Pyridine and nitro-phenyl linked 1,3,4-thiadiazoles as MDR-TB inhibitors

Article abstract of DOI:10.1016/j.ejmech.2019.01.073

In the present study, a series of substituted 1,3,4-thiadiazole derivatives 4(a–o), 5(a–m) and 6(a–j) were synthesized and characterized by IR, ¹H NMR, ¹³C NMR and mass spectroscopic technique. The synthesized compounds were evaluate

Full text of DOI:10.1016/j.ejmech.2019.01.073

Accepted Manuscript
Pyridine and nitro-phenyl linked 1,3,4-thiadiazoles as MDR-TB inhibitors
Harun Patel, Harsha Jadhav, Iqrar Ansari, Rahul Pawara, Sanjay Surana
PII:
S0223-5234(19)30093-5
DOI:
https://doi.org/10.1016/j.ejmech.2019.01.073
EJMECH 11080
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 14 May 2018
Revised Date: 26 January 2019
Accepted Date: 28 January 2019
Please cite this article as: H. Patel, H. Jadhav, I. Ansari, R. Pawara, S. Surana, Pyridine and nitro-phenyl
linked 1,3,4-thiadiazoles as MDR-TB inhibitors, European Journal of Medicinal Chemistry (2019), doi:
https://doi.org/10.1016/j.ejmech.2019.01.073.
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ACCEPTED MANUSCRIPT
Graphical Abstract
Pyridine and Nitro-phenyl linked 1,3,4-thiadiazoles as MDR-TB Inhibitors
Harun Patel, Harsha Jadhav Iqrar Ahmad Rahul Pawara, Sanjay Surana
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Pyridine and Nitro-phenyl linked 1,3,4-
thiadiazoles as MDR-TB Inhibitors
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Harun Patel*, Harsha Jadhav**, Iqrar Ansari, Rahul Pawara, Sanjay Surana
Department of Pharmaceutical Chemistry, R. C. Patel Institute of Pharmaceutical Education and
Research, Shirpur
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Corresponding Author*
* Harun Patel; Email: hpatel_38@yahoo.com; Tel: +91-8806621544; Fax: + 91-2563251808
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Abstract
In the present study, a series of substituted 1,3,4-thiadiazole derivatives 4(a–o), 5(a-m) and
6(a-j) were synthesized and characterized by IR, 1H NMR, 13C NMR and mass spectroscopic
technique. The synthesized compounds were evaluated for their in vitro anti-mycobacterial
activity against the Mycobacterium tuberculosis H37Rv and resistance MDR-TB strain.
Among the compounds tested N-(5-(4-nitrophenyl)-1,3,4-thiadiazol-2-yl)furan-2-
carboxamide (4h) showed significant inhibitory activity with MIC of 9.87 µM (H37Rv
strain) and 9.87 µM (MDR-TB strain) compared to isoniazide [MIC of 3.64 µM (H37Rv) and
> 200 µM (MDR-TB strain)] and rifampin [MIC of 0.152 µM (H37Rv) and 128 µM (MDR-
TB strain)]. In addition, these compounds have also been assessed for their cyto-toxicity to a
mammalian Vero cell line using the MTT assay. The result shows that these compounds
exhibit anti-tubercular activity at non-cytototoxic concentrations.
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Keywords: Synthesis;1,3,4-thiadiazole; MDR-TB; Cyto-toxicity
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1. Introduction
Multi-drug-resistant tuberculosis (MDR-TB) is a tuberculosis infection caused by bacteria
that is resistant to the most capable first-line anti-TB drugs, i.e. isoniazide and rifampin [1].
MDR-TB has been an issue of growing significance in the most recent decade. Regardless of
its global significance, the problem has not been addressed effectively [2-5]. According to
the WHO, about 33 percent of the 40 million people with HIV/AIDS are co-infected with
Mycobacterium tuberculosis [6]. About 80 percent of TB cases are found in 23 countries; the
highest occurrence rates are found in Africa and South-East Asia [7,8]. After the presentation
of RIF in 1967, bedaquiline is the merely novel chemical compound developed for the
treatment of TB that has reached the market, yet confined to the treatment of MDR and XDR-
TB [9-11]. Shockingly, after its presentation in the clinical practice, the number of abnormal
deaths has been accounted due to cardiac arrhythmia [12-13]. A nitroimidazole derivative
delamanid: the mycolic acid biosynthesis inhibitor is another novel drug approved for MDR-
TB by the European Medicines Agency (EMA) in 2014 [14]. When delamanid was used in
combination with INH or fluoroquinolones, serious side effects such as cardiac arrhythmia
and CNS toxicity were shown [15]. These findings have settled down the preliminary
excitement as a result of the arrival of this novel anti-TB agent in therapy. Similarly,
mutations inside Mtb genome causing resistance to bedaquiline and delamanide were recently
reported [16]. Hence efforts are still needed to develop new anti-TB therapeutic alternatives,
which are safe and effective for drug-resistant Mtb. The 1,3,4-thiadiazole and their
derivatives are extensively recognized for their antimicrobial profile because of the
occurrence of toxophoric N-C-S moiety, which reveals a wide range of biological activities
[17-23]. Currently, there are numerous reports referring thiadiazoles as capacity anti-
tubercular agent [24-31]. Roused by the references and in continuation of our research, we
herein converse the synthesis and MDR TB-inhibitory report of 1,3,4-thiadiazoles.
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According to the updated guidelines of the world health organization, the medications
effective against MDR-TB are confined because only a limited selection of drugs are
available; therefore, the development of novel or re-purposed drugs with activity against
MDR is strongly desired [32]. The emergence of multidrug-resistant tuberculosis coupled
with the increasing overlap of the AIDS and tuberculosis pandemics has brought tuberculosis
to the forefront as a major global health concern. Number of 1,3,4-thiadiazoles have been
reported for the anti-mycobacterial activity [18-25]. Mahajan et al., identified and reported a
substituted pyridinyl-thiadiazole (I) to have MDR inhibitory activity of 0.06 µM [33].
Foroumadi et al., synthesized 2-(5-nitro-2-furyl)-and 2-(1-methyl-5-nitro-1H-imidazol-2-yl)-
1,3,4-thiadiazole derivatives and screened for their in vitro anti-mycobacterial activity
against Mycobacterium tuberculosis H37Rv using alamar-blue susceptibility test [34].
Compounds with 5-nitro-2-furyl (II) have shown the highest activity against M. tuberculosis
(MIC =3.13 µg/mL). Among nitrofuran derivatives, substitution of the thioester group at C-5
of 1,3,4-thiadiazole ring with a benzyl analog (III) was found to be the most active (MIC =
0.78 µg/mL) [35]. Among nitrofuran derivatives, thioester substitution at C-5 of 1,3,4-
thiadiazole ring with benzyl analog (III) was found to be the most active (MIC=0,78 µg /
mL).We deduce the common pharmacophore after studying these three potent compounds (I,
II and III). The pharmacophoric features indicate that 1,3,4-thiadiazole should be linked with
aromatic ring via a linker (in case of Compound I; its amidal linker). Other aromatic ring
should be directly attached to the 5th position of 1,3,4-thiadiazole. Presence of nitro group on
either of aromatic ring is favourable for the anti-mycobacterial activity. Based upon this
assumption, we designed our compounds as shown in Fig.1, where we incorporated all the
pharmacophoric features.
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Fig. 1 Pharmacophoric features of the reported compounds and structural correlation between the reported and proposed compounds
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2. Result and Discussion
The desired compounds 4(a-o), 5(a-m) and 6(a–j) were synthesized in significant yield as given in
Scheme 1-3. 5-substituted-1,3,4-thiadiazol-2-amine 3(a-c) were synthesized according to the
procedure specified in our earlier research work by the Oxidative cyclization mechanism [17].
