Design, synthesis, and evaluation of isoniazid derivatives acting as potent anti-inflammatory and anthelmintic agents via Betti reaction

Article abstract of DOI:10.1007/s00044-013-0693-2

A novel synthesis of isoniazid derivatives achieved by the condensation of aldehydes, isoniazid, and phenols via Betti reaction has been described. The reactions were carried out at room temperature using fluorite as catalyst. The catalyst is efficient, benign, reusable, cost-effective, and ecofriendly. The novel synthesized moieties were characterized on the basis of ¹H NMR, ¹³C NMR, mass spectrometry, and elemental analysis. All the synthesized agents 4(a-j) were examined for their potential in vivo anti-inflammatory activity on Wistar albino rats using a standard reference drug, diclofenac. These synthesized derivatives were further screened for their potent in vitro anthelmintic activity using a standard reference drug, albendazole on Indian earthworms, Pheretima posthuma. A correlation of structure and activity relationship of these compounds with respect to Lipinski's rule of five, drug likeness, toxicity profiles, and other physico-chemical properties of drugs is described.

Full text of DOI:10.1007/s00044-013-0693-2

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2014) 23:939–947
DOI 10.1007/s00044-013-0693-2
ORIGINAL RESEARCH
Design, synthesis, and evaluation of isoniazid derivatives acting
as potent anti-inflammatory and anthelmintic agents via Betti
reaction
•
Ipsita Mohanram Jyotsna Meshram
Received: 13 March 2013 / Accepted: 6 August 2013 / Published online: 21 August 2013
Ó Springer Science+Business Media New York 2013
Abstract A novel synthesis of isoniazid derivatives
achieved by the condensation of aldehydes, isoniazid, and
phenols via Betti reaction has been described. The reac-
tions were carried out at room temperature using fluorite as
catalyst. The catalyst is efficient, benign, reusable, cost-
effective, and ecofriendly. The novel synthesized moieties
current clinical usage suffer from a common drawback of
gastrointestinal toxicity due to direct contact of free car-
boxylic group with gastrointestinal mucosa and inhibition
of cyclooxygenase enzyme non-selectively (Akhter et al.,
2010). Microbial infections often produce pain and
inflammation besides none of the drugs possesses chemo-
therapeutic and anti-inflammatory activities in a single
component. However, studies have shown that many active
antimicrobial agents demonstrate very good anti-inflam-
matory activity (Nath et al., 2005; Bekhit and Aziem,
2004; Bekhit et al., 2003). Hydrazide derivatives are of
wide interest because of their diverse biological and clin-
ical applications. Hydrazides have been demonstrated to
possess antidepressant (Mohareb et al., 2010), anti-
inflammatory (Bhandari et al., 2008), antimalarial (Gemma
et al., 2006), antimicrobial (Bayrak et al., 2009), antimy-
cobacterial (Nayyar et al., 2007), antitumor (Lembege
et al., 2008), antiviral (Osama et al., 2009), antitubercu-
losis (Kaymakcioglu et al., 2006), and analgesic (Ali et al.,
2005) activities. Similarly, isoniazid (INH) derivatives also
play an important starting material for the preparation of
other biological active compounds. Isoniazid has the
greatest bactericidal activity and is used almost from the
outset of tuberculosis chemotherapy (Banerjee et al., 1994;
Deretic et al., 1996; Rozwarski et al., 1998).
1
were characterized on the basis of H NMR, 13C NMR,
mass spectrometry, and elemental analysis. All the syn-
thesized agents 4(a–j) were examined for their potential
in vivo anti-inflammatory activity on Wistar albino rats
using a standard reference drug, diclofenac. These syn-
thesized derivatives were further screened for their potent
in vitro anthelmintic activity using a standard reference
drug, albendazole on Indian earthworms, Pheretima post-
huma. A correlation of structure and activity relationship of
these compounds with respect to Lipinski’s rule of five,
drug likeness, toxicity profiles, and other physico-chemical
properties of drugs is described.
Keywords Anti-inflammatory ꢀ Anthelmintic ꢀ
Betti reaction ꢀ Green synthesis ꢀ Virtual screening
Introduction
The non-steroidal anti-inflammatory drugs (NSAIDs) like
ibuprofen, fenoprofen, diclofenac, and fenbufen under
Betti (1941) reaction has gained importance due to their
application in pharmaceutical chemistry. They have been
encountered with antibacterial (Holla et al., 1998), anti-
cancer (Holla et al., 2003), analgesic and anti-inflammatory
(Gokce et al., 2005), anticonvulsant (Dimmock et al.,
1992), antimalarial (Lopes et al., 2004), antiviral (Sriram
et al., 2005), and CNS depressant (Knabe et al., 1983)
activities. Due to the remarkable importance of isoniazid
derivatives and its isomers, the aim of this article is to
synthesize novel compounds of isoniazid derivatives by
I. Mohanram (&)
Department of Chemistry, Rashtrasant Tukadoji Maharaj Nagpur
University, Nagpur 440033, Maharashtra, India
e-mail: ipsita.mohanram@gmail.com
J. Meshram
Department of Organic Chemistry, School of Chemical Sciences,
North Maharashtra University, Jalgaon 425001, India
123

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Med Chem Res (2014) 23:939–947
three-component Betti reaction using fluorite (Wada and
Suzuki, 2003) as a catalyst under ambient conditions and
evaluate for their potential anti-inflammatory and anthel-
mintic activities.
yield economy. Consequently, this is a reaction among
isoniazid, aldehydes, and phenols. Betti reaction is a spe-
cial case of Mannich condensation reaction (Mannich and
Krosche, 1912) which consists of an amino alkylation of an
acidic proton placed next to a carbonyl functional group
with aldehydes and ammonia or any primary or secondary
amine. The reaction proceeds by a nucleophilic addition of
an amine to a carbonyl group followed by dehydration to
the Schiff base. The Schiff base is an electrophile which
reacts in the second step in an electrophilic addition with a
compound containing an acidic proton. The structures of
The experimental protocol was approved by the Insti-
tutional Animal Ethics Committee (IAEC) as per the
guidelines of Committee for the Purpose of Control and
Supervision of Experiments on Animals (CPCSEA), Min-
istry of Social Justice and Empowerment, Government of
India. (Grant no. SPCP/2013/595).
compounds 4(a–j) were characterized from IR, 1H and 13
C
Results and discussion
NMR, elemental and mass spectral data. The IR spectra of
these compounds reveal a characteristic aromatic stretch
between 3010 and 3058 cm-1. Carbonyl (C=O) stretching
Chemistry
vibrations for amide were seen around 1640–1664 cm-1
.
