Green synthesis of 5-substituted-1,3,4-thiadiazole-2-thiols as new potent nitrification inhibitors [1]

Article abstract of DOI:10.1002/jhet.345

(Chemical Equation Presented) A fast, efficient synthesis of 5-substituted-1,3,4-thiadiazole-2-thiols was successfully developed, assessed using green chemistry matrices, and compounds were screened for their in vitro nitrification inhibitory activity. The greener method was superior with higher energy efficiency, E(nvironmental) factor, atom economy, atom efficiency, carbon efficiency, and reaction mass efficiency.

Full text of DOI:10.1002/jhet.345

838
Green Synthesis of 5-Substituted-1,3,4-thiadiazole-2-thiols as
New Potent Nitrification Inhibitors [1]
Vol 47
Ajoy Saha, Rajesh Kumar,* Rajendra Kumar, and C. Devakumar
Division of Agricultural Chemicals, Indian Agricultural Research Institute, New Delhi 110 012,
India
*E-mail: rkb_1973@yahoo.co.in or rkb1973@gmail.com.
Received June 20, 2009
DOI 10.1002/jhet.345
Published online 4 June 2010 in Wiley InterScience (www.interscience.wiley.com).
A fast, efficient synthesis of 5-substituted-1,3,4-thiadiazole-2-thiols was successfully developed,
assessed using green chemistry matrices, and compounds were screened for their in vitro nitrification
inhibitory activity. The greener method was superior with higher energy efficiency, E(nvironmental)
factor, atom economy, atom efficiency, carbon efficiency, and reaction mass efficiency.
J. Heterocyclic Chem., 47, 838 (2010).
INTRODUCTION
effective nitrification inhibitors, and the economics of
their use in field conditions [11]. Various heterocyclics
being an important bioactive class are used as nitrifica-
tion inhibitors [12], but their use is restrained because
of the complex synthetic procedures involving hazardous
chemicals and subsequent cost. This problem can be
solved by synthesizing potential nitrification inhibiting
heterocyclics with minimum cost in an environment
friendly manner. Keeping in view the aforementioned
consideration, this study was taken up to design and
synthesize 5-substituted-1,3,4-thiadiazole-2-thiols in a
greener and cost effective way as potential nitrification
inhibitors.
With increasing global population, demands have
been increased to meet the requirements and conse-
quently the industrial pollution. To control this detri-
mental phenomenon, the term ‘‘green chemistry’’ has
come into existence [2]. Green chemistry is a package
of technologies, design principles, and tools to reduce
toxicity, resource energy use, and pollution of chemicals
[2,3]. Due to environmental awareness [4], chemists
have focused their attention to examine bioactive prod-
ucts such as heterocycles and processes in terms of envi-
ronment friendliness. The 1,3,4-thiadiazoles are impor-
tant bioactive heterocyclic moieties and have been
reported to have wide range of bioactivities [5] such as
neuroprotective, antibacterial, antidepressant, and antitu-
berculour. One of the title compound, 5-methyl-1,3,4-
thiadiazole-2-thiol is the side chain at C-3 position of a
well known antibiotic Cefazolin sodium [6].
RESULTS AND DISCUSSION
5-Substituted-1,3,4-thiadiazole-2-thiols have been syn-
thesized starting from carboxylic acids following the
conventional (protocol 1) and improved greener syn-
thetic protocol (protocol 2). Reaction pathway is
depicted in Scheme 1. In protocol 1, carboxylic acids
were esterified by refluxing with ethyl alcohol for 3–4 h
in the presence of sulfuric acid. These ester yielded cor-
responding hydrazide on refluxing with hydrazine
hydrate for 3–5 h [13]. The analytical and spectral data
of hydrazides were in complete agreement with those
given in literature [13–17]. Hydrazides on reaction with
carbon disulphide and potassium hydroxide afforded thi-
ocarbazate salts, which are cyclized in the presence of
concentrated sulfuric acid to afford 5-substituted-1,3,4-
The low nitrogen use efficiency accounts to US$ 17
Billion annual nitrogen losses worldwide along with
environmental and health hazards [7,8]. There is, thus,
an urgent call for improving the efficiency of N-fertil-
izer use to achieve higher food production for catering
the ever increasing population and also to minimize fer-
tilizer related pollution of the environment [9]. Despite
a great interest in the development and use of nitrifica-
tion inhibitors to date, only a few compounds have been
adopted for agricultural use [10]. The main problems
associated with these inhibitors are the high cost
involved in the development, subsequent registration of
C
V
2010 HeteroCorporation

July 2010
Green Synthesis of 5-Substituted-1,3,4-thiadiazole-2-thiols as New Potent
Nitrification Inhibitors
839
Scheme 1. Synthesis of 5-substituted-1,3,4-thiadiazole-2-thiols.