These substituted 5-substituted-1,3,4-thiadiazol-2-amines 3(a-c) were further reacted with different
aryl and aliphatic carbonyl chlorides in THF with prolong stirring for 3 hrs at 0°C to get the preferred
compounds by the Schotten-Baumann reaction mechanism.
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A series of nitro-phenyl and pyridine clubbed 1,3,4-thiadiazole derivatives 4(a–o), 5(a-m) and 6(a-
j) have been synthesized in better yields using the synthetic route outlined in Scheme 1-3.
Substituted 1,3,4-thiadiazol-2-amine (3) was prepared by reacting aromatic carboxylic acid (1) with
thiosemicarbazide (2) in the presence of excess phosphorus oxychloride (POCl3) by oxidative
cyclization. Unreacted phosphorus oxychloride was eliminated by neutralizing with saturated NaOH
solution. Substituted 1,3,4-thiadiazol-2-amine (3) was further reacted with different aryl and
aliphatic carbonyl chlorides in tetrahydrofuran (THF) to obtain the preferred compounds 4(a–o),
5(a-m) and 6(a-j). The structures of the synthesized final compounds were confirmed based on IR,
1H NMR, 13C NMR and mass spectroscopy data.
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The IR spectrum of title compounds was confirmed by a single lobed absorption band at ~3400 cm-1
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due to secondary amine (-NH-), which was further substantiated by the H-NMR singlet at
approximately δ 12.00 ppm. In 13C NMR, the amidal carbonyl peak was observed at~170 ppm and it
was further validated by the IR spectrum peak of the carbonyl group at ~1700 cm-1, indicating the
substitution of different aromatic/aliphatic carbonyl chloride on title compound.
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Scheme 1. Synthesis of compounds 4(a-o), 5(a-m) and 6(a-j)
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Compound Code
R1
R2
Yield
Compound Code
R1
R2
Yield
64%
42%
52%
72%
4k
4l
4a
4b
80%
81%
78%
65%
4c
4m
4n
4d
81%
81%
75%
50%
50%
36%
60%
64%
50%
78%
70%
87%
4e
4f
4o
5a
5b
5c
5d
5e
4g
4h
4i
4j
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Compound Code
R1
R2
Yield
Compound Code
R1
R2
Yield
75%
50%
76%
81%
62%
69%
65%
75%
55%
64%
75%
51%
54%
74%
5f
5g
5h
5i
6b
6c
6d
6e
6f
5j
5k
5l
6g
6h
75%
75%
68%
76%
5m
6a
6i
6j
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In this study a new series of pyridine and nitro phenyl linked 1,3,4-thiadiazole derivatives 4(a-
o), 5(a-m) and 6(a–j) were synthesized and evaluated against M. tuberculosis and resistant
phenotypic MDR-TB strain. The result of anti-mycobacterial and MDR-TB inhibitory activity is
presented in Table 1. It is observed that the activity is significantly influenced by various
substituents at the 2nd position of 1,3,4-thiadiazole. The tested compounds 4d, 4g, 4h, 4j, 4k,
4m, 5d, 5h, 5j and 6e displayed significant MDR inhibitory activity as shown in Table 1.
Compound 4h was found to be the potent compound of the series having MIC of 9.87 µM
against the MDR-TB strain as compared to the standard isoniazid (> 200 µM).
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A concise analysis of the structure–activity reveals that the anti-mycobacterial activity is
significantly influenced by different substituents on the thiadiazole ring. Electron withdrawing
group on aliphatic side chain at 2nd position of 1,3,4-thiadiazole has diminishing effect on anti-
mycobacterial and MDR inhibitory activity (4i, 5e, 5f, 5i, 6g, 6h, 6i) compared to the
unsubstituted aliphatic side chain (4c, 4d, 4g, 5d, 6d). In case of aromatic substitution on 1,3,4-
thiadiazole; nitro substitution favors activity followed by the methoxy group at para position (4j,
4k, 5j, 5k, 6e). In addition, we have seen that the heterocyclic furan ring has a significant
contribution to anti-mycobacterial activity compared to the other compounds (4h, 5h). The
synthesized potent compounds of the series were further screened for cyto-toxicity (IC50) in a
mammalian Vero cell line (Table 1). All the tested derivatives showed lower toxicity with IC50
values >250 µM and none of the synthesized compounds displayed significant activity against
the mammalian Vero cell line at concentrations <100 mM.
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These outcomes are vital, as compounds with increased cytoliability are appealing in the
development of new chemical entities for the treatment of tuberculosis. This is mainly because
the management of tuberculosis requires extensive course of drug treatment, leading to a number
of side-effects with a high margin of safety.
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Table 1. Anti-mycobacterial and MDR inhibitory profile of synthesized compound
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Compound
Code
H37rv MIC
(µM)
MDR-TB
MIC (µM)
Cyto-toxicity
IC50 (µM)
Compound
Code
H37rv MIC
(µM)
MDR-TB
MIC (µM)
Cyto-toxicity
IC50 (µM)
R
LogP
R
LogP
168 ± 6.24
204 ± 5.23
89 ± 3.23
40 ± 3.45
> 200
> 200
---
2.32
16.84 ± 2.67
35 ± 2.98
> 200
67 ± 3.12
140 ± 4.12
> 200
290 ± 6.34
278 ± 5.74
---
1.76
4a
4b
4c
4d
4j
4k
4l
---
---
3.41
2.59
3.65
2.22
3.32
3.87
O
> 200
S
163 ± 6.34
278 ± 6.64
18.38 ± 3.78
147 ± 4.76
310 ± 6.24
4m
153 ± 3.86
> 200
---
2.86
117 ± 4.34
> 200
---
4.46
4e
4n
135 ± 3.75
42 ± 3.45
271 ± 5.23
171 ± 4.23
9.87 ± 1.12
---
3.75
3.15
2.68
38 ± 3.86
21 ± 3.65
204 ± 6.23
306 ± 7.12
> 200
---
---
---
3.42
2.29
3.39
4f
4g
4h
4o
5a
5b
301 ± 5.89
306 ± 7.21
9.87 ± 1.56
> 200
273 ± 5.34
> 200
---
3.56
89 ± 4.12
> 200
---
2.56
4i
5c
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Table 1. Continue….
