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

ORIGINAL RESEARCH

Design, synthesis, and evaluation of isoniazid derivatives actingas potent anti-inflammatory and anthelmintic agents via Bettireaction

Ipsita Mohanram • Jyotsna Meshram

Received: 13 March 2013 / Accepted: 6 August 2013 / Published online: 21 August 2013

� 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

were characterized on the basis of 1H 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

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).

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: [email protected]

J. Meshram

Department of Organic Chemistry, School of Chemical Sciences,

North Maharashtra University, Jalgaon 425001, India

123

Med Chem Res (2014) 23:939–947

DOI 10.1007/s00044-013-0693-2

MEDICINALCHEMISTRYRESEARCH

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.

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).

Results and discussion

Chemistry

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

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

compounds 4(a–j) were characterized from IR, 1H and 13C

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

vibrations for amide were seen around 1640–1664 cm-1.

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

synthesized molecules. The 1H 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

FluoriteH

O

N

OHN

H2N

OH

N

OH

RRT,

OH

HN NH

O

N

R

OH

HN NH

O

N

R

N

1 2

3a

3b

4 (f-j)

4 (a-e)

15-20 min

R

4a 4f 3-NO2

4b 4g 4-Cl

4c 4h 4-OH

4d 4i 4-OCH3

4e 4j 4-N(CH3)2

Scheme 1 Synthesis of isoniazid derivatives using Betti reaction with fluorite catalyst

940 Med Chem Res (2014) 23:939–947

123

and peaks of 1H 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.

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.

In vitro anthelmintic investigation

The anthelmintic activity of the synthesized agents was

carried out on Indian earthworms, Pheretima posthuma

(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.

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

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 26.4 ± 0.5 (0.0) 22.3 ± 0.3 (0.0) 18.4 ± 0.2 (0.0) 14.5 ± 0.6 (0.0)

4a 37.5 ± 0.3 (13.78) 34 ± 0.1 (20.81) 32.1 ± 0.5 (35.53) 31.9 ± 0.3* (30.12)

4b 14.3 ± 0.4 (20.16) 12.5 ± 0.2* (41.32) 10.6 ± 0.2 (64.15) 7.67 ± 0.3* (76.57)

4c 13.1 ± 0.1* (23.32) 12.9 ± 0.3 (55.53) 11.7 ± 0.5 (68.21) 10.8 ± 0.2* (70.47)

4d 14.2 ± 0.6 (25.71) 13.6 ± 0.3 (38.25) 12.2 ± 0.8 (44.36) 10.8 ± 0.9* (62.11)

4e 33.1 ± 0.4 (11.05) 32.6 ± 0.5 (22.17) 34.1 ± 0.2* (38.25) 33.8 ± 0.7 (40.10)

4f 32.6 ± 0.5* (33.06) 31.2 ± 0.4 (20.13) 30.5 ± 0.9 (18.51) 31.4 ± 0.4 (34.42)

4g 13.5 ± 0.7 (21.42) 12.5 ± 0.5 (33.12) 11.6 ± 0.7 (57.22) 10.3 ± 0.6* (68.61)

4h 12.6 ± 0.6 (36.21) 11.3 ± 0.4 (54.33) 10.8 ± 0.7 (62.32) 9.23 ± 0.2* (74.53)

4i 14.6 ± 0.8 (30.52) 12.9 ± 0.6 (56.46) 10.6 ± 0.3* (67.32) 8.20 ± 0.08 (75.24)

4j 32.9 ± 0.1 (28.12) 31.6 ± 0.4 (18.53) 35.3 ± 0.6 (11.63) 30.5 ± 0.1* (32.46)

DLF 11.7 ± 0.3* (65.26) 10.3 ± 0.1* (70.14) 8.61 ± 0.2* (78.12) 5.51 ± 0.6* (83.65)

Standard drug diclofenac (DLF) was used at 10 mg/kg. % inhibition was denoted in bracket

* Significantly different from control at P \ 0.05a Results are expressed as mean ± SEM and compared with one-way ANOVA followed by Dunnett’s test, with the level of significance at

