INTRODUCTION
Imides are the compounds in which nitrogen atom get linked to two carbonyl
groups and consists of-CO-NH-CO structural grouping. These compounds are structurally
related to derivatives of ammonia. These compounds can easily cross the biological
membranes as they are neutral and hydrophobic in nature (Prado
et al., 2004). The two hydrogen atoms of ammonia are replaced by
carbonyl groups. Nitrogen atom in the imide moiety plays a significant role.
Imide moiety plays an integral part in various important molecules such as thalidomide,
isogranulatimide and rebeccamycin etc. These are the compounds which possess
various pharmacological activities such as analgesic, anti-inflammatory, anti-depressant
and anti-viral etc. (Abdel-Aziz, 2007). Imides and their
derivatives are also used in polymer chemistry. Today various routes are available
for the synthesis of imides which involve either Lewis-acid mediated condensation
of an amine with maleic or phthalic anhydride or N-alkylation of the corresponding
imide with halides or alcohols (Barchin et al., 2002).
Various methods are used for the synthesis of imides and these methodologies
and starting materials plays a significant role in their synthesis. The starting
material, reaction conditions, reagents and selective methods leads to the better
yield of these organic compounds which are more efficient. In synthetic organic
chemistry, the development of selective methods with readily or easily available
materials is the key task in their synthesis (Shinde et
al., 2011). The discovery and development of Schiff bases of imide moiety
are the most powerful and successful achievements of modern science and technology.
A hetero cyclic compound plays an important role in regulating the biological
activities. Schiff bases contain carbon-nitrogen double bonds in which nitrogen
atom get linked to aryl and alkyl atoms. Schiff bases can possess different
pharmacological activities and also have industrial applications. Schiff bases
can be derived from aniline or o-amino phenol. These are the compounds containing
characteristic -C = N- group. Schiff bases of imides possess different pharmacological
activities such as antimicrobial, anti-inflammatory, antidepressant, antipyretic
etc. (Vora et al., 2009; Da
Silva et al., 2011). Schiff bases derived from ortho-hydroxyaryl
aldehydes and aromatic or hetero aromatic amines have been synthesized in high
yields via condensation in ethanol in the presence of catalytic amounts of sulfuric
acid (Roman and Andrei, 2001; Ashraf
et al., 2011). The development and synthesis of novel Schiff base
derivatives as potential chemotherapeutics still attract the attention of organic
and medicinal chemist. Besides their potential use as biologically active agents,
Schiff bases and their metal complexes have been often used as chelating ligands
in the coordination chemistry of transition metals and as radiopharmaceuticals
for cancer targeting and agrochemicals (Hranjec et al.,
2011). Compounds with the structure of AC = NB are known as Schiff bases,
in which A and B are reacting material which are usually synthesized from the
condensation of primary amines and active carbonyl groups (Shi
et al., 2007). Schiff bases derived from aromatic ortho-hydroxy aldehydes
have recently attracted considerable attention as new organic materials which
could be utilised for designing various novel molecular devices (Koll
et al., 2000). The synthesized Schiff bases can be used for their
therapeutic potential. So the present study was carried out to synthesize Schiff
bases of imides and to evaluate them for their analgesic and anti-inflammatory
potential.
MATERIALS AND METHODS
Melting points were measured using Buchi melting point apparatus and are uncorrected.
The 1H-NMR spectra were recorded on a Bruker AC-300F, 300 MHz instrument
using DMSO-d6 as solvent and Tetramethyl Silane (TMS) as internal reference
standard. Thin-layer chromatography was performed on silica gel thin layer chromatographic
plates using ethylacetate and hexane (3:7).
Drugs and chemicals: Carrageenan was obtained from Central Drug House
Pvt. Ltd., Mumbai, India. 4-amino benzaldehyde was obtained from Oceanic laboratories
Mumbai. Diclofenac sodium was obtained from Jackson Laboratories Pvt. Ltd.,
Amritsar. Silica gel G and acetone were obtained from E-Merk Pvt. Ltd., Mumbai.
All other chemical reagents used were of analytical grade which were procured
from different companies (Loba Chem, Mumbai and Merck Limited, Mumbai).
