Some novel Schiff bases of [1,2,4]triazole bearing haloarene moiety - Synthesis and evaluation of antituberculosis properties and neutrophil function test

Article abstract of DOI:10.1007/s00044-015-1461-2

A series of novel Schiff bases of [1,2,4]triazole-bearing haloarene moiety (5a-l) were synthesized from 4-amino-3-methyl-5-mercapto-4H-[1,2,4]triazole (3) and were confirmed by FT-IR, ¹H-NMR, ¹³C-NMR, LC-Mass spectra and elemental an

Full text of DOI:10.1007/s00044-015-1461-2

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-015-1461-2
ORIGINAL RESEARCH
Some novel Schiff bases of [1,2,4]triazole bearing haloarene
moiety—synthesis and evaluation of antituberculosis properties
and neutrophil function test
Prakash Anil Castelino1 Jagadeesh Prasad Dasappa1
•
•
Kishore G. Bhat2 Sumith Ashok Joshi3 Sunil Jalalpure3
•
•
Received: 13 May 2015 / Accepted: 2 September 2015
Ó Springer Science+Business Media New York 2015
Abstract A series of novel Schiff bases of [1,2,4]tria-
zole-bearing haloarene moiety (5a–l) were synthesized
from 4-amino-3-methyl-5-mercapto-4H-[1,2,4]triazole (3)
Introduction
The compounds with c group (–CH=N–) are popularly
known as the Schiff bases (March, 1985). They are syn-
thesized by the condensation of primary amines and active
carbonyl groups. In general, a Schiff base is a nitrogen
analogue of an aldehyde or ketone in which the >C=O
group is replaced by [C=N–R group. Schiff bases have
drawn the attention of researchers and have surfaced to
prominence due to ease in synthesis and possession of
important pharmacological activities. Schiff bases derived
from different heterocyclic compounds have been found to
possess antibacterial, antifungal, anticancer and antiviral
(Holla et al., 1998; Yadav and Singh, 2001; Vinci et al.,
2003; Ashok et al., 2007), anticonvulsant (Chaubey and
Pandeya, 2012), antiproliferative (Bayrak et al., 2009),
herbicidal (Holla et al., 2000) and antituberculosis activity
(Fokes et al., 2005). In addition to all these, they play vital
role in inorganic chemistry as ligands, e.g.: copper (II)
complex of the Schiff base salicylaldoxime, salen (a
common tetra-dentate ligand) and Jacobson’s catalyst
(Molina and Mederos, 2003).
1
and were confirmed by FT-IR, H-NMR, 13C-NMR, LC-
Mass spectra and elemental analyses and screened for
in vitro antituberculosis properties and for neutrophil
function test, where they exhibited moderate neutrophil
functions and antituberculosis properties. Compounds (5a),
(5c), (5f), (5h) and (5i) were moderately sensitive to anti-
tuberculosis activity, while (5a), (5b), (5g) and (5h) were
sensitive for neutrophil functions in the series. The acute
oral toxicity studies on the Swiss albino mice according to
the OECD/OCDE guidelines 423 confirmed that the com-
pounds were slightly toxic and hence they may be con-
sidered as drug candidates for microbial pathogens. The
antituberculosis studies by both ZOI and MIC tests con-
firmed that the novel derivatives are moderately sensitive
towards Mycobacterium tuberculosis (H37RV) strain.
Keywords [1,2,4]triazole Á Schiff bases Á
Haloarene moiety Á Antituberculosis studies Á
Neutrophil function test
In addition to the observation on Schiff bases, it is
observed that numerous research articles have reported of
[1,2,4]triazoles as possessing excellent antibacterial, anti-
fungal and anticancer (Holla et al., 2006), antitubercular
(Walczak et al., 2004), analgesic and anti-inflammatory
properties (Amir and Shikha, 2004). Moreover, [1,2,4]tri-
azole core is incorporated in wide variety of therapeutically
interesting molecules to transform them into better drugs.
Some of the present day drugs with triazole nucleus include
ribavirin (antiviral agent), rizatriptan (antimigraine agent),
alprazolam (anxiolytic agent), fluconazole, itraconazole
(antifungal agent) (Karthikeyan et al., 2006), etc.
& Jagadeesh Prasad Dasappa
jprasad2003@gmail.com
1
Department of Chemistry, Mangalore University,
Mangalagangothri, Karnataka State 574 199, India
2
Maratha Mandal’s NGH Institute of Dental Sciences and
Research Centre, Belagavi, Karnataka State 590 010, India
3
Department of Pharmacognosy, Dr Kore Basic Science
Research Centre, KLE University, Belagavi, Karnataka State
590 010, India
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Med Chem Res
Gathering the facts together, it was decided to synthesize
novel Schiff bases bearing [1,2,4]triazole and haloarene
moiety. Earlier, a number of biologically active Schiff bases
have been reported from our laboratory; hence, to continue
the legacy of synthesizing novel Schiff bases, 4-amino-3-
methyl-5-mercapto-4H-[1,2,4]triazole was chosen as the
starting material. The synthesis, results and biological activ-
ities of the novel Schiff bases (5a–l) are discussed herein.
Experimental section
Melting points of the novel Schiff bases, namely 4-[(benzyli-
dene)amino]-3-methyl-5-mercapto-4H-[1,2,4]triazole (5a–l)
were determined in an open capillary tubes and are uncor-
rected. The purity of synthesized compounds was checked by
TLC observing a single spot on Merck silica gel 60 F254-coated
alumina plates. The structures of novel Schiff bases (5a–l)
were confirmed by spectral studies. The FT-IR spectra (cm-1
)
were recorded on a Thermo Nicolet Avatar 370 Infrared
spectrophotometer in KBr pellets. The 400 MHz 1H-NMR and
13C-NMR spectra were recorded on a Bruker Avance III
400 MHz spectrometer using DMSO-d6 solvent and TMS as
the internal standard. All the chemical shift values are reported
in d-scale downfield from TMS. The Mass spectra were
recorded on PerkinElmer 018444-Y Triple Quadruple LC/MS
Spectrometer, and the elemental analyses were carried out on
an Elementar Vario EL III analyzer.
Scheme 1 Preparation of novel Schiff bases (5a–l)
Procedure for the preparation of 4-amino-3-methyl-
5-mercapto-4H-[1,2,4]triazole (3) (Reid and Heindel,
1976)
(0.01 mol) was refluxed for 12 h in DMF and ethanol
solvent (1:2) and a catalytic amount of Conc. H2SO4 acid.