Compound
Code
H37rv MIC
(µM)
MDR-TB
MIC (µM)
Cyto-toxicity
IC50 (µM)
Compound
Code
H37rv MIC
(µM)
MDR-TB
MIC (µM)
Cyto-toxicity
IC50 (µM)
R
LogP
R
LogP
40 ± 2.86
163 ± 3.23
> 200
289 ± 5.34
3.65
196 ± 4.12
> 200
> 200
---
1.07
5d
6a
153 ± 4.32
---
2.82
238 ± 3.15
---
2.17
5e
6b
O
135 ± 3.68
42 ± 4.76
> 200
> 200
---
---
3.72
3.12
21 ± 1.76
95 ± 2.10
> 200
---
---
1.43
2.40
5f
6c
S
381 ± 5.21
5g
6d
19 ± 2.87
> 200
39 ± 2.43
> 200
312 ± 6.23
---
2.66
2.92
3.36
9.93 ± 1.12
> 200
76 ± 2.10
> 200
369 ± 6.23
2.13
1.70
2.50
5h
5i
6e
6f
---
---
33 ± 2.87
134 ± 2.86
268 ± 7.23
192 ± 3.78
> 200
5j
6g
35 ± 2.98
301 ± 6.87
38 ± 2.76
280 ± 4.75
> 200
---
---
---
3.46
3.30
3.40
221 ± 3.86
> 200
> 200
> 200
---
---
1.61
2.31
5k
5l
6h
6i
O
S
> 200
201 ± 4.65
---
> 200
---
---
1.90
---
5m
6j
---
---
---
---
---
-0.7
2.7
---
0.028 ± 0.008
Isoniazid
Rifampin
Staurosporine
3.64 ± 0.68
0.152 ± 0.09
---
> 200
128 ± 2.86
---
0.028 ± 0.008
Staurosporine
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3
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The inhibitory activity (MIC50) was determined against M. tuberculosis H37Rv and clinically isolated phenotype MDR-TB strain. Assay were carried out at least three times n = 3
LogP value is calculated using ChemBioDraw 14
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3. Conclusion
In the current research paper, we detailed synthesis and anti-mycobacterial activity of a new
series of sustituted-1,3,4-thiadiazoles. The in vitro results of the anti-mycobacterial activities
against resistant MDR strain suggest that the presence of furoyl substitution on 1,3,4-thiadiazole
N-(5-(4-nitrophenyl)-1,3,4-thiadiazol-2-yl)furan-2-carboxamide (4h) has demonstrated the
significant inhibitory activity with MIC of 9.87 µM against MTB-H37Rv and MDR-TB
respectively as compared to the isoniazid and rifampin. Structure–activity reveals that the
electron withdrawing group on aliphatic side chain at 2nd position of 1,3,4-thiadiazole has
diminishing effect on the anti-mycobacterial and MDR inhibitory activity (4i, 5e, 5f, 5i, 6g, 6h,
6i) as compared to the unsubstituted aliphatic side chain (4c, 4d, 4g, 5d, 6d). In case of aromatic
substitution on 1,3,4-thiadiazole; nitro substitution favors the activity followed by the methoxy
groups at para position (4j, 4k, 5j, 5k, 6e). We additionally have seen that among the
heterocyclic substitution furan ring has the significant contribution on anti-mycobacterial
activity as compared to the others (4h, 5h). The synthesized compounds, which demonstrated
significant anti-mycobacterial activity, additionally evaluated for their cyto-toxicity (IC50)
against the mammalian Vero cell line, utilizing the MTT assay. The outcomes demonstrated that
these compounds confirmed anti-mycobacterial activity at non-cytotoxic level. In conclusion,
compound 4h was found to be the most potent compound of the series and could be exploited
further for developing safe antimicrobial agents by slight modifications at second position of
1,3,4-thiadiazole ring and/or extensive additional functionalization that necessitate further
investigations.
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4. Experimental
All the solvents and chemicals have been provided by Spectrochem and Sigma-Aldrich. The
reactions were supervised with the aid of pre-coated silica gel TLC aluminium sheets. Melting
points were established using an Analab Scientific Melting point apparatus. FTIR spectrum was
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documented utilizing FTIR-8400S Shimadzu spectrometer. HNMR (DMSO/CDCl3) spectra of
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the compounds have been determined by Bruker Avance-II spectrometer at 400 MHz (Punjab
University-Chandigarh). Chemical shift were assessed relative to the internal standard TMS and
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are reported in δ ppm. Mass spectra of the compounds were determined at Oxygen Heath Care
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Pvt.Ltd.at Ahmadabad, Gujarat.
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4.1. Preparation of 5-(4-nitrophenyl)-1,3,4-thiadiazol-2-amine (3a)
This compound is prepared as per the method given by Patel et al [17].
4.2. General procedure for the synthesis of (5-(4-nitrophenyl)-1,3,4-thiadiazol-2-yl)substituted
carbonyl chloride 4(a-o)
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An equimolar amount of 5-(4-nitrophenyl)-1,3,4-thiadiazol-2-amine (3a) dissolve in 10-15 mL of
tetrahydrofuran (THF), add 2-3 drops of triethylamine, to this solution add 1.5 mole different aryl
chlorides gradually, continuous stirring for 3 hrs on ice bath (0°C). After completion of reaction
solid will precipitate out, washed with sodium bicarbonate solution to remove excess of chloride and
recrystallized from ethanol to get pure compound.
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4.2.1. 2-Chloro-N-(5-(4-nitrophenyl)-1,3,4-thiadiazol-2-yl)acetamide (4a)
64% yield; mp 184-186°C; IR (KBr) ѵmax: 3283 (N-H stretch), 3045 (Arom.CH strech), 2956
(Alip.CH strech), 1698 (amide C=O) cm-1; 1H NMR (DMSO-d6) 12.51(s, 1H, -NH), 7.79 (d, 2H,
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J = 8.8 Hz, Ar-H), 8.27 (d, 2H, J = 8.8 Hz, Ar-H), 4.29 (s, 2H, CH2); C NMR (DMSO-d6)
δ
170.9, 169.2, 153.1, 148.5, 136.0, 124.1, 125.8, 45.7; HRMS (ESI) m/z calcd. C10H7ClN4O3S:
297.9927; found: 297.9923 [Mass Fragments: 223, 297].
4.2.2. N-(5-(4-nitrophenyl)-1,3,4-thiadiazol-2-yl)pivalamide (4b)
42% yield; mp 156-158 °C; IR (KBr) ѵmax: 3296 (N-H stretch), 2924 (Arom.CH strech), 2856
(Alip.CH strech), 1658 (amide C=O) cm-1; 1H NMR (DMSO-d6) 12.58(s, 1H, -NH), 7.69 (d, 2H,
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J = 8.8 Hz, Ar-H), 8.36 (d, 2H, J = 8.8 Hz, Ar-H), 1.29 (s, 9H, CH3); C NMR (DMSO-d6)
δ
177.3, 170.2, 154.2, 148.4, 135.9, 128.7, 125.1, 45.7, 26.9; HRMS (EI) m/z calcd.
C13H14N4O3S: 306.0787; found: 329.0660 [M+Na+] [Mass Fragments: 223, 329].
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4.2.3. N-(5-(4-nitrophenyl)-1,3,4-thiadiazol-2-yl)propionamide (4c)
80% yield; mp 219-221°C; IR (KBr) ѵmax: 3352 (N-H stretch), 3008 (Arom.CH strech), 2956
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(Alip.CH strech), 1698 (amide C=O) cm-1; 1H NMR (DMSO-d6) 12.55(s, 1H, -NH), 7.76 (d, 2H,
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J = 8.8 Hz, Ar-H), 8.21 (d, 2H, J = 8.8 Hz, Ar-H), 2.29 (q, 2H, J = 7.4, CH2), 1.45 (t, 3H, J =
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7.4 CH3); C NMR (DMSO-d6)
δ
169.8, 178.4, 154.2, 149.7, 137.9, 128.9, 123.2, 31.7, 10.0;
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HRMS (ESI) m/z calcd. C11H10N4O3S: 278.0474; found: 278.0479 [Mass Fragments: 223,
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278].
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4.2.4. N-(5-(4-nitrophenyl)-1,3,4-thiadiazol-2-yl)pentanamide (4d)
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81% yield; mp 172-174°C; IR (KBr) ѵmax: 3292 (N-H stretch), 3139 (Arom.CH strech), 2946
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(Alip.CH strech), 1664 (amide C=O) cm-1; 1H NMR (DMSO-d6) 12.40(s, 1H, -NH), 7.70 (d, 2H,
J = 8.8 Hz, Ar-H), 8.39 (d, 2H, J = 8.8 Hz, Ar-H), 2.29 (m, 2H, CH2), 1.38-1.55 (m, 4H, CH2
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Aliphatic chain), 1.05 (t, 3H, CH3); C NMR (DMSO-d6)
δ 171.7, 170.03, 153.1, 147.8, 140.2,
137.9,130.2, 38.3, 31.7, 21.0, 14.0; HRMS (ESI) m/z calcd. C13H14N4O3S: 306.0787; found:
306.0795 (86.0732, 223.0133, 306.0795) [Mass Fragments: 223, 306].