using various green chemistry measures [19–21]. The
developed greener protocol showed 4.7–11.4% increase
in overall yield (Table 1). 5-Phenyl-1,3,4-thiadiazole-2-
thiol was taken as a case for all calculations and related
data for both the protocols (1 and 2) are described in
Table 3.
The overall yield, i.e., starting from benzoic acid to
5-phenyl-1,3,4-thiadiazole-2-thiol, following protocol 2
was 69.3% as compared with 59.6% from conventional
protocol. There was 9.7% increase in the yield. The
number of steps was also reduced to three as compared
with four in protocol 1. This resulted in the higher
energy efficiency and reduction of time, as total heating
time was just 60–200 s under microwaves in protocol 2
as compared with 6–9 h in protocol 1. Among the vari-
ous green chemistry matrices, E-factor values for proto-
cols 1 and 2 were 20.1 and 14.8 kg waste/kg product,
respectively, showing the reduction of 26.4% in the
waste produced over the production of 1 kg of the prod-
uct. Atom economy was increased by 5% as it was 43.3
and 48.3% for protocols 1 and 2, respectively. Atom ef-
ficiency, which considers both atom economy and yield
parameters, showed an improvement of 7.7%. Carbon
efficiency was found to be 28.5 and 34.7% for protocols
1 and 2, respectively, and it was improved by 6.2%.
Reaction mass efficiency possessed the values of 31.5
(protocol 1) and 36.1 (protocol 2) as well as an increase
of 4.6% was observed.
Results obtained in the in vitro soil incubation study
are described in the Table 4. All the test compounds
showed significantly higher ammonium-N and lower ni-
trate-N content as compared with urea alone The nitrite-
N content remained insignificant (<0.5 mg/kg) in all the
samples on all the sampling days.
thiadiazole-2-thiols. In protocol 2, our new greener one-
pot method was used for the synthesis of acid hydra-
zides [18], and hydrazides were further processed simi-
larly as in protocol 1 to yield 5-substituted-1,3,4-thiadia-
zole-2-thiols. This innovation improved the overall syn-
thetic protocol for 5-substituted-1,3,4-thiadiazole-2-thiols
in terms of yield, time, energy, atom economy and effi-
ciency, number of steps, and other green chemistry
measures (Table 3). All these compounds were charac-
terized by analytical and spectral data (Tables 1 and 2).
The structures of 5-substituted-1,3,4-thiadiazole-2-thi-
All the compounds have been found to be effective
nitrification inhibitors showing 49.5–79.7%, 36.8–
78.8%, 42.4–78.5%, and 24.6–76.85% nitrification inhi-
bition (NI) at 7th, 14th, 21st, and 28th days, respec-
tively (Table 4). 4-Amino-1,2-4-triazole (ATC), the ref-
erence inhibitor at 10% dose, showed 75.3%, 75.7%,
77.2%, and 73.7% inhibitory activity on 7th, 14th, 21st,
and 28th days, respectively, whereas the corresponding
data on 5% dose were 65.9%, 65.5%, 65.3%, and
59.2% as well as at 1% dose were 60.9%, 57.4%,
54.6%, and 39.0% on 7th, 14th, 21st, and 28th days,
respectively.
1
ols were established on the basis of their IR, H-NMR,
13C-NMR, CHNS data. The analytical and spectral data
of 5-substituted-1,3,4-thiadiazole-2-thiols are given in
Tables 1 and 2. The structure was supported by the ab-
sence of IR bands at 3200–3300, 1650–1670 cmꢀ1 due
to NHNH2 and CONH groups, respectively, and appear-
ance of 1550–1590 cmꢀ1 due to C¼¼N groups. It was
Among the series, 5-heptyl-1,3,4-thiadiazole-2-
thiol (ii), 5-(2-chloro phenyl)-1,3,4-thiadiazole-2-thiol
(vi), 5-(2,4,-dichloro phenyl)-1,3,4-thiadiazole-2-thiol
(vii), 5-(2-methyl phenyl)-1,3,4-thiadiazole-2-thiol (viii),
5-(3-methyl phenyl)-1,3,4-thiadiazole-2-thiol (ix), 5-(3,4,-
dimethoxy phenyl)-1,3,4-thiadiazole-2-thiol (xii), 5-(2-
hydroxy phenyl)-1,3,4-thiadiazole-2-thiol (xiii), and 5-
(4-hydroxy-3-methoxy phenyl)-1,3,4-thiadiazole-2-thiol
1
also supported by the H-NMR data showing signals at
d 11.0–14.2 due to SH and 13C-NMR data showing sig-
nals at d 160.0–174.0 and 176–192 due to 5 CAN and 2
CAN, respectively.