The progress of the reaction was monitored by TLC. The
reaction mixture was poured into crushed ice. The solid
mass obtained was filtered, washed with excess of water,
dried and recrystallized from ethanol and DMF mixture to
yield the title compound. Melting points of the novel Schiff
bases (5a–l) were noted (Scheme 1). The characterization
data are given in Table 1.
The synthesis of 4-amino-3-methyl-5-mercapto-4H-[1,2,4]-
triazole (3) was done according to the well-described pro-
cedure in the literature (Reid and Heindel, 1976). A mixture
of thiocarbohydrazide (1) (1eq) was fused with an excess of
acetic acid (2) at reflux temperature for 8 h. The progress of
the reaction and the purity of the compound were checked
by TLC. The reaction mixture was cooled and poured into
crushed ice. The solid separated was collected by filtration
and recrystallized from ethanol to yield needle-shaped
crystals. The melting point was noted which was in con-
formity with the literature value (m. p. 202–204 °C, yield
55 %) (CAS No. 20939-15-5) (Scheme 1).
The spectroscopic data
4-Amino-3-methyl-5-mercapto-4H-[1,2,4]triazole (3) IR
(KBr, cm-1): 3342, 3270 (asymmetric and symmetric
stretch, –NH2), 2942 ([C–H stretch, –CH3 of 1,2,4-tria-
zole), 2580 (–SH stretch), 1579, 1507 ([C=N–), 1476
([N–N\ stretch) and 1214 ([C–S– stretch). 1H-NMR
(DMSO-d6, d ppm): 2.24 (3H, s, –CH3 of 1,2,4-triazole),
5.46 (2H, s, –NH2 of 1,2,4-triazole) and 13.38 (1H, s,
–SH of 1,2,4-triazole). 13C-NMR (d ppm): 10.35 (–CH3,
1,2,4-triazole), 149.12 ([C–CH3, 1,2,4-triazole) and
165.01 ([C–SH, 1,2,4-triazole). LC-Mass, [M??1], (m/z):
131.00. Anal. Cald for C3H6N4S: C, 27.68; H, 4.65; N,
Procedure for the preparation of novel Schiff bases:
4-[(benzylidene)amino]-3-methyl-5-mercapto-4H-
[1,2,4]triazole (5a–l) (Karthikeyan et al., 2006)
A mixture of 4-amino-3-methyl-5-mercapto-4H-[1,2,4]tri-
azole (3) (0.01 mol) and substituted aromatic aldehyde (4)
123

Med Chem Res
Table 1 Characterization data of Schiff bases (5a–l)
Compound
R
Mol. formula (mol. wt)
C10H8F2N4S (254.29)
m. p. (°C) (yield %)
% Composition found (Cald)
C
H
N
5a
5b
5c
5d
5e
5f
2,4-F2
178–180 (74)
214–216 (66)
212–214 (72)
188–190 (68)
224–226 (65)
204–206 (69)
176–178 (68)
184–186 (72)
214–216 (70)
220–222 (68)
200–202 (75)
230–232 (73)
47.23 (47.24)
38.10 (38.11)
47.23 (47.24)
38.10 (38.11)
44.35 (44.37)
44.36 (44.37)
44.11 (44.12)
44.10 (44.12)
44.36 (44.37)
44.36 (44.37)
41.81 (41.82)
41.81 (41.82)
3.19 (3.17)
2.57 (2.56)
3.18 (3.17)
2.57 (2.56)
2.99 (2.98)
2.99 (2.98)
2.60 (2.59)
2.60 (2.59)
2.99 (2.98)
2.99 (2.98)
2.82 (2.81)
2.82 (2.81)
22.02 (22.04)
17.77 (17.78)
22.03 (22.04)
17.77 (17.78)
20.69 (20.70)
20.69 (20.70)
20.57 (20.58)
20.58 (20.58)
20.69 (20.70)
20.69 (20.70)
19.51 (19.51)
19.50 (19.51)
4-Br-2-F
2,5-F2
C
10H8BrFN4S (314.25)
C10H8F2N4S (254.29)
C10H8BrFN4S (314.25)
3-Br-4-F
2-Cl-5-F
4-Cl-3-F
3,4,5-F3
2,3,4-F3
3-Cl-2-F
2-Cl-4-F
2,4-Cl2
C
10H8ClFN4S (270.26)
10H8ClFN4S (270.26)
C
5g
5h
5i
C10H7F3N4S (272.24)
C10H7F3N4S (272.24)
C
10H8ClFN4S (270.26)
5j
C10H8ClFN4S (270.26)
5k
5l
C10H8Cl2N4S (286.26)
C10H8Cl2N4S (286.26)
3,4-Cl2
43.04. Found: C, 27.67; H, 4.66; N, 43.03. m.
([C–H stretch, 4-bromo-2-fluorophenyl moiety), 2946
([C–H stretch, –CH3 attached to 1,2,4-triazole), 2577
(–SH stretch), 1588, 1491, 1342 ([C=N–, [C=C\), 1409
([N–N\stretch), 1285 ([C–S– stretch), 1021 and 748 (C–
p. 202–205 °C; yield: 55 %.
4-[(2,4-Difluorobenzylidene)amino]-3-methyl-5-mercapto-
4H-[1,2,4]triazole (5a) IR (KBr, cm-1): 3070 ([C–H
stretch, 2,4-difluorophenyl moiety), 2936 ([C–H stretch,
–CH3 attached to 1,2,4-triazole), 2652 (–SH stretch), 1594,
1502, 1382 ([C=N–, [C=C\), 1428 ([N–N\ stretch),
1273 ([C–S– stretch), 1095 and 965 (C–F, 2,4-difluo-
1
F and C–Br of 4-bromo-2-fluorophenyl moiety). H-NMR
(DMSO-d6, d ppm): 2.38 (3H, s, –CH3 of 1,2,4-triazole),
7.61 (1H, d, J = 8.4 Hz, 4-bromo-2-fluorophenyl moiety),
7.78 (1H, t, 4-bromo-2-fluorophenyl moiety), 8.01 (1H, t,
4-bromo-2-fluorophenyl moiety), 10.56 (1H, s, benzylidene
proton, –N=CH–Ar), 13.79 (1H, s, –SH of 1,2,4-triazole).
13C-NMR (d ppm): 10.63 (–CH3 of 1,2,4-triazole), 148. 80
(C–CH3 of 1,2,4-triazole), 119.65, 120.01, 126.64, 128.54,
128.66 and 152.99 (6C atoms, 4-bromo-2-fluorophenyl
moiety), 159.97 ([C–S–, 1,2,4-triazole) and 162.54 (C
atom, benzylidene, –N=CH–Ar). LC-Mass, [M??1], (m/
z): 314.10/315.90 (Br-79 and Br-81, ratio 1:1 due to iso-
topic abundance). [M??3], (m/z): 316.60.