P \ 0.05b The group was injected with 1 ml of 0.5 % aqueous saline water

Med Chem Res (2014) 23:939–947 941

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http://www.osiris.com

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

Table 2 Anthelmintic activity of compounds 4(a–j) on Pheretima posthuma

Test

compounds

25 mg/ml 50 mg/ml 100 mg/ml 150 mg/ml

Time of

paralysis (min)

Time of

death (min)

Time of

paralysis (min)

Time of

death (min)

Time of

paralysis (min)

Time of

death (min)

Time of

paralysis (min)

Time of

death (min)

Control – – – – – – – –

ALB 15 ± 0.3 15 ± 0.6 13 ± 0.5 14 ± 0.8 11 ± 1.3 12 ± 0.6 10 ± 0.5 10 ± 1.4

4a 20 ± 0.9* 21 ± 0.3 17 ± 0.8 18 ± 1.4 15 ± 1.2 16 ± 0.7* 13 ± 0.4 14 ± 1.0

4b 16 ± 1.1 16 ± 1.6 14 ± 0.3 14 ± 1.2 12 ± 0.6 13 ± 0.5 11 ± 0.9* 12 ± 0.1

4c 18 ± 0.5 20 ± 0.8* 16 ± 1.0 16 ± 1.6 13 ± 0.4 15 ± 1.6* 10 ± 0.8 11 ± 0.3*

4d 22 ± 0.3 24 ± 1.4 20 ± 0.2 20 ± 0.3* 18 ± 0.5 18 ± 1.2 15 ± 0.1 15 ± 1.0

4e 17 ± 1.6* 18 ± 0.5 15 ± 0.2 15 ± 1.1 14 ± 0.7* 15 ± 1.0* 12 ± 0.8 13 ± 0.2

4f 19 ± 1.3 21 ± 1.0 18 ± 0.5* 20 ± 0.9 17 ± 0.5 18 ± 0.6 16 ± 0.2* 16 ± 0.9

4g 15 ± 0.2* 15 ± 1.1 14 ± 1.0 14 ± 1.8* 13 ± 1.5 14 ± 0.2 12 ± 0.8 12 ± 1.2*

4h 21 ± 1.5 22 ± 0.4 18 ± 0.2 19 ± 0.6 17 ± 0.3 18 ± 0.4 15 ± 0.6 17 ± 0.1

4i 17 ± 0.4 17 ± 1.4 16 ± 0.5 16 ± 0.7 13 ± 0.1* 13 ± 1.2 11 ± 0.7 11 ± 1.2

4j 20 ± 1.2 20 ± 1.0* 18 ± 0.2 18 ± 1.4 16 ± 0.7 16 ± 0.1* 15 ± 1.0 16 ± 0.9

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 Molecular properties calculation

MUT TUMO IRRI REP CLP Log S MW DL DS

4a ? ? - - 3.29 -5.42 414 -5.32 0.18

4b ? ? - - 4.03 -5.69 403 1.64 0.3

4c ? ? - - 3.12 -4.66 385 0.55 0.35

4d ? ? - - 3.31 -4.97 399 0.35 0.31

4e ? ? - - 3.42 -4.99 412 -0.76 0.19

4f ? - - - 2.42 -4.52 415 -2.11 0.3

4g - - - - 3.16 -4.79 404 4.81 0.63

4h - - - - 2.25 -3.76 386 3.74 0.76

4i - - - - 2.44 -4.07 400 3.53 0.72

4j - ? - - 2.55 -4.09 413 2.42 0.41

DLF - - - ? 4.4 -4.64 295 2.06 0.36

ALB ? - - ? 3.48 -4.1 265 -2.11 0.15

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

942 Med Chem Res (2014) 23:939–947

123

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:

DS ¼ P1

2þ 1

2Si

� ��Pti ð1Þ

S ¼ 1

1þ eapþbð2Þ

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.

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

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.