Both conventional and greener synthetic protocols for
5-substituted-1,3,4-thiadiazole-2-thiols were assessed
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

840
A. Saha, R. Kumar, R. Kumar, and C. Devakumar
Vol 47
Table 1
Physical and spectral data of 5-substituted-1,3,4-thiadiazole-2-thiols.
Melting Point
Obs./Lit.
% Yield Protocol 1IR (cmꢀ1
)
1H NMR
(DMSO-d6 þ CDCl3 d, ppm)
13C NMR
(DMSO-d6 þ CDCl3 d, ppm)
S. no.
(Protocol 2)
(C¼N)
i.
182–184/184–187 [22]
97–98/95–96 [22]
53.1 (79.9)
1553 2.43 (s, 3H, CH3),
14.16 (s,1H, 2-SH)
1548 0.89 (t, 3H, 70-CH3),
1.29 (m, 8H, 4 ꢁ CH2),
1.63 (m, 2H, 20-CH2),
2.35 (t, 2H, 10-CH2),
19.9 (CH3), 160 (5 C¼N),
190 (2 C¼N)
ii.
58.4 (63.2)
49.5 (65.4)
14.0 (CH3), 23.1 (CH2),
32.5 (CH2), 30.0 (CH2),
29.9 (CH2), 32.1 (CH2),
29.6, (CH2) 155 (5 C¼N),
187 (2 C¼N)
12.9 (s, 1H, 2-SH)
iii. 105–108/105–107 [22]
1540 0.88 (t, 3H, 90CH3),
1.27 (m, 12H, 6 ꢁ CH2),
1.61 (m, 2H, 20-CH2),
2.34 (t, 2H, 10-CH2),
14.2 (CH2), 23.3 (CH2),
32.6 (CH2), 30.0 (CH2),
29.3 (CH2), 30.2 (CH2),
31.2 (CH2), 31.5 (CH2),
34.1 (CH2), 154 (5C¼N),
182 (2 C¼N)
12.8 (s, 1H, 2-SH)
iv. 123–126/123–125 [22]
51.7 (60.3)
59.4 (77.0)
1535 0.88 (t, 3H, 110-CH3),
1.26 (m, 16H, 8 ꢁ CH2),
1.62 (m, 2H, 20-CH2),
2.34 (t, 2H, 10 CH2),
15.1 (CH3), 24.1 (CH2), 30.1 (CH2),
31.2 (CH2), 29.5 (CH2),
31.2 (CH2), 33.0 (CH2),
31.2 (CH2), 30.5 (CH2),
12.7 (s, 1H, 2-SH)
32.1 (CH2), 34.1 (CH2), 151 (5 C¼N),
180 (2 C¼N)
v.
214–216/215–216 [23]
1572 7.51–7.55 (m, 1H, ArAH),
7.59 (dd, 2H, ArAH,
122.81 (ArAC),
126.42 (ArAC) 126.91(ArAC),
129.82 (ArAC), 132.0 (ArAC),
160.90 (ArAC), 177.8 (5 C¼N),
192 (2 C¼N)
J ¼ 8.0 MHz), 7.94–7.96
(m, 2H, ArAH) 11.0 (s, 1H, SH)
vi. 212–214/210–212 [24]
57.9 (68.3)
57.1 (60.2)
1575 7.90 (d, 2H, ArAH),
7.48 (d, 2H, ArAH),
13.16 (s, 1H, SH)
129.1 (ArAC), 130.0 (2 ArAC),
131.5 (2 ArAC), 138.2 (ArAC),
166.9 (5 C¼N), 179.23 (2 C¼N)
126.58 (ArAC), 127.20 (ArAC),
131.47 (ArAC), 133.55 (ArAC),
136.02 (ArAC), 139.62 (ArAC),
169.60 (5 C¼N), 182.46 (2 C¼N)
21.19 (CH3), 125.59 (ArAC),
129.71 (ArAC), 131.98 (2ArAC),
132.79 (ArAC), 141.4 (ArAC),
173.76 (5 C¼N), 186.43 (2 C¼N)
vii.