4.2.5. 4-Chloro-N-(5-(4-nitrophenyl)-1,3,4-thiadiazol-2-yl)butanamide (4e)
81% yield; mp 164-166°C; IR (KBr) ѵmax : 3251 (N-H stretch), 3125 (Arom.CH strech), 2986
(Alip.CH strech), 1674 (amide C=O) cm-1; 1H NMR (DMSO-d6) 12.40(s, 1H, -NH), 7.56 (d, 2H,
J = 8.8 Hz, Ar-H), 8.40 (d, 2H, J = 8.8 Hz, Ar-H), 3.50 (t, 2H, J = 7.4 Hz, CH2), 2.38 (t, 2H, J =
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7.4 Hz, CH2) 1.55 (m, 2H, CH2 Aliphatic chain); C NMR (DMSO-d6)
δ 174.0, 173.0, 153.1,
147.8, 137.9, 128.9, 124.7, 47.1, 33.7, 26.0; HRMS (ESI) m/z calcd. C12H11ClN4O3S: 326.0240;
found: 326.0248 [Mass Fragments: 223, 326].
4.2.6. 6-((5-(4-Nitrophenyl)-1,3,4-thiadiazol-2-yl)amino)-6-oxohexanoyl chloride (4f)
75% yield; mp 159-161°C; IR (KBr) ѵmax : 3321 (N-H stretch), 3078 (Arom.CH strech), 2956
(Alip.CH strech), 1697 (amide C=O) cm-1; 1H NMR (DMSO-d6) 12.45(s, 1H, -NH), 7.90 (d, 2H,
J = 8.8 Hz, Ar-H), 8.40 (d, 2H, J = 8.8 Hz, Ar-H), 2.82 (t, 2H, J = 7.4 Hz, CH2), 2.38 (t, 2H, J =
13

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13
1
2
7.4 Hz, CH2) 1.55 (t, 2H, J = 7.4 Hz, CH2 Aliphatic chain); C NMR (DMSO-d6)
δ
172.8,
171.9, 152.7, 147.5, 137.9, 128.0, 126.2, 124.9, 46.8, 35.9, 26.9, 24.8; HRMS (ESI) m/z calcd.
C14H13ClN4O3S: 368.0346; found: 368.0353 [Mass Fragments: 223, 368].
4.2.7. N-(5-(4-nitrophenyl)-1,3,4-thiadiazol-2-yl)butyramide (4g)
3
4
5
50% yield; mp 239-241°C; IR (KBr) ѵmax : 3464 (N-H stretch), 3115 (Arom.CH strech), 2992
6
(Alip.CH strech), 1664 (amide C=O) cm-1; 1H NMR (DMSO-d6) 12.40(s, 1H, -NH), 7.70 (d, 2H,
J = 8.8 Hz, Ar-H), 8.30 (d, 2H, J = 8.8 Hz, Ar-H), 2.29 (t, 2H, J = 7.4 Hz, CH2), 1.45 (m, 2H,
7
8
CH3), 1.01 (t, 3H, J = 7.4 Hz, CH3); 13C NMR (DMSO-d6)
δ 169.8, 178.4, 154.2, 149.7, 137.9,
9
128.9, 128.5, 123.2, 31.7, 10.0; HRMS (ESI) m/z calcd. C12H12N4O3S: 292.0630; found:
292.0638 [Mass Fragments: 223, 292].
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
4.2.8. N-(5-(4-nitrophenyl)-1,3,4-thiadiazol-2-yl)furan-2-carboxamide (4h)
50% yield; mp 220-222°C; IR (KBr) ѵmax : 3153 (N-H stretch), 2920 (C-H Strech), 1701 (amide
C=O) cm-1; 1H NMR (DMSO-d6) 12.92 (s, 1H, NH), 6.74 (1H, t, J = 7.5 Hz, Furoyl), 7.37 (1H,
d, J = 7.5, 0.9 Hz, Furoyl), 8.03 (1H, d, J = 7.8, 0.9 Hz, Furoyl), 8.23 (d, 2H, J = 8.8 Hz, Ar-H),
8.24 (d, 2H, J = 8.8 Hz, Ar-H); 13C NMR (DMSO-d6)
δ 171.3, 160.1, 149.8, 148.6, 143.3, 140.1,
128.3, 124.9, 116.3, 111.3; HRMS (ESI) m/z C13H8N404S: 316.0266; found: 317.0272 [M+1]
[Mass Fragments: 223, 291, 317].
4.2.9. 2,2,2-Trichloro-N-(5-(4-nitrophenyl)-1,3,4-thiadiazol-2-yl)acetamide (4i)
36% yield; mp 151-153°C; IR (KBr) ѵmax : 3367 (N-H stretch), 3018 (Arom.CH strech), 2909
(Alip.CH strech), 1697 (amide C=O) cm-1; 1H NMR (DMSO-d6) 12.70(s, 1H, -NH), 7.84 (d, 2H,
J = 8.8 Hz, Ar-H), 8.34 (d, 2H, J = 8.8 Hz, Ar-H); 13C NMR (DMSO-d6)
δ 175.4, 160.2, 154.29,
148.4, 135.9, 128.0, 125.9, 94.2; HRMS (EI) m/z calcd. C10H5Cl3N4O3S: 365.9148; found:
365.9154 [Mass Fragments: 223, 365]
4.2.10. 4-Nitro-N-(5-(4-nitrophenyl)-1,3,4-thiadiazol-2-yl)benzamide (4j)
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2
52% yield; mp 240-242°C; IR (KBr)
ѵ
max : 3354 (N-H stretch), 3034 (Arom.CH strech), 2911
(Alip.CH strech), 1707 (amide C=O) cm-1; H NMR (DMSO-d6) 12.50 (s, 1H, -NH), 8.24-8.39
1
3
(m, 8H, Ar-H); 13C NMR (DMSO-d6)
δ 174.5, 164.2, 152.7, 147.2, 138.5, 134.3, 132.67, 130.5,
4
129.8, 128.4, 124.3; HRMS (ESI) m/z calcd. C15H9N5O5S: 371.0324; found: 371.0330 [Mass
Fragments: 223, 371]
5
6
4.2.11. 4-Methoxy-N-(5-(4-nitrophenyl)-1,3,4-thiadiazol-2-yl)benzamide (4k)
7
65% yield; mp 224-226°C; IR (KBr)
ѵ
max : 3287 (N-H stretch), 3134 (Arom.CH strech), 2957
1
8
(Alip.CH strech), 1687 (amide C=O) cm-1; H NMR (DMSO-d6) 12.69 (s, 1H, -NH), 7.08-8.27
13
9
(m, 8H, Ar-H), 3.80 (s, 3H, OCH3); C NMR (DMSO-d6)
δ
174.0, 165.2, 164.3, 151.3, 148.9,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
134.2, 132.5, 130.8, 129.8, 124.3, 114.2, 55.6 ; HRMS (ESI) m/z calcd. C18H12N4O4S: 356.0579;
found: 356.0584 [Mass Fragments: 223, 356]
4.2.12. N-(5-(4-nitrophenyl)-1,3,4-thiadiazol-2-yl)thiophene-2-carboxamide (4l)
72% yield; mp 247-249°C; IR (KBr) ѵmax : 3291 (N-H stretch), 2971 (C-H Strech),1691 (amide
1
C=O) cm-1; H NMR (DMSO-d6) 12.64 (s, 1H, NH), 8.01-8.40 (m, 4H, Ar-CH), 7.20-7.98 (m,
3H, Ar-CH thiophene); 13C NMR (DMSO-d6)
δ 174.3, 161.0, 152.7, 148.6, 150.3, 146.7, 139.5,
138.9, 132.9, 131.2, 130.3, 128.4, 124.9; HRMS (ESI) m/z C13H8N4O3S2: 332.0038; found:
332.0038 [Mass Fragments: 223, 332]
4.2.13. 4-Methyl-N-(5-(4-nitrophenyl)-1,3,4-thiadiazol-2-yl)benzamide (4m)