1
rophenyl moiety). H-NMR (DMSO-d6, d ppm): 2.37 (3H,
s, –CH3 of 1,2,4-triazole), 7.27–7.32 (1H, m, 2,4-difluo-
rophenyl moiety), 7.45–7.51 (1H, m, 2,4-difluorophenyl
moiety), 8.12–8.18 (1H, m, 2,4-difluorophenyl moiety),
10.46 (1H, s, benzylidene proton, –N=CH–Ar), 13.77 (1H,
s, –SH of 1,2,4-triazole). 13C-NMR (d ppm): 10.57 (–CH3
of 1,2,4-triazole), 148.64 (C–CH3 of 1,2,4-triazole),
116.82, 116.92, 129.02, 129.12, 153.63 and 160.74
(6C atoms, 2,4-difluorophenyl moiety), 163.42 ([C–S–,
1,2,4-triazole) and 163.69 (C atom, benzylidene,
–N=CH–Ar). LC-Mass, [M??1], (m/z): 255.06 and
[M??3], (m/z): 257.05.
4-[(2,5-Difluorobenzylidene)amino]-3-methyl-5-mer-
capto-4H-[1,2,4]triazole (5c) IR (KBr, cm-1): 3072
([C–H stretch, 2,5-difluorophenyl moiety), 2940 ([C–H
stretch, –CH3 attached to 1,2,4-triazole), 2581 (–SH
stretch), 1583, 1502, 1383 ([C=N–, [C=C\), 1431 ([N–
4-[(4-Bromo-2-fluorobenzylidene)amino]-3-methyl-5-mer-
capto-4H-[1,2,4]triazole (5b) IR (KBr, cm-1): 3065
123

Med Chem Res
N\ stretch), 1275 ([C–S– stretch), 1093 and 990 (C–F,
2,5-difluorophenyl moiety). H-NMR (DMSO-d6, d ppm):
4-[(4-Chloro-3-fluorobenzylidene)amino]-3-methyl-5-mer-
capto-4H-[1,2,4]triazole (5f) IR (KBr, cm-1): 3063
([C–H stretch, 4-chloro-3-fluorophenyl moiety), 2954
([C–H stretch, –CH3 attached to 1,2,4-triazole), 2576
(–SH stretch), 1586, 1487, 1366 ([C=N–, [C=C\), 1461
([N–N\stretch), 1267 ([C–S– stretch), 1018 and 778 (C–
F and C–Cl, 4-chloro-3-fluorophenyl moiety). 1H-NMR
(DMSO-d6, d ppm): 2.38 (3H, s, –CH3 of 1,2,4-triazole),
7.61 (1H, t, J = 7.2 Hz, 4-chloro-3-fluorophenyl moiety),
7.79 (1H, t, J = 2.4 Hz, 4-chloro-3-fluorophenyl moiety),
8.03 (1H, dd, J = 6.4 Hz, 4-chloro-3-fluorophenyl moi-
ety), 10.59 (1H, s, benzylidene proton, –N = CH–Ar),
13.68 (1H, s, –SH of 1,2,4-triazole). 13C-NMR (d ppm):
10.59 (-CH3 of 1,2,4-triazole), 149. 80 (C-CH3 of 1,2,4-
triazole), 119.69, 123.15, 127.44, 130.74, 132.56 and
153.99 (6C atoms, 4-chloro-3-fluorophenyl moiety),
159.77 ([C–S–, 1,2,4-triazole) and 164.14 (C atom, ben-
zylidene, –N=CH–Ar,). LC-Mass, [M??1], (m/z): 271.10,
[M??3], (m/z): 272.90/274.90 (Cl-35 and Cl-37, ratio 3:1
due to isotopic abundance). [M??5], (m/z): 279.10.
1
2.39 (3H, s, –CH3 of 1,2,4-triazole), 7.47–7.56 (2H, m, 2,5-
difluorophenyl moiety), 7.82–7.87 (1H, m, 2,5-difluo-
rophenyl moiety), 10.64 (1H, s, benzylidene proton,
–N=CH–Ar), 13.80 (1H, s, –SH of 1,2,4-triazole).
13C-NMR (d ppm): 10.61 (–CH3 of 1,2,4-triazole), 148. 96
(C–CH3 of 1,2,4-triazole), 118.24, 118.56, 121.13, 121.47,
152.36 and 156.69 (6C atoms, 2,5-difluorophenyl moiety),
159.59 ([C–S–, 1,2,4-triazole) and 161.22 (C atom, ben-
zylidene, –N=CH–Ar). LC-Mass, [M??1], (m/z): 255.06.
[M??3], (m/z): 257.05.
4-[(3-Bromo-4-fluorobenzylidene)amino]-3-methyl-5-mer-
capto-[1,2,4]triazole (5d) IR (KBr, cm-1): 3063 ([C–H
stretch, 3-bromo-4-fluorophenyl moiety), 2941 ([C–H
stretch, –CH3 attached to 1,2,4-triazole), 2565
(–SH stretch), 1591, 1495, 1356 ([C=N–, [C=C\), 1407
([N–N\ stretch), 1281 ([C–S– stretch), 1018 and 738
(C–F and C–Br, 3-bromo-4-fluorophenyl moiety).
1H-NMR (DMSO-d6, d ppm): 2.37 (3H, s, –CH3 of 1,2,4-
triazole), 7.39 (1H, t, J = 6.4 Hz, 3-bromo-4-fluorophenyl
moiety), 8), 7.63 (1H, dd, J = 2.4 Hz, 3-bromo-4-fluo-
rophenyl moiety), 8.18 (1H, dd, J = 6.4 Hz 3-bromo-4-
fluorophenyl moiety), 10.61 (1H, s, benzylidene proton,
–N = CH–Ar), 13.68 (1H, s, –SH of 1,2,4-triazole).
13C-NMR (d ppm): 10.58 (-CH3 of 1,2,4-triazole), 148. 85
(C–CH3 of 1,2,4-triazole), 120.65, 121.71, 126.84, 129.68,
130.76 and 151.97 (6C atoms, 3-bromo-4-fluorophenyl
moiety), 159.59 ([C–S–, 1,2,4-triazole) and 163.51 (C
atom, benzylidene, –N=CH–Ar). [M??1], (m/z): 314.10/
315.90 (Br-79 and Br-81, ratio 1:1 due to isotopic abun-
dance). [M??3], (m/z): 316.60.