197–201
142–143
1581 7.32–7.39 (m, 1H, ArAH),
7.57 (d, 1H, ArAH),
8.2 (d, 1H, ArAH),
13.98 (s, 1H, SH)
viii.
54.5 (59.3)
53.3 (61.2)
1574 2.70 (s, 3H, CH3),
8.12–8.31 (m, 1H, ArAH),
7.92–8.1 (m, 1H, ArAH),
7.46–7.49 (m, 1H, ArAH),
7.29–7.32 (m, 1H, ArAH),
12.28 (s, 1H, SH)
ix.
x.
161–162
180–183
1569 2.43 (s, 3H,CH3),
7.26 (d, H, ArAH), 7.41 (dd,
ArAH, J ¼ 9.6 MHZ) 7.49
21.28 (CH3), 127.39 (ArAC),
128.39 (ArAC), 129.22 (ArAC),
130.72 (ArAC), 134.63 (ArAC),
(d, 1H, ArAH), 7.94 (d, 1H, ArAH), 138.32 (ArAC), 172.65 (5 C¼N),
12.68 (s, 1H, SH)
1561 2.28 (s, 3H, CH3), 7.19
183.24 (2 C¼N)
57.1 (67.4)
56.2 (65.3)
53.6 (58.2)
21.42 (CH3), 128.48 (ArAC),
129.43 (2 ArAC), 129.75 (2 ArAC),
143.34 (ArAC), 167.8 (5 C¼N),
176.34 (2 C¼N)
(d, 2H, ArAH), 7.81 (d, 2H,
ArAH), 12.65 (s, 1H, SH)
xi. 223–225/222–224 [24]
1573 3.72 (s, 3H, CH3), 7.31
(d, 2H, ArAH), 7.68
48.24 (OCH3), 111.30 (2 ArAC),
121.35 (2 ArAC), 139.75 (ArAC),
147.34 (ArAC), 164.8 (5 C¼N),
178.34 (2 C¼N)
(d, 2H, ArAH),
12.36 (s, 1H, SH)
xii.
240–242
232–235
1577 3.9 (s, 3H, OCH3), 4.8 (s, 3H,
OCH3), 6.92 (d, 1H, ArAH),
7.59 (s, 1H, ArAH), 7.76–7.78
(dd, ArAH, J ¼ 6.81),
55.98 (OCH3), 56.04 (OCH3),
110.30 (ArAC), 112.22 (ArAC),
121.70 (ArAC), 124.59 (ArAC),
148.61 (ArAC), 153.45 (ArAC),
172.03 (5C¼N), 183.23 (2 C¼N)
113.26 (ArAC), 117.20 (ArAC),
119.47 (ArAC), 130.67 (ArAC),
135.96 (ArAC), 161.16 (ArAC),
172.47 (5 C¼N), 183.24 (2 C¼N)
12.76 (s, 1H, SH)
xiii.
55.5 (67.3)
1582 5.2 (s, 1H, OH), 6.83–6.91
(m, 1H, ArAH), 7.41–7.45
(m, 1H, ArAH), 7.76 (d, 1H,
ArAH), 7.78 (d, 1H, ArAH),
11.38 (s, 1H, SH)
(Continued)
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

July 2010
Green Synthesis of 5-Substituted-1,3,4-thiadiazole-2-thiols as New Potent
Nitrification Inhibitors
841
Table 1
(Continued)
Melting Point
Obs./Lit.
% Yield Protocol 1IR (cmꢀ1
)
1H NMR
13C NMR
S. no.
xiv.
(Protocol 2)
(C¼N)
(DMSO-d6 þ CDCl3 d, ppm)
(DMSO-d6 þ CDCl3 d, ppm)
210–214
57.9 (59.3)
1579 3.89 (s, 1H, ArAOH), 3.8
(s, 3H, OCH3), 6.63 (d, 1H,
ArAH), 6.83–6.99 (m, 1H),
7.03 (d, 1H), 9.82 (s, 1H, OH),
12.43 (s, 1H, SH)
55.87 (CH3), 113.05 (ArAC),
115.46 (ArAC), 122.03 (ArAC),
151.45 (ArAC), 167.72 (5C¼N),
188.7 (2C¼N)
xv.
