Synthesis and pharmacological evaluation of condensed heterocyclic 6-substituted 1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazole and 1,3,4-oxadiazole derivatives of isoniazid

Article abstract of DOI:10.1016/j.bmcl.2010.06.125

The significance of this study was to prepare various isoniazid derivatives by introducing the isoniazid core into several molecules to explore the possibilities of some altered biological activities. Series of 6-substituted-1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazole (3a-g) and 1,3,4-oxadiazole (4a-g and 5) derivatives of isoniazid were synthesized in satisfactory yield and pharmacologically evaluated for their anti-inflammatory, analgesic, ulcerogenic, and lipid peroxidation activities by known experimental models.

Full text of DOI:10.1016/j.bmcl.2010.06.125

Bioorganic & Medicinal Chemistry Letters 20 (2010) 4762–4765
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and pharmacological evaluation of condensed heterocyclic
6-substituted 1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazole and 1,3,4-oxadiazole
derivatives of isoniazid
*
Sadaf J. Gilani , Suroor A. Khan, Nadeem Siddiqui
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Jamia Hamdard (Hamdard University), New Delhi 110062, India
a r t i c l e i n f o
a b s t r a c t
Article history:
The significance of this study was to prepare various isoniazid derivatives by introducing the isoniazid
core into several molecules to explore the possibilities of some altered biological activities. Series of 6-
substituted-1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazole (3a–g) and 1,3,4-oxadiazole (4a–g and 5) derivatives
of isoniazid were synthesized in satisfactory yield and pharmacologically evaluated for their anti-inflam-
matory, analgesic, ulcerogenic, and lipid peroxidation activities by known experimental models.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 20 January 2010
Revised 10 June 2010
Accepted 24 June 2010
Available online 27 June 2010
Keywords:
Isoniazid
Triazolo-thiadiazole
1,3,4-Oxadiazole
Anti-inflammatory
Analgesic
Ulcerogenic
Lipid peroxidation
Isoniazid, an organic compound, is a prodrug and must be acti-
vated by bacterial catalase. It is activated by catalase-peroxidase
hemoprotein, KatG which couples the isonicotinic acyl with nico-
tinamide adenine dinucleotide (NADH) to form isonicotinic acyl-
NADH complex. This complex indirectly inhibits the synthesis of
mycolic acid required for the mycobacterial cell wall.1 It is bacteri-
cidal to rapidly-dividing mycobacteria but is bacteriostatic if the
mycobacterium is slow-growing.2
Similarly, 1,3,4-oxadiazoles is a class of heterocycle which have at-
tracted significant interest in medicinal chemistry and they have a
wide range of pharmaceutical and biological activities.12,13 There-
fore, it was planned to synthesize hybrid compounds that comprise
both the isoniazid and the aforementioned heterocyclic ring
systems. Such hybridization was designed in order to investigate
the effect of structural variation on the anticipated anti-inflamma-
tory-analgesic, ulcerogenic and lipid peroxidation activities.
Isoniazid is metabolized by the liver mainly by acetylation and
dehydrazination. The N-acetylhydrazine metabolite is believed to
be responsible for the hepatotoxic effects seen in patients treated
with isoniazid. CYP2E1 is reportedlyinvolvedinINH-induced hepato-
toxicity in humans.3 For these reasons, in our study, we tried to elim-
inate in vivo acetylation by arylamine N-acetyltransferase (NAT) to
form inactive acetylated drug by replacing hydrazide moiety of INH
with triazolo-thiadiazole and 1,3,4-oxadiazole heterocycles.
A survey of literature revealed that substituted-1,2,4-triazolo-
[3,4-b]-1,3,4-thiadiazole rings, have received much attention during
recent years on account of their prominent potential as antifungal,4
anti-inflammatory,5 anti-tumor,6 analgesic,7 and antibacterial
agents.8 A triazolo-thiadiazole system may be viewed as a cyclic
analog of two very important components-thiosemicarbazide9,10
and biguanide,11 which often display diverse biological activities.
