INTRODUCTION:
Heterocyclic compounds are acquiring more importance in recent years due to the pharmacological activities. Nitrogen, sulphur, oxygen, containing five/six member heterocyclic compounds has occupied enormous significance in the field of drug discovery process. Substituted oxazole, pyrazole and their analogs have been used as precursors for synthesis of various biologically active molecules, oxazole derivatives as brain-derived neurotrophic factor inducers1, analgesic2, trypanocidal activity3, anti-mitotic agents with pro-apoptotic activity4, antifungal activity5, anti-inflammator6, anti-depressant7, anti-cancer8, anti-microbial, anti-diabetic and anti-obesity 9-10.
An antibiotic is a drug extracted from fungus or other microorganism that kills or slows the growth of bacteria. Antibiotics are one class of antimicrobials, a larger group which also includes anti-viral, anti-fungal and anti-parasitic drugs.
They are relatively harmless to the host and therefore can be used to treat infections11. Antibiotic resistance occurs when bacteria change in some way that reduces or eliminates the effectiveness of drugs, chemicals or other agents designed to cure or prevent infections. Obviously, if a bacterial pathogen is able to develop or acquire resistance to an antibiotic then that substance becomes useless in the treatment of infectious disease caused by that pathogen. So, as pathogens develop resistance, we must find novel drugs to fill the place of the old ones in treatment regimes.12.
To achieve the above-mentioned target, scientists have utilized the concept of bioisosterism. The concept and study of bioisosterism is a central theme in drug design and development. It is now known that many heterocycles, when appropriately substituted, display bioisosterism.
Pyrazole and pyrimidine derivatives attracted organic chemists very much due to their biological and chemotherapeutic importance. Pyrazolopyrimidines and related fused heterocycles are of interest as potential bioactive molecules because there is not much difference in the basic structures of pyrazolopyrimidines and purines. In many cases, heterocyclic fusion of pyrimidine ring and pyrazole ring resulted in compounds with wide spectrum of biological activities. Some of the activities possessed by heterofused pyrazolopyrimidines include CNS depressant13, neuroleptic13, tuberculostatic13 ,antimicrobial13,14, anticancer14,15, adenosine receptor antagonistic16-18, antinociceptive19, anti-inflammatory19,20, non-narcotic analgesic20 and leishmanicidal21 activity.
However, literature survey showed that a few attempts were made to synthesize heterofused pyrazolopyrimidine ring system for the evaluation of antibacterial activity22, 23. In the recent years, the efficiency of microwave chemistry in dramatically reducing reaction times has recently been proven in several different fields of organic chemistry24, microwave-assisted organic synthesis has shown significant improvement in the generation of combinatorial libraries of small molecules25. In general, microwave irradiation was found to be very useful to accelerate the rate of reaction of various thermally conducted reactions and also this technique was found to be very useful to improve an overall yield and reaction selectivity. Moreover, microwave chemistry assures safe and reproducible experimental procedures. Thus, in the present investigation, along with the conventional method, we decided to develop microwave-assisted one-pot facile method for the synthesis of 6-substituted1-phenyl-1 H-pyrazolo[3,4-pyrimidin-4[5H]-one, as a new class of antimicrobial agents.
MATERIAL AND METHODS:
All the chemicals were of synthetic grade and commercially procured from S.D. fine. Melting points were determined in open capillary tubes and are uncorrected. IR spectra carried out by spectrophotometer. Peak values are shown in ppm, in the d scale. All reactions were followed and checked by TLC (Chloroform: Ehtyl acetate (7:3)).
Synthesis Protocol:
Synthesis of 5-amino-1-phenyl pyrazole-4-carboxamide (APPC) (1a-c)
Synthesis of 5-amino-1-phenyl pyrazole-4-carboxamide was carried out using a literature procedure and their spectral data are given below.
1. Synthesis of Ethoxymethylenemalanonitrile (EMMN): (1a)
Rf 0.64 (Chloroform : Ethyl acetate (7 : 3)), IR
(cm-1): IR (KBr) : 3025.2 cm–1 (=C–H); 2941.2 cm–1
(–CH2–); 2228.3 cm–1 (–C
N); 1615.8 cm–1
(C=C); 1012.6 cm–1 (=C–O); C6H6N2O
(M.Wt. 122).