167–169
53.3 (60.9)
1576 1.34 (s, 9H, t-C4H9), 7.46 (d,
2H, ArAH), 8.03 (d, 2H, ArAH),
13.39 (s, 1H, SH)
14.3 (Aliph-C),
29.7 (Aliph-C), 31.1 (Aliph-C),
35.2 (Aliph-C), 125.3 (ArAC),
125.5 (ArAC), 126.5 (ArAC),
129.4 (ArAC), 130.1 (ArAC),
157.58 (ArAC), 172.15 (5 C¼N),
183.92 (2 C¼N)
(xiv) were found to be the promising nitrification
inhibitors.
inhibitor ATC. Among them, least active was compound
(iv) with activity of 58% on the 28th day at 10% dose.
Among the active series compound (vi) was most
active at 5% dose with NI 74.1, 73.5, 72.3, and 65.4 on
7th, 14th, 21st, and 28th days, respectively (Table 4). It
performed better than ATC (5%) and statistically at par
with ATC (10%) on 7th and 14th day. The next active
compound was (vii) at 5% dose with NI 72.5%, 71.5%,
72.1%, and 63.1% on 7th, 14th, 21st, and 28th days,
respectively. Other active compounds showed NI in the
range of 56.1–73.8% during the entire period of incuba-
tion. All these compounds were statistically similar to
ATC (5%). Remaining compounds were found to be
inferior to reference inhibitor ATC (5%).
Among the active series, 5-(3-methyl phenyl)-1,3,4-
thiadiazole-2-thiol (ix) was most active at 10% dose
with NI 78.2%, 77.1%, 77.3%, and 76.8% on 7th, 14th,
21st, and 28th days, respectively. The next active com-
pound was 5-(2-chloro phenyl)-1,3,4-thiadiazole-2-thiol
(vi) with activity at 10% was 79.7%,78.8%, 77.7%, and
75.1% on 7th, 14th, 21st and 28th days, respectively
(Table 4).
Other active compounds (vii), (xii), (xiii), (viii), (ii),
and (xiv) were statistically at par with ATC as evident
from least significant difference (LSD) values. Com-
pound (xv) showed 77.3%, 76.6%, 78.5%, and 68.4%
NI on 7th, 14th, 21st, and 28th days, respectively. It per-
formed statistically at par with ATC on 7th, 14th, 21st,
but its activity was lower on 28th day. Rest of the com-
pounds was less active at 10% doses than the reference
Compound (vi) at 5% dose performed best with NI
66.3%, 62.9%, 54.4%, and 41.4% on 7th, 14th, 21st,
and 28th days, respectively. The next in performance
were (x), (xiv), (viii), (vii), (ii), and (xii). All these
Table 2
Elemental-analytical data of 5-substituted-1,3,4-thiadiazole-2-thiols.
C (%)
H (%)
N (%)
S (%)
S. no.
Molecular formula
FW
Cal
Obs
Cal
Obs
Cal
Obs
Cal
Obs
i.
C3H4N2S2
C9H16N2S2
C11H20N2S2
C13H24N2S2
C8H6N2S2
132
216
244
272
194
228.5
263
208
208
208
224
254
210
240
250
27.25
49.96
54.05
57.30
46.46
42.01
36.51
51.89
51.89
51.89
48.19
47.23
45.69
44.98
57.56
28.2
47.73
52.5
3.04
7.45
8.25
8.88
3.11
2.20
1.53
3.87
3.87
3.87
3.59
3.96
2.88
3.36
5.64
2.99
7.23
8.99
7.59
2.97
2.09
1.60
3.19
3.64
3.61
3.32
3.41
3.19
2.98
5.31
21.19
12.95
11.46
10.28
14.42
12.25
10.64
13.45
13.45
13.45
12.49
11.01
13.32
11.66
11.19
20.83
13.32
11.21
9.73
13.63
12.85
9.83
14.05
12.86
12.32
12.93
12.51
14.26
12.59
10.70
48.51
29.64
26.24
23.54
33.01
28.04
24.37
30.79
30.79
30.79
28.59
25.22
30.50
26.69
25.61
47.21
30.12.
27.16
22.24
31.42
26.52
23.56
29.23
31.03
31.63
26.35
24.89
29.12
25.36
25.13
ii.
iii.
iv.
v.
