INTRODUCTION:
Now a day’s wide interest has been developed in
synthesis of heterocyclic molecules which possess wide range of therapeutic
applications. The anti- inflammatory, analgesic, and antipyretic drugs are a
heterogeneous group of compounds, often chemically unrelated (although most of
them are organic acids), which nevertheless share certain therapeutic actions
and side effects. The prototype is aspirin; hence these compounds are also
referred to as aspirin- like drugs; they are also frequently called as Non
Steroidal anti-inflammatory drugs, or NSAID’s. Aspirin apart from its
anti-inflammatory and analgesic activity is used in long term prophylaxis
against myocardial infarction and shock due to its anti-thrombotic properties.
On the basis of this fact we have reported the synthesis and anti-platelet
activity of three pyrrole derivative conjugates of aspirin. Aspirin was taken as
reference drugs for testing the anti-platelet activity of the synthesized
compounds.
MATERIALS AND METHODS:
All the chemicals were of laboratory grade and were
purified by the established methods. Melting points were determined in open
capillary tubes and are uncorrected. Purity and homogeneity of the synthesized
compounds was routinely ascertained by TLC on glass plates using silica gel G
as adsorbent and solvent system benzene: methanol (1:1). Spots were visualized
by iodine vapor or by irradiation with UV light (254 nm). IR spectra were
recorded using KBr disc on FTIR 8010 Shimadzu model.
Mass spectra of the synthesized compounds were recorded
using double focusing mass spectrometer. Anti-platelet aggregatory activity was
performed on aggregated albino rat platelet rich plasma (PRP) using aspirin as
reference drug.
EXPERMENTAL:
1.
Synthesis of
Pyrrole-2-Carboxylic acid:
a) Synthesis of pyrrole-2-carbaldehyde:
b)
Synthesis of
Pyrrole-2-carboxylic acid:
2. Synthesis of Pyrrole derivative conjugates of
Aspirin:
a) [5(2-acetyloxy) phenyl keto pyrrole-2-carboxylic
acid]. [Compound-I]:
Protection of the phenolic “-OH” group of salicylic
acid by acetylation:
Chlorination of acetyl salicylic acid to its acid
chloride:
Treatment of the acid chloride with
pyrrole-2-carboxylic acid to get compound-I
b)
[5(2-acetyloxy)
phenyl keto-N-methyl pyrrole-2-carboxylic acid] [Compound-II]:
c)
[5(2-acetyloxy)
phenyl keto-N-acetyl pyrrole-2-carboxylic acid] [Compound-III]
1.
Synthesis of
Pyrrole-2-Carboxylic acid:
The synthesis of pyrrole-2-carboxylic acid was achieved
in two steps:
a) Synthesis of pyrrole-2-carbaldehyde:
50 ml of chloroform was taken in a 250 ml conical flask
and to it slight excess KOH solution (55ml) was added. The mixture was stirred
on a magnetic stirrer without external warming. 25ml pyrrole was then added
drop wise and mixture was allowed to stir on a magnetic stirrer for about 2.5
hrs. After the completion of stirring the mixture was extracted thrice with
ether (10ml). The organic layer was separated and the ether was evaporated.
Finally the residue was distilled off to get pyrrole-2-carbaldehyde. Yield=66%.
b)
Synthesis of
Pyrrole-2-carboxylic acid:
About 20ml of pyrrole-2-carbaldehyde was taken in a
250ml conical flask and to it 15ml of saturated KMnO4 solution and
0.5 gm of Na2CO3 were added. The mixture was then warmed
on a water bath for 20 minutes. Then the resulting mixture was acidified using
conc. HCl, and then 25% solution of sodium sulphite was added until the
precipitated manganese dioxide got re-dissolved. The mixture was then cooled to
give the precipitates of pyrrole-2-carboxylic acid. Yield=63%.
2. Synthesis of Pyrrole derivative conjugates of
Aspirin:
a) [5(2-acetyloxy) phenyl keto pyrrole-2-carboxylic
acid]. [Compound-I]:
Compound I was synthesized in four steps:
Protection of the phenolic “-OH” group of salicylic
acid by acetylation:
10 gm of salicylic acid was dissolved in 7ml of dry
pyridine in a 250ml conical flask. After that 7.5ml of acetyl chloride was
added (adding 1ml each time) and the mixture was shaken continuously. The
mixture was then heated on a water bath for 5 minutes and than after cooling it
was poured into 300ml of cold water. The crude acetyl salicylic acid separated
as a white precipitate, which was than filtered off and then recrystalized from
equimolar mixture of water and acetic acid. The acetyl salicylic acid was
obtained as white crystals. Yield=70%.