Animals: The Wistar albino rats (200-250 g) and Swiss albino mice (25-30
g) of either sex were obtained from Punjab Agricultural University Ludhiana.
They were kept at standard laboratory diet, environmental temperature and humidity.
The experimental protocol was duly approved by Institutional Animal Ethics Committee
(IAEC) and care of the animals was carried out as per the guidelines of Committee
for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA).
Synthesis
4-(1,3-dioxoisoindolin-2-yl)benzaldehyde (1): A solution of 4-amino
benzaldehyde (12.1 g, 0.1 mol) was prepared in 25 mL of dichloromethane and
was added with stirring dropwise to a solution of phthalic anhydride (18.4 g,
0.1 mol) in 100 mL of dichloromethane. The reaction mixture was stirred for
8 h at 15-20°C. Then the mixture was treated with sodium bicarbonate (2
mg), water (5 mL) and solvent was removed under reduced pressure to obtain a
residue. The product obtained was then poured in crushed ice and filtered. Then
the product was crystallized in solvent ether to yield 1. The completion of
reaction was monitored by TLC. Yield = 58%, m.p = 348.49°C, IR (KBr, cm-1):
1756 (C = O), 1878 (-NH), 1510 (C = C), 1725 (-CHO) 1H-NMR (DMSO-d6,
δ, ppm): 7.3-7.6 (m, 2H, ArH), 7.8 (d, 2H, ArH), 7.93 (d, 2H, ArH), 8.17
(d, 2H, ArH), 9.97 (s, 1H, CHO).
Synthesis of Schiff bases
Schiff base 2-(4-(phenylimino) methyl) phenyl) isoindoline-1,3-dione
(2) using aniline: A solution of compound 1 (0.1 mol) was prepared in ethanol
20 mL and was treated with aniline (10 mL) and 2-3 drops of conc. sulfuric acid
in a 250 mL round bottom flask. The mixture was then refluxed for 8 h and then
checked for completion by TLC. After cooling the obtained product was filtered
then washed with water and dried. Recrystallization was done by using ethanol.
Yield = 52%, m.p = 396.55°C, IR (KBr cm-1): 1715 (C = O), 2200
(-NH), 1527 (C = C), 2700 (HC = N). 1H-NMR (DMSO-d6, δ, ppm):
7.1-7.46 (m, 5H, ArH), 7.66 (d, 2H, ArH), 7.86 (d, 2H, ArH), 7.96 (m, 2H, ArH),
8.13 (d, 2H, ArH), 8.39 (s, 1H, -CH = N-).
The steps involved in synthesis of Schiff bases of imide are presented in Fig.
1.
Schiff base 2-(4-[(4-nitrophenylimino) methyl) phenyl) isoindoline-1, 3-dione
(3) using p-nitro aniline: Equimolar quantities of compound 1 (0.1 mol)
and p-nitro aniline (0.1 mol) were added into 20 mL of absolute ethanol containing
a few drops of glacial acetic acid in a 250 mL round bottom flask. The reaction
mixture was refluxed for half an hour and then checked for completion by TLC.
The solvent was stripped off and the product was recrystallized from ethanol.
Yield = 48%, m.p. = 247.98°C, IR (KBr cm-1): 1787 (C = O), 1827
(-NH), 1384 (C = NH), 1498 (NO2), 1585 (C = C).
|
Fig. 1: |
Steps involved in the synthesis of Schiff bases of imide |
1H-NMR (DMSO-d6, δ, ppm): 7.5 (d, 2H, ArH), 7.63 (d, 2H, ArH),
7.65 (d, 2H, ArH), 7.73 (m, 2H, ArH), 7.84 (d, 2H, ArH), 8.19 (d, 2H, ArH),
8.39 (s, 1H, -CH = N-).
Schiff base (4) using p-hydroxy aniline: Equimolar quantities of compound
1 (0.1 mol) and p-hydroxy aniline (0.1 mol) were added into 20 mL of absolute
ethanol containing a few drops of glacial acetic acid in a 250 mL round bottom
flask. The reaction mixture was refluxed for half an hour and then checked for
completion by TLC. The solvent was stripped off and the product was recrystallized
from ethanol.