59.29
44.70
40.26
34.76
50.36
49.12
49.64
46.21
45.06
44.26
42.91
55.63
vi.
vii.
viii.
ix.
x.
C8H5N2S2Cl
C8H4N2S2Cl2
C9H8N2S2
C9H8N2S2
C9H8N2S2
xi.
xii.
xiii.
xiv.
xv.
C9H8ON2S2
C10H10O2N2S2
C8H6ON2S2
C9H8O2N2S2
C12H14N2S2
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

842
A. Saha, R. Kumar, R. Kumar, and C. Devakumar
Vol 47
Table 3
Comparative assessment of protocols 1 and 2 using green chemistry measures.
Matrix
Protocol 1 (Conventional)
Protocol 2 (Greener)
Improvement
Overall yield (%)
Number of steps
Heating time
Energy consumption (KWh)
E(nvironmental) factor (Kg waste/Kg product)
Atom economy (%)
Atom efficiency (%)
Carbon efficiency (%)
Reaction mass efficiency (%)
59.6
Four
6–9 h
6–9
69.3
Three
60–200 s
0.015–0.050
14.8
9.7% increase
25% reduction
162–360 times decrease
180–400 times reduction
26.4% reduction
5% increase
7.7% increase
6.2% increase
4.6% increase
20.1
43.3
25.8
28.5
31.5
48.3
33.5
34.7
36.1
EXPERIMENTAL
compounds were statistically at per with each other (Ta-
ble 4). All the compounds showed an increase in NI
with the increase in dose. Enhancement in NI is more
from 1 to 5% increases in dose as compared with 5 to
10%.
Thin layer chromatography was performed on 200-lm thick
aluminum sheets having silica gel 60 F254 as adsorbent. Differ-
ent solvent systems were used for developing the TLC plates.
Spots were visualized either by UV-light or iodine vapors.
Melting points were determined by using electro thermal melt-
ing point apparatus and are uncorrected. Microwave irradiation
was carried out by using Samsung microwave oven model
CE118KF operating at 2450 MHz. A Christ Lypholizer model
Alpha 1-2 LD was used for lyophilizing purpose. Nuclear
Structure-activity relationship. In general, aryl sub-
stituted thiadiazoles performed better than the alkyl sub-
stituted 1,3,4-thiadiazole-2-thiols at all the doses. The 5-
substitution in the 1,3,4-thiadiazole with aliphatic chains
of 1, 7, 9, and 11 carbon atoms were used. The chain al-
iphatic seven of carbon atoms performed the best as evi-
dent from their performance on 28th day of incubation.
The overall effect in minimizing NI was significantly
higher with seven carbon atoms with 69.8 and 63.1% as
compared with 58.1–64.4% and 52.9–56.2% with others
at 10 and 5% doses, respectively (Fig. 1). At 1% dose,
maximum NI, 36.2%, was observed with seven carbon
atoms, but it was superior to NI with one carbon atom,
25.7% only and performed at par with others.
magnetic resonance (NMR) spectra were recorded on
a
Brucker Abamce, 400 MHz instrument, after dissolving the
samples in CDCl3/DMSO(d6) using tetramethylsilane (TMS)
as an internal standard. Chemical shifts were reported in d val-
ues relative to TMS and the notations used are s-singlet, d-
doublet, t-triplet, m-multiplet, and brs-broad singlet. Infrared
(IR) spectra were recorded on a Nicolet Fourier Transform
Infrared Spectrophotometer, Model Impact 400 (FTIR) in ei-
ther nujol mull or KBr disc. A Varian, Series 634, UV–Vis
double beam spectrophotometer was used. Elemental analysis
was carried out using EuroVector CHNS analyzer. All the
chemicals and reagents were purchased from S D Fine Chemi-
cals, Qualigens Fine Chemicals, and Merck India.
Synthesis of test chemicals. 5-Substituted-1,3,4-thiadiazole-
2-thiols were synthesized following the Scheme 1 using the
both conventional (protocol 1) and greener synthesis (protocol
2). Protocol 2 used the new greener method [18] for synthesiz-
ing the intermediate hydrazides which otherwise required 6–9
h heating under protocol 1 [13].
Conventional method of synthesis of carboxylic acid hydra-
zides (Method-A). The organic acids were esterified in the
presence of ethyl alcohol and catalytic amount of sulfuric acid.
