Hz, m, Ar-H),
8.10 (1H, s, NH); 13C NMR δ (ppm): 118.3, 121.2, 125.8,
126.1, 127.5, 128.7, 129.2, 129.8, 132.3, 151.3, 164.5, 169.3,
174.5; Mass (m/z): 328 (M+). Analysis for C15H9 ClN4OS
(328.78): C; 54.82, H; 2.78, N; 17.08, S; 9.77. Found: C;
54.80, H; 2.76, N; 17.04, S; 9.75.
(7b). IR (KBr) νmax (cm−1): 624 (C-S-C benzothiazole),
821 (C-Cl ), 1456 (C-N benzothiazole), 1491 (C-O-C ox-
adiazole), 1512 (C = C aromatic), 3317 (N-H).1H NMR
(DMSO-d6) (300 MHz) δ (ppm) 7.22–7.26 (7H, J = 12 Hz,
m, Ar-H), 8.14 (1H, s, NH); 13C NMR δ (ppm): 118.3, 121.2,
125.8, 127.3, 128.9, 129.3, 129.8, 130.1, 132.2,132.3, 136.9,
151.3, 164.5, 169.3, 174.5; Mass m/z: 363 (M+). Analysis
for C15H8 Cl2N4OS (363.22): C; 49.64, H; 2.24, N; 15.45, S;
8.85. Found: C; 49.60, H; 2.22, N; 15.42, S;8.83.
(7c). IR (KBr) νmax (cm−1): 628 (C-S-C benzothiazole), 826
(C-Cl), 1461 (C-N benzothiazole), 1497 (C-O-C oxadiaz-
ole), 1516 (C = C aromatic), 3321 (N-H). 1H NMR (DMSO-
d6) (300 MHz) δ (ppm) 7.26–7.30 (6H, J = 12 Hz, m, Ar-H),
8.20 (1H, s, NH); 13C NMR δ (ppm): 118.3, 121.2, 125.8,
128.8, 129.8, 130.2, 130.3, 130.7, 132.3, 132.9, 138.3, 151.3,
164.5, 169.3, 174.5; Mass (m/z): 397 (M+).Analysis for
General procedure for the synthesis of 5-(6-Chlorobenzo[d]
thiazol-2-ylamino)-1,3,4-oxadiazole-2-thiol (8)
A mixture of 3 (0.005 mol), KOH (0.005 mol) and carbon
disulphide (5 mL) in ethanol (50 mL) was refluxed on a
steam bath for 12 h. e solution was then concentrated,
cooled and acidified with dilute HCl. e solid mass that
separated out was filtered, washed with ethanol, dried
and recrystallized from ethanol.
(8). IR (KBr) νmax (cm−1): 614 (C-S-C benzothiazole), 826
(C-Cl), 1421 (C-N), 1505 (C-O-C oxadiazole), 1514 (C = C),
1614 (C = O), 2516 (SH), 3316 (N-H); 1H NMR (DMSO-d6)
(300 MHz) δ (ppm) 7.17–7.19 (3H, J= 6 Hz, m, Ar-H), 8.05
(1H, s, NH), 10.41(1H, s, SH); 13C NMR δ (ppm): 118.3,
121.2, 125.8, 129.8, 132.3, 151.3, 169.3, 174.5; Mass (m/z):
284 (M+). Analysis for C9H5ClN4OS2 (284.75): C; 37.98, H;
1.80, N; 19.71, S; 22.56. Found: C; 37.96, H; 1.77, N; 19.68,
S; 22.52.
© 2011 Informa UK, Ltd.

336 Sadaf J. Gilani et al.
Antimicrobial activity
Antimicrobial activity
Antibacterial activity of the synthesized compounds
were determined in vitro by using serial plate dilution
method25,26 against E. coli (ATCC-25922), S. aureus (ATCC-
25923), P. aeruginosa (ATCC-27853) and K. pneumoniae
(ATCC-700603) at 100 µg/mL, 50 µg/mL, 25 µg/mL and
12.5 µg/mL concentrations, respectively, in the nutrient
agar media. Standard antibiotic ofloxacin was used as
reference drug at 25 µg/mL, 12.5 µg/mL and 6.25 µg/mL
concentrations.
e newly synthesized compounds were screened for
their antimicrobial activity. e results of antimicrobial
effect of all the tested compounds were reported as mini-
mal inhibitory concentrations (MICs, µg/mL).e inves-
tigation of antibacterial and antifungal screening data
revealed that all the tested compounds 6a–g, 7a–g and 8
showed moderate to good inhibition at 12.5–100 µg/mL
in DMSO.