3-Methyl-4-[(3,4,5-trifluorobenzylidene)amino]-5-mer-
capto-4H-[1,2,4]triazole (5g) IR (KBr, cm-1): 3067
([C–H stretch, 3,4,5-trifluorophenyl moiety), 2948 ([C–H
stretch, –CH3 attached to 1,2,4-triazole), 2575 (–SH
stretch, 1,2,4-triazole), 1594, 1497, 1362 ([C=N–,
[C=C\), 1470 ([N–N\ stretch), 1268 ([C–S– stretch),
1063, 1019 and 978 (C–F, 3,4,5-trifluorophenyl moiety).
1H-NMR (DMSO-d6, d ppm): 2.38 (3H, s, –CH3 of 1,2,4-
triazole), 7.67–7.72 (1H, m, 3,4,5-trifluorophenyl moiety),
8.23–8.29 (1H, m, 3,4,5-trifluorophenyl moiety), 10.58
(1H, s, benzylidene proton, –N=CH–Ar), 13.74 (1H, s,
–SH of 1,2,4-triazole). 13C-NMR (d ppm): 10.56 (–CH3 of
1,2,4-triazole), 149.81 (C–CH3 of 1,2,4-triazole), 121.21,
123.68, 133.49, 136.27, 153.93 and 160.41 (6C atoms,
2,3,4-trifluorophenyl moiety), 162.92 ([C–S–, 1,2,4-tria-
zole) and 164.89 (C atom, benzylidene, –N=CH–Ar). LC-
Mass, [M??1], (m/z): 273.31 and [M??3], (m/z): 275.40.
4-[(2-Chloro-5-fluorobenzylidene)amino]-3-methyl-5-mer-
capto-4H-[1,2,4]triazole (5e) IR (KBr, cm-1): 3073
([C–H stretch, 2-chloro-5-fluorophenyl moiety), 2920
([C–H stretch, –CH3 attached to 1,2,4-triazole), 2581
(–SH stretch), 1588, 1499, 1399 ([C=N–, [C=C\), 1461
([N–N\stretch), 1270 ([C–S– stretch), 1091 and 824 (C–
F and C–Cl, 2-chloro-5-fluorophenyl moiety). 1H-NMR
(DMSO-d6, d ppm): 2.39 (3H, s, –CH3 of 1,2,4-triazole),
7.39–7.44 (2H, m, 2-chloro-5-fluorophenyl moiety), 7.66
(1H, dd, J = 2.4 Hz, 2-chloro-5-fluorophenyl moiety),
8.23 (1H, dd, J = 6.4 Hz, 2-chloro-5-fluorophenyl moi-
ety), 10.81 (1H, s, benzylidene proton, –N = CH–Ar),
13.79 (1H, s, –SH of 1,2,4-triazole). 13C-NMR (d ppm):
10.65 (–CH3 of 1,2,4-triazole), 148.91 (C–CH3 of 1,2,4-
triazole), 115.85, 117.70, 126.92, 129.63, 136.27 and
155.28 (6C atoms, 2-chloro-5-fluorophenyl moiety),
159.13 ([C–S–, 1,2,4-triazole) and 165.33 (C atom, ben-
zylidene, –N=CH–Ar). LC-Mass, [M??1], (m/z): 271.20,
[M??3], (m/z): 273.10/275.10 (Cl-35 and Cl-37, ratio 3:1
due to isotopic abundance). [M??5], (m/z): 279.10.
3-Methyl-4-[(2,3,4-trifluorobenzylidene)amino]-5-mer-
capto-4H-[1,2,4]triazole (5h) IR (KBr, cm-1): 3068
([C–H stretch, 2,3,4-trifluorophenyl moiety), 2945 ([C–H
stretch, –CH3 attached to 1,2,4-triazole), 2574 (–SH
stretch, 1,2,4-triazole), 1592, 1498, 1372 ([C=N–,
[C=C\), 1468 ([N–N\ stretch), 1271 ([C–S– stretch),
1075, 1021 and 985 (C–F, 2,3,4-trifluorophenyl moiety).
1H-NMR (DMSO-d6, d ppm): 2.39 (3H, s, –CH3 of 1,2,4-
triazole), 7.28–7.33 (1H, m, 2,3,4-trifluorophenyl moiety),
7.47–7.53 (1H, m, 2,3,4-trifluorophenyl moiety), 10.51
(1H, s, benzylidene proton, –N=CH–Ar), 13.79 (1H, s,
–SH of 1,2,4-triazole). 13C-NMR (d ppm): 10.51 (–CH3 of
1,2,4-triazole), 149.78 (C–CH3 of 1,2,4-triazole), 121.32,
123.72, 133.52, 136.48, 154.73 and 161.71 (6C atoms,
2,3,4-trifluorophenyl moiety), 163.72 ([C–S–, 1,2,4-
123

Med Chem Res
triazole) and 165.39 (C atom, benzylidene, –N=CH–Ar).
LC-Mass, [M??1], (m/z): 273.30 and [M??3], (m/z):
275.40.
([C–S– stretch), 795 and 732 (C–Cl, 2,4-dichlorophenyl
moiety). H-NMR (DMSO-d6, d ppm): 2.39 (3H, s, –CH3
1
of triazole), 7.60 (1H, dd, J = 2 Hz, 2,4-dichlorophenyl
moiety), 7.82 (1H, d, J = 2 Hz, 2,4-dichlorophenyl moi-
ety), 8.16 (1H, d, J = 8.4 Hz, 2,4-dichlorophenyl moiety),
10.89 (1H, s, benzylidene proton, –N = CH–Ar), 13.80
(1H, s, –SH of 1,2,4-triazole). 13C-NMR (d ppm): 10.65
(–CH3 of 1,2,4- triazole), 137.54 (C–CH3 of 1,2,4-triazole),
118.97, 128.25, 128.68, 129.19, 129.72 and 135.74
(6C atoms, 2,4-dichlorophenyl moiety), 154.53 ([C–S–,
1,2,4-triazole) and 161.17 (C atom, benzylidene,
–N = CH–Ar). LC-Mass, [M?], (m/z): 286.60, [M??1],
(m/z): 287.20/287.80 (Cl-35 and Cl-37, ratio 3:1 due to
isotopic abundance). [M??2], (m/z): 288.80/290.60. Anal.
Cald for C10H8Cl2N4S: C, 41.82; H, 2.81; N, 19.51. Found:
C, 41.81; H, 2.82; N, 19.51. m. p. 200–202 °C; yield:
75 %.