The resulted esters were hydrazinolysed with hydrazine
hydrate to afford the hydrazides [13].
Greener method for synthesis of carboxylic acid hydrazides
(Method B). Carboxylic acid (0.01 mol) and hydrazine hydrate
(0.012 mol) were irradiated under microwaves for 60–200 s at
900 W. Then, the reaction mixture was cooled to ꢀ20ꢂC and
lyophilized at ꢀ50ꢂC. The product obtained was recrystallized
from methyl alcohol. The hydrazides were characterized on
the basis of physical and spectral data [13–17].
5-Aryl substitution in the 1,3,4-thiadiazole ring with
chloro atoms in phenyl ring was used. Introduction of
chlorine atoms in the phenyl ring resulted in the
increase of activity and found to be significantly supe-
rior to phenyl substitution with no chlorine atom (Fig.
2). Their effect on NI was similar for both mono and
dichloro phenyl derivatives (Fig. 2), which were supe-
rior to phenyl derivative. Both chloro derivative showed
36.2–41.4%, 63.8–65.4%, and 73.6–75.1% NI at 1, 5,
and 10% dose, respectively. The phenyl derivative
showed 24.6%, 43.6%, and 60.8% NI at respective 1, 5
and 10% doses.
5-(3-Methyl phenyl)-1,3,4-thiadiazole-2-thiol, 5-(4-
chloro phenyl)-1,3,4-thiadiazole-2-thiol and 5-(2,4-
dichlorophenyl)-1,3,4-thiadiazole-2-thiol emerged as
potent nitrification inhibitors. The study revealed the
cost effective and environment friendly synthesis of the
potent 1,3,4-thiadizole-2-thiols. These compounds hold
promise to be used as nitrification inhibitors and also as
prototypes for the discovery of potent analogues through
the process of compound library design and screening.
Preparation
of
5-substituted-1,3,4-thiadiazole-2-
thiols. Potassium hydroxide (0.11 mol) was dissolved in mini-
mum amount of ethanol, and hydrazide (0.1 mol) was added
to it. The reaction mixture was cooled to 0–5ꢂC followed by
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

July 2010
Green Synthesis of 5-Substituted-1,3,4-thiadiazole-2-thiols as New Potent
Nitrification Inhibitors
843
Table 4
Effect of 5-substituted-1,3,4-thiadiazole-2-thiols on nitrification inhibition (NI).
Nitrification inhibition (%)
Comp.
i.
R
Dose
7th Day
14th Day
21st Day
28th Day
CH3A
1
5
54.7
69.3
76.8
59.1
70.1
76.1
52.7
65.3
73.9
49.5
69.8
75.8
58.0
61.9
65.5
66.3
74.1
79.7
61.3
72.5
78.4
65.8
64.6
72.4
63.4
72.9
78.2
68.5
73.7
79.4
53.8
62.6
68.0
58.9
70.6
77.3
58.3
72.3
73.3
65.3
68.8
76.9
58.1
73.8
77.3
60.9
65.9
75.3
4.7
36.8
66.9
74.1
53.9
67.9
76.0
46.3
61.1
69.4
40.6
64.7
73.0
44.3
58.4
65.0
62.9
73.5
78.8
54.1
71.5
76.6
61.2
64.4
71.2
60.1
72.3
77.1
62.2
72.3
77.7
51.7
64.2
69.4
52.9
68.7
75.1
51.6
69.9
71.4
57.0
67.7
75.3
51.6
71.5
76.6
57.4
65.5
75.7
5.0
44.1
63.9
75.4
51.2
68.6
77.0
46.9
59.0
68.0
42.4
57.8
74.7
48.0
56.3
67.6
54.4
72.3
77.7
53.5
72.1
76.7
54.6
65.3
72.4
52.7
70.7
77.3
55.1
68.7
76.0
48.8
63.0
71.6
51.3
68.1
75.7
49.4
67.0
71.2
51.5
61.3
72.4
44.8
69.8
78.5
54.6
65.3
77.2
4.6
25.7
56.1
64.4
36.2
63.1
69.8
32.8
52.9
63.0
32.8
56.2
58.1
24.6
43.6
60.8
41.4
65.4
75.1
36.2
63.8
73.6
37.9
59.0
70.4
39.1
63.7
76.8
33.2
61.7
67.1
29.7
52.8
67.0
34.4
63.2
71.9
32.7
59.2
70.2
38.3
62.5
70.0
32.9
60.1
68.4
39.0
59.2
73.7
5.0
10
1
ii.