78% yield; mp 169-171°C; IR (KBr) ѵmax : 3297 (N-H stretch), 3084 (Arom.CH strech), 2948
1
(Alip.CH strech), 1690 (amide C=O) cm-1; H NMR (DMSO-d6) 12.55 (s, 1H, -NH), 7.24-8.39
13
(m, 8H, Ar-H), 2.41 (s, 3H, CH3); C NMR (DMSO-d6)
δ 174.0, 165.2, 151.3, 148.9, 139.5,
131.7, 130.2, 129.7, 128.3, 129.8, 124.3, 21.5; HRMS (ESI) m/z calcd. C18H12N4O3S: 340.0630;
found: 340.0638 [Mass Fragments: 223, 340]
4.2.14. 2-(Heptan-4-yl)-N-(5-(4-nitrophenyl)-1,3,4-thiadiazol-2-yl)benzamide (4n)
15

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1
2
65% yield; mp 163-165°C; IR (KBr)
ѵ
max : 3185 (N-H stretch), 3014 (Arom.CH strech), 2950
(Alip.CH strech), 1690 (amide C=O) cm-1; H NMR (DMSO-d6) 12.73 (s, 1H, -NH), 8.24-7.11
1
13
3
(m, 8H, Ar-H), 1.54-2.58 (m, 15H, CH aliphatic branch); C NMR (DMSO-d6)
δ
174.0, 165.5,
4
152.2, 148.2, 147.0, 145.9, 138.5, 139.7, 137.7, 130.2,128.3, 129.1, 124.9, 41.8, 38.9, 20.9,
14.3; HRMS (ESI) m/z calcd. C22H24N4O3S: 424.1569; found: 424.1574 [Mass Fragments: 223,
424]
5
6
7
4.2.15. N-(5-(4-nitrophenyl)-1,3,4-thiadiazol-2-yl)benzamide (4o)
8
60% yield; mp 129-131°C; IR (KBr) ѵmax : 3227.92 (N-H stretch), 3024.01 (Arom.CH strech),
9
2961.59 (Alip.CH strech), 1695.96 (amide C=O) cm-1; 1H NMR (DMSO-d6) 12.78 (s, 1H, -NH),
13
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
7.54-8.27 (m, 8H, Ar-H); C NMR (DMSO-d6)
δ
174.1, 165.7, 152.4, 147.0, 142.3, 139.2,
132.3, 130.2, 129.6, 128.5, 129.9; HRMS (ESI) m/z calcd. C15H10N4O3S: 326.0474; found:
326.0480 [Mass Fragments: 223, 326]
4.3. Preparation of 5-(3-nitrophenyl)-1,3,4-thiadiazol-2-amine (3b)
This compound is prepared as per the method given by Patel et al [17].
4.4. General procedure for the synthesis of N-(5-(3-nitrophenyl)-1,3,4-thiadiazol-2-yl)
substituted carbonyl chloride 5(a-m)
An equimolar amount of 5-(3-nitrophenyl)-1,3,4-thiadiazol-2-amine (3b) dissolve in 10-15 mL
of tetrahydrofuran (THF), add 2-3 drops of triethylamine, to this solution add 1.5 mole different
aryl chlorides gradually, continuous stirring for 3 hrs on ice bath(0°C). After completion of
reaction solid will precipitate out, washed with sodium bicarbonate solution to remove excess of
chloride and recrystallized from ethanol to get pure compound
4.4.1. 2-Chloro-N-(5-(3-nitrophenyl)-1,3,4-thiadiazol-2-yl)acetamide (5a)
64% yield; mp 192-194°C; IR (KBr) ѵmax : 3283 (N-H stretch), 3038 (Arom.CH strech), 2956
(Alip.CH strech), 1698 (amide C=O) cm-1; 1H NMR (DMSO-d6) 12.51(s, 1H, -NH), 7.59 (t, 1H,
J = 8.3 Hz, Ar-H), 8.05 (d, 1H, J = 8.3 Hz, Ar-H), 8.19 (d, 1H, J = 8.3 Hz, Ar-H), 8.52 (s, 1H,
16

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1
2
Ar-H), 4.29 (s, 2H, CH2); C NMR (DMSO-d6) δ 170.9, 169.2, 153.1, 148.5, 136.0, 129.7,
128.5, 124.1, 125.8, 45.7; HRMS (ESI) m/z calcd. C10H7ClN4O3S: 297.9927; found: 297.9933
[Mass Fragments: 223, 297].
3
4
4.4.2. N-(5-(3-nitrophenyl)-1,3,4-thiadiazol-2-yl)pivalamide (5b)
5
50% yield; mp 162-164°C; IR (KBr) ѵmax : 3253 (N-H stretch), 3038 (Arom.CH strech), 2909
6
(Alip.CH strech), 1658 (amide C=O) cm-1; 1H NMR (DMSO-d6) 12.51(s, 1H, -NH), 7.50 (t, 1H,
7
J = 8.3 Hz, Ar-H), 8.10 (d, 1H, J = 8.3 Hz, Ar-H), 8.21 (d, 1H, J = 8.3 Hz, Ar-H), 8.42 (s, 1H,
13
8
Ar-H), 1.29 (s, 9H, CH3); C NMR (DMSO-d6)
δ
177.3, 169.2, 154.2, 148.5, 135.9, 128.8,
9
128.0, 125.9, 125.9, 39.7, 26.9; HRMS (EI) m/z calcd. C13H14N4O3S: 306.0787; found: 306.0794
[Mass Fragments: 223, 306].
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
4.4.3. N-(5-(3-Nitrophenyl)-1,3,4-thiadiazol-2-yl)propionamide (5c)
78% yield; mp 168-170°C; IR (KBr)
ѵ
max : 3275 (N-H stretch), 3022 (Arom.CH strech), 2922
1
(Ali C-H Strech),1691 (amide C=O); H NMR (DMSO-d6) 12.58 (s, 1H, NH), 8.04 (t, 1H, J =
8.3 Hz, Ar-H), 8.21 (d, 1H, J = 8.3 Hz, Ar-H), 8.24 (s, 1H, Ar-H), 8.31 (d, 1H, J = 8.3 Hz, Ar-
13
H), 3.07(q, 2H, J = 7.4 Hz,CH2), 1.29 (t, 3H, J = 7.4 Hz,CH3); C NMR (DMSO-d6)
δ 170.4,
154.5, 148.4, 132.9, 133.3, 131.6, 124.0, 121.9, 120.9, 30.5, 10.8; HRMS (EI) m/z calcd.
C11H10N4O3S: 278.0474; found 279.0480 [Mass Fragments: 223, 278].
4.4.4. N-(5-(3-Nitrophenyl)-1,3,4-thiadiazol-2-yl)pentanamide (5d)
70% yield; mp 202-204 °C; C13H14N4O3S °C; IR (KBr) ѵmax : 3290 (N-H stretch), 3045
(Arom.CH strech), 2974 (Alip.CH strech), 1694 (amide C=O) cm-1; 1H NMR (DMSO-d6) 12.40
(s, 1H, -NH), 7.60 (t, 1H, J = 8.3 Hz, Ar-H), 7.90 (d, 1H, J = 8.3 Hz, Ar-H), 8.01 (d, 1H, J = 8.3
Hz, Ar-H), 8.31 (s, 1H, Ar-H), 1.40-2.30 (m, 9H, CH2 Aliphatic chain); 13C NMR (DMSO-d6)
δ
171.7, 169.0, 153.1, 147.8, 137.9,133.2, 132.6, 130.8, 128.9, 31.7, 27.2, 21.0, 14.0; HRMS (ESI)
m/z calcd. C13H14N4O3S: 306.0787; found: 306.0794 [Mass Fragments: 223, 306].