6-Substituted-1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazole
(3a–g)
and 1,3,4-oxadiazole (4a–g and 5) were prepared according to
the procedure outlined in Scheme 1. The required dithiocarbazi-
nate14 was synthesized by reacting acid hydrazide with carbon
disulfide and potassium hydroxide in ethanol. This salt underwent
ring closure with an excess of 99% hydrazine hydrate to give the 4-
amino-5-(pyridin-4-yl)-4H-1,2,4-triazole-3-thiol (2).15Hence re-
sulted triazole (2) was then converted to the title compounds16
(3a–g) in a one-pot reaction, by condensation with aromatic acids
in the presence of POCl3. The synthesis of compounds17 (4a–g) was
accomplished in a single step by reacting the acid hydrazide of
starting drug, isoniazid with aromatic acids in the presence of
POCl3 and compound18 5 by reacting with carbon disulfide and
potassium hydroxide in ethanol, respectively. The structure of syn-
thesized compounds was confirmed by elemental analysis and
spectral data (IR, 1H NMR, and LRMS).19
The anti-inflammatory activities20 of the synthesized com-
pounds 3a–g, 4a–g, and 5 were evaluated. The compounds were
* Corresponding author. Tel.: +91 9891128162; fax: +91 11 26059663x5612.
E-mail address: gilanisadaf@gmail.com (S.J. Gilani).
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.06.125

S. J. Gilani et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4762–4765
4763
N
N
N
KOH /C2H5OH/CS2
Stirring, Room Temp.
N
CS2/KOH
SH
O
N
(5)
S-K+
NH
CONHNH2
NH
O
C
S
RCOOH
3
(1)
Isoniazid
N
N
POCl
R
O
Reflux
NH2NH2.H2O
N
(4)
N
N
N
N
RCOOH
N
SH
N
S
Reflux,
POCl3
N
N
NH2
N
R
(3)
(2)
Scheme 1. Reagents: 3, 4 [(a) R = C6H5; (b) R = 2-C6H4Cl; (c) R = 2,4-C6H3Cl2; (d) R = 2-C6H4CH3; (e) R = 2-C6H4OCOCH3; (f) R = OC6H5(phenoxy); (g) R = 4-C6H4NO2].
Table 1
Biological data of 1,2,4-triazolo-[3,4-b]-1,3,4-thiadiazole and 1,3,4-oxadiazole derivatives of isonicotinic acid hydrazide (3a–g, 4a–g, and 5)
Compound
Anti-inflammatory activity
Analgesic activity
Ulcerogenic activity
nmol MDA
(% inhibition SEM after 4 h)–
(% inhibition SEM after 4 h)#
(severity index SEM)–
content SEM/100 mg tissue–
3a
3b
3c
3d
3e
3f
3g
4a
4b
4c
4d
4e
4f
4g
5
66.36 2.56a
45.41 3.10b
69.32 2.17a
79.79 1.75a
39.80 3.73b
63.55 2.23a
86.32 1.09
82.63 2.59
37.74 3.20b
84.00 1.66
43.38 3.94b
63.36 2.00a
71.23 2.83a
54.01 3.22b
62.88 2.45a
51.62 3.20b
82.69 1.65
—
56.06 3.25d
37.27 3.73d
61.81 2.45d
62.54 2.27d
25.00 3.94d
52.27 2.81d
74.32 1.20c
68.21 1.65c
29.54 3.27d
70.37 1.67c
31.81 3.71d
57.57 2.45d
62.14 3.74d
44.05 3.76d
54.28 3.96d
45.90 2.05d
73.52 1.00
—
0.666 0.10b
1.417 0.08b
0.750 0.17b
0.471 0.11a
2.250 0.24
1.500 0.00b
0.324 0.08a
1.667 0.13b
2.000 0.10b
0.386 0.10a
2.266 0.20
0.831 0.16b
0.731 0.24b
1.762 0.26b
1.327 0.21b
3.75 0.11b
2.315 0.18
0.00 0.00
5.78 0.10b
8.80 0.06b
6.71 0.16b
4.16 0.17b
8.82 0.14b
7.32 0.19b
3.47 0.19a
4.87 0.26b
8.11 0.13b
3.29 0.15
7.33 0.16b
5.19 0.22b
3.33 0.19
6.85 0.14b
5.78 0.10b
9.10 0.14b
7.51 0.16
3.26 0.01
Isoniazid
Ibuprofen
Control
–
Relative to their respective standard and data were analyzed by ANOVA followed by Dunnett’s multiple comparison test for n = 6; aP < 0.05; bP < 0.01.