CH3(CH2)5CH2A
CH3(CH2)7CH2A
CH3(CH2)9CH2A
C6H5A
5
10
1
iii.
5
10
1
iv.
5
10
1
5
v.
10
1
vi.
4-ClC6H4A
5
10
1
vii.
viii.
ix.
2,4-(Cl)2C6H3A
2-CH3C6H4A
3-CH3C6H4A
4-CH3C6H4A
4-CH3C6H4A
3,4-(CH3O)2C6H3A
2-HOC6H4A
5
10
1
5
10
1
5
10
1
x.
5
10
1
xi.
5
10
1
5
xii.
xiii.
xiv.
xv.
10
1
5
10
1
4-HO, 3-CH3OC6H3A
4-t-C4H9C6H4A
–
5
10
1
5
10
1
ATC
LSD (5%)
5
10
–
drop wise addition of carbon disulphide (0.11 mol). After addi-
tion, the reaction mixture was stirred for 30 min to afford solid
potassium dithiocarbazate salt. It was filtered, washed with
chilled acetone, dried, and used as such for further reaction.
Potassium dithiocarbazate salt (0.1 mol) was added slowly in
small lots to conc sulfuric acid (2.5 times of salt) at 5ꢂC with
constant stirring. The reaction mixture was stirred for 30 min,
and the resulting viscous liquid was poured over crushed ice
slowly. The solid obtained was filtered and washed with excess
of water till the filtrate become neutral to litmus paper. The
wet solid was suspended in water at 40ꢂC and 25% sodium hy-
droxide solution was added slowly with stirring to adjust the
pH of the solution in the range of 8.0–9.0. The resulting solu-
tion was filtered through a charcoal bed, and pH was adjusted
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

844
A. Saha, R. Kumar, R. Kumar, and C. Devakumar
Vol 47
mixed thoroughly, and distilled water was added to each
beaker for maintaining the moisture at 50% of water holding
capacity [25] of the soil. The experiment was conducted in
triplicate with concomitant controls. For ATC, the soil was
prepared in similar way. All the beakers were accurately
weighed, labeled, and incubated in a BOD incubator at 28 6
1ꢂC, and 98% relative humidity. Soil moisture was maintained
by adding distilled water every alternate day (if required) after
taking the difference of weight. Samples (5 g) were drawn on
7th, 14th, 21st, and 28th day of incubation. Before sampling,
distilled water was added to make up for the loss in weight
because of evaporation of water and mixed thoroughly. Am-
monium, nitrite, and nitrate-N were extracted in 50 mL 2M
aqueous sodium sulfate solution. The soil with extracting solu-
tion was shaken for an hour on a reciprocal shaker and filtered.
Ammonium, nitrite, and nitrate-N were estimated following
Indophenol blue, sulfanilic acid, and phenol disulfonic acid
method, respectively [26,27].
Figure 1. Effect of number of aliphatic C-atoms at 5-position of 1,3,4-
thiadiazole-2-thiol on nitrification inhibition.
to 3.0–5.0 by drop wise addition of 50% hydrochloric acid.
White solid obtained was filtered, washed with water, and
dried. All the compounds were characterized on the basis of
physical and spectral data as depicted in Tables 1 and 2.
Assessment by green chemistry matrices. The developed
synthetic protocol for 5-substituted-1,3,4-thiadiazole-2-thiols
was assessed by following green chemistry matrices, which
were calculated as reported [19–21].
The contents of ammonium nitrate and nitrite-N were
obtained from the standard curves and expressed in mg kgꢀ1
.
The nitrification rate for a constant period of incubation was
calculated using Sahrawat’s [28] formula. The data were statis-
tically analyzed following the procedure laid out by Gomez
and Gomez [29]. The analysis of variance was computed using
Statistical Package for Social Services (SPSS version 10.0),
and treatment means were compared by LSD at 5% levels.
Nitrification inhibitory activity. The soil with following
properties was collected from the farm of the institute for
in vitro incubation experiments. Sand 60.8%, silt 18.7%, clay
20.5%, water holding capacity 35.5%, bulk density 1.51 mg/
kg, organic C 0.5%, available N 553.72%, ammonium-N 3.2
mg/kg, nitrite-N traces, nitrate-N 8.54 mg/kg, pH (soil:
REFERENCES AND NOTES
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845
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