2. Synthesis of 5-amino-4-cyano-1-phenyl pyrazole (ACPP): (1b)
Rf 0.57 (Chloroform : Ethyl acetate (7 : 3)), IR
(cm-1): IR (KBr) : 3324.7 cm–1,3221.3 cm–1
(–NH2); 3053.2 cm–1 (=C–H, aromatic); 2218.5 cm–1
(–CN); 1638.5 cm–1
(–C=N); 763.6 cm–1 (–CH oop). Note: oop – out of plane; C10H8N4
(M.Wt. 184)
3. Synthesis of 5 -amino-1-phenyl pyrazole-4-carboxamide (APPC): (1c)
Rf 0.20 Chloroform : Ethyl acetate (7 : 3)), IR (cm-1): IR (KBr) : 3322.1 cm–1, 3180.1 cm–1 (–NH2); 1647.6 cm–1 (–C=O); 1557.8 cm–1 (–C=N); 1336.7 cm–1 (=C–N); 734.8 cm–1 (–CH oop); C10H10N4O (M.Wt. 202)
Method A:
Compound (1) (APPC) (0.01 M; 2.02 g) and Ethyl formate (2) (0.02 M; 1.48 g/1.6 ml) were added in the presence of sodium ethoxide (prepared from 1.1 g of sodium in 60 ml of ethanol). The reaction mixture was heated to reflux for 14 hours in an oil bath. The resulting solution was concentrated under reduced pressure to dryness and the residue was dissolved in 500 ml of water. The insoluble impurities were removed by filtration; the filtrate was neutralized with dilute hydrochloric acid. The solid thus obtained was filtered, dried and recrystallized from methanol to obtain compound, 6-substituted1-phenyl-1 H-pyrazolo[3,4-pyrimidin-4[5H]-one (3).
Method B:
Compound (1) (APPC) (0.01 M; 2.02 g) and Ethyl formate (2) (0.02 M; 1.48 g/1.6 ml) were added in the presence of sodium ethoxide (prepared from 1.1 g of sodium in 60 ml of ethanol). The reaction mixture was taken in a test tube and irradiated for 3 minutes at 960 watt using domestic microwave oven (Whirlpool 20G(M)). The reaction mixture was poured onto crushed ice and insoluble impurities were removed by filtration. The filtrate was neutralized with dilute hydrochloric acid. The solid thus obtained was filtered, dried and recrystallized from methanol to obtain compound 6-substituted1-phenyl-1 H-pyrazolo[3,4-pyrimidin-4[5H]-one (3).
1-phenyl-1 H-pyrazolo[3,4-d]pyrimidin-4[5H]-one (3a):
Rf 0.21 (Chloroform : Ethyl acetate (7 : 3)), IR (cm-1): IR (KBr) : 3165.3 cm–1 (–NH, 2° amide); 3018.2 cm–1 (=C–H, aromatic); 1733.6 cm–1 (–C=O); 1507.7 cm–1 (C=C, aromatic); 783.2 cm–1 (–CH oop), C11H8N4O (M.Wt. 212).
6-methyl-1-phenyl-1 H-pyrazolo[3,4-d]pyrimidin-4[5H]-one (3b):
Rf 0.24 (Chloroform : Ethyl acetate (7 : 3)), IR (cm-1): R (KBr) : 3127.3 cm–1 (–NH, 2° amide); 3014 cm–1 (=C–H, aromatic); 2930.6 cm–1 (–CH2–); 1684.9 cm–1 (–C=O); 1507.7 cm–1 (C=C, aromatic); 752.4 cm–1 (–CH oop), C12H10N4O (M.Wt. 226).
6-ethyl-1-phenyl-1 H-pyrazolo[3,4-d]pyrimidin-4[5H]-one (3c):
Rf 0.30 (Chloroform : Ethyl acetate (7 : 3)), IR (cm-1): IR (KBr) : 3127.3 cm–1 (–NH, 2° amide); 3019.6 cm–1 (=C–H, aromatic); 2935.6 cm–1 (–CH2–); 1683.2 cm–1 (–C=O); 1504.9 cm–1 (C=C, aromatic); 752.4 cm–1 (–CH oop), C13H12N4O (M.Wt. 240).
6-propyl-1-phenyl-1 H-pyrazolo[3,4-d]pyrimidin-4[5H]-one (3d):
Rf 0.36 (Chloroform : Ethyl acetate (7 : 3)), IR (cm-1): IR (KBr) : 3090.9 cm–1 (–NH, 2° amide); 3019.6 cm–1 (=C–H, aromatic); 2963.6 cm–1 (–CH2–); 1684.8 cm–1 (–C=O); 1504.3 cm–1 (C=C, aromatic); 758.1 cm–1 (–CH oop), C14H14N4O (M.Wt. 254).