Yield = 59%, m.p. = 321.34°C, IR (KBr cm-1): 3677 (OH), 1857
(C = O), 1582 (C = NH), 1624 (C = C), 2700 (C-H). 1H-NMR (DMSO-d6,
δ, ppm): 5.0 (s, 1H, OH), 6.7 (d, 2H, ArH), 7.1 (d, 2H, ArH), 7.60 (d,
2H, ArH), 7.67 (d, 2H, ArH), 7.69-7.79 (m, 2H, ArH), 8.13 (d, 2H, ArH), 8.39
(s, 1H, -CH = N-).
Schiff base 2-(4-((o-tolylimino) methyl) phenyl) isoindoline-1, 3-dione
(5) using 2-methyl aniline: Equimolar quantities of compound 1 (0.1 mol)
and 2-methyl aniline (0.1 mol) were added into 20 mL of absolute ethanol containing
a few drops of glacial acetic acid in a 250 mL round bottom flask. The reaction
mixture was refluxed for half an hour and then checked for completion by TLC.
The solvent was stripped off and the product was recrystallized from ethanol.
Yield = 44%, m.p. = 376.44°C, IR (KBr cm-1): 1475 (-CH3), 1856
(C = O), 1810 (-NH), 1525 (C = C). 1H-NMR (DMSO-d6, δ, ppm):
2.5 (T, 3H, -CH3), 7.1-7.49 (m, 4H, ArH), 7.60 (d, 2H, ArH), 7.72 (d, 2H, ArH),
7.91 (m, 2H, ArH), 8.13 (d, 2H, ArH), 8.39 (s, 1H, -CH = N-).
Schiff base 2-(4-(3-chlorophenylimino)methyl)phenyl) isoindoline-1,3-dione(6)
using 3-chloro aniline: Equimolar quantities of compound (1) (0.1 mol) and
3-chloro aniline (0.1 mol) were added into 20 mL of absolute ethanol containing
a few drops of glacial acetic acid in a 250 mL round bottom flask. The reaction
mixture was refluxed for half an hour and then checked for completion by TLC.
The solvent was stripped off and the product was recrystallized from ethanol.
Yield = 53%, m.p. = 242.23°C, IR (KBr cm-1): 785 (Cl), 1787
(C = O), 2067 (-NH). 1524 (C = C). 1H-NMR (DMSO-d6, δ, ppm):
6.96 (s, 1H, ArH), 7.3-7.50 (m, 1H, ArH), 7.58 (d, 2H, ArH), 7.63 (d, 2H, ArH),
7.79 (d, 2H, ArH), 7.81-7.83 (m, 2H, ArH), 8.13 (d, 2H, ArH), 8.39 (s, 1H, -CH
= N-).
Pharmacological evaluation: Ethanolic solution of synthesized compound
was evaluated for its anti-inflammatory and analgesic activities using following
model:
Anti-inflammatory activity
Carrageenan-induced rat paw edema: The carrageenan-induced rat paw edema
assay was carried out according to Winter et al.
(1962). Wistar rats were divided into 5 groups each consisting of 6 animals:
Group I: |
Control: Carboxymethyl cellulose (1% CMC, p.o.)+carrageenan |
Group II: |
Standard: Diclofenac sodium (12.5 mg kg-1, p.o.)+carrageenan |
Group III: |
Dose 1: Ethanolic solution of synthesized compound (100 mg kg-1,
p.o.)+carrageenan |
Group IV: |
Dose 2: Ethanolic solution of synthesized compound (200 mg kg-1,
p.o)+carrageenan |
Group V: |
Dose 3: Ethanolic solution of synthesized compound (300 mg kg-1,
p.o.)+carrageenan |
Edema was induced on the left hind paw of the rats by sub plantar injection
of 0.1 mL of a solution of 1% (w/v) carrageenan in a 0.9% NaCl (w/v). The paw
volume was measured at intervals of 60, 120, 180 min by the mercury displacement
method using a plethysmograph after administration of the extract/drug orally.
The percentage inhibition of paw edema in drug treated group was compared with
the carrageenan control group and calculated according to the following formula:
where, Vc is the inflammatory increase in paw volume of control
group of animals and Vt is the inflammatory increase in paw volume
of drug-treated animals.
