INTRODUCTION:

Azomethine group (–CH=N–) containing compounds typically known as Schiff bases, have been synthesized by the condensation of primary amines with active carbonyls. Compounds with the azomethine linkage were shown to possess biological effects such as anti-fungal,anti-bacterialand anti-inflammatoryactivities^1,2. In view of this all observations, here an attempt is made to synthesize a new series of hydrazide Schiff’s base derivatives of o-iodo benzoic acid for evaluation of analgesic and anti-inflammatory effects by reaction of 2-iodo benzohydrazide (1) with appropriately substituted aromatic aldehyde in glacial acetic acid. Thus the present communication describes synthesis of 2-Iodo-N'-[(1E)-Substituted Phenylmethylidene] Benzohydrazides and their subsequent evaluation for analgesic, anti- inflammatory activity.

MATERIALS AND METHODS

Unless otherwise noted, starting materials were procured from commercial suppliers and were used without further purification. All the melting points of newly synthesized compounds were determined on ‘Veego’ VMP-D apparatus and were found uncorrected. Silica gel G plates of 3x8 cm (Sigma-Aldrich) were used for TLC and spots were located by iodine vapors. Physical data for compounds is noted in Table 01. The structures of the synthesized compounds were confirmed by spectral data^3,4.

The IR spectra were recorded on FTIR 8400 F- Shimadzu spectrometer using KBr disc pellet method. ¹H NMR spectra were recorded on Varian Mercury (300MHz) using DMSO as solvent and TMS as internal standard, values are expressed in δ ppm. GC-MS spectra were recorded on GCMS-QP-5050 Schimadzu. Analytical data for synthesized compounds is recorded in Table 02.

Synthesis of o-iodo benzoic acid^5,6

Anthranillic acid was dissolved in water and concentrated sulphuric acid. Diazotization was carried by gradual addition of cold solution of sodium nitrite checked with starch iodide paper at end point. Into the clear solution added the solution of potassium iodide in sulphuric acid. Reaction mixture was heated, filtered and recrystallized from hot water. Yield 85 % w/w, M. P 161⁰C.

Synthesis of 2-iodo benzohydrazide (1)

In methanol o-iodo benzoic acid was dissolved & added 2-3 drops of concentrated sulphuric acid, refluxed for 4-5 hours to form ester. Added hydrazine hydrate and refluxed for 4 hours. Removed methanol to obtain residue. Recrystallized by mixture of cold water and alcohol. Yield 75 % w/w, M. P. 260⁰C.

Formation of 2-iodo-N'-[(1E)-substituted phenylmethylidene] benzohydrazides(2)

General procedure

2-iodo benzohydrazide (1) (0.0025 mole) was dissolved in methanol, added (0.0025 mole) aromatic aldehyde and 3 drops of glacial acetic acid, mixture was refluxed for 5 hr. Recrystallized by mixture of cold water and alcohol.

Animals:

Albino mice of either sex (weighing 20-30 gms) were used for toxicity and analgesic activity respectively. The animals were housed in standard laboratory conditions with natural light and dark place cycle. They were fed on standard pellets and ad libitum. Animals were acclimatized to their environment for one week prior to experimentation. (Ethical Committee Approval no.1197/c/08/CPCSEA)

Toxicity Studies:^7,8

Healthy Swiss albino mice weighing between 20-30 gms were used in the present investigation. The samples were tested from 500 mg to 3000 mg /kg body weight (as suspended in propylene glycol) in groups of 06 animals by Intraperitoneal route administration. The control group of animals received only the vehicle (propylene glycol). The animals were observed for 48 hrs after the time of administration of test compound to record the mortality. LD₅₀ observed was 3000mg/kg of body weight.

Analgesic Activity:

Acetic Acid Induced Writhing Test^9-11

The test compounds and standard diclofenac at dose of 1/10^thof LD₅₀and at 10mg per kg body weight were administered in suspension form by intra-peritoneal route to respective mice groups (6 animals in each group), 30min before induction of writhing. Control group animals received 10ml/kg body weight of volume of vehicle. Writhing was induced by an injection of 0.6% w/v aqueous acetic acid. Number of writhing episodes occurring between 5 and 20 min after acetic acid injection was recorded. The results are recorded in Table No.03

Tail Immersion Method:^9-11

The mice were selected by immersing tail in hot water at temperature 55±5 ⁰C and basal reaction time was noted. The animals that showed positive response within 5 second (withdrawal of tail clearly out of water) were selected. The test compounds 1/10^th of LD₅₀dose were administered in suspension form by intra-peritoneal route to respective mice groups (6 animals in each group) and standard pentazocine 5 mg/kg body weight was administered intra-peritonealy to standard group. Animals in control group received propylene glycol 10 ml per kg body weight as vehicle. Observations were made up to 3 hrs after administration of the test compounds. Perce protection was calculated. Results are noted in Table No.03

Anti-Inflammatory Activity:^9-11

Edema was induced in rats by injecting carrageenan (0.1ml) into the sub-plantar region of the right hind paw of each rat. The test compounds and standard ibuprofen was administered at dose 1/10^th of the LD₅₀intra-peritonealy to respective groups (6 rats in each group), 30 min before carrageenan injection. Rats under control group received an equal volume of vehicle. The volume of edema was measured by plethysmometer at 0, 1, 2, 3 and 4 hrs after carrageenan injection. Results are noted in Table No. 04.