In this paper, the isoniazid derivatives were synthesized by
using different aldehydes and varying phenols viz.
8-hydroxyquinoline and 2-naphthol (Scheme 1). In an
approach, 1 equiv of isoniazid, 1 equiv substituted alde-
hydes, and 1 equiv of phenols were dissolved in 95 % of
ethanol and magnetically stirred at room temperature in
presence of fluorite as a catalyst. The progress of the
reaction was duly checked by TLC. The products were
isolated by simple and usual work up with 80–92 % of
Stretching frequency vibrations between 3210 and
3330 cm-1 confirm the presence of phenolic O–H in the
structure. All other peaks are in well agreement with the
1
synthesized molecules. The H NMR spectra were recor-
ded in DMSO-d6 at room temperature using TMS as an
internal standard. The spectra showed characteristic singlet
around 5.14–5.24 ppm for –CH moiety in the structures.
Presence of singlet around 4.98–8.37 ppm reveals the
presence of phenolic O–H on the ring. The other signals
O
OH
N
HN NH
3a
H
OH
N
O
H2N
R
O
4 (a-e)
Fluorite
RT,
H
O
OH
15-20 min
N
N
R
N
HN NH
1
2
N
OH
3b
R
4 (f-j)
R
4a
4f
4g
4h
4i
3-NO2
4-Cl
4b
4c
4d
4e
4-OH
4-OCH3
4j
4-N(CH3)2
Scheme 1 Synthesis of isoniazid derivatives using Betti reaction with fluorite catalyst
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Med Chem Res (2014) 23:939–947
941
Table 1 Anti-inflammatory activity of compounds 4(a–j) using carrageenin-induced paw edema in Wistar albino rats
Test compounds
% Inhibition of paw edema at different time (h) intervalsa
250 mg/kg
0
1
3
5
Controlb
4a
26.4 0.5 (0.0)
22.3 0.3 (0.0)
18.4 0.2 (0.0)
14.5 0.6 (0.0)
37.5 0.3 (13.78)
14.3 0.4 (20.16)
13.1 0.1* (23.32)
14.2 0.6 (25.71)
33.1 0.4 (11.05)
32.6 0.5* (33.06)
13.5 0.7 (21.42)
12.6 0.6 (36.21)
14.6 0.8 (30.52)
32.9 0.1 (28.12)
11.7 0.3* (65.26)
34 0.1 (20.81)
12.5 0.2* (41.32)
12.9 0.3 (55.53)
13.6 0.3 (38.25)
32.6 0.5 (22.17)
31.2 0.4 (20.13)
12.5 0.5 (33.12)
11.3 0.4 (54.33)
12.9 0.6 (56.46)
31.6 0.4 (18.53)
10.3 0.1* (70.14)
32.1 0.5 (35.53)
10.6 0.2 (64.15)
11.7 0.5 (68.21)
12.2 0.8 (44.36)
34.1 0.2* (38.25)
30.5 0.9 (18.51)
11.6 0.7 (57.22)
10.8 0.7 (62.32)
10.6 0.3* (67.32)
35.3 0.6 (11.63)
8.61 0.2* (78.12)
31.9 0.3* (30.12)
7.67 0.3* (76.57)
10.8 0.2* (70.47)
10.8 0.9* (62.11)
33.8 0.7 (40.10)
31.4 0.4 (34.42)
10.3 0.6* (68.61)
9.23 0.2* (74.53)
8.20 0.08 (75.24)
30.5 0.1* (32.46)
5.51 0.6* (83.65)
4b
4c
4d
4e
4f
4g
4h
4i
4j
DLF
Standard drug diclofenac (DLF) was used at 10 mg/kg. % inhibition was denoted in bracket
* Significantly different from control at P \ 0.05
a
Results are expressed as mean SEM and compared with one-way ANOVA followed by Dunnett’s test, with the level of significance at
P \ 0.05
b
The group was injected with 1 ml of 0.5 % aqueous saline water
1
and peaks of H NMR and IR are in complete agreement
with the assigned structures. The mass spectra of these
compounds displayed a molecular ion peak at appropriate
m/z values. All the compounds have given the satisfactory
elemental analysis.
(Gbolade and Adeyemi, 2008). Anthelmintic activities of
all prototypes were tested in this bioassay at various con-
centrations of 25, 50, 100, and 150 mg/ml described in
Table 2. Compounds 4b and 4g had shown significant
activity at 25 mg/ml for time taken to paralysis and death
when compared to the standard drugs albendazole. At the
concentration of 50 mg/ml, compounds 4b, 4g, and 4e
exhibited their significant action for time taken to paralysis
and death. Compounds 4d and 4h showed their moderate
action for time taken to paralysis. While at 100 mg/ml,
compounds 4c and 4i significantly reduced the paralysis
and death time as well. Rather compounds 4b, 4c, and 4i
exhibited their highly significant action for time taken to
paralysis and death and which is almost an equipotent
action when compared to standard reference drug. How-
ever at higher concentration of 150 mg/ml compounds
4(a–j) showed time taken to paralysis and death was sig-
nificantly reducing.