Analgesic activity: Swiss albino mice of either sex were divided into
5 groups each consisting of 6 animals:
Group I: |
Control: Carboxymethyl cellulose suspension (1% CMC,
p.o.) |
Group II: |
Standard: Diclofenac sodium at a dose of 10 mg kg-1
p.o. |
Group III: |
Dose 1: Ethanolic solution of synthesized compound at a dose of
100 mg kg-1 p.o. |
Group IV: |
Dose 2: Ethanolic solution of synthesized compound at a dose of
200 mg kg-1 p.o. |
Group V: |
Dose 3: Ethanolic solution of synthesized compound at a dose of
300 mg kg-1 p.o. |
Tail immersion test: The procedure is based on the observation that
diclofenac sodium like drugs selectively prolongs the reaction time of the typical
tail withdrawal reflex in mice (Toma et al., 2003).
The tail of mice was immersed in warm water kept constant at 52.5±0.5°C.
The reaction time of the tail-withdrawal response was determined at 60, 120
and 180 min after the administration of drugs. A cut off time of 15 sec was
maintained to prevent tissue damage (Grotto and Sulman,
1967).
Hot plate method: The animals were divided into six groups of six animals
each. Group I served as control and group II as standard and were injected diclofenac
sodium (10 mg kg-1) intraperitoneally. Group III, IV and V were treated
orally with ethanolic solution of drug in doses of 100, 200 and 300 mg kg-1.
The animals were individually placed on the hot plate maintained at 55°C,
one hour after their respective treatments. The animals were placed on the hot
plate and the time until either licking or jumping occurs is recorded by a stop-watch.
The latency is recorded before and after 60, 120 and 180 min following oral
or subcutaneous administration of the standard or the test compound (Shukla
et al., 2010).
Statistical analysis: All the results were expressed as Mean±Standard
Error of Means (SEM). The data was statistically analyzed by one way Analysis
of Variance (ANOVA) followed by Tukeys
multiple range tests by using Sigmastat Version-2.0 Software. The p<0.05
was considered to be statistically significant.
RESULTS
In the present study, novel Schiff base derivatives of imides moiety have been
synthesized by multistep reaction.
Synthesized compounds with their spectral data
4-(1,3-dioxoisoindolin-2-yl)benzaldehyde (1): 1H-NMR spectrum
exhibited multiplet at 7.3-7.6 (m, 2H, ArH), doublet at 7.8 (d, 2H, ArH), 7.93
(d, 2H, ArH), 8.17 (d, 2H, ArH) and singlet at 9.97 (s, 1H, CHO). IR spectrum
bands at 1756, 1878, 1510 and 1725 confirms the presence of (C = O) (-NH) (C
= C) and (-CHO), respectively.
2-(4-(phenylimino) methyl) phenyl) isoindoline-1,3-dione (2): 1H-NMR
spectrum exhibited multiplet at 7.1-7.46 (m, 5H, ArH), 7.96 (m, 2H, ArH) and
doublet at 7.66 (d, 2H, ArH), 7.86 (d, 2H, ArH), 8.13 (d, 2H, ArH). IR spectrum
bands at 1715, 2200, 1527, 2700 confirms the presence of (C = O), (-NH), (C
= C) and (HC = N), respectively.
2-(4-[(4-nitrophenylimino) methyl) phenyl) isoindoline-1, 3-dione (3):
1H-NMR spectrum exhibited doublet at 7.5 (d, 2H, ArH), 7.63 (d, 2H,
ArH), 7.65 (d, 2H, ArH), multiplet at 7.73 and singlet at 8.39 (s, 1H, -CH =
N-). IR spectrum showed bands at 1787, 1827, 1384, 1498, 1585 for (C = O) (-NH)
(C = NH) (NO2) and (C = C).
2-(4-((4-hydroxyphenylimino)methyl)phenyl)isoindoline-1,3-dione (4):
1H-NMR spectra showed multiplet at 7.69-7.79 (m, 2H, ArH), doublet
at 6.7 (d, 2H, ArH), 7.1 (d, 2H, ArH), 7.60 (d, 2H, ArH), 7.67 (d, 2H, ArH)
and singlet at 5.0 (s, 1H, OH), 8.39 (s, 1H, -CH = N-). IR spectrum showed bands
at 3677, 1857, 1582, 1624, 2700 for (OH), (C = O), (C = NH), (C = C), (C-H),
respectively.