4-[(3-Chloro-2-fluorobenzylidene)amino]-3-methyl-5-mer-
capto-4H-[1,2,4]triazole (5i) IR (KBr, cm-1): 3073
([C–H stretch, 3-chloro-2-fluorophenyl moiety), 2934
([C–H stretch, –CH3 attached to 1,2,4-triazole), 2583
(–SH stretch, 1,2,4-triazole), 1592, 1504, 1380 ([C=N–,
[C=C\), 1453 ([N–N\ stretch), 1278 ([C–S– stretch),
1021 and 790 (C–F and C–Cl, 3-chloro-4-fluorophenyl
1
moiety). H-NMR (DMSO-d6, d ppm): 2.38 (3H, s, –CH3
of 1,2,4-triazole), 7.41 (1H, t, J = 8 Hz, 3-chloro-2-fluo-
rophenyl moiety), 7.82–7.86 (1H, m, 3-chloro-2-fluo-
rophenyl moiety), 8.02–8.06 (1H, m, 3-chloro-2-
fluorophenyl moiety), 10.62 (1H, s, benzylidene proton,
–N = CH–Ar), 13.81 (1H, s, –SH of 1,2,4-triazole).
13C-NMR (d ppm): 10.62 (–CH3 of 1,2,4-triazole), 149.71
(C–CH3 of 1,2,4-triazole), 120.78, 121.97, 126.11, 134.33,
152.04 and 153.21 (6C atoms, 3-chloro-2-fluorophenyl
moiety), 158.05 ([C–S–, 1,2,4-triazole) and 161.22 (C
atom, benzylidene –N=CH–Ar). LC-Mass, [M??1], (m/z):
271.20, [M??3], (m/z): 273.10/275.10 (Cl-35 and Cl-37,
ratio 3:1 due to isotopic abundance). [M??5], (m/z):
279.10.
4-[(3,4-Dichlorobenzylidene)amino]-3-methyl-5-mer-
capto-4H-[1,2,4]triazole (5l) (CAS No. 4666-44-95-1).
IR (KBr, cm-1): 3056 ([C–H stretch, 3,4-dichlorophe-
nyl moiety), 2934 ([C–H stretch, –CH3 attached to 1,2,4-
triazole), 2580 (–SH stretch, 1,2,4-triazole) 1578, 1504,
1375 ([C=N–, [C=C\), 1467 ([N–N\ stretch), 1273
([C–S– stretch), 793 and 736 (C–Cl, 3,4-dichlorophenyl
1
moiety). H-NMR (DMSO-d6, d ppm): 2.38 (3H, s, –CH3
4-[(2-Chloro-4-fluorobenzylidene)amino]-3-methyl-5-
mercapto-4H-[1,2,4]triazole (5j) IR (KBr, cm-1): 3061
([C–H stretch, 2-chloro-4-fluorophenyl moiety), 2936
([C–H stretch, –CH3 attached to 1,2,4-triazole), 2579
(–SH stretch, 1,2,4-triazole), 1595, 1501, 1402 ([C=N–,
[C=C\), 1430 ([N–N\ stretch), 1274 ([C–S– stretch),
1019 and 811 (C–F and C–Cl, 2-chloro-4-fluorophenyl
of triazole), 7.19 (1H, d, J = 7.6 Hz, 3,4-dichlorophenyl
moiety), 7.42 (1H, s, 3,4-dichlorophenyl moiety), 7.50 (1H,
d, J = 7.6 Hz, 3,4-dichlorophenyl moiety), 10.88 (1H, s,
benzylidene proton, –N = CH–Ar), 13.79 (1H, s, –SH of
1,2,4-triazole). 13C-NMR (d ppm): 10.67 (–CH3 of 1,2,4-
triazole), 137.27 (C–CH3 of 1,2,4-triazole), 119.25, 128.63,
128.97, 129.53, 130.32 and 135.87 (6C atoms, 3,4-
dichlorophenyl moiety), 153.93 ([C–S–, 1,2,4-triazole)
and 160.87 (C atom, benzylidene, –N=CH–Ar). LC-Mass,
[M?], (m/z): 286.60, [M??1], (m/z): 287.20/287.80 (Cl-35
and Cl-37, ratio 3:1 due to isotopic abundance). [M??2],
(m/z): 288.80/290.60. Anal. Cald for C10H8Cl2N4S: C,
41.82; H, 2.81; N, 19.51. Found: C, 41.81; H, 2.82; N,
19.50. m. p. 230–232 °C; yield: 73 %.
1
moiety). H-NMR (DMSO-d6, d ppm): 2.39 (3H, s, –CH3
of 1,2,4-triazole), 7.38–7.43 (1H, m, 2-chloro-4-fluo-
rophenyl moiety), 7.64 (1H, dd, J = 2.4 Hz, 2-chloro-4-
fluorophenyl moiety), 8.22 (1H, dd, J = 6.4 Hz 2-chloro-
4-fluorophenyl moiety), 10.80 (1H, s, benzylidene proton,
–N = CH–Ar), 13.79 (1H, s, –SH of 1,2,4-triazole).
13C-NMR (d ppm): 10.62 (–CH3 of 1,2,4-triazole), 148.92
(C–CH3 of 1,2,4-triazole), 115.84, 117.65, 126.90, 129.61,
136.27 and 159.10 (6C atoms, 2-chloro-4-fluorophenyl
moiety), 162.80 ([C–S–, 1,2,4-triazole) and 165.32 (C
atom, benzylidene, –N=CH–Ar). LC-Mass, [M??1], (m/
z): 271.10, [M??2], (m/z): 272.10/272.70 (Cl-35 and Cl-
37, ratio 3:1 due to isotopic abundance). [M??5], (m/z):
275.10.
Pharmacology
The novel Schiff bases (5a–l) were synthesized with ease
and in excellent yields, confirmed by the spectroscopic data
and evaluated for in vitro antituberculosis activity studies
using disc diffusion method (ZOI test) and microplate
Alamar Blue assay (MABA) method (MIC test). They were
also subjected to the test of various components of neu-
trophil functions, namely neutrophil chemotaxis, NBT test,
phagocytosis and microbicidal (candidacial) assay test
according to the simple and well-established procedure.
4-[(2,4-Dichlorobenzylidene)amino]-3-methyl-5-mer-
capto-4H-[1,2,4]triazole (5k) (CAS No. 4666-44-93-9).
IR (KBr, cm-1): 3058 ([C–H stretch, 2,4-dichlorophe-
nyl moiety), 2924 ([C–H stretch, –CH3 attached to 1,2,4-
triazole), 2582 (–SH stretch, 1,2,4-triazole), 1584, 1500,
1381 ([C=N–, [C=C\), 1469 ([N–N\ stretch), 1277
123

Med Chem Res
Antituberculosis activity
Neutrophil function test
All the newly synthesized Schiff bases (5a–l) were evalu-
ated for in vitro antituberculosis activity by disc diffusion
method (ZOI test) (Isenberg, 1992 and Bauer et al., 1966)
and microplate Alamar Blue assay (MABA) method (MIC
test) (Lourenco et al., 2007) according to the standard
procedure. Three drugs, namely pyrazinamide, cipro-
floxacin and streptomycin, were the reference standards for
the comparison in both the methods (ZOI and MIC tests).