e compounds 6c, 6d, 6g, 7a, 7c, 7f and 8 showed
comparatively good activity against all the bacterial
strains (Table 2). e good activity is attributed to the
presence of pharmacologically active 2,4-dichloro (6c),
methyl (6d), 4-nitro (6g) substituent attached to phenyl
group at position 6 of the triazolo-thiadiazole ring (MIC
12.5 µg/mL & 25 µg/mL), whereas phenyl (7a), 2,4-
dichlorophenyl (7c), phenoxy groups (7f) and mercapto
(8), attached at second position of 1,3,4-oxadiazole moi-
ety (MIC 12.5 µg/mL & 25 µg/mL). When this group was
replaced by 2-chlorophenyl (6band 7b) and acetyl groups
(6e and 7e) it caused sharp decrease in activity against
most of the strains (MIC 100 µg/mL). e compounds
6a, 6f, 7d and 7g, exhibited moderate activity compared
to that of standard against all the bacterial strains (MIC
25 µg/mL & 50 µg/mL). Further, the result also shows that
gram-negative (E. coli, P. aeruginosa, K. pneumoniae)
exhibited better activity than gram positive (S. aureus)
organisms.
Newly synthesized compounds were screened in vitro
against pathogenic fungal strains C. albicans (ATCC
2091), A. niger (MTCC 281), Aspergillus flavus (MTCC
277), M. purpureus (MTCC 369) and P. citrinum (NCIM
768) by serial plate dilution method27,28 at 100 µg/mL,
50 µg/mL, 25 µg/mL and 12.5 µg/mL concentrations in
sabouraud dextrose medium. Ketoconazole was used as
standard drug at 25 µg/mL, 12.5 µg/mL and 6.25 µg/mL
concentrations. Solutions of required concentrations of
test compounds were prepared by dissolving the com-
pounds in DMSO. e minimum inhibitory concentra-
tion (MIC) obtained for the test compounds and refer-
ence drugs are reported in Tables 2 and 3. e minimum
inhibitory concentration (MIC) was defined as the lowest
concentration of the compounds that inhibited visible
growth of microorganisms on the plate.
Results and discussion
e compounds 6c, 6d, 6f, 7e, 7g and 8 showed com-
paratively good activity against all the fungal strains
(Table 3). e structure of these compounds contains
biologically active 2,4-dichloro phenyl, methyl phe-
nyl and phenoxy group attached at position 6 of the
triazolo-thiadiazole ring (MIC 12.5 µg/mL & 25 µg/mL)
and mercapto, acetyl and 4-nitro phenyl group attached
at second position of oxadiazole ring (MIC 12.5 µg/mL
& 25 µg/mL), respectively. e compounds 6a, 6g, 7a,
7c and 7d showed moderate activity compared to that
of standard (6.25 µg/mL) against all the fungal strains
(MIC 25 µg/mL & 50 µg/mL). It has been observed that
triazolo-thiadiazole derivatives are found to be more
active than 1,3,4-oxadiazole derivatives against all
pathogenic bacterial and fungal strains.
e antimicrobial activity study revealed that all the
compounds tested showed good to moderate antibac-
terial and antifungal activities against all pathogenic
strains (MIC 12.5 µg/mL, 25 µg/mL and 50 µg/mL).
Structure and biological activity relationship of title
compounds showed that the presence of 2,4-dichloro
phenyl, methyl phenyl,4-nitro phenyl group at sixth
position of triazolo-thiadiazole (MIC 12.5 µg/mL and
25 µg/mL) and 2-mercapto, phenyl, 2,4-dichlorophenyl
and phenoxy group at second position of oxadiazole nu-
cleus (MIC 12.5 µg/ mL and 25 µg/mL) are responsible
for good antibacterial activity.
Chemistry
6-substituted-1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazole
(6a–g) and 1,3,4-oxadiazole (7a–g, 8) were prepared ac-
cording to the procedure outlined in Schemes 1 and 2. e
requireddithiocarbazinate(4)wassynthesizedbyreacting
N-(6-chlorobenzo[d]thiazol-2-yl) hydrazine carboxamide
withcarbondisulfideandpotassiumhydroxideinethanol.
is salt underwent ring closure with an excess of 99% hy-
drazine hydrate to give the 4-amino-5-(6-chlorobenzo[d]
thiazol-2-ylamino)-4H-1,2,4-triazole-3-thiol (5). Hence
resulted triazole (5) was then further converted to the
6-chloro-N-(6-substituted-[1,2,4]triazolo[3,4-b][1,3,4]
thiadiazol-3-yl)benzo[d] thiazol-2-amine (6a–g) in a one-
pot reaction, by condensation with aromatic acids in the
presence of POCl3. Phosphorus oxychloride was neces-
sary for this condensation, which activate the carbonyl
group of aromatic acids and increases its electrophilicity
to enhance the addition of triazole to it. e synthesis of
compounds (7a–g) was accomplished in a single step by
reacting the benzothiazole carboxamide with aromatic
acids in the presence of POCl3 and compound (8) by re-
acting with carbon disulfide and potassium hydroxide in
ethanol, respectively. e structure of synthesized com-
pounds was confirmed by elemental analysis and spectral
data (IR, 1H NMR, Mass).