Table 4 Molinspiration bioactivity score of compounds 4(a–j)

Compounds GPCRL ICM KI NRL PI EI

4a -0.21 -0.27 -0.18 -0.34 -0.24 -0.17

4b -0.08 -0.23 -0.08 -0.28 -0.15 -0.09

4c -0.06 -0.20 -0.04 -0.22 -0.10 -0.04

4d -0.11 -0.27 -0.09 -0.28 -0.15 -0.09

4e -0.07 -0.23 -0.04 -0.24 -0.14 -0.08

4f -0.21 -0.23 -0.15 -0.49 -0.19 -0.10

4g -0.08 -0.19 -0.05 -0.44 -0.11 -0.02

4h -0.06 -0.18 -0.01 -0.37 -0.07 0.02

4i -0.11 -0.24 -0.06 -0.43 -0.11 -0.03

4j -0.08 -0.20 -0.00 -0.39 -0.10 -0.01

DLF 0.14 0.20 0.17 -0.09 -0.10 0.25

ALB -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

Med Chem Res (2014) 23:939–947 943

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Experimental section

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; 1H NMR spectra and 13C

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 in vivo anti-inflammatory investigation

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.

Protocol for in vitro anthelmintic investigation

Adult Indian earthworms of the genus and species,

Pheretima posthuma (family: Megascolecidae), were used

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.

Protocol for the synthesis of isoniazid derivatives using

Betti reaction 4(a–j)

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.

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,

944 Med Chem Res (2014) 23:939–947

123

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-


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.


chlorophenyl)methyl)isonicotinohydrazide (4b)



str.), 1648 (C=O str.), 1620 (C=N str.), 721 (C–Cl str.). 1H

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-










100 %). Anal. Calcd: C23H18ClN3O2: C, 67.46; H, 4.53; N,

10.45. Found: C, 68.48; H, 4.50; N, 10.43.

N’-((2-Hydroxynaphthalen-1-yl)(4-

Hydroxyphenyl)methyl)isonicotinohydrazide (4c)



str.), 1652 (C=O str.), 1610 (C=N str.). 1H 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,



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),

123.8 (C6, Phenyl), 164.6 (C, Amide), 141.5 (C1, Pyri-

dine), 123.8 (C2, Pyridine), 150.1 (C3, Pyridine), 150.6


100 %). Anal. Calcd: C23H19N3O3: C, 70.36; H, 4.76; N,

10.75. Found: C, 70.38; H, 4.79; N, 10.71.


methoxyphenyl)methyl)isonicotinohydrazide (4d)



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):




116.7 (C8, 2-naphthol), 133.5 (C9, 2-naphthol), 129.3

(C10, 2-naphthol), 52.2 (C, Methine), 135.6 (C1, Phenyl),


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.


(dimethylamino)phenyl)methyl)isonicotinohydrazide (4e)



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),




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.

Med Chem Res (2014) 23:939–947 945

123

N0-((8-Hydroxyquinolin-7-yl)(3-

nitrophenyl)methyl)isonicotinohydrazide (4f)

Yellow crystals; Yield 92 %; Mp. 143–145 �C; FTIR


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),


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.


chlorophenyl)methyl)isonicotinohydrazide (4g)



str.), 1655 (C=O str.), 1628 (C=N str.), 720 (C–Cl str.). 1H

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



(C9, Quinoline), 52.7 (C, Methine), 142.8 (C1, Phenyl),


nyl), 130.5 (C5, Phenyl), 129.5 (C6, Phenyl), 165.6 (C,



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.


hydroxyphenyl)methyl)isonicotinohydrazide (4h)



str.), 1664 (C=O str.), 1623 (C=N str.). 1H 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

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,








14.45. Found: C, 68.25; H, 4.71; N, 14.48.


methoxyphenyl)methyl)isonicotinohydrazide (4i)



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):




Quinoline), 137.4 (C9, Quinoline), 52.7 (C, Methine),


nyl), 158.5 (C4, Phenyl), 56.3 (C5, Methoxy), 115.8 (C6,

Phenyl), 129.7 (C7, Phenyl), 164.5 (C, Amide), 141.3 (C1,




14.06. Found: C, 69.03; H, 5.09; N, 14.02.


(dimethylamino)phenyl)methyl)isonicotinohydrazide (4j)

Yellow crystals; Yield 87 %; Mp. 174–176 �C; FTIR


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



(C8, Quinoline), 138.2 (C9, Quinoline), 52.6 (C, Methine),


nyl), 148.7 (C4, Phenyl), 42.3 (C5, Methyl), 42.3 (C6,

Methyl), 116.6 (C7, Phenyl), 130.2 (C8, Phenyl), 165.4 (C,


946 Med Chem Res (2014) 23:939–947

123


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,

16.92.

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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Documents

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