4.4.5. 4-Chloro-N-(5-(3-nitrophenyl)-1,3,4-thiadiazol-2-yl)butanamide (5e)
17

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1
2
87% yield; mp 218-220°C; IR (KBr) ѵmax : 3261 (N-H stretch), 3035 (Arom.CH strech), 2966
(Alip.CH strech), 1676 (amide C=O) cm-1; 1H NMR (DMSO-d6) 12.75 (s, 1H, -NH), 7.62 (t, 1H,
J = 8.3 Hz, Ar-H), 8.05 (d, 1H, J = 8.3 Hz, Ar-H), 8.18 (d, 1H, J = 8.3 Hz, Ar-H), 8.40 (s, 1H,
3
4
Ar-H), 3.50 (t, 2H, J = 7.4 Hz, CH2 Aliphatic chain), 2.38 (t, 2H, J = 7.4 Hz, CH2 Aliphatic
13
5
chain) 1.55 (p, 2H, J = 7.4 Hz, CH2 Aliphatic chain); C NMR (DMSO-d6)
δ
174.8, 172.0,
6
153.4, 147.8, 135.9, 132.6, 130.7, 128.9, 124.7, 47.1, 33.8, 26.7 ; HRMS (ESI) m/z calcd.
C12H11ClN4O3S: 326.0240; found: 326.0248 [Mass Fragments: 223, 326].
7
8
4.4.6. 6-((5-(3-Nitrophenyl)-1,3,4-thiadiazol-2-yl)amino)-6-oxohexanoyl chloride (5f)
9
75% yield; mp 132-134°C; IR (KBr) ѵmax : 3221 (N-H stretch), 3082 (Arom.CH strech), 2982
10
11
12
13
14
(Alip.CH strech), 1687 (amide C=O) cm-1; 1H NMR (DMSO-d6) 12.44 (s, 1H, -NH), 7.70 (t, 1H,
J = 8.3 Hz, Ar-H), 7.98 (d, 1H, J = 8.3 Hz, Ar-H), 8.14 (d, 1H, J = 8.3 Hz, Ar-H), 8.28 (s, 1H,
Ar-H), 1.55-2.38 (m, 8H, CH2-CH2 Aliphatic chain); 13C NMR (DMSO-d6)
δ 173.8, 172.9,
152.2, 147.7, 137.4,134.8, 132.8, 131.9, 130.1, 128.1, 125.1, 46.7, 35.3, 26.8, 24.3; HRMS
(ESI) m/z calcd. C14H13ClN4O3S: 368.0346; found: 368.0354 [Mass Fragments: 223, 368].
4.4.7. N-(5-(3-nitrophenyl)-1,3,4-thiadiazol-2-yl)butyramide (5g)
15
16
17
18
19
20
21
22
23
24
25
26
50% yield; mp 144-146°C; IR (KBr) ѵmax : 3495 (N-H stretch), 3078 (Arom.CH strech), 2996
(Alip.CH strech), 1669 (amide C=O) cm-1; 1H NMR (DMSO-d6) 12.40(s, 1H, -NH), 7.69 (t, 1H,
J = 8.3 Hz, Ar-H), 7.92 (d, 1H, J = 8.3 Hz, Ar-H), 8.12 (d, 1H, J = 8.3 Hz, Ar-H), 8.38 (s, 1H,
Ar-H), 3.83 (t, 2H, 7.4 Hz, CH2), 1.45-2.13 (m, 5H, CH3-CH2) ; 13C NMR (DMSO-d6)
δ 169.8,
177.6, 155.2, 149.7, 137.9,132.8, 128.9, 128.5, 123.2, 31.7, 20.1, 10.0; HRMS (ESI) m/z calcd.
C12H12N4O3S: 292.0630; found: 292.0638 [Mass Fragments: 223, 292].
4.4.8. N-(5-(3-nitrophenyl)-1,3,4-thiadiazol-2-yl)furan-2-carboxamide (5h)
76% yield; mp 268-270°C; IR (KBr) ѵmax : 3263 (N-H stretch), 2996 (C-H Strech),1761 (amide
1
13
C=O) cm-1; H NMR (DMSO-d6) 12.75 (s, 1H, NH), 7.37-8.11 (m, 7H, Ar-CH); C NMR
(DMSO-d6) 170.5, 164.2, 159.1, 152.1, 148.8, 148.6, 137.1, 130.1, 128.3, 126.2, 124.9, 116.3,
111.3 ; HRMS (ESI) m/z C13H8N404S: 316.0266; found: 316.0274 [Mass Fragments: 223, 316].
18
δ

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1
2
3
4
5
6
7
8
9
4.4.9. 3-Chloro-N-(5-(3-nitrophenyl)-1,3,4-thiadiazol-2-yl)butanamide (5i)
81% yield; mp 139-141°C; IR (KBr) ѵmax : 3394 (N-H stretch), 3045 (Arom.CH strech), 2985
(Alip.CH strech), 1694 (amide C=O) cm-1; 1H NMR (DMSO-d6) 12.40(s, 1H, -NH), 7.72 (t, 1H,
J = 8.3 Hz, Ar-H), 8.12 (d, 1H, J = 8.3 Hz, Ar-H), 8.28 (d, 1H, J = 8.3 Hz, Ar-H), 8.35 (s, 1H,
Ar-H), 1.45-3.01 (m, 6H, CH2) ; 13C NMR (DMSO-d6)
δ 178.4, 169.8, 154.2, 149.7,
137.9,130.2, 129.8, 128.9, 128.5, 123.2, 40.1, 31.7, 10.0; HRMS (ESI) m/z calcd. C12H12N4O3S:
326.0240; found: 326.0248 [Mass Fragments: 223, 326].
4.4.10. 4-Nitro-N-(5-(3-nitrophenyl)-1,3,4-thiadiazol-2-yl)benzamide (5j)
62% yield; mp 222-224°C; IR (KBr)
ѵ
max : 3394 (N-H stretch), 3035 (Arom.CH strech), 2911
1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
(Alip.CH strech), 1684 (amide C=O) cm-1; H NMR (DMSO-d6) 12.50 (s, 1H, -NH), 7.23-8.29
13
(m, 8H, Ar-H); C NMR (DMSO-d6)
δ 174.5, 166.2, 164.2, 158.1, 152.7, 148.2, 147.2, 140.2,
138.5,136.7,129.8,124.3,122.1;HRMS (ESI) m/z calcd. C15H9N5O5S: 371.0324; found: 371.0332
[Mass Fragments: 223, 371].
4.4.11. 4-Methoxy-N-(5-(3-nitrophenyl)-1,3,4-thiadiazol-2-yl)benzamide (5k)
69% yield; mp 217-219°C; IR (KBr) ѵmax : 3287(N-H stretch), 3034 (Arom.CH strech), 2957
1
(Alip.CH strech), 1687 (amide C=O) cm-1; H NMR (DMSO-d6) 12.69 (s, 1H, -NH), 7.08-8.27
13
(m, 8H, Ar-H), 3.80 (s, 3H, OCH3); C NMR (DMSO-d6)
δ 174.0, 165.2, 164.3, 151.3,149.8,
148.9,146.2,143.1,140.3,132.7,129.8, 124.3, 114.2, 55.6; HRMS (ESI) m/z calcd. C18H12N4O4S:
356.0579; found: 356.0584 [Mass Fragments: 223, 356].