Physicochemical data for the N-(6-chlorobenzo[d]
thiazol-2-yl) hydrazine carboxamide derivatives (6a–g,
7a–g, 8) is given in Table 1. e synthetic route for the
title compounds is shown in Schemes 1 and 2.
Similarly, presence of 2,4,-dichloro phenyl, methyl
phenyl and phenoxy group attached at sixth position of
triazolo-thiadiazole (MIC 12.5µg/mL and 25µg/mL),
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis and in vitro antimicrobial activity of novel N-(6-chlorobenzo[d]thiazol-2-yl) hydrazine carboxamide derivatives 337
Table 1. Physicochemical data of N-(6-chlorobenzo[d]thiazol-2-yl)hydrazine carboxamide derivatives (6a–g, 7a–g, 8).
Comp.
6a
R
Yield (%)
74
M.P. (°C)
240
Molecular Weight
384.87
Molecular Formula
C16H9ClN6S2
Rf
0.7
6b
6c
6d
6e
6f
Cl
71
68
71
66
76
254
248
282
288
218
419.31
453.76
398.89
442.90
400.87
C16H8Cl2N6S2
C16H17Cl3N6S2
C16H17Cl3N6S2
C18H11ClN6O2S2
C16H9ClN6OS2
0.6
0.9
0.8
0.7
0.6
Cl
Cl
CH3
OCOCH3
O
6g
7a
7b
63
67
64
116
262
234
429.86
328.78
363.22
C16H17Cl3N6S2
C15H9 ClN4OS
C15H8 Cl2N4OS
0.7
0.8
0.6
O2N
Cl
7c
7d
7e
Cl
66
65
64
249
232
246
397.67
342.80
386.81
C15H7 Cl3N4OS
C16H11 ClN4OS
C17H11 ClN4O3S
0.7
0.8
0.7
Cl
CH3
OCOCH3
Table 1. continued on next page
© 2011 Informa UK, Ltd.

338 Sadaf J. Gilani et al.
Table 1. Continued.
Comp.
7f
R
Yield (%)
73
M.P. (°C)
261
Molecular Weight
344.78
Molecular Formula
C15H9ClN4O2S
Rf
0.8
O
7g
67
74
223
234
373.77
284.75
C15H8ClN5O3S
C9H5ClN4OS2
0.6
0.8
O2N
8
—
Elemental analysis were found to be within 0.4% of theoretical values.
Table 2. Antibacterial activity of the title compounds (6a–g, 7a–g and 8).
MIC inµg/mL and zone of inhibition in mm
Compounds
Escherichia coli
25 (16–20)
100 (<10)
Staphylococcus aureus
Pseudomonas aeruginosa
50 (11–15)
Klebsiella pneumoniae
50 (11–15)
6a
50 (11–15)
100 (<10)
6b
100 (<10)
100 (<10)
6c
12.5 (21–25)
12.5 (21–25)
100 (<10)
25 (16–20)
25 (16–20)
100 (<10)
12.5 (21–25)
12.5 (21–25)
100 (<10)
12.5 (21–25)
12.5 (21–25)
100 (<10)
6d
6e
6f
25 (16–20)
12.5 (21–25)
12.5 (21–25)
100 (<10)
25 (16–20)
12.5 (21–25)
25 (16–20)
100 (<10)
50(11–15)
25 (16–20)
6g
12.5 (21–25)
25 (16–20)
25 (16–20)
7a
25 (16–20)
7b
100 (<10)
100 (<10)
7c
12.5 (21–25)
25 (16–20)
100 (<10)
25 (16–20)
50 (11–15)
100 (<10)
12.5 (21–25)
50 (11–15)
12.5 (21–25)
50 (11–15)
7d
7e
100 (<10)
100 (<10)
7f
12.5 (21–25)
25 (16–20)
12.5 (21–25)
6.25 (26–30)
12.5 (21–25)
50 (11–15)
12.5 (21–25)
6.25 (26–30)
12.5 (21–25)
50 (11–15)
25 (16–20)
7g
50 (11–15)
8
12.5 (21–25)
6.25 (26–30)
12.5 (21–25)
6.25 (26–30)
Ofloxacin
e figures in the table show the MIC (Minimum Inhibitory Concentration) values in µg/mL and the corresponding zone of inhibition
in mm.
Table 3. Antifungal activity of the title compounds (6a–g, 7a–g and 8).