2-(4-((o-tolylimino)methyl)phenyl)isoindoline-1,3-dione (5): 1H-NMR
spectra showed doublet at 7.60 (d, 2H, ArH), 7.72 (d, 2H, ArH), 8.13 (d, 2H,
ArH), multiplet at 7.91 (m, 2H, ArH) and singlet at 8.39(s, 1H, -CH = N-). IR
showed bands at 1475, 1856, 1810, 1525 (-CH3), (C = O), (-NH) and (C = C), respectively.
2-(4-(3-chlorophenylimino)methyl)phenyl)isoindoline-1,3-dione (6): 1H-NMR
spectra showed singlet at 6.96 (s, 1H, ArH), 8.39 (s, 1H, -CH = N-), doublet
at 7.58 (d, 2H, ArH), 7.63 (d, 2H, ArH), 7.79 (d, 2H, ArH) and multiplet at
7.3-7.50 (m, 1H, ArH), 7.81-7.83 (m, 2H, ArH). IR spectrum showed bands at 785,
1787, 2067, 1524 for (Cl), (C = O), (-NH) and (C = C). The Schiff bases with
imide moiety were evaluated for anti-inflammatory and analgesic property.
Physical properties: Various physical properties of the synthesized
compounds were analyzed and are shown in Table 1.
Anti-inflammatory activity: Table 2 shows the results
of the anti-edematous effect of orally administered ethanolic solution of synthesized
compound on carrageenan induced paw edema in rats. The ethanolic solution of
synthesized compound showed dose dependent anti-inflammatory activity in carrageenan
induced paw edema in rats. At a maximum dose of 300 mg kg-1, ethanolic
solution of synthesized compound caused 58.8% reduction in paw edema. At other
doses of 100 and 200 mg kg-1 the compound showed 14.70 and 44.11%
inhibition of edema, respectively. All the values were significant (p<0.05)
in comparison with control and standard.
Analgesic activity
Tail immersion test: Table 3 depicts the analgesic
activity shown by ethanolic solution of synthesized compound by tail immersion
method.
Table 1: |
Physical properties of prepared compounds |
 |
Table 2: |
Effect of ethanolic solution of Schiff bases of imides on
carrageenan-induced paw edema in rats |
 |
Values are Mean±SEM of 6 animals in each group, ap<0.05
compared with disease control group, bp<0.05 compared with
diclofenac sodium treated group |
Table 3: |
Analgesic effect of Schiff bases of imides by tail immersion
test |
 |
Values are Mean±SEM of 6 animals in each group, ap<0.05
compared with disease control group, bp<0.05 compared with
diclofenac sodium treated group |
Ethanolic solution of synthesized compound showed dose dependent analgesic
activity against conduction of heat-induced algesia in mice. After 3 h a maximum
dose of 300 mg kg-1 showed significant difference in the analgesic
activity when compared with control group in which the reaction time of 10.47±0.04
sec was observed. At lower doses of 100 and 200 mg kg-1 the extract
showed the reaction time of 5.73±0.04 and 8.38±0.18 sec, respectively
after 3 h. All the values were significant (p<0.05) in comparison with control
and standard.
Hot plate method: Table 4 shows the results of analgesic
activity shown by ethanolic solution of synthesized compound by hot plate method.
The given compound showed maximum analgesic activity after 3 hours at a maximum
dose of 300 mg kg-1 with the reaction time of 12.24±0.01 seconds.
At lower doses of 100 mg kg-1 and 200 mg kg-1 the reaction
time of 7.13±0.01 and 10.33±0.05 sec, respectively was noted after
3 h. All the values were significant (p<0.05) in comparison with control
and standard.