#
Relative to normal and data were analyzed by paired Student’s t-test for n = 6; cP < 0.0001; dP < 0.005.
tested at an equimolar oral dose relative to 70 mg/kg ibuprofen.
The tested compounds showed anti-inflammatory activity ranging
from 37.74 3.20% to 86.32 1.09%, whereas the standard and par-
ent drug ibuprofen and isoniazid showed 82.69 1.65% and
51.62 3.20% inhibition, respectively, after 4 h (Table 1). It was ob-
served that the triazolo-thiadiazole derivatives having 4-nitro-
phenyl (3g) at sixth position possess highest activity
(86.32 1.09%) comparable to that of ibuprofen (82.69 1.65%).
Further it was observed that the presence of 2-chlorophenyl moi-
ety (3b) at C-6 showed decrease of activity (45.41 3.10%) and
replacement of this group by acetyl phenyl group (3e) resulted in
sharp decrease of anti-inflammatory activity (39.80 3.73%). In
oxadiazole derivatives highest activity (84.00 1.66%) was found
in 4c having 2,4-dichlorophenyl group at second position. When
the phenyl group was replaced by mercapto group 5 the activity
was found to be moderate (62.88 2.45%).
All compounds were further tested for their analgesic activity21
at the same oral dose and showed good to moderate analgesic
activity in comparison to their respective standard and parent
drugs. The compounds showed analgesic activity ranging from
25.00 3.94% to 74.32 1.20% inhibition, whereas parent isoniazid
and standard drug ibuprofen showed 45.90 2.05% and
73.52 1.00% inhibition, respectively (Table 1).
The compound 3g having 4-nitrophenyl group at C-6 position of
triazolo-thiadiazole ring (74.32 1.20%), and 2,4-dichlorophenyl
group, present at the second position on oxadiazole ring 4c,
showed the maximum activity (70.37 1.67%), equivalent to that
of the standard drug ibuprofen (73.52 1.00%). The 2-mercapto
group 5 at second position of oxadiazole ring showed moderate
analgesic activity (54.28 3.96%).
The results showed that an electron-donating group (3g) in-
creases the analgesic activity of the triazolo-thiadiazole derivatives

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S. J. Gilani et al. / Bioorg. Med. Chem. Lett. 20 (2010) 4762–4765
Table 2
Effect of compounds 3g and 4c on serum enzymes, total proteins, and total albumin
Compound
SGOTa (U/mL)
SGPTa (U/mL)
Alkaline phosphatasea
Total proteina (g/dL)
Total, albumina (g/dL)
Control
Isoniazid
Ibuprofen
3g
143.71 1.10
151.25 1.56
32.46 0.76
40.21 0.64
34.26 0.72
12.17 0.16
8.31 0.21
11.67 0.18
12.76 0.13
1.72 0.02
1.55 0.05
1.74 0.06
1.78 0.08
1.76 0.02
1.63 0.02
1.48 0.07
1.71 0.01
1.77 0.15
1.72 0.05
**
140.14 1.12
138.32 1.22
136.24 1.20
**
**
**
29.34 0.64
**
**
4c
28.67 0.67
13.79 0.19
a
Relative to control and data were analyzed by ANOVA followed by Dunnett’s multiple comparison test, for n = 6; **P < 0.01.
ulcerogenic activity also showed reduced malondialdehyde (MDA)
content, a byproduct of lipid peroxidation.23 Therefore, an attempt
was made to correlate the decrease in ulcerogenic activity of the
compounds with that of lipid per oxidation.