Chlorination of acetyl salicylic acid to its acid
chloride:
An equimolar quantity of acetyl
salicylic acid was taken in a 250ml conical flask and to it about 20ml of
chloroform was added. Than to it 1gm K2CO3 was added and
the mixture was stirred at room temperature. Than to it 5ml of thionyl chloride
was added drop wise and the mixture was allowed to stir for 30 min. After that
the excess of thionyl chloride and chloroform was evaporated to get the acid
chloride. Yield=59.5%.
Treatment of the acid chloride with
pyrrole-2-carboxylic acid to get compound-I
6gm of finely powdered aluminum chloride was taken in a
250ml RBF fitted with a reflux condenser. Equimolar quantities of the acid
chloride and pyrrole-2-carboxylic acid were dissolved in about 20ml chloroform
and this mixture was added in the RBF. The reaction mixture was then refluxed
on the water bath for 30 minutes at 500C. Steady evolution of
hydrogen chloride gas occurred. When the evolution of hydrogen chloride gas
ceased the mixture was then poured into about 50ml of cold water. Immediately
the yellow colored protected pyrrole derivative separated off. It was
then washed with dilute NaOH to remove the traces of hydrogen chloride. The
product was then filtered off and was recrystallized from ethanol. Yield=56.2%.
b) [5(2-acetyloxy) phenyl keto-N-methyl
pyrrole-2-carboxylic acid] [Compound-II]:
6gm of compound (I) was taken in a conical flask and to
it 25ml of 10% NaOH solution was added. The solution was diluted with about
30ml of water and then 10ml of dimethyl sulphate was added. The flask was then
stoppered and was shaken vigoursly for 30 minutes. The mixture became warm and
then compound (II) started to separate as a yellowish solid. When the
separation was complete the solid was filtered through suction. The solid was
then washed with little 10% NaOH solution to remove excess dimethyl sulphate
and traces of compound (I).
It was then recrystallized from methylated spirit to
give [5(2-acetyloxy) phenyl keto-N-methyl pyrrole-2-carboxylic acid] (II) as
the final product. Yield=56%.
c) [5(2-acetyloxy) phenyl keto-N-acetyl
pyrrole-2-carboxylic acid] [Compound-III]
7 gm of compound (I) was taken in a conical flask and
to it 13.89 ml of triethyl amine was added. The resulting solution was stirred
on ice bath followed by the drop wise addition of slightly excess acetyl
chloride (about 12ml). The resulting mixture was allowed to stir on an ice bath
for about 45 minutes when pale brown colored compound (III) separated as a fine
solid. It was filtered through suction and was washed with water to remove
excess of acetyl chloride. Finally the compound was recrystallized from
chloroform. Yield =60%.
PHARMACOLOGICAL SCREENING:
Blood samples were taken from the caudal vein of rats
that had not taken any food or received any drug for the past 2 weeks. Platelet
rich Plasma (PRP) was prepared by centrifugation of citrated blood for 20
minutes at 1000rpm. Aliquots (300µL) of PRP was added into the aggregometer
(Elvis) cuvettes and aggregation was recorded as increased light transmission
under continuous stirring at 1000rpm at 370C for 5 minutes after the
addition of the stimulus. Collagen (2.5 mg/ml) was used as platelet activator
in PRP. The inhibitory activity of the compounds was tested by the addition of
compounds to PRP 10 minutes before the addition of the stimulus (collagen).
Drug vehicle (<0.5% DMSO) added to PRP served as control and did not effect
platelet function.
The anti-platelet activity of the compounds was
evaluated as % inhibition (pIC50) of platelet aggregation compared
to control samples. When inhibition of aggregation at maximal inhibitory
concentration exceeded 50%, pIC50 values were calculated by non linear
regression analysis.
CONCLUSION:
From anti-platelet screening of the
synthesized compounds it is clear that platelet inhibition increases in case of
conjugates with N-methyl pyrrole or N-acetyl pyrrole residues then in compounds
with pyrrole residue when compared with that of Aspirin as the standard. Thus
there are tremendous possibilities that such conjugates may be useful in the
treatment of various thrombotic disorders, with lower incidence of GI
ulceration. This may be due to the presence of pyrrole ring which acts as the
basic template of COX-2 inhibition and not COX-1 which protects the GI tract.
ACKNOWLEDGEMENT:
The authors are highly thankful to the
Medicinal Chemistry division and Pharmacology division of Central Drug Research
Institute (CDRI), Lucknow for their valuable and continuous support throughout
this work.
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