In vivo anti-inflammatory investigation
Winter et al., (1962) method was used to induce inflam-
mation by injecting carrageenin in hind paw of Wistar
albino rats. Plethysmometer (Bhatt et al., 1977) was used to
measure increase in paw volume. Paw volume was recor-
ded at interval of 0, 1, 3, and 5 h illustrated in Table 1. At
initial hour, compounds 4c, 4g, and 4h have shown sig-
nificant anti-inflammatory activity compared with standard
reference drug, diclofenac. After 1, 3, and 5 h of oral
administration of synthesized agents, compounds 4b, 4c,
4d, 4g, 4h, and 4i have shown increase in inhibition of paw
edema. No significant activity was observed in compounds
4a, 4e, 4f, and 4j. Hence it has been interpreted from the
data obtained from Table 1 that moieties bearing –OH,
–OCH3, and –Cl functional groups at 3 or 4 positions
possess potential anti-inflammatory activity (Bukhari et al.,
2013) with respect to the standard drug, diclofenac.
Virtual screenings and molecular properties
calculations
Osiris calculations
Toxicity risks (mutagenicity, tumorigenicity, irritation, and
reproduction) and physico-chemical properties [clog P,
solubility, drug likeness, and drug score (DS)] of com-
pounds 4(a–j) were calculated by the methodology devel-
oped by Osiris (www.osiris.com). Toxicity risk prediction
In vitro anthelmintic investigation
The anthelmintic activity of the synthesized agents was
carried out on Indian earthworms, Pheretima posthuma
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Med Chem Res (2014) 23:939–947
Table 2 Anthelmintic activity of compounds 4(a–j) on Pheretima posthuma
Test
compounds
25 mg/ml
50 mg/ml
100 mg/ml
Time of
paralysis (min) death (min) paralysis (min) death (min) paralysis (min) death (min) paralysis (min) death (min)
150 mg/ml
Time of
Time of
Time of
Time of
Time of
Time of
Time of
Control
ALB
4a
–
–
–
–
–
–
–
–
15 0.3
20 0.9*
16 1.1
18 0.5
22 0.3
17 1.6*
19 1.3
15 0.2*
21 1.5
17 0.4
20 1.2
15 0.6
21 0.3
16 1.6
20 0.8*
24 1.4
18 0.5
21 1.0
15 1.1
22 0.4
17 1.4
20 1.0*
13 0.5
17 0.8
14 0.3
16 1.0
20 0.2
15 0.2
18 0.5*
14 1.0
18 0.2
16 0.5
18 0.2
14 0.8
18 1.4
14 1.2
16 1.6
20 0.3*
15 1.1
20 0.9
14 1.8*
19 0.6
16 0.7
18 1.4
11 1.3
15 1.2
12 0.6
13 0.4
18 0.5
14 0.7*
17 0.5
13 1.5
17 0.3
13 0.1*
16 0.7
12 0.6
16 0.7*
13 0.5
15 1.6*
18 1.2
15 1.0*
18 0.6
14 0.2
18 0.4
13 1.2
16 0.1*
10 0.5
13 0.4
11 0.9*
10 0.8
15 0.1
12 0.8
16 0.2*
12 0.8
15 0.6
11 0.7
15 1.0
10 1.4
14 1.0
12 0.1
11 0.3*
15 1.0
13 0.2
16 0.9
12 1.2*
17 0.1
11 1.2
16 0.9
4b
4c
4d
4e
4f
4g
4h
4i
4j
Standard drug albendazole (ALB) was used at 20 mg/ml. Results are expressed as mean SEM and compared with one-way ANOVA followed
by Dunnett’s test, with the level of significance at P \ 0.05. ‘–’ indicates absence of activity in 24 h of administration
* Significantly different from ALB at P \ 0.05
Table 3 Osiris calculation of compounds 4(a–j)
Compounds
Toxicity risk
MUT
Molecular properties calculation
TUMO
IRRI
REP
CLP
Log S
MW
DL
DS
4a
?
?
?
?
?
?
-
-
-
-
-
?
?
?
?
?
?
-
-
-
-
?
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
?
?
3.29
4.03
3.12
3.31
3.42
2.42
3.16
2.25
2.44
2.55
4.4
-5.42
-5.69
-4.66
-4.97
-4.99
-4.52
-4.79
-3.76
-4.07
-4.09
-4.64
-4.1
414
403
385
399
412
415
404
386
400
413
295
265
-5.32
1.64
0.18
0.3
4b
4c
0.55
0.35
0.31
0.19
0.3
4d
4e
0.35
-0.76
-2.11
4.81
4f
4g
0.63
0.76
0.72
0.41
0.36
0.15
4h
4i
3.74
3.53
4j
2.42
DLF
ALB
2.06
3.48
-2.11
DLF diclofenac, ALB albendazole, MUT mutagenic, TUMO tumorigenic, IRRI irritant, REP reproductive effective, MW molecular weight in
g/mol, CLP c log P, S solubility, DL drug likeness, DS drug score
indicates that compounds may be harmful concerning the
risk category specified. The Osiris calculations were tab-
ulated in Table 3. Osiris data indicate that all compounds
are non-mutagenic and non-tumorigenic, non-irritating
with no reproductive effects when run through the muta-
genicity assessment system comparable with standard
drugs used except 4(a–e) which show mild mutagenic and
tumorigenic effects. Also compound 4f shows mild muta-
genicity while compound 4j shows mild tumorigenic
effect. The hydrophilicity of a compound is calculated by
clog P value which is the logarithm of its partition
coefficient between n-octanol and water. The clog P value
of a compound must not be greater than 5.0 to have a
probability of being well absorbed. It is evident from
Table 3, all compounds 4(a–j) have clog P values under
tolerable limits. Log S value is the logarithm of the solu-
bility of a compound measured in mol/liter. Log S value
should be greater than -4 for better solubility of a com-
pound which significantly affects its absorption and dis-
tribution characteristics of the drug. Our calculated log
S values for all compounds are greater than -4 except for
compound 4h. Drug likeness of compounds 4(a–j) are in
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Med Chem Res (2014) 23:939–947
943
the comparable zone with that of standard drugs used. The
overall calculated DS for compounds 4(a–j) matches with
the standard drug used in this study. Drug likeness, clog P,
log S, molecular weight, and toxicity risks together con-
stitutes to the DS. DS is calculated by the following
equation:
Table 4 Molinspiration bioactivity score of compounds 4(a–j)
Compounds GPCRL ICM KI NRL PI EI
4a
-0.21
-0.08
-0.06
-0.11
-0.07
-0.21
-0.08
-0.06
-0.11
-0.08
0.14
-0.27 -0.18 -0.34 -0.24 -0.17
-0.23 -0.08 -0.28 -0.15 -0.09