4.4.12. N-(5-(3-nitrophenyl)-1,3,4-thiadiazol-2-yl)thiophene-2-carboxamide (5l)
65% yield; mp 298-300°C; IR (KBr) ѵmax : 3281 (N-H stretch), 2991 (C-H Strech),1709 (amide
C=O) cm-1; H NMR (DMSO-d6) 12.64 (s, 1H, NH), 7.98-8.40 (m, 7H, Ar-CH); C NMR
1
13
(DMSO-d6) 174.3, 161.0, 152.7, 148.6, 139.5, 138.2, 136.1, 135.7, 134.1, 132.7, 131.2, 128.4,
δ
124.9; HRMS (ESI) m/z C13H8N4O3S2: 332.0038; found: 332.0046 [Mass Fragments: 223, 332].
4.4.13. N-(5-(3-nitrophenyl)-1,3,4-thiadiazol-2-yl)benzamide (5m)
19

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1
2
75% yield; mp 263-265°C; IR (KBr) ѵmax : 3395 (N-H stretch), 3093(Arom.CH strech), 2955
1
(Alip.CH strech), 1662 (amide C=O) cm-1; H NMR (DMSO-d6) 12.55 (s, 1H, -NH), 7.28-8.42
13
3
(m, 9H, Ar-H), C NMR (DMSO-d6)
δ
174.0, 165.2, 151.3, 148.9, 144.1, 142.6, 140.1, 139.5,
4
136.8, 134.2, 131.7, 128.3, 129.8; HRMS (ESI) m/z calcd. C18H12N4O3S: 326.0474; found:
326.0480 [Mass Fragments: 223, 326].
5
6
4.5. Preparation of 5-(pyridin-4-yl)-1,3,4-thiadiazol-2-amine (3c)
7
This compound is prepared as per the method given by Patel et al [17].
4.6. General procedure for the synthesis of N-(5-(pyridin-4-yl)-1,3,4-thiadiazol-2-yl)
substituted carbonyl choloride 6(a-j)
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
An equimolar amount of of 5-(pyridin-4-yl)-1,3,4-thiadiazol-2-amine (3c) dissolve in 10-15 mL
of tetrahydrofuran (THF), add 2-3 drops of triethylamine, to this solution add 1.5 mole different
aryl chlorides gradually, continuous stirring for 3 hrs on ice bath(0°C). After completion of
reaction solid will precipitate out, washed with sodium bicarbonate solution to remove excess of
chloride and recrystallized from ethanol to get pure compound
4.6.1. 2-Chloro-N-(5-(pyridin-4-yl)-1,3,4-thiadiazol-2-yl)acetamide (6a)
75% yield; mp 138-140°C; 75% yield; mp 258-261°C; IR (KBr) ѵmax : 3345 (N-H stretch), 3053
(Arom.CH strech), 2945 (Alip.CH strech), 1692 (amide C=O) cm-1; 1H NMR (DMSO-d6) 12.80
(s, 1H, NH), 8.14 (d, 2H, J = 5.2 Hz, Ar-H), 8.63 (d, 2H, J = 5.2, Hz, Ar-H), 4.18 (s, 2H, CH2);
13C NMR (DMSO-d6)
δ 174.1, 173.2, 152.4, 149.9, 131.5, 121.2, 42.7; HRMS (ESI) m/z calcd.
C9H7ClN4OS: 254.0029; found: 254.0035 [Mass Fragments: 179, 254].
4.6.2. N-(5-(pyridin-4-yl)-1,3,4-thiadiazol-2-yl)pivalamide (6b)
75% yield; mp 264-266°C; 59% yield; mp 148-151°C; IR (KBr) ѵmax : 3384 (N-H stretch), 3083
(Arom.CH strech), 2956 (Alip.CH strech), 1680 (amide C=O) cm-1; 1H NMR (DMSO-d6) 12.72
(s, 1H, NH), 8.10 (d, 2H, J = 5.2 Hz, Ar-H), 8.39 (d, 2H, J = 5.2, Hz, Ar-H), 1.38 (s, 9H, CH3);
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13C NMR (DMSO-d6)
δ
174.5, 173.6, 152.4, 149.9, 131.4, 121.2, 45.7, 26.9; HRMS (ESI) m/z
calcd. C12H14N4OS: 262.0888; found: 262.0896 [Mass Fragments: 179, 262].
3
4.6.3. N-(5-(pyridin-4-yl)-1,3,4-thiadiazol-2-yl)thiophene-2-carboxamide (6c)
4
55% yield; 158-160°C; 75% yield; mp 258-261°C; IR (KBr) ѵmax : 3394 (N-H stretch), 3083
(Arom.CH strech), 2945 (Alip.CH strech), 1662 (amide C=O) cm-1; 1H NMR (DMSO-d6) 12.82
(s, 1H, NH), 8.09 (d, 2H, J = 5.2 Hz, Ar-H), 8.43 (d, 2H, J = 5.2, Hz, Ar-H), 7.77 (d, 2H, J = 5
Hz, thiophene), 7.21 (t, 2H, J = 5 Hz, thiophene), 7.95 (d, 2H, J = 5 Hz, thiophene); 13C NMR
5
6
7
8
(DMSO-d6) δ 174.8, 173.3, 152.4, 149.9, 148.2, 146.3, 140.1, 121.2, 116.3, 111.3 HRMS (ESI)
9
m/z calcd. C12H14N4OS: 288.0140; found: 288.0149 [Mass Fragments: 179, 288].
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4.6.4. N-(5-(pyridin-4-yl)-1,3,4-thiadiazol-2-yl)pentanamide (6d)
64% yield; mp 154-156°C; 75% yield; mp 258-261°C; IR (KBr) ѵmax : 3375 (N-H stretch), 3083
(Arom.CH strech), 2945 (Alip.CH strech), 1672 (amide C=O) cm-1; 1H NMR (DMSO-d6) 12.89
(s, 1H, NH), 8.14 (d, 2H, J = 5.2 Hz, Ar-H), 8.32 (d, 2H, J = 5.2, Hz, Ar-H), 1.05-2.29 (m, 9H,
13
CH2 Aliphatic chain); C NMR (DMSO-d6)
δ 174.6, 173.2, 152.3, 149.9, 146.1, 140.1, 121.2,
31.7, 21.0, 14.0 HRMS (ESI) m/z calcd. C12H14N4OS: 262.0888; found: 262.0894 [Mass
Fragments: 179, 262].
4.6.5. 4-Nitro-N-(5-(pyridin-4-yl)-1,3,4-thiadiazol-2-yl)benzamide (6e)
75% yield; mp 254-256°C; 75% yield; mp 258-261°C; IR (KBr) ѵmax : 3375 (N-H stretch),
3093(Arom.CH strech), 2945 (Alip.CH strech), 1662 (amide C=O) cm-1; H NMR (DMSO-d6)
1
12.92 (s, 1H, NH), 7.45-8.50 (m, 8H, Ar-H); 13C NMR (DMSO-d6)
δ 174.0, 173.2, 152.3, 149.9,
146.2, 140.2, 136.7, 132.1, 130.2, 121.2 ; HRMS (ESI) m/z calcd. C14H9N5O3S: 327.0426;
found: 328.0432 [M+1] [Mass Fragments: 179, 328].
4.6.6. 3-Chloro-N-(5-(pyridin-4-yl)-1,3,4-thiadiazol-2-yl)butanamide (6f)
51% yield; mp 187-189°C; 75% yield; mp 258-261°C; IR (KBr) ѵmax : 3495 (N-H stretch), 3093
(Arom.CH strech), 2975 (Alip.CH strech), 1692 (amide C=O) cm-1; 1H NMR (DMSO-d6) 12.52
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(s, 1H, NH), 8.12 (d, 2H, J = 5.2 Hz, Ar-H), 8.42 (d, 2H, J = 5.2, Hz, Ar-H), 1.45-3.29 (m, 6H,
CH2 aliphatic side chain); 13C NMR (DMSO-d6)
δ 173.9, 173.5, 152.9, 148.9, 130.1, 121.2, 51.5,
3
31.7, 10.1; HRMS (ESI) m/z calcd. C14H9N5O3S: 282.0342; found: 282.0350 [Mass Fragments:
179, 282].
