Synthesis of new bi(pyrazolo[1,5-a ]pyrimidinyl)-7-one derivatives from dehydroacetic acid and its analogues as antibacterial agents

. Synthesis of some new bi(pyrazolo[1,5-a ]pyrimidinyl)-7-ones 3 was accomplished by the reaction of 3-and/or 4-substituted 5-aminopyrazoles 2 with 3-acetyl-4-hydroxy-6-methyl-2 H -pyran-2-one (dehydroacetic acid, DHAA) 1a and its analogues 1b-c in refluxing ethanol. A tentative mechanism for the formation of 3 is proposed. These compounds were evaluated for their antibacterial activity against two Gram positive bacteria i.e., Bacillus subtilis (MTCC 8509) and Bacillus stearothermophilus (MTCC 8505) and two Gram negative bacteria i.e. Escherichia coli (MTCC 119) and Pseudomonas aeruginosa (MTCC 741) using agar well diffusion assay technique and minimum inhibitory concentration (MIC) method. One of the compound 3d displayed significant antibacterial activity.


Results and Discussion
The reaction of 5-amino-4-phenyl-1H-pyrazole 2a, obtained by the formylation of benzylcyanide followed by the reaction with hydrazine hydrate, was initially carried out with an equimolar amount of DHAA 1a in refluxing ethanol for 6 h which afforded 5,5'-dimethyl-3,3'-diphenyl-4H-[6,7']bi(pyrazolo[1,5-a]pyrimidinyl)-7-one 3a (Scheme 1).The yield of the reaction was very low as some DHAA was left unreacted, however, on using two equivalents of 5-aminopyrazole 2a there was a tremendous improvement in the yield of the reaction from 35% to 79% and the tlc showed disappearance of DHAA.Also, using a catalytic amount of acetic acid in ethanol resulted in the reduction of reaction time to 5 h.The structure of the compound 3a was identified on the basis of the IR, 1 H, 13 C NMR and mass spectra.The IR spectrum of 3a showed absorption bands at 3472 and 1637 cm -1 due to N-H and C=O stretch, respectively.The 1 H NMR spectrum of 3a shows the presence of two singlets at δ 2.28 and 2.72 ppm due to the presence of two methyl protons at positions 5 and 5' respectively.6] The 1 H NMR spectrum of 3a also showed one exchangeable proton at δ 13.05 ppm due to NH present at position-4.
To generalize the reaction, different substituted bi(pyrazolo[1,5-a]pyrimidines) 3b-3e were prepared by reacting differently substituted 5-amino-4-phenyl-1H-pyrazoles 2a-b with DHAA 1a and its analogues 1b-c in a ratio of 2:1 (Scheme 1).All the compounds were identified on the basis of the IR, 1 H, 13 C NMR and mass spectral data.In the 1 H NMR spectrum of 3b, one of the methyl of DHAA is replaced by ethyl group and a typical pattern of triplet at δ 1.14 ppm and quartet at δ 3.06 ppm appeared along with the other signals as were obtained in the spectrum of 3a.Proton present at 6'-H disappears in 3d as this proton is replaced by bromine atom.Also, two singlets at about δ 7.99 and 8.35 ppm, which appeared for two pyrazole ring 2 and 2'-H in 3a and 3b, were replaced by that of two singlets of methyl protons between δ 2.39-2.51ppm and 2.55-2.65 ppm in 3c-3d for 2 and 2'-CH3, respectively.The 13 C NMR spectra of 3a-e also showed 12 signals for the basic skeleton along with other indispensable signals for the substituents, thus confirming the condensation of two moles of pyrazole moiety with one mole of DHAA, and its analogues.
An extensive literature survey of the chemical behavior of N-unsubstituted 5-amino-1Hpyrazoles 2 ring having vacant 4-position revealed that it may react with electrophiles such as βdiketones 4 to yield a mixture of structural isomers pyrazolo[1,5-a]pyrimidines 5 and pyrazolo [3,4-b]pyridines 6 (Scheme 2) depending upon the nature of substituent on β-diketones. 27Solvent also plays a crucial role in controlling the structure of the product as acetic acid leads to the formation of a mixture of pyrazolo [3,4-b]pyridine 6 and pyrazolo[1,5a]pyrimidine 5. Pyrazolopyrimidine 5 was formed as the only reaction product when DMSO was used as solvent whereas on boiling the reactants in ethanol in the presence of TEA yielded pyrazolopyridine 6 predominantly. 28R and/or After having performed reactions with 2a and 2b, it was thought of investigating the structure of products on treating 5-amino-3-aryl-1H-pyrazoles 2c-g (unsubstituted at position 4) with DHAA and its analogues 1a-b (Scheme 1).5-Amino-3-aryl-1H-pyrazoles 2c-g, obtained from the condensation of α-cyanoacetophenones and hydrazine hydrate in ethanol, on refluxing with DHAA and its analogues 1a-b resulted in the formation of single products which were characterized on the basis of their IR, 1 H, 13 C NMR and mass spectra as bi(pyrazolopyrimidinyl)-7-ones 3f-o.No trace of pyrazolo [3,4-b]pyridine was detected in the tlc and the NMR of the crude reaction mixture.