RESULTS AND DISCUSSION:

All the synthesized compounds did not cause mortality up to 3000mg/kg in acute oral toxicity (OECD-423 guidelines) and were considered at safe. The synthesized compounds A, D and E, H significantly (p< 0.05) reduced the number of writhing which showed significant analgesic activity, compounds G, B reduced number of writhing (p< 0.01) that showed moderate analgesic activity when compared to that of standard diclofenac sodium (Table 03). In tail immersion method compound E, H, D, C, F showed significant (p< 0.05) analgesic activity, compound G, A showed moderate (p< 0.01) analgesic activity as compared to standard pentazocine (Table 03). Compounds A, B, C, F showed significant reduction in paw edema, compound E, H showed moderate reduction, while compound D and G showed not significant inhibition of rat paw volume when compared to the standard ibuprofen (Table 04).

Table No. 01. Physical data for 2-iodo-N'-[(1E)-substituted phenylmethylidene] benzohydrazides

Comp.	Ar	Mol. formula	Mol. weight	M.P	Yield
A	p-methoxy phenyl	C₁₅H₁₃N₂O₂I₁	279.9	240⁰C	36%
B	m-nitro phenyl	C₁₄H₁₀N₂O₁I₁	380.9	245⁰C	41%
C	Furan	C₁₂H₉N₂O₂I₁	339.9	95⁰C	55%
D	o-hydroxy phenyl	C₁₄H₁₁N₂O₂I₁	365.9	220⁰C	32%
E	Phenyl	C₁₄N₂H₁₁I₁	249.9	290⁰C	94%
F	Cinnamaldehyde	C₁₆N₂H₁₃I₁O₁	375.9	140⁰C	68%
G	p-hydroxy phenyl	C₁₄N₂H₁₁O₂I₁	365.9	240⁰C	66%
H	p-dimethyl amino phenyl	C₁₆H₁₆N₃O₁I₁	378.9	255⁰C	55%

Table No. 02. Sophisticated analytical data for 2-iodo-N'-[(1E)-substituted Phenylmethylidene benzohydrazides

Comp	Ar	IR, ¹H NMR and GC-MS spectra
A	p-OCH₃ phenyl	IR: 2677(w, SH, Str),2358 (s,CH₂, Str), 1596(w, C=N, Str) ¹H NMR: 7.6-7.9(s, 2H each for Ph) 8.3(s,1H, Imino),8.0(s,1H hydrazide) 7.8(for Ph), 3.8(s,3H methoxy gr.) GC-MS: M⁺¹ 279.9, 261.96, 230.93,202.94,177.07,149.93,120.06
B	m-nitro phenyl	IR: 2677 (w, SH, Str), 2358 (s, CH₂, Str), 1596 (w, C=N, Str) ¹H NMR: 7.6-7.9(s, 2H each for Ph) 8.3(s,1H, Imino),8.0(s,1H hydrazide) 7.8(for Ph), 8.2(s, for nitro gr.)
C	Furan	IR: 2677(w, SH, Str),2358(s, CH₂,Str),1596 (w, C=N, Str) ¹H NMR: 6.5-7.8 (s, Ph), 8.4(s,1H,imino), 7.0(s,Hydrazide),6.5-7.7(furan)
D	o-OH phenyl	IR: 2677 (w, SH, Str), 2358 (s, CH₂, Str), 1596 (w, C=N, Str) ¹H NMR: 7.6-7.9(s, 2H each for Ph) 8.3(s,1H, Imino), 8.0(s,1H hydrazide) 6.8-7.7(for Ph)5.3(s, 1H hydroxy gr.)
E	phenyl	IR: 2677 (w, SH, Str), 2358 (s, CH₂, Str), 1596 (w, C=N, Str) ¹H NMR:7.6-7.9(s,2H each for ph)8.3(s,1H,Imino),8.0(s,1H hydrazide)7.8(Ph).
F	Cinnamadehyde	IR: 2677(w, SH,Str), 2358(s, CH₂,Str),1596 (w, C=N, Str) ¹H NMR: 6.5-7.8(s,Ph),7.0 (s, hydrazide) 7.5(s,1H imino gr.)7.2(s, 2H ethylene gr.), 6.8-7.7(for Ph)
G	p-OH phenyl	IR: 2677 (w, SH, Str), 2358 (s,CH₂, Str), 1596 (w, C=N, Str) ¹H NMR: 7.6-7.9(s, 2H each for Ph) 8.3(s,1H, Imino),8.0(s,1H hydrazide), 6.8-7.7(for Ph), 5.3(s,1H,hydroxy)
H	p-dimethyl amino phenyl	IR: 2677 (w, SH, Str), 2358 (s, CH₂, Str), 1596 (w, C=N, Str) ¹H NMR: 7.6-7.9(s, 2H each for Ph) 8.3(s,1H, Imino),8.0(s,1H hydrazide) 7.8(for Ph), 3.0(s, 6H dimethyl )

Table No. 03. Analgesic activity for some 2-iodo-N'-[(1E)-substituted Phenylmethylidene benzohydrazides.