Table 4: |
Analgesic effect of Schiff bases of imides by hot plate method |
 |
Values are Mean±SEM of 6 animals in each group, ap<0.05
compared with disease control group, bp<0.05 compared with
diclofenac sodium treated group |
DISCUSSION
According to the synthetic methodology of imides, various methods, reagents
and reaction conditions have been already described for the synthesis of imides
which exhibit various pharmacological activities. For example: the b-alkylidene
succinanilic acids on treatment with cyanuric chloride in the presence of triethylamine
yields b-alkylisomaleimides. The Wittig condensation of alkyl substituted isomaleimides/maleimides
with aliphatic aldehydes gave the desired dialkyl substituted maleimides had
already been described (Haval and Argade, 2006). Formamide
was used as reagent for the synthesis of various aliphatic and aromatic imides
with cyclic carboxylic anhydrides. The reaction was carried out at 170-180°C
for 5-6 h (Chiriac et al., 2007). In the present
study, the imide 4-(1,3-dioxoisoindolin-2-yl)benzaldehyde has been synthesized
by reacting phthalic anhydride and 4-amino benzaldehyde in the presence of dichloromethane.
Dichloromethane was used as a reagent to form product with better yield. From
the above mentioned imide moiety, different derivatives of Schiff bases of imides
have been synthesized. Various other methods of synthesis of imide derivatives
are discovered which are carried out in different conditions in which primary
and secondary alcohols are converted to esters (Benjamin
and Hijji, 2008). Various pharmacological activities have been noticed from
imide derivatives. Schiff bases of 4-amino benzaldehyde showed anti-bacterial
activity (Parekh et al., 2005). Some novel cyclic-imides
were found to possess hypoglycaemic, anti-hyperlipidemic activity. Various pharmacological
activities have been shown by halogenated cyclic imides which are derived from
N-substituted phthalimide moiety (Abdel-Aziz et al.,
2011). The cyclic anhydrides and imides are the compounds of choice for
all chemists from both the basic and applied point of view for multiple purposes
(Haval and Argade, 2006). Since imides are a valuable
group of bioactive compounds showing various pharmacological activities. Therefore,
the target of present research work has been directed towards the synthesis
of novel Schiff bases of imide moiety with expected biological activities (Orzeszko
et al., 2001). For the discovery of new analgesic drugs, cyclic imides
like 1,8-naphthalimide and 1,4,5,8-naphthalene diimide were prepared and their
analgesic properties were evaluated by using the writhing test in mice (Andricopulo
et al., 2000). According to literature review, imides have been evaluated
for various pharmacological activities, so in this research Schiff bases of
imides have been evaluated for their anti-inflammatory and analgesic potential
(Rana et al., 2012; Okunrobo
et al., 2006; Gaikwad et al., 2010).
Tail immersion and hot plate methods were carried out to evaluate the analgesic
potential of Schiff bases of imides. Results of the study indicate the significant
decrease in the reaction time of tail withdrawal by ethanolic solution of Schiff
bases of imides. It shows that ethanolic solution of Schiff bases of imides
possesses analgesic property. This analgesic activity may be due to its free
radical scavenging activity as these free radicals are involved during pain
stimulation and antioxidants show reduction in such pain (Kim
et al., 2004). The synthesized compound was further evaluated for
its in vivo anti-inflammatory potential. Carrageenan induced rat paw
edema test has frequently been used to assess the anti-edematous effect of the
synthesized compound. Carrageenan is used to cause inflammation and it helps
in releasing various inflammatory mediators like prostaglandins, leukotrienes,
histamine, bradykinin etc. (Crunkhorn and Meacock, 1971).
Decrease of edema in rat paw indicated that the ethanolic solution of Schiff
bases of imides possess anti-inflammatory activity. Thus, the synthesized Schiff
bases of imides can be employed as analgesic and anti-inflammatory agent.
CONCLUSION
The present study demonstrated that synthesized Schiff bases of imides possess
various significant pharmacological activities like analgesic and anti-inflammatory
properties at high dose level which was comparable to that of standard drug.
Therefore, these compounds can be used in the treatment of various pains and
inflammation.
ACKNOWLEDGMENTS
Thanks to Professor A.C. Rana and all faculty members of Rayat Institute of
Pharmacy for their encouragement and support. We are also grateful to Rayat
and Bahra Educational and Research Trust for their unconditional help to carry
out this project.