-0.20 -0.04 -0.22 -0.10 -0.04
-0.27 -0.09 -0.28 -0.15 -0.09
-0.23 -0.04 -0.24 -0.14 -0.08
-0.23 -0.15 -0.49 -0.19 -0.10
-0.19 -0.05 -0.44 -0.11 -0.02
4b
4c
ꢀ
ꢁ
4d
4e
1
2
1
2
DS ¼ P
S ¼
þ
Si ꢀ Pti
ð1Þ
ð2Þ
4f
1
4g
1 þ eapþb
4h
4i
-0.18 -0.01 -0.37 -0.07
0.02
-0.24 -0.06 -0.43 -0.11 -0.03
-0.20 -0.00 -0.39 -0.10 -0.01
where, P is drug likeness, Si is calculated from clog P,
log S, molecular weight, and pi using equation (2). a and
b are (1, -5), (1, 5), (0.012, -6) and (1, 0) for clog P, log
S, molecular weight and drug likeness, respectively. p is
the potency of the drug candidate. Higher potency is
desirable in a drug as it reduces the risk of non-specific,
off-target pharmacology at a given concentration. ti is
calculated from the mutagenicity, tumorigenicity, irritant,
and reproductive effects. The ti values assigned 1.0 to no
risk, 0.8 to medium risk, and 0.6 to higher risk for assessing
toxicity risk of compounds. The reported compounds 4(a–
j) showed moderate to good DS as compared with standard
drugs used. Lipinski formulated a molecular descriptor,
Rule of five (Lipinski et al. 2001) which states that most
drug-like molecules have partition coefficient, log P B 5,
molecular weight B500, number of hydrogen bond
acceptors B10, and number of hydrogen bond donors B5.
Molecules violating more than one of these rules may have
problems with bioavailability (Veber et al. 2002). It is
evident from Table 3 that all the synthesized compounds
does not violate the Lipinski’s rule of five and proved to be
significant biological moieties.
4j
DLF
ALB
0.20
0.17 -0.09 -0.10
0.25
-0.11
-0.10 -0.04 -0.62 -0.18 -0.02
DLF diclofenac, ALB albendazole, GPCRL GPCR ligand, ICM ion
channel modulator, KI kinase inhibitor, NRL nuclear receptor ligand,
PI protease inhibitor, EI enzyme inhibitor
the diclofenac score whereas when compared with alben-
dazole score all drug classes show similar score except
score of nuclear receptor ligand. Consequently, this study
shows the probability that the synthesized compounds
possess similar structural feature with that of albendazole
while only few structural features of synthesized com-
pounds are similar to diclofenac.
Conclusions
In conclusion, we have successfully achieved a rapid and
facile synthesis of biologically potent novel isoniazid deriv-
atives via multicomponent Betti reaction using fluorite as
catalyst. The screening results of anti-inflammatory and
anthelmintic activities revealed that the compounds 4b, 4c,
4h, and 4i possess significant anti-inflammatory activity when
compared with standard drug, diclofenac. However com-
pounds 4b, 4c, and 4i were found to be equally potent
anthelmintic when comparable with standard drug, albenda-
zole. A correlation of structure and activity relationship of
compounds with respect to Lipinski’s rule of five, drug like-
ness, toxicity tolerance, and other physico-chemical proper-
ties of drugs was further proved that the synthesized
compounds were in the tolerable criteria except compound
4(a–e) which shows mild mutagenic and tumorigenic effects.
Thus in vivo and in vitro studies revealed that the synthesized
compounds possess potential anti-inflammatory and anthel-
mintic activities with respect to the standard drugs used.
Moreover theoretical physico-chemical studies showed mild
mutagenic and tumorigenic effects in few of the synthesized
whereas rest of the parameters is in tolerable limits.
Molinspiration bioactivity prediction
A probable prediction of bioactivity or drug likeness of
compounds 4(a–j) was calculated by using online molin-
spiration software program (www.molinspiration.com) and
compared the activity with the values obtained from stan-
dard drug used in this study viz., diclofenac and albenda-
zole. Drug likeliness is a qualitative means of analysis to
check whether the compound is similar to the known drug.
Activity of all the ten compounds and standard drugs was
rigorously analyzed under six criteria of drug classes such
as GPCR ligand, ion channel modulation, kinase inhibition,
nuclear receptor ligand, protease inhibitor, and enzyme
inhibition activities. Results are shown in Table 4 by
means of numerical assignment. Only nuclear receptor
ligand score and protease inhibitor score of all test com-
pounds were similar to diclofenac score. Rest of the drug
classes score of the test compounds does not matches with
123

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Med Chem Res (2014) 23:939–947
Experimental section
to study the anthelmintic activity. The earthworms were
washed with normal saline to remove all the fecal matter
surrounding their body. The earthworms are 3–5 cm in
length and 0.1–0.2 cm in width were used for all experi-
mental protocols. The worms were divided into six earth-
worms in each group of ten compounds, control and
standard drug. All compounds and standard drug solution
were freshly prepared before start of the experiments.
Albendazole solution was used as reference standard drug
and saline water as control. The test compounds 4(a–j) and
albendazole were dissolved in minimum quantity of 2 %
dimethyl sulfoxide (DMSO) and the volume was adjusted
to 20 ml with saline water for making the concentration of
25, 50, 100, and 150 mg/ml. The earthworms were
observed for their spontaneous motility and evoked
responses. Observations were made for time taken to
paralysis and death of individual worms. Paralysis was said
to occur when the worms do not revive in saline water.