Compound	Analgesic Activity
	Tail Immersion Method		Writhing Test
	Mean ± S.E.	% Inhibition	Mean ± S.E	% Protection
A	3.667 ± 0.2108	74.00^*	2.667± 0.3333	38.40^@
B	3.941 ± 0.3073	72.01^*	1.833± 0.3073	57.74^*
C	3.412 ± 0.2108	78.00^@	1.667± 0.2108	73.34^NS
D	2.833 ± 0.1667	86.01^@	2.812± 0.4014	34.91^@
E	1.433 ± 0.2667	90.14^@	2.743± 0.3073	35.62^@
F	3.547 ± 0.1547	76.01^@	1.467± 0.4014	75.06^NS
G	3.873 ± 0.4014	72.06^*	2.500±0.3416	42.27^*
H	2.737 ± 0.1667	87.59^@	2.667±0.2108	40.45^@
Control	1.007 ± 0.2108	------	4.500 ±0.2234	------
Pentazocine	1.833 ± 0.1667	98.01	-------	------
Diclofenac Sodium	------	------	1.333±0.2108\	78.11

All values are mean±SEM from 6 animals in each group, % inhibition and % protection shown in parenthesis. Comparison Groups test vs. standard, ^@ P< 0.05, ^* P< 0.01, ^NS 0.001- Non significant.

Table No.04 Anti-inflammatory activity of some 2-iodo-N'-[(1E)-substituted Phenylmethylidene benzohydrazides.

Compound	Anti-inflammatory activity
Compound	Mean + S.E.	% Inhibition
A	3.331± 0.2108	16.67^@
B	3.243± 0.5102	16.60^@
C	3.167± 0.1667	19.62^@
D	3.833±0.1667	04.71^NS
E	3.500± 0.2236	12.50^*
F	2.823± 0.1047	29.71^@
G	3.667± 0.1132	08.32^NS
H	3.415± 0.4231	12.75^*
Control	4.000 ± 0.0012	------
Ibuprofen	3.000 ± 0.2001	40.00

All values are mean±SEM from 6 animals in each group, % inhibition and % protection shown in parenthesis, Comparison Groups test vs. standard, ^@ P< 0.05, ^* P< 0.01, ^NS 0.001- Non significant.

CONCLUSION:

In conclusion, the results of analgesic and anti-inflammatory activity testing revealed the compounds to possess significant in-vivo activity. Therefore, this study would be fruitful matrix for development of novel class of analgesic and anti-inflammatory agents. It is convincing that, derivatives showing significant biological activity can be further modified to exhibit better potency as that of standard drugs.

REFERENCES:

1. Sahu A, Srinivasa R. M. and Venugopala K. N. Synthesis, characterization and determination of partition coefficient of some hydrazide derivatives for their anti-microbial activity, Asian J Chem., 19(1), 2007,73-78

2. Rao G. K. and Rajasekaran S. Synthesis and anti-microbial activity of some 5-phenyl-4-substituted amino -3- mercapto-4(H)-1, 2, 4-triazoles. Indian J. Pharm. Sciences 2000, 475-477.

3. Silverstein R. M, Bassler G. C and Morril T. C. Spectrophotometric identification of some compounds, John Wiley and Son’s, New York, 5^th edn, 1979, 328.

4. Indian Pharmacopoeia, Controller of publications, Vol. II, 1996, A-100, A-113.

5. Korolkovas A. Essentials of Medicinal Chemistry, Jhon Wiley and Son’s, New York, 2^nd edn, 1988, 03.

6. Williams D. A. and Lekme T. L, Foye’s Principles of Medicinal Chemistry, 5^th edn, Lippincott Williams and Wilkins, Philadelphia, 2002,827.

7. OECD Guidelines – 423, for testing of animals, 1996.

8. Ecobichon, D.J. The Basis of Toxicity Testing, 2^nd edⁿ, 1997, 43-86.

9. Udupi, R. H., Narayanrao, S. and Bhat, A. R. Synthesis of some new pyrazoline derivatives as anti-microbial, anti-inflammatory and analgesic agents Indian J. Heterocyclic Chemistry, (7), 1998, 217-220.

10. Chandrasekar, K. S., Satyanarayana, D and Joshi, A. B. Analgesic activity of Leucas levandulaefolia rees, Journal of Ethnopharmacology, (94), 2004, 77-83.

11. Kulkarni, S.K. Handbook of Experimental Pharmacology Vallabh Prakashan, New Delhi, 3^rd edⁿ, 1997, 43-86.

Received on 18.11.2009 Modified on 29.12.2009

Asian J. Research Chem. 3(2): April- June 2010; Page 504-507