Neutrophils are major contributors to the host defence
against invading microorganisms (especially bacteria and
fungi) at the various body sites including oral cavity.
Advance studies have shown that there is an impairment of
neutrophil functions in aggressive and chronic form peri-
odontitis, and hence the concept of hyperactive or primed
neutrophil has dawned which is highlighting the destruc-
tive aspect of neutrophil mediated tissue damage and bone
resorption through the release of cytokines, especially in
aggressive periodontitis. The new concept regarding neu-
trophil has opened up new avenues of research for the
diagnosis and treatment of aggressive as well as chronic
periodontitis (Lord, 1989; Matzner, 1987; Boqomolski-
Yahalom and Matzner, 1995; Nathan, 2006).
Disc diffusion method (ZOI test) (Isenberg, 1992 and Bauer
et al., 1966)
Ten microlitres (10 mg/mL) of the drug (test com-
pounds/standards) was loaded over Whatman filter paper
no. 1 discs of diameter 5 mm and dried. Discs were placed
in a dish plated with 4-week-old bacteria culture My-
cobacterium tuberculosis (H37RV) strain on 7H11 agar
media supplemented with OADC (growth supplement) and
incubated at 37 °C for 4 weeks for bacterial growth. Zone
of inhibition was measured using Digital Caliper (Mitu-
toyo, Japan). Experiments were performed in triplicates,
and the standard error was calculated.
Neutrophils are found in good numbers in circulation
system of gingival/periodontal tissue. In normal circum-
stances, in the absence of infection, neutrophils roll along
the vascular endothelium, transiently attaching to and
detaching from their surface. This is mediated by lectin-
binding glycoproteins, L-selectin on the surface of neu-
trophils and E-selectins on the endothelial cell.
In relation to infections, the neutrophils respond to
chemoattractants such as complement components, IL-8,
platelet activating factor and microbial peptides generated
at the infection site leading to activation of integrins pre-
sent on the surface of neutrophils. These integrins subse-
quently induce tighter adherence of neutrophils to vascular
endothelial cells and cessation of rolling. Neutrophils then
detach from endothelium and migrate through a gap in the
endothelial lining created by contraction of juxtaposed
vascular endothelial cells. After their migration into the
extravascular space, neutrophils proceed with the ingestion
and killing of microbes. This proceeds in three steps,
namely adhesion, opsonization and ingestion and intracel-
lular killing.
Microplate Alamar Blue dye assay method (MIC test)
(Lourenco et al., 2007)
All the synthesized Mannich bases were screened for
in vitro antituberculosis activity against M. tuberculosis
(H37RV) strain by microplate Alamar Blue assay method
(MABA) to determine the minimum inhibitory concentra-
tion (MIC test). This methodology is non-toxic and uses a
thermally stable reagent and shows a good correlation with
proportional and BACTEC radiometric method.
Two hundred microlitres of the sterile deionized water
was added to all the outer perimeter wells of sterile 96-well
plate to minimize evaporation of the medium in the test
wells during incubation. One hundred microlitres of the
Middlebrook 7H9 broth was added to the 96-well plate.
Serial dilution of the compounds was made directly on the
plate. The final drug concentrations tested were
100–0.2 lg/mL. The plates were covered and sealed with
parafilm and incubated at 37 °C for 5 days. Then, 25 lL of
freshly prepared 1:1 mixture of Alamar Blue dye reagent
and 10 % Tween-80 was added to the plate and incubated
for 24 h. The change in the colour (i.e. blue to pink) was
scored as bacterial growth. The no change in blue colour
was interpreted as no growth in bacteria. The minimum
inhibitory concentration (MIC) was defined as the lowest
drug concentration which prevented the colour change
from blue to pink.
The various components of neutrophil functions
(Wilkinson, 1981; Metcalf et al., 1986; Ryder, 2010;
Kunhs, 2008, Dale et al., 2008) are tested in the laboratory
by using several and simple economical tests which yield
valuable information regarding neutrophil functional
abnormalities in periodontitis.
Isolation of the neutrophils
For laboratory assay of neutrophils, a relatively pure pop-
ulation of granulocytes or at least all white blood cells with
minimum manipulation of blood are preferred. The blood
sample is collected either with heparin or EDTA as anti-
coagulant. Most of the isolation protocols use differences
in cell density as the basis for separation. A 3 % solution of
123

Med Chem Res
gelatin or high molecular weight dextran in saline is the
most preferred method because it gives a relatively pure
population of WBCs with the minimal contamination with
the RBCs. To obtain granulocytes, Ficoll-Hypaque is most
commonly used.
wells were loaded with Hanks solution on one side and
FMLP a known chemotactic agent on the other. The slides
are incubated at 37 °C for 2 h and fixed first with methanol
and then with formaldehyde. The agarose layer was
scraped off, and the slide was stained with Giemsa stain.
The movements of the cells were observed as a tongue-
shaped structure with the narrow end away from the central
well and towards the peripheral well containing FMLP.
The distance migrated was measured under the microscope
using oculometer.
For the separation of WBCs, the blood sample is diluted
with saline and equal amount of 3 % gelatin or dextran is
added. The tubes are made to stand upright for 45 min till
all the RBCs settle down. The supernatant is collected in a
fresh tube, centrifuged and washed with saline, and the
WBCs are counted in Neubauer’s chamber and then diluted
with Hanks balanced salt solution to get a specific con-
centration depending on the type of test to be performed.
Generation of reactive oxygen intermediate (NBT assay
test)
Neutrophil adherence
The release of reactive oxygen intermediates (ROIs) such
as superoxide radicals and hydrogen peroxide is an
important component of neutrophil’s bactericidal machin-
ery. These reactions are mediated by NADPH oxidase, and
the neutrophils defective in this enzyme cannot generate
ROI leading to defective microbial killing.
Adherence of neutrophils to the endothelium is a prereq-
uisite step to a migration of neutrophils into the tissues.
The adhesive function is commonly assessed by the pas-
sage of 1 mL of the whole blood through the column filled
with nylon wool. Adherence is measured as the difference
in the absolute neutrophil count before and after the pas-
sage of the sample through the column. Alternatively,
isolated neutrophils are induced as to bind to plastic using a
96-well plate coated with foetal bovine serum, fibrinogen
or fibronectin. The adherent activity of the cells can be
promoted by treatment with phorbol myristate acetate, and
the results can be analysed after staining.