Death was concluded when the worms lost their motility
followed with fading away of their body color. All the
results were expressed as mean SEM. Statistical signif-
icance was determined by ANOVA followed by Dunnett’s
test, with the level of significance at P \ 0.05.
General
All the reagents and solvents used are of analytical grade and
purchased from a commercial source and used directly. All
the melting points were determined by open tube capillaries
method and are uncorrected. The purity of compounds was
checked routinely by TLC (0.5 mm thickness) using silica
gel-G coated Al-plates (Merck) and spots were visualized by
exposing the dry plates in iodine vapors. IR spectra (tmax in
cm-1) were recorded on a Schimadzu-IR Prestige 21 spec-
trometer using KBr technique; H NMR spectra and 13C
1
NMR spectra of the synthesized compounds were recorded
on
a Bruker-Avance II (400 MHz), Varian-Gemini
(100 MHz) spectrophotometer using DMSO-d6 solvent and
TMS as an internal standard. Mass spectra were recorded on
a Micromass Q–T of high-resolution mass spectrometer
equipped with electrospray ionization (ESI) on Masslynx 4.0
data acquisition system. A hexapole collision cell, between
the two mass analyzers, is used to induce fragmentation to
study the structural investigations while using instrument in
MS/MS mode. The elemental analysis (C, H, N, and S) of
compounds was performed on Carlo Erba-1108 elemental
analyzer. The results were found to be in good agreement
with the calculated values.
Protocol for the synthesis of isoniazid derivatives using
Betti reaction 4(a–j)
Protocol for in vivo anti-inflammatory investigation
A mixture of substituted aldehyde (0.01 mol) 1, isoniazid
(0.01 mol) 2, and phenols (0.01 mol) 3 was dissolved in
10 ml of 95 % ethanol in one-pot and was magnetically
stirred at room temperature in presence of fluorite catalyst
(2 % weight with respect to all reactants) Scheme 1. The
reaction mixture was stirred for 15–20 min. The comple-
tion of the reaction was monitored by TLC (Merck Silica
gel 60F254) by using mixture of ethyl acetate and hexane
as mobile phase. After completion, the reaction mixture
was poured into crushed ice. The crude product and cata-
lyst was collected on a Buchner funnel by filtration. The
crude product was purified by recrystallization from hot
ethanol to get pure product. All products were character-
ized by elemental analysis, IR, 1H and 13C NMR, and mass
spectral data. The following are the characterization data of
the synthesized compounds.
Wistar albino rats were used to investigate anti-inflammatory
activity of synthesized agents. Rats of either sex weighing
between 150 and 200 g were used for the present study. A
test containing ten compounds, control and a standard drug
was performed in a group of six animals each. Freshly pre-
pared 0.1 ml of 1 % carrageenin was used to induce edema
into the left hind limb of each rat under the subplantar apo-
neurosis. 250 mg/kg of the synthesized drugs and 10 mg/kg
reference drug, diclofenac, were orally administered to each
rat. Normal saline water was used as control. Measurement
of paw volume was done by means of volume displacement
technique using plethysmometer. Paw volume was recorded
at the interval of 0, 1, 3, and 5 h after carrageenin injection.
Results were expressed as an increase in paw volume in
comparison to the initial paw volumes and in comparison
with control group. All the results were expressed as
mean SEM. Statistical significance was determined by
one-way analysis of variance (ANOVA) followed by Dun-
nett’s test with the level of significance at P \ 0.05.
N0-((2-Hydroxynaphthalen-1-yl)(3-
nitrophenyl)methyl)isonicotinohydrazide (4a)
Pale yellow crystals; Yield 92 %; Mp. 165–167 °C; FTIR
(cm-1): 3218 (–OH str.), 3430 (–NH str.), 3010 (Ar–H
str.), 1640 (C=O str.), 1615 (C=N str.), 1520 (–NO2 str.);
1H NMR (DMSO-d6, ppm): 5.04 (s, 1H, OH), 5.22 (s, 1H,
CH), 3.15 (s, 1H, NH), 8.12 (s, 1H, CONH), 6.84–7.25 (m,
6H, Ar–H), 6.40–7.86 (m, 4H, Ar–H), 7.91–8.91 (m, 4H,
Protocol for in vitro anthelmintic investigation
Adult Indian earthworms of the genus and species,
Pheretima posthuma (family: Megascolecidae), were used
123