The lipid peroxidation is measured as nmol of MDA/100 mg of
tissue. The isoniazid (parent drug) and ibuprofen (standard drug)
showed the maximum lipid per oxidation (9.10 0.14 and
7.51 0.16), whereas the control group showed 3.26 0.01. It was
found that all the condensed derivatives showing less ulcerogenic
activity also showed reduction in lipid peroxidation (Table 1). Thus
these studies showed that synthesized compounds have inhibited
the induction of gastric mucosal lesions and the results further sug-
gested that their protective effect might be related to the inhibition
of lipid per oxidation in the gastric mucosa. Further it has been
reported that isoniazid causes hepatotoxicity due to the presence
of hydrazine moiety in their molecule. Hence, conversion of hydra-
zine group of isoniazid to triazolo-thiadiazole and 1,3,4-oxadiazole
moietymight have caused COX-2 inhibitory activity, resulting in sig-
nificant anti-inflammatory activity with less ulcerogenicity.
The compounds 3g and 4c, derivatives of triazolo-thiadiazole
and 1,3,4-oxadiazole, respectively, showing potent anti-inflamma-
tory and analgesic activities with reduced ulcerogenicity and lipid
peroxidation, were further studied for their hepatotoxic effect in
comparison with parent isoniazid and standard ibuprofen drugs.
Both compounds were studied for their effect on biochemical
parameters24,25 (serum enzymes, total proteins, and total albu-
min), as shown in Table 2 and liver histopathological26 testing
were also carried out. The studies of the liver samples do not show
any significant pathological changes in comparison to control
group (Figs. 1 and 2).
Figure 1. Compound 3g: section of liver showing hepatocytes with mild swelling
and clearing of cytoplasm. SS, sinusoidal space; BD, bile duct (100Â).
In Summary, various triazolo-thiadiazole and 1,3,4-oxadiazole
derivatives of isoniazid were prepared. Among these the com-
pound
6-(4-nitrophenyl)-3-(pyridin-4-yl)-[1,2,4]triazolo[3,4-b]
Figure 2. Compound 4c: section of liver, showing hepatocytes with mild clearing of
cytoplasm. Hep, hepatocyte; SS, sinusoidal space; BD, bile duct (100Â).
[1,3,4]thiadiazole (3g) and 2-(2,4-dichlorophenyl-5-(pyridin-4-
yl)-1,3,4-oxadiazole (4c) showed maximum anti-inflammatory
and analgesic activity. Therefore, it was concluded that triazolo-
thiadiazole and 1,3,4-oxadiazole derivatives of isoniazid might
afford a safer alternative to isoniazid for the treatment of inflam-
matory disease, pain and hepatotoxicity caused by acetyl hydra-
zine, a metabolite of isoniazid.
whereas electron-withdrawing group (4c) increases the analgesic
activity of the 1,3,4-oxadiazole derivatives of isoniazid. It was
noted that the compound 3g and 4c showing highest anti-inflam-
matory activity also exhibited highest analgesic activity
(74.32 1.20% and 70.37 1.67%) as well as those exhibiting low-
est anti-inflammatory activity 3e and 4b, also exhibited lowest
analgesic activity (25.00 3.94% and 29.54 3.27%). It was further
interestingly noted that parent isoniazid does not show any fruitful
results for both anti-inflammatory (51.62 3.20%) and analgesic
activity (45.90 2.05%) as compared to their triazolo-thiadiazole
and 1,3,4-oxadiazole derivatives which showed interesting results
for both the activities.
Acknowledgments
The authors are thankful to Central Drug Research Institute
(CDRI) for spectral analysis of the compounds and Dr. A. Mukher-
jee, M.D., Department of Pathology, All India Institute of Medical
Sciences (AIIMS), New Delhi, for the histopathological studies.