1,6-diphenyl-1 H-pyrazolo[3,4-d]pyrimidin-4[5H]-one (3e):
Rf 0.42 (Chloroform : Ethyl acetate (7 : 3)), IR (cm-1): IR (KBr) : 3200 cm–1 (–NH, 2° amide); 3081 cm–1 (=C–H, aromatic); 2924.4 cm–1 (–CH2–); 1684.5 cm–1 (–C=O); 1507.7 cm–1 (C=C, aromatic); 758.1 cm–1 (–CH oop).,C17H12N4O (M.Wt. 288).
Using Method A and B, total 5 derivatives were prepared. The spectral data of compound (3a-e) obtained by Method A and B were found to be identical.
Procedure for determination of Antimicrobial activity:
Antimicrobial activity is determined based on the in-vitro activity in pure cultures. In-vitro susceptibility tests are done by agar diffusion method26, 27. In this technique petridishes of agar are prepared by pouring method. The agar is inoculated with microorganisms. In the agar dilution method different antibiotic concentrations are incorporated in to an agar medium for both aerobes and anaerobes. The plates are incubated at a temperature of 37ºC for 24 hours. The antimicrobial substance diffuses through the agar and produces a clear zone of inhibition. The diameter of this zone can be measured and an estimation of the degree of activity of the antimicrobial substance can be obtained.
Here, responses of microorganisms to the synthesized compounds were measured and compared with the response of the standard reference drug. The standard reference drug used in the present work was Streptomycin. Four microorganisms used were Klebsiella (Gram –ve), Escherichia coli (Gram –ve), Staphylococcus aureus (Gram +ve) and Bacillus subtillis (Gram +ve)28. Each test compound was dissolved in Dimethyl sulfoxide (DMSO) to get a concentration of 500 μg/ml. This concentration was used for testing antibacterial activity. Mueller Hinton agar media 29, 30 was prepared of the composition as beef infusion 300 g, casamino acids 17.5 g, starch 1.5 g, agar 17 g, distilled water 1,000 ml. The beef extract was taken in a 1000 ml beaker and made up the volume to 1000 ml with water. To this mixture known quantities of beef infusion, agar, starch and casamino acids were added and dissolved by heating the mixture. The pH was adjusted to 7.3. Finally the media was sterilized by autoclaving at 121ºC for 15 minutes at 15-PSI (Pound per square inch) pressure. Afterwards the mixture was cooled to 45ºC and then inoculums were added to the above cooled media, mixed properly and poured into the sterile petridishes for solidifying. Bores were made on the medium using sterile borer. 0.1 ml of test solution and standard solution at a concentration of 50 μg/0.1 ml were taken. A standard (streptomycin) was maintained with same concentration in each plate and a control having only DMSO in one plate. Then the petridishes were incubated at 37ºC for 24 hours and zones of inhibition were observed and measured.
The suspensions (inoculums) of all the organisms were prepared as per standard procedure (Mcfarland Nephelometer Standard). A 24 hour-old culture was used for the preparation of bacterial suspension. Suspensions of organisms were made in sterile isotonic solution of sodium chloride (0.9 % w/v).
RESULTS AND DISCUSSION:
In this work total 5 derivatives of pyrazolopyrimidines containing 6-substituted1-phenyl-1 H-pyrazolo[3,4-pyrimidin-4[5H]-one (3a-e) were prepared by both conventional and one pot microwave synthesis. In the first method (Method A), ester (2) was added to 5 -amino-1-phenyl pyrazole-4-carboxamide (APPC) (1) in a solvent such as sodium ethoxide. The reaction mixture was heated to reflux for 6-14 hours in an oil bath followed by filtration and neutralization with dilute hydrochloric acid to obtain 6-substituted-1-phenyl-1H-pyrazolo[3,4-d] pyrimidin-4[5H]-ones (3a-e) in 70-85 % yield. In second method (Method B), ester (2) was added to 5 -amino-1-phenyl pyrazole-4-carboxamide (APPC) (1) in a solvent such as sodium ethoxide in a test tube and irradiated for 3 minutes at 960 watt using domestic microwave oven followed by filtration and neutralization with dilute hydrochloric acid to obtain 6-substituted-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4[5H]-ones (3a-e) in 76-85 % yield.
All the titled compounds (3a-e) were characterized by their analytical and spectral data. The IR data of compounds (3a-e) showed characteristic C=O vibrations around 1700 cm-1 and –NH (2° amide) vibrations around 3175-3100 cm-1. This indicated the formation of the compound.
As shown in table no. 1, when the synthesis of compounds (3a-e) were attempted using conventional method (Method A), it took 6-14 hrs for the completion of reaction and the overall yield were found in the range of 70-85 %. When the same reaction was attempted using the one-pot microwave-assisted synthesis (Method B), reaction was successfully completed within 10-20 minutes and the overall yields were found in the range of 76-85%.