4
5
4.6.7. 6-Oxo-6-((5-(pyridin-4-yl)-1,3,4-thiadiazol-2-yl)amino)hexanoyl chloride (6g)
6
54% yield; mp 153-155°C; IR (KBr) ѵmax: 3395 (N-H stretch), 3043 (Arom.CH strech), 2955
7
(Alip.CH strech), 1692 (amide C=O) cm-1; 1H NMR (DMSO-d6) 12.80 (s, 1H, NH), 8.09 (d, 2H,
J = 5.2 Hz, Ar-H), 8.14 (d, 2H, J = 5.2, Hz, Ar-H), 1.55-2.82 (m, 8H, CH2 Aliphatic chain); 13C
8
9
NMR (DMSO-d6) δ 174.7, 173.2, 152.4,149.9,132.1,130.4, 121.9, 46.8, 35.9, 26.9, 24.8; HRMS
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(ESI) m/z C13H13ClN4O2S: 324.0448; found: 324.0454 [Mass Fragments: 179, 324].
4.6.8. 4-Chloro-N-(5-(pyridin-4-yl)-1,3,4-thiadiazol-2-yl)butanamide (6h)
74% yield; mp 148-150°C; 75% yield; mp 258-261°C; IR (KBr) ѵmax: 3345 (N-H stretch), 3043
(Arom.CH strech), 2963 (Alip.CH strech), 1692 (amide C=O) cm-1; 1H NMR (DMSO-d6) 12.81
(s, 1H, NH), 8.10 (d, 2H, J = 5.2 Hz, Ar-H), 8.43 (d, 2H, J = 5.2, Hz, Ar-H), 1.45-3.56 (m, 6H,
13
CH2 Aliphatic chain); C NMR (DMSO-d6)
δ 174.2, 173.6, 153.9, 149.9,130.1, 121.2, 47.1,
33.7, 26.1 ; HRMS (ESI) m/z C11H11ClN4OS: 282.0342; found: 282.0350 [Mass Fragments:
179, 282].
4.6.9. 2,2,2-Trichloro-N-(5-(pyridin-4-yl)-1,3,4-thiadiazol-2-yl)acetamide (6i)
68% yield; mp 258-260°C; 75% yield; mp 258-261°C; IR (KBr) ѵmax : 3485 (N-H stretch), 3043
(Arom.CH strech), 2956 (Alip.CH strech), 1692 (amide C=O) cm-1; 1H NMR (DMSO-d6) 12.56
(s, 1H, NH), 8.10 (d, 2H, J = 5.2 Hz, Ar-H), 8.31 (d, 2H, J = 5.2, Hz, Ar-H), 13C NMR (DMSO-
d6)
δ 174.2, 173.9, 152.6, 149.9,125.9, 95.2 HRMS (ESI) m/z C9H5Cl3N4OS: 321.9250; found:
321.9258 [Mass Fragments: 179, 321].
4.6.10. N-(5-(pyridin-4-yl)-1,3,4-thiadiazol-2-yl)butyramide (6j)
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76% yield; mp 287-290°C; 75% yield; mp 258-261°C; IR (KBr) ѵmax : 3405 (N-H stretch), 3003
(Arom.CH strech), 2855 (Alip.CH strech), 1692 (amide C=O) cm-1; 1H NMR (DMSO-d6) 12.62
(s, 1H, NH), 8.26 (d, 2H, J = 5.2 Hz, Ar-H), 8.77 (d, 2H, J = 5.2, Hz, Ar-H), 2.79 (t, 2H, J =7.3
Hz, CH2 Aliphatic chain), 1.79 (h, 2H, J = 7.3 Hz, CH2 Aliphatic chain), 0.94 (d, 3H, J =7.3 Hz,
3
4
5
CH3 Aliphatic chain); 13C NMR (DMSO-d6)
δ 174.2, 176.2, 152.4, 149.5,125.3, 94.2, 45.1, 31.7,
6
10.0; HRMS (ESI) m/ C11H12N4OS: 248.0732; found 249.0738 [M+1] [Mass Fragments: 188,
249].
7
8
4.7. Anti-mycobacterial activity
9
In vitro evaluation of the anti-mycobacterial activity of the newly synthesized compounds 6 (a-
o), 5 (a-m) and 6 (a–j) was carried out at the Micro Care Laboratory and Tuberculosis Research
Center Surat, India screening program. The antitubercular screening for test compounds was
conducted using M. Tuberculosis H37Rv strain and the clinically isolated phenotype MDR-TB
strain by adopting L. J. (conventional Lowenstein and Jensen) agar dilution method for the
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measurement of MIC, and is defined as the lowest concentration of drug, which inhibits
of bacterial population present at the beginning of the assay. Stock solutions of 200, 100, 62.5,
50, 25, 12.5, 6.25, 3.12, and 1.56 g /mL dilutions of each test compound in DMSO were added
≥ 99 %
µ
in the liquid L. J. Medium, and then media were sterilized by inspissations method. A culture of
M. tuberculosis (H37Rv strain and MDR-TB strain) growing on L. J. Medium was harvested in
0.85 % saline in bijou bottles. These tubes were then incubated at 37 ºC for 24 h followed by
4
streaking of M. tuberculosis (H37Rv strain and MDR-TB strain) [(5 x 10 ) bacilli per tube].
These tubes were then incubated at 37 ºC. Growth of bacilli was seen after 12 days, 22 days, and
finally at 28th day of incubation. Tubes having the compounds were compared with control
tubes where medium alone was incubated with M. tuberculosis (H37Rv strain and MDR-TB
strain). The concentration at which no development of colonies occurred or < 20 colonies was
taken as MIC concentration of test compound. The standard strain M. tuberculosis (H37Rv strain
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and MDR-TB strain) was tested with known drugs Isoniazid and Rifampin. MIC values µg /mL
are converted to
µM/mL [36].
3
4.8. Cyto-toxicity
4
The selected set of compounds, which showed potent activity against MTB (H37Rv) and
MDR-TB strains were also evaluated for their cyto-toxicity on VERO cell Lines by MTT assay.
Mammalian VERO cells were cultured in Dulbecco Modified Eagle Medium (DMEM)
containing 2 mM Na2CO3 supplemented with 10% (v/v) fetal bovine serum (FBS). The cells
were incubated at 37 °C under 5% CO2 and 95% air in a humidified atmosphere until confluent
and then diluted with phosphate-buffered saline to 106 cells/ml. Stock solutions were prepared in
dimethyl sulfoxide (DMSO) and further dilutions were made with fresh culture medium. The
concentration of DMSO in the final culture medium was 1%, which had no effect on the cell
viability. In a transparent 96-well plate, serially diluted stock solutions were placed at 37 °C for
72 h then the medium was removed and monolayer was washed twice with 100 µL of warm
Hanks’ balanced salt solution (HBSS). 100 µL of warm medium and 20 µL of freshly made
MTS-PMS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-
tetrazolium and phenylmethasulfazone] (Promega) were added to each well, plates were
incubated for 3 h and absorbance was determined at 490 nm using a plate reader. The same
experimental conditions were provided for all compounds and analysis was repeated three times
for the cell line tested using the standard compound Staurosporine.
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Acknowledgements
The authors would like to thank “Science and Engineering Research Board (SERB) of
Department of Science and Technology (DST) Govt. of India” (Grant No. YSS/2015/002017) for
funding the project.
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