Med Chem Res (2014) 23:939–947
945
Ar–H); 13C NMR (DMSO-d6, ppm): 126.7 (C1, 2-naph-
thol), 125.5 (C2, 2-naphthol), 128.4 (C3, 2-naphthol),
128.6 (C4, 2-naphthol), 127.6 (C5, 2-naphthol), 119.4 (C6,
2-naphthol), 154.2 (C7, 2-naphthol), 115.4 (C8, 2-naph-
thol), 134.2 (C9, 2-naphthol), 123.5 (C10, 2-naphthol),
52.6 (C, Methine), 144.6 (C1, Phenyl), 124.2 (C2, Phenyl),
149.5 (C3, Phenyl), 118.4 (C4, Phenyl), 131.4 (C5, Phe-
nyl), 133.8 (C6, Phenyl), 163.9 (C, Amide), 141.3 (C1,
Pyridine), 123.5 (C2, Pyridine), 151.4 (C3, Pyridine), 150.6
(C4, Pyridine), 123.8 (C5, Pyridine); MS: 414.13 (M?,
100 %); Anal. Calcd: C23H18N4O4: C, 67.56; H, 4.51; N,
14.15. Found: C, 67.55; H, 4.51; N, 14.18.
123.8 (C6, Phenyl), 164.6 (C, Amide), 141.5 (C1, Pyri-
dine), 123.8 (C2, Pyridine), 150.1 (C3, Pyridine), 150.6
(C4, Pyridine), 123.6 (C5, Pyridine); MS: 385.14 (M?,
100 %). Anal. Calcd: C23H19N3O3: C, 70.36; H, 4.76; N,
10.75. Found: C, 70.38; H, 4.79; N, 10.71.
N0-((2-Hydroxynaphthalen-1-yl)(4-
methoxyphenyl)methyl)isonicotinohydrazide (4d)
Pale yellow crystals; Yield 80 %; Mp. 151–153 °C; FTIR
(cm-1): 3216 (–OH str.), 3431 (–NH str.), 3012 (Ar–H
str.), 1656 (C=O str.), 1616 (C=N str.), 2820 (–OCH3 str.);
1H NMR (DMSO-d6, ppm): 4.98 (s, 1H, OH), 5.21 (s, 1H,
CH), 3.12 (s, 1H, NH), 8.23 (s, 1H, CONH), 3.75 (s, 3H,
OCH3), 6.67–7.59 (m, 6H, Ar–H), 6.80–7.12 (m, 4H, Ar–
H), 7.72–8.65 (m, 4H, Ar–H); 13C NMR (DMSO-d6, ppm):
126.6 (C1, 2-naphthol), 123.8 (C2, 2-naphthol), 128.1 (C3,
2-naphthol), 128.8 (C4, 2-naphthol), 127.5 (C5, 2-naph-
thol), 118.5 (C6, 2-naphthol), 154.6 (C7, 2-naphthol),
116.7 (C8, 2-naphthol), 133.5 (C9, 2-naphthol), 129.3
(C10, 2-naphthol), 52.2 (C, Methine), 135.6 (C1, Phenyl),
129.8 (C2, Phenyl), 115.5 (C3, Phenyl), 158.4 (C4, Phe-
nyl), 56.7 (C, Methoxy), 115.4 (C5, Phenyl), 123.2 (C6,
Phenyl), 163.6 (C, Amide), 140.7 (C1, Pyridine), 122.6
(C2, Pyridine), 151.3 (C3, Pyridine), 150.5 (C4, Pyridine),
123.3 (C5, Pyridine); MS: 399.16 (M?, 100 %); Anal.
Calcd: C24H21N3O3: C, 73.12; H, 5.26; N, 10.55. Found: C,
73.18; H, 5.28; N, 10.51.
N0-((2-Hydroxynaphthalen-1-yl)(4-
chlorophenyl)methyl)isonicotinohydrazide (4b)
Pale yellow crystals; Yield 88 %; Mp. 154–156 °C; FTIR
(cm-1): 3210 (–OH str.), 3433 (–NH str.), 3015 (Ar–H
1
str.), 1648 (C=O str.), 1620 (C=N str.), 721 (C–Cl str.). H
NMR (DMSO-d6, ppm): 5.08 (s, 1H, OH), 5.20 (s, 1H,
CH), 3.10 (s, 1H, NH), 8.16 (s, 1H, CONH), 6.81–7.29 (m,
6H, Ar–H), 6.05–7.16 (m, 4H, Ar–H), 7.85–8.81 (m, 4H,
Ar–H); 13C NMR (DMSO-d6, ppm): 126.9 (C1, 2-naph-
thol), 124.4 (C2, 2-naphthol), 128.1 (C3, 2-naphthol),
128.9 (C4, 2-naphthol), 127.5 (C5, 2-naphthol), 119.2 (C6,
2-naphthol), 154.8 (C7, 2-naphthol), 116.1 (C8, 2-naph-
thol), 134.6 (C9, 2-naphthol), 123.7 (C10, 2-naphthol),
51.9 (C, Methine), 141.6 (C1, Phenyl), 128.7 (C2, Phenyl),
129.5 (C3, Phenyl), 132.6 (C4, Phenyl), 130.2 (C5, Phe-
nyl), 130.8 (C6, Phenyl), 163.5 (C, Amide), 141.4 (C1,
Pyridine), 123.3 (C2, Pyridine), 150.7 (C3, Pyridine), 149.2
(C4, Pyridine), 123.5 (C5, Pyridine); MS: 403.11 (M?,
100 %). Anal. Calcd: C23H18ClN3O2: C, 67.46; H, 4.53; N,
10.45. Found: C, 68.48; H, 4.50; N, 10.43.
N0-((2-Hydroxynaphthalen-1-yl)(4-
(dimethylamino)phenyl)methyl)isonicotinohydrazide (4e)
Pale yellow crystals; Yield 84 %; Mp. 162–164 °C; FTIR
(cm-1): 3213 (–OH str.), 3432 (–NH str.), 3022 (Ar–H
str.), 1645 (C=O str.), 1618 (C=N str.), 2870 (–CH3 str.).
1H NMR (DMSO-d6, ppm): 5.05 (s, 1H, OH), 5.18 (s, 1H,
CH), 3.22 (s, 1H, NH), 8.10 (s, 1H, CONH), 2.88 (s, 6H,
N(CH3)2), 6.64–7.15 (m, 6H, Ar–H), 7.41–7.78 (m, 4H,
Ar–H), 7.93–8.82 (m, 4H, Ar–H); 13C NMR (DMSO-d6,
ppm): 126.4 (C1, 2-naphthol), 123.7 (C2, 2-naphthol),
128.5 (C3, 2-naphthol), 128.9 (C4, 2-naphthol), 127.4 (C5,
2-naphthol), 119.1 (C6, 2-naphthol), 153.8 (C7, 2-naph-
thol), 116.7 (C8, 2-naphthol), 134.5 (C9, 2-naphthol),
122.9 (C10, 2-naphthol), 50.8 (C, Methine), 132.5 (C1,
Phenyl), 129.7 (C2, Phenyl), 116.3 (C3, Phenyl), 148.8
(C4, Phenyl), 42.4 (C5, Methyl), 42.4 (C6, Methyl), 116.6
(C7, Phenyl), 130.1 (C8, Phenyl), 164.5 (C, Amide), 141.7
(C1, Pyridine), 123.2 (C2, Pyridine), 149.5 (C3, Pyridine),
150.1 (C4, Pyridine), 122.8 (C5, Pyridine); MS: 412.19
(M?, 100 %). Anal. Calcd: C25H24N4O2: C, 73.06; H, 5.72;
N, 13.45. Found: C, 73.08; H, 5.80; N, 13.49.