The 1 H NMR spectra of bi(pyrazolopyrimidines) 3f-o were almost identical with those of bi(pyrazolopyrimidines) 3a-e.One important difference is that the spectra showed three singlets of one proton intensity each between δ 6.71-6.82ppm, 7.02-7.09ppm and 7.08-7.19ppm corresponding to 6', 3 and 3'-H, respectively, showing the presence of two pyrazolopyrimidine nuclei having a vacant 3-position.The 1 H NMR spectra of 3f-o displayed only one exchangeable proton at about δ 13.05 ppm due to NH present at position-4.Had the structure been pyrazolopyridine then the signals for 3 and 3'-H would not have appeared rather exchangeable signals for three NH protons would have been observed in the NMR and IR spectra.
A plausible mechanism leading to the formation of bi(pyrazolo[1,5-a]pyrimidinyl)-7-ones 3a-o is depicted in Scheme 3.An amino group of 5-aminopyrazole 2 attacks on carbonyl of acetyl side chain of 1 to generate a Schiff's base intermediate 1', followed by the subsequent nucleophilic attack of the endo nitrogen on the lactone carbonyl of DHAA to provide a pyrazolo[1,5-a]pyrimidinyl-β-diketone 7 through an intramolecular pyran ring opening (Scheme 3).] Efforts were made to isolate pyrazolo[1,5-a]pyrimidinyl-β-diketone 7 by performing the reaction at room temperature or in the cold or by adding the solution of 5-aminopyrazole 2b drop wise to the solution of DHAA 1 or by using two equivalent of DHAA 1 and one equivalent of 5aminopyrazole 2b but the reaction did not stop at this level as the resulting β-diketones 7 has greater reactivity towards nucleophiles and the second mole of 5-aminopyrazole 2 reacted further with the β-diketone 7 to yield the bi(pyrazolo[1,5-a]pyrimidinyl)-7-ones 3.
The antimicrobial activities of the compounds were compared against the standard drugs, ampicillin, chloramphenicol (25 µg/mL) (+ve control) and DMSO (-ve control).These tests were performed in triplicate and the mean of diameter of zone of growth inhibition was taken.From the Table 1, it may be concluded that the compounds bearing phenyl substituent at position-3 of pyrazolopyrimidine ring i.e. 3,3'-diphenyl-4H-[6,7']bi(pyrazolo[1,5-a]pyrimidinyl)-7-ones 3a-d showed good result as compared to compounds having vacant 3-position 3f-o.More importantly, compound 3d bearing bromine at position-6' exhibited the maximum zone of inhibition followed by that for compounds 3c and 3b at concentration of 100, 50 and 10 µg/mL against both Gram-positive and Gram-negative bacteria as compared to other compounds (3a, 3e-o).Compounds 3g and 3l having p-methylphenyl at 2 and 2'-position showed no zone of inhibition at concentrations 10, 50, 100 µg/mL.The antibacterial activity of these compounds was also compared with two commercially available antibiotics namely Chloramphenicol and Ampicillin.It is clear from the Table 1 that the compound 3d has the zone of inhibition compared to the standard antibiotic Chloramphenicol when used at the concentration of 25 µg/mL whereas no compound is as effective as the Ampicillin when used at the concentration of 25 µg/mL.
In the whole series, the MIC value of various synthesized compounds was evaluated between 200-0.19 μg/mL against Gram-positive and Gram-negative bacteria (Table 2).The compound 3d was found to be the most effective against all Gram-positive and Gram-negative bacteria having the lowest MIC value of 3.12 µg/mL against B. subtilis and P. aeruginosa, 6.25 μg/mL against B. stearothermophilus and E. coli.Compound 3c showed MIC value of 6.25 μg/mL against B. stearothermophilus, P. aeruginosa and E. coli while 25 μg/mL against B. subtilis comparable with commercial antibiotics Chloramphenicol and Ampicillin which showed MIC value of <0.19 and 0.19 μg/mL against all the bacteria.Whereas the compounds 3g and 3l are having the MIC value greater than 200 μg/mL.

Conclusions
A series of 5'-methyl-4H-[6,7']bi(pyrazolo[1,5-a]pyrimidinyl)-7ones 3a-o were synthesized from the reaction of substituted 5-amino-1H-pyrazoles with DHAA and its analogues and were evaluated for their antibacterial activity against both Gram-positive and Gram-negative bacteria.Treatment of 5-aminopyrazoles with or without a substituent at position-4 with DHAA and its analogues afforded single compound having two prazolopyrimidine nuclei.Compounds bearing phenyl substituent at position-3 of pyrazolopyrimidine ring 3a-d showed good result as compared to compounds having vacant 3-position 3f-o.It is interesting to note that compound 3d displayed significant antibacterial activity compared to the standard antibiotic Chloramphenicol.

Table 1 .
In vitro antibacterial activity of compounds 3a-o by using well diffusion method a "-" means not determined or no activity whatever applies.b Chloramphenicol and Ampicillin has been used as a standard drugs.