MIC inµg/mL and zone of inhibition in mm
Compounds Candida albicans
Aspergillus niger
25 (16–20)
100 (<10)
Aspergillus flavus
50 (11–15)
100 (<10)
Monascus purpureus
50 (11–15)
100 (<10)
Penicillium citrinum
50 (11–15)
100 (<10)
6a
6b
6c
6d
6e
6f
50 (11–15)
100 (<10)
12.5 (21–25)
12.5 (21–25)
100 (<10)
25 (16–20)
25 (16–20)
100 (<10)
12.5 (21–25)
12.5 (21–25)
100 (<10)
25 (16–20)
12.5 (21–25)
100 (<10)
12.5 (21–25)
12.5 (21–25)
100 (<10)
12.5 (21–25)
50 (11–15)
25 (16–20)
100 (<10)
12.5 (21–25)
50 (11–15)
50 (11–15)
100 (<10)
12.5 (21–25)
25 (16–20)
25 (16–20)
100 (<10)
25 (16–20)
25 (16–20)
25 (16–20)
100 (<10)
25 (16–20)
50 (11–15)
50 (11–15)
100 (<10)
6g
7a
7b
7c
7d
7e
7f
50 (11–15)
50 (11–15)
12.5 (21–25)
100 (<10)
25 (16–20)
50 (11–15)
12.5 (21–25)
100 (<10)
25 (16–20)
50 (11–15)
25 (16–20)
100 (<10)
50 (11–15)
25 (16–20)
25 (16–20)
100 (<10)
50 (11–15)
50 (11–15)
12.5 (21–25)
100 (<10)
7g
8
25(16–20)
12.5 (21–25)
25 (16–20)
12.5 (21–25)
6.25 (26–30)
12.5 (21–25)
12.5 (21–25)
6.25 (26–30)
12.5 (21–25)
12.5 (21–25)
6.25 (26–30)
12.5 (21–25)
12.5 (21–25)
6.25 (26–30)
Ketoconazole 6.25 (26–30)
e figures in the table show the MIC (minimum inhibitory concentration) values in µg/mL and the corresponding zone of inhibition
in mm.
Journal of Enzyme Inhibition and Medicinal Chemistry

Synthesis and in vitro antimicrobial activity of novel N-(6-chlorobenzo[d]thiazol-2-yl) hydrazine carboxamide derivatives 339
N
g.a.a
Br2
NH2
Cl
KSCN
NH2
+
S
Cl
(1)
p-Chloroamline
g.a.a
NaOCN
O
O
N
NH2.NH2.H2O
NaOH/EtOH
N
S
NH NH
NH2
NH2
NH
S
Cl
(3)
Cl
(2)
Scheme 1. Synthetic pathways to N-(6-chlorobenzo[d]thiazol-2-yl) hydrazine carboxamide.
N
S
O
NH
O
N
S
SH
CS2/KOH
Cl
NH
NH
(3)
NH2
N
N
(8)
Cl
KOH/CS2/C2H5OH
Stirring; Roomtemp.
RCOOH/PO Cl3
Reflux
N
NH
S
O
O
R
S
Cl
N
S
N
N
S−K+
NH NH NH
(4)
(7a-g)
Cl
NH2NH2H2O
Reflux
NH2
N
SH
N
S
N
H
N
N
(5)
Cl
RCOOH/PO Cl3
Reflux
N
N
NH
N
N
S
S
N
Cl
R
(6a-g)
Scheme 2. Synthetic route for the title compounds.
whereasmercapto(–SH),acetyland4-nitrophenylattached
at second position of oxadiazole nucleus (MIC 12.5µg/mL
and 25µg/mL) are responsible for good antifungal activity.
us, various triazolo-thiadiazole (6a–g) and oxadi-
azole (7a–g, 8) derivatives of benzothiazole hydrazine
carboxamide were prepared with the objective of
developing better antimicrobial agents. e triazolo-
thiadiazole and oxadiazole derivatives were found to
have a promising class of compounds with an interesting
pharmacological profile. Further, it is clear from structure
© 2011 Informa UK, Ltd.

340 Sadaf J. Gilani et al.
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Eur J Med Chem 2007; 42:823–840.
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Acknowledgements
e authors are thankful to the Head of the Department,
Pharmaceutical Chemistry for providing laboratory facil-
ities, Central Drug Research Institute (CDRI) for spectral
analysis of the compounds. Authors are also thankful to
Dr. Qadir, Head, IGIB, New Delhi for providing bacterial
and fungal strains.
Declaration of interest
15. Amir M, Kumar H, Javed SA. Synthesis and pharmacological
evaluation of condensed heterocyclic 6-substituted-1,2,4-
triazolo[3,4-b]-1,3,4-thiadiazole derivatives of naproxen. Bioorg
Med Chem Lett 2007; 17:4504–4508.
e authors report no conflict of interest.
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and bioassay of a new class of heterocycles pyrrolyl oxadiazoles/
thiadiazoles/triazoles. Eur J Med Chem 2009; 44:2313–2321.
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