N’-((2-Hydroxynaphthalen-1-yl)(4-
Hydroxyphenyl)methyl)isonicotinohydrazide (4c)
Pale yellow crystals; Yield 91 %; Mp. 146–148 °C; FTIR
(cm-1): 3220 (–OH str.), 3428 (–NH str.), 3028 (Ar–H
1
str.), 1652 (C=O str.), 1610 (C=N str.). H NMR (DMSO-
d6, ppm): 5.05 (s, 1H, OH), 5.19 (s, 1H, CH), 3.16 (s, 1H,
NH), 8.14 (s, 1H, CONH), 6.82–7.60 (m, 6H, Ar–H),
6.63–6.82 (m, 4H, Ar–H), 7.83–8.75 (m, 4H, Ar–H); 13C
NMR (DMSO-d6, ppm): 127.2 (C1, 2-naphthol), 123.5
(C2, 2-naphthol), 127.9 (C3, 2-naphthol), 128.2 (C4,
2-naphthol), 127.1 (C5, 2-naphthol), 118.3 (C6, 2-naph-
thol), 154.7 (C7, 2-naphthol), 116.2 (C8, 2-naphthol),
133.8 (C9, 2-naphthol), 129.5 (C10, 2-naphthol), 51.3 (C,
Methine), 134.8 (C1, Phenyl), 129.1 (C2, Phenyl), 119.5
(C3, Phenyl), 157.4 (C4, Phenyl), 119.8 (C5, Phenyl),
123

946
Med Chem Res (2014) 23:939–947
N0-((8-Hydroxyquinolin-7-yl)(3-
NMR (DMSO-d6, ppm): 152.3 (C1, Quinoline), 121.5 (C2,
Quinoline), 136.2 (C3, Quinoline), 128.5 (C4, Quinoline),
120.8 (C5, Quinoline), 128.6 (C6, Quinoline), 121.2 (C7,
Quinoline), 148.9 (C8, Quinoline), 137.5 (C9, Quinoline),
53.6 (C, Methine), 136.3 (C1, Phenyl), 128.9 (C2, Phenyl),
117.6 (C3, Phenyl), 156.3 (C4, Phenyl), 116.5 (C5, Phe-
nyl), 128.8 (C6, Phenyl), 164.7 (C, Amide), 141.5 (C1,
Pyridine), 123.4 (C2, Pyridine), 150.5 (C3, Pyridine), 150.8
(C4, Pyridine), 122.6 (C5, Pyridine); MS: 386.14 (M?,
100 %). Anal. Calcd: C22H18N4O3: C, 68.15; H, 4.66; N,
14.45. Found: C, 68.25; H, 4.71; N, 14.48.
nitrophenyl)methyl)isonicotinohydrazide (4f)
Yellow crystals; Yield 92 %; Mp. 143–145 °C; FTIR
(cm-1): 3315 (–OH str.), 3410 (–NH str.), 3052 (Ar–H
str.), 1650 (C=O str.), 1621 (C=N str.), 1580 (–NO2 str.).
1H NMR (DMSO-d6, ppm): 8.36 (s, 1H, OH), 5.24 (s, 1H,
CH), 3.18 (s, 1H, NH), 8.12 (s, 1H, CONH), 7.12–8.65 (m,
5H, Ar–H), 7.11–7.16 (m, 4H, Ar–H), 7.81–8.72 (m, 4H,
Ar–H); 13C NMR (DMSO-d6, ppm): 151.4 (C1, Quino-
line), 122.6 (C2, Quinoline), 134.3 (C3, Quinoline), 127.7
(C4, Quinoline), 121.6 (C5, Quinoline), 129.2 (C6, Quin-
oline), 122.5 (C7, Quinoline), 149.4 (C8, Quinoline), 138.4
(C9, Quinoline), 51.8 (C, Methine), 143.1 (C1, Phenyl),
124.5 (C2, Phenyl), 148.6 (C3, Phenyl), 119.8 (C4, Phe-
nyl), 130.5 (C5, Phenyl), 134.7 (C6, Phenyl), 165.1 (C,
Amide), 142.6 (C1, Pyridine), 121.3 (C2, Pyridine), 149.8
(C3, Pyridine), 149.6 (C4, Pyridine), 121.8 (C5, Pyridine);
MS: 415.13 (M?, 100 %). Anal. Calcd: C22H17N5O4: C,
63.56; H, 4.26; N, 16.75. Found: C, 63.58; H, 4.21; N,
16.79.
N0-((8-Hydroxyquinolin-7-yl)(4-
methoxyphenyl)methyl)isonicotinohydrazide (4i)
Pale yellow crystals; Yield 85 %; Mp. 169–171 °C; FTIR
(cm-1): 3320 (–OH str.), 3413 (–NH str.), 3051 (Ar–H
str.), 1660 (C=O str.), 1625 (C=N str.), 2822 (–OCH3 str.).