The compounds were further tested for their acute ulcerogenic
activity22 at an equimolar oral dose relative to 210 mg/kg ibuprofen
and parent drug isoniazid. The tested compounds showed low
ulcerogenic activity ranging from 0.324 0.08 to 2.266 0.20, com-
pared to standard ibuprofen and parent drug isoniazid having high
severity index of 2.315 0.18 and 3.750 0.11, respectively (Table
1). It has been reported in literature that compounds showing less
References and notes
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4765
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(20 mL) and refluxed for 20 h. The reaction mixture was slowly poured over
crushed ice and kept overnight. The solid thus separated out was filtered,
washed with water, dried and recrystallised from ethanol, yield: 65–75%.
18. General method for the synthesis of 5-(pyridin-4-yl)-1,3,4-oxadiazole-2-thiol:
a mixture of isonicotinic acid hydrazide (0.005 mol), KOH (0.005 mol) and
carbon disulfide (5 mL) in ethanol (50 mL) was refluxed on a steam bath for
12 h. The solution was then concentrated, cooled and acidified with dilute HCl.
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recrystallized from ethanol. Yield: 74%, mp 166 °C; IR (KBr,
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cmÀ1): 1638
11. Haglind, J. Chem. Abstr. 1966, 64, 16509.
(C@N), 1521 (C@C aromatic), 1430 (C–O–C oxadiazole), 1164 (SH); 1H NMR
(300 MHz, CDCl3): d 7.94, 8.74 (m, 4H, Py), 13.03 (s, 1H, SH); MS: m/z 179 (M+).
Anal. Calcd for C7H5N3OS: C, 46.95; H, 2.83; N, 23.48; S, 17.91. Found: C, 46.92;
H, 2.81; N, 23.45; S, 17.89.
12. Amir, M.; Shikha, K. Eur. J. Med. Chem. 2004, 39, 535.
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m
cmÀ1): 3300 (NH), 1670
19. Physical and analytical data of the selected compounds: Compound 3g: yield
(C@O), 1088 (C@S); 1H NMR (DMSO-d6): d 10.76 (br s, 1H, CONH), 9.23 (br s,
1H, NH),7.76, 8.64 (m, 4H, Py) MS: m/z 250 (M+). Anal. Calcd for C7H6KN3OS2: C,
33.40; H, 2.36; N, 16.76; S, 25.48. Found: C, 33.45; H, 2.41; N, 16.72; S, 25.51.
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70%, mp 204 °C; IR (KBr, m
cmÀ1): 3164 (C–H), 1238 (N–N@C triazolo-
thiadiazole), 1674 (C@N), 1544 (C@C), 696 (C–S–C), 1316 (NO2); 1H NMR
(300 MHz, CDCl3): d 7.92, 8.76 (m, 4H, Py), 7.24–7.28 (m, J = 12 Hz, 4H Ar-H);
MS: m/z 324 (M+); Anal. Calcd for C14H8N6O2S: C, 51.87; H, 2.52; N, 25.94; S,
9.92. Found: C, 51.85; H, 2.49; N, 25.91; S, 9.89.
68%, mp 198 °C; IR (KBr,
m
cmÀ1): 3320 (NH), 2598 (SH), 1610 (C@N), 1572
Compound 4c: yield 71%, mp 164 °C; IR (KBr, m
cmÀ1): 1646 (C@N), 1548
(C–H); 1H NMR (300 MHz,CDCl3): d 5.15 (s, 2H, NH2), 13.18 (br s, 1H, SH), 7.73
(m, 2H, Py, H3 and 5), 8.63 (m, 2H, Py, H2 and 6); MS: m/z 193 (M+). Anal. Calcd
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14.38.
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on to crushed ice with stirring. The mixture was allowed to stand overnight
and the solid separated out was filtered, treated with dilute sodium hydroxide
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and aromatic acids (0.001 mol) was dissolved in phosphorus oxychloride