The production of superoxide can be tested by reduction
of cytochrome C, and the extracellular release of H2O2 can
be measured by horse radish peroxidase-induced oxidation
of phenol red. The most common test used for this purpose
is nitro blue tetrazolium (NBT) assay. The cells are
exposed to the yellow dye NBT. Unstimulated neutrophils
do not ingest this dye, but if the cells are stimulated to
phagocytic activity, then they take the dye into the
phagosomes and intracellular reduction of the dye and
convert it to an insoluble blue crystalline form (formazan
crystals). These blue crystals are visible in the light
microscope. This test gives information about phagocytic
function since the dye is not taken into cells except by
phagocytosis and also about metabolic function since the
intracellular reduction depends upon the production of
ROIs.
Neutrophil chemotaxis
Neutrophil migration is a prerequisite for these cells to
accumulate at sites of inflammation. Chemotaxis in vitro is
generally measured by one of the two methods—Boyden’s
chamber technique or agarose assay. The Boyden’s
chamber includes three components: a lower compartment
containing chemoattractant agent, middle nitrocellulose
membrane filter layer and an upper chamber for neu-
trophils. After adding cell suspension to the upper chamber
and incubation for a specified period of time, the mem-
brane filter is removed, stained and studied under the
microscope to find out the number of cells that have
adhered to the membrane and trying to pass through the
narrow pores.
In NBT test, two aliquots of whole blood were mixed
separately with Hanks’ salt solution and 0.34 % NBT dye.
Escherichia coli endotoxin was added to one aliquot to
stimulate the neutrophils, and the other was left unstimu-
lated to act as a control. After incubation for about 45 min,
smears are prepared from both test and control samples and
stained with Giemsa and the number of cells with formazan
crystals was noted. [In a normal sample, only few cells in
control (unstimulated) and[60 % cells in test (stimulated)
should show the presence of crystals.]
The agarose assay is popular, because it is simpler to
perform and the results are comparable to those of Boy-
den’s assay. Here, 0.24 % agarose is prepared, melted and
mixed with pooled human serum. Hanks balanced salt
solution and sodium bicarbonate solution poured onto
microscopic slides when still hot. Once the slides were set,
a series of wells of 3–5 mm were cut in the agarose layer.
The central well was charged with cells, and the peripheral
The method employed nitro blue tetrazolium (NBT)
assay to analyse the reactive oxygen intermediates, hence
the name NBT assay. Escherichia coli endotoxin/test
compound was the positive control (PC), and the test
compound without E. coli endotoxin was the negative
control (NC).
123

Med Chem Res
Phagocytosis
4-Amino-3-methyl-5-mercapto-4H-[1,2,4]triazole
(3)
was condensed with different aromatic aldehydes (4) to
yield the resultant series of novel Schiff bases, namely
4-[(benzylidene)amino]-3-methyl-5-mercapto-4H-[1,2,4]-
triazole (5a–l) (Scheme 1). The FT-IR spectra in KBr
pellets gave peaks in the region 3040–3080, 2920–2960
and 2650–2760 cm-1 corresponding to [C–H stretch of
aryl group, –C–H stretch of –CH3 group and –SH stretch,
respectively. In the novel Schiff bases (5a–l), the absence
of peaks for –NH2 of the compound (3) was the indication
for the formation of Schiff bases. In addition to that, the
formation of benzylidene moiety (–N=CH–Ar) was con-
firmed which was further supported by 400 MHz 1H-NMR
and 13C-NMR recorded in DMSO-d6, d ppm downfield
from TMS. The benzylidene proton appeared in the region
d 10.50–10.90 ppm. The benzylidene carbon resonated at d
160.00–165.00 ppm. The LC-Mass spectra were in con-
formity with the corresponding molecular formulae of the
novel compounds. The structures of the novel compounds
(5a–l) were confirmed by spectral and elemental analyses.
Most methods for measuring phagocytosis rely on the
uptake of particles by phagocytes over a brief period of
time. The number of particles are then counted under the
microscope and expressed as mean particle number (MPN).
In this assay, neutrophil suspension was mixed with
equal portions of cooled normal human serum, Hanks’
solution and Candida albicans or latex particles and
incubated at 37 °C for 30 min. The tubes were then cen-
trifuged; smears were made from the deposit. Most meth-
ods for measuring phagocytosis relied on the uptake of
particles by phagocytes over a brief period of time. The
number of particles was then counted under the microscope
and expressed as mean particle number (MPN).
Microbicidal (candidacial) assay
This test is performed to assess the microbicidal or intra-
cellular killing capacity of neutrophils. Here, the WBC
suspension was mixed with Hanks’ solution, pooled serum
and Candida cells as mentioned above, and the test and
control samples were incubated for an hour at 37 °C. Equal
portion of 2.5 % sodium deoxycholate was then added and
mixed properly. This leads to lyses of neutrophil mem-
branes and releases ingested candida to the exterior. The
samples were then mixed with 0.01 % methylene blue
solution; centrifuged and wet preparations were made from
the deposit and examined under the microscope. Viable
candida remained unstained where as dead cells took up
the dye and appeared blue (In a healthy person, the normal
range of killing should be around 20–35 %). The cooled
human serum/test compound was the positive control (PC),
and the negative control (NC) was test compound without
serum.
Pharmacological screening
Antituberculosis activity
The Schiff bases (5a–l) possess antituberculosis activity for
in vitro test system but with different sensitivity (Tables 2,
3). The antituberculosis activity by MABA method (MIC
test) revealed that all the compounds of the novel series
exhibited excellent sensitivity up to 50 lg/mL, and in the
ZOI tests, the tested compounds exhibit moderate activity
almost half to the reference standards. The compounds
(5a), (5c), (5f), (5h) and (5i) are most sensitive in the
series, but are moderately active compared to the reference
standards. These compounds were subjected to the acute
oral toxicity studies, where they belong to the class of
slightly toxic. The compounds should be tested further to
consider them as potential antituberculosis drug candidate.