1H NMR (DMSO-d6): 8.32 (s, 1H, OH), 5.14 (s, 1H, CH),
3.11 (s, 1H, NH), 8.20 (s, 1H, CONH), 3.78 (s, 3H, OCH3),
7.63–7.85 (m, 5H, Ar–H), 6.46–7.32 (m, 4H, Ar–H),
7.75–8.65 (m, 4H, Ar–H); 13C NMR (DMSO-d6, ppm):
152.2 (C1, Quinoline), 121.5 (C2, Quinoline), 135.7 (C3,
Quinoline), 127.3 (C4, Quinoline), 121.8 (C5, Quinoline),
129.5 (C6, Quinoline), 122.4 (C7, Quinoline), 149.5 (C8,
Quinoline), 137.4 (C9, Quinoline), 52.7 (C, Methine),
135.8 (C1, Phenyl), 129.6 (C2, Phenyl), 115.5 (C3, Phe-
nyl), 158.5 (C4, Phenyl), 56.3 (C5, Methoxy), 115.8 (C6,
Phenyl), 129.7 (C7, Phenyl), 164.5 (C, Amide), 141.3 (C1,
Pyridine), 123.2 (C2, Pyridine), 151.6 (C3, Pyridine), 151.8
(C4, Pyridine), 122.4 (C5, Pyridine); MS: 400.15 (M?,
100 %). Anal. Calcd: C23H20N4O3: C, 69.02; H, 5.11; N,
14.06. Found: C, 69.03; H, 5.09; N, 14.02.
N0-((8-Hydroxyquinolin-7-yl)(4-
chlorophenyl)methyl)isonicotinohydrazide (4g)
Pale yellow crystals; Yield 90 %; Mp. 158–160 °C; FTIR
(cm-1): 3328 (–OH str.), 3418 (–NH str.), 3056 (Ar–H
1
str.), 1655 (C=O str.), 1628 (C=N str.), 720 (C–Cl str.). H
NMR (DMSO-d6, ppm): 8.33 (s, 1H, OH), 5.16 (s, 1H,
CH), 3.21 (s, 1H, NH), 8.18 (s, 1H, CONH), 7.14–8.85 (m,
5H, Ar–H), 7.14–7.21 (m, 4H, Ar–H), 7.78–8.81 (m, 4H,
Ar–H); 13C NMR (DMSO-d6, ppm): 150.6 (C1, Quino-
line), 121.5 (C2, Quinoline), 135.8 (C3, Quinoline), 127.3
(C4, Quinoline), 120.8 (C5, Quinoline), 128.5 (C6, Quin-
oline), 121.7 (C7, Quinoline), 148.3 (C8, Quinoline), 137.5
(C9, Quinoline), 52.7 (C, Methine), 142.8 (C1, Phenyl),
130.4 (C2, Phenyl), 129.6 (C3, Phenyl), 132.6 (C4, Phe-
nyl), 130.5 (C5, Phenyl), 129.5 (C6, Phenyl), 165.6 (C,
Amide), 141.3 (C1, Pyridine), 122.5 (C2, Pyridine), 152.3
(C3, Pyridine), 151.2 (C4, Pyridine), 121.4 (C5, Pyridine);
MS: 404.1 (M?, 100 %). Anal. Calcd: C22H17ClN4O2: C,
66.13; H, 4.26; N, 13.85. Found: C, 66.08; H, 4.21; N,
13.81.
N0-((8-Hydroxyquinolin-7-yl)(4-
(dimethylamino)phenyl)methyl)isonicotinohydrazide (4j)
Yellow crystals; Yield 87 %; Mp. 174–176 °C; FTIR
(cm-1): 3325 (–OH str.), 3416 (–NH str.), 3054 (Ar–H
str.), 1653 (C=O str.), 1626 (C=N str.), 2870 (–CH3 str.).
1H NMR (DMSO-d6, ppm): 8.37 (s, 1H, OH), 5.19 (s, 1H,
CH), 3.18 (s, 1H, NH), 8.12 (s, 1H, CONH), 2.87 (s, 6H,
N(CH3)2), 7.12–8.73 (m, 5H, Ar–H), 6.40–7.16 (m, 4H,
Ar–H), 7.91–8.83 (m, 4H, Ar–H); 13C NMR (DMSO-d6,
ppm): 152.7 (C1, Quinoline), 120.7 (C2, Quinoline), 135.9
(C3, Quinoline), 127.3 (C4, Quinoline), 120.5 (C5, Quin-
oline), 129.1 (C6, Quinoline), 121.5 (C7, Quinoline), 148.5
(C8, Quinoline), 138.2 (C9, Quinoline), 52.6 (C, Methine),
133.4 (C1, Phenyl), 129.3 (C2, Phenyl), 116.4 (C3, Phe-
nyl), 148.7 (C4, Phenyl), 42.3 (C5, Methyl), 42.3 (C6,
Methyl), 116.6 (C7, Phenyl), 130.2 (C8, Phenyl), 165.4 (C,
Amide), 141.5 (C1, Pyridine), 123.6 (C2, Pyridine), 150.8
N0-((8-Hydroxyquinolin-7-yl)(4-
hydroxyphenyl)methyl)isonicotinohydrazide (4h)
Pale yellow crystals; Yield 83 %; Mp. 155–157 °C; FTIR
(cm-1): 3330 (–OH str.), 3415 (–NH str.), 3058 (Ar–H
1
str.), 1664 (C=O str.), 1623 (C=N str.). H NMR (DMSO-
d6, ppm): 8.34 (s, 1H, OH), 5.20 (s, 1H, CH), 3.15 (s, 1H,
NH), 8.14 (s, 1H, CONH), 7.18–8.55 (m, 5H, Ar–H),
6.64–6.87 (m, 4H, Ar–H), 7.82–8.74 (m, 4H, Ar–H); 13C
123

Med Chem Res (2014) 23:939–947
947
antimalarial agents active against CQ resistant P. falciparum
strains. Bioorg Med Chem Lett 16:5384–5388
(C3, Pyridine), 151.3 (C4, Pyridine), 123.2 (C5, Pyridine);
MS: 413.19 (M?, 100 %). Anal. Calcd: C24H23N5O2: C,
69.62; H, 5.51; N, 16.86. Found: C, 69.66; H, 5.59; N,
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Acknowledgments The authors are thankful to Rajiv Gandhi
National Fellowship [RGNF-SC-UTT-2299], University Grants
Commission, New Delhi for financial support. The authors thank
Head, Department of Chemistry, RTM, Nagpur University for pro-
viding laboratory facilities, Director, SAIF, Chandigarh for spectral
data and Head, Sharad Pawar College of Pharmacy, RTM Nagpur
University for assistance in biological screening.
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