The presence of halogens on phenyl ring may be the
additional factor for the excellent activity of the com-
pounds. The structure activity relationships (SAR) of
numerous FDA-approved drugs confirmed that the pres-
ence of electronegative halogen atoms (especially fluorine
or chlorine) augments or contributes to the biological
activity. It is proved by number of researchers that halogen
compounds exhibit excellent pharmacological results due
to its electronegativity factor. The reference standards
pyrazinamide (specific antituberculosis drug), strepto-
mycin and ciprofloxacin (antibiotics and broad-spectral
antituberculosis drugs) were highly sensitive against M.
tuberculosis (H37RV) strain. However, tested compounds
cannot directly be compared with the reference standards
for their efficacy since it depends on therapeutic index,
Results and discussion
Chemistry
Thiocarbohydrazide (1) and acetic acid (2) were refluxed
for 8 h (Scheme 1) to yield 4-amino-3-methyl-5-mercapto-
4H-[1,2,4]triazole (3). The FT-IR spectra in KBr pellets
confirmed the asymmetric and symmetric stretches for
–NH2 at 3342 and, 3270 cm-1. The –C–H stretch of –CH3
was at 2942 cm-1 and –SH stretch at 2580 cm-1. The
400 MHz 1H-NMR and 13C-NMR recorded in DMSO-d6, d
ppm downfield from TMS confirmed the formation of
4-amino-3-methyl-5-mercapto-4H-[1,2,4]triazole
(3)
which was further confirmed by LC-Mass spectra, where
the compound signalled protonated molecular ion peak, i.e.
[M??1] peak at (m/z): 131.00.
123

Med Chem Res
established procedure. The tested novel compounds pos-
sess excellent activity with different sensitivity under dif-
ferent segments. Tabulating the results of the different
components under the collective title neutrophil function
test, the novel Schiff bases (5a–l) exhibited moderate to
excellent activities. Considering the individual compo-
nents, i.e. chemotaxis ([1.0), NBT ([25 %), phagocytosis
([3) and candidacial assay (25 %) possessing excellent
broad-spectrum properties, the compounds (5a), (5b), (5g)
and (5h) exhibited excellent neutrophil function test and
can be considered potential drug candidates after being
subjected to further tests. The data are given in Table 4.
Table 2 Antituberculosis activity of Schiff bases (5a–l) by disc
diffusion method (ZOI test)
Samples
Diameter of inhibition
zone (mm SD)a
Mycobacterium
tuberculosis (H37RV) strain
5a
5b
5c
5d
5e
5f
13.00 0.25
9.00 0.35
13.50 0.25
11.25 0.35
8.50 0.30
13.50 0.40
12.60 0.20
13.00 0.35
13.50 0.40
12.00 0.35
11.50 0.30
12.00 0.45
5g
5h
5i
Toxicity studies
The novel Schiff bases, (5a), (5c), (5f), (5h) and (5i) which
exhibited moderate antituberculosis activity and (5a), (5b),
(5g) and (5h) which showed excellent neutrophil function
test, were analysed for acute oral toxicity considering their
future potential utilities and safer mode of exposures. The
acute oral toxicity was done according to the OECD/OCDE
423 guidelines (retrieved on 10.08.2014). Handling and
animal experimentation were done according to the
guidelines of Committee for the Purpose of Control and
Supervision on Experiments on Animals (CPCSEA, 2001),
India (EPA 815-R-08-003, retrieved on 18.02.2014), with
the ethical clearance certificate KLECOP/IAEC/Res.
18–19/05/2014. The acute toxicity was tested as per the
OECD 423 guidelines for the period of 14 days on Swiss
albino mice (Mus musculus) in administering the doses of
5, 50, 300 and 2000 mg/kg body weight where the test
animals showed the normal behavioural pattern without
developing any complications. Finally from the acute
5j
5k
5l
‘0’ indicating no sensitivity (zone of inhibition \5 mm)
Reference standards: pyrazinamide: 30.00 0.65 mm, ciprofloxacin:
27.00 0.45 mm, streptomycin: 27.00 0.55 mm
a
Mean values of three trials
which varies for different bioactive compounds depending
upon the ratio between lethal dose and therapeutic dose.
Neutrophil function test
The novel Schiff bases (5a–l) were subjected to the various
components of neutrophil function, namely neutrophil
chemotaxis, NBT test, phagocytosis and microbicidal
(candidacial) assay test according to the simple and well-
Table 3 Antituberculosis activity of Schiff bases (5a–l) by microplate Alamar Blue assay method (MABA) (MIC test)
Samples
100 lg/mL
50 lg/mL
25 lg/mL
12.5 lg/mL
6.25 lg/mL
3.12 lg/mL
5a
5b
5c
5d
5e
5f
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
S
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
R
5g
5h
5i
5j
5k
5l
Bacterial strain: M. tuberculosis (H37RV) strain
Reference standards: pyrazinamide: 3.125 lg/mL, ciprofloxacin: 3.125 lg/mL, streptomycin: 6.25 lg/mL
S sensitive, R resistant
123

Med Chem Res
Table 4 Neutrophil function test of Schiff bases (5a–l)
Samples
Neutrophil
chemotaxis
(mm)
NBT (%)
Phagocytosis
(MPN)
Microbicidal
(candidacial)
assay (%)
5a
5b
5c
5d
5e
5f
2.2
2.1
2.4
2.6
2.3
2.2
2.4
2.7
2.3
2.4
2.3
2.3
0.5
2.2
30
30
19
21
20
22
30
32
18
18
15
19
20
65
3
32
30
30
34
32
31
30
37
30
31
28
30
16
30
4
3.5
3.5
3
2.5
3
5g
5h
5i
4
3
5j
3
5k
5l
3.5
2.5
3
NC
PC
5
For neutrophil chemotaxis: PC—test compound/FMLP, NC—WBC suspended in the media. For NBT: PC—E. coli endotoxin/test compound,
NC—test compound without E. coli endotoxin. For phagocytosis: PC—cooled normal human serum/test compound, NC—test compound without
serum. For candidacial assay: PC—serum/test compound, NC—test compound without serum
Control: NC negative control, PC positive control
Acknowledgments The authors are grateful to UGC, New Delhi, for
toxicity studies, it is revealed that the compounds (5a),
BSR fellowship, Mr. Dominic Harold Castelino, for the financial assis-
tance, the Head, NMR Research Centre, SAIF, Cochin-22, for 1H-NMR,
(5b), (5c), (5f), (5g), (5h) and (5i) were safe even at the
13C-NMR spectroscopy, FT-IR and CHN analyses, and Dr. Akash Nav-
ilebasappa, Acquity Labs, Bengaluru, for LC-Mass spectroscopy.
dose of 2000 mg/kg body weight. Hence, as per the Hodge
and Sterner scale toxicity (CPCSEA, 2001), these com-
pounds can be classified as slightly toxic as far as mam-
malian oral route of exposure is concerned. The acute
toxicity study values obtained are of use for determination
of therapeutic index in view of their relatively good anti-
tuberculosis activity and neutrophil function test.
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