Facile synthesis of new pyrazolo[4′,3′:5,6]pyrano[2,3-d]pyrimidin-5(1H)-ones via the tandem intramolecular Pinner–Dimroth rearrangement and their antibacterial evaluation

1 Introduction

The synthesis of nitrogen- and oxygen-containing heterocyclic compounds has received considerable attention due to their presence in a large number of natural products and wide biological and pharmaceutical activities [1], [2], [3], [4]. The presence of different biologically active heterocyclic moieties in a single molecule may unite the properties of all structural motifs and enhance the biological activity. Therefore, the synthesis of new polycyclic heterocyclic compounds formed from a combination of two or more heterocyclic scaffolds has been a subject of increasing interest.

Pyrazoles, pyrans, and pyrimidines have occupied unique positions in the design and synthesis of novel biologically active agents due to their broad spectrum of activities. Literature reports had already established that certain pyrazoles exhibit significant biological properties such as anti-HCV [5], antipyretic [6], antiviral [7], anti-inflammatory [8], antiangiogenic [9], and antitumor [10] activities. The success of COX-2 inhibitors containing a pyrazole moiety [11], [12] has highlighted the importance of this motif in medicinal chemistry [13]. A number of these compounds are also used as potential inhibitors of P38 kinase [14], hMAO-B [15], and TNF-α [16]. On the other hand, structures containing pyrimidine and pyran scaffolds have important biological activities such as antimalarial [17], anti-inflammatory [18], antitumor [19], [20], antibacterial [21], anti-osteosarcoma [22], and antiproliferative [23] properties. Recently, pyrimidines have been known as inhibitors of tyrosine kinase [24], cyclin-dependent kinase 4 [25], and epidermal growth factor receptor tyrosine kinase [26]. Furthermore, a number of pyrans have been employed as apoptosis-inducing agents [27] and constitute the structural unit of a series of natural products [28]. Because of the importance of these heterocycles we became interested in the synthesis of some new heterocyclic compounds containing pyrazole, pyran, and pyrimidine scaffolds.

Among various pyrazolopyranopyrimidine scaffolds, pyrazolo[4′,3′:5,6]pyrano[2,3-d]pyrimidines have been of more interest because of reported interesting biological properties such as antifungal [29], antiviral [30], and antimicrobial [31] activities. A number of methods have already been reported for the synthesis of these compounds starting from pyranopyrazoles or by a one-pot multi-component reaction [29], [30], [31], [32], [33], [34].

In view of the above-mentioned properties, and in continuation of our previous studies on the synthesis of heterocyclic compounds with potential biological activities [35], [36], [37], [38], [39], [40], [41], [42], [43], [44], we report here the synthesis of new 7-alkyl-4,6-dihydropyrazolo[4′,3′:5,6]pyrano[2,3-d]pyrimidin-5(1H)-one derivatives 3a–3f by the reaction of 6-amino-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitriles 1a–1d with aliphatic carboxylic acid 2a or 2b in the presence of phosphoryl chloride (POCl₃) (Scheme 1). To the authors’ knowledge, compounds 3a–3f are new and have not been reported in the literature. The antibacterial assay of the synthesized compounds was also evaluated.

Scheme 1:

Synthesis of new compounds 3a–3f with yields given in parentheses.

2 Results and discussion

6-Amino-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitriles 1a–1d were prepared according to methods cited in the literature [45], [46]. At first, in a model reaction, 6-amino-4-(4-methylphenyl)-3-phenyl-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitrile 1a (1 mmol) was allowed to interact with acetic acid 2a (1 mL) under reflux without the addition of any catalyst and solvent. After 3 h, the product 3a was obtained only in 27% yield. Next, the reaction was conducted in acetic acid (1 mL) in the presence of POCl₃ (0.2 mL) as the well-known chlorinating agent. After refluxing the reaction mixture for 3 h, monitoring with thin-layer chromatography (TLC) showed that the reaction efficiently proceeded in these conditions leading to 91% yield of the product identified as 7-methyl-4-(4-methylphenyl)-3-phenyl-4,6-dihydropyrazolo[4′,3′:5, 6]pyrano[2,3-d]pyrimidin-5(1H)-one 3a. Consequently, all subsequent processes for the synthesis of compounds 3b–3f were carried out under similar conditions. The structural elucidation of the isolated products 3a–3f was based upon spectral and microanalytical data. For example, the ¹H NMR spectrum of compound 3a in [D₆]dimethyl sulfoxide (DMSO) demonstrated two singlets at δ=2.15 and 2.29 ppm for two methyl groups, a sharp singlet at δ=5.33 ppm belonging to the CH in pyran ring, the characteristic signals at δ=6.96–7.58 ppm for the aromatic protons, and two single broad bands at δ=12.47 and 12.95 ppm for two NH groups, which indicated the formation of the tricyclic compound 3a. The IR spectrum of 3a was devoid of the CN absorption band at 2199 cm⁻¹ of the precursor 1a, which shows the inclusion of the nitrile moiety in the cyclization process. Furthermore, this compound gave satisfactory ¹³C NMR spectrum and elemental analysis data corresponding to the molecular formula C₂₂H₁₈N₄O₂ (Experimental section and Supplementary Information).

On the basis of similar transformations [47], [48], [49], [50], a plausible pathway for the formation of compounds 3a–3f is proposed, as depicted in Scheme 2. Formation of these compounds probably proceeds via the tandem intramolecular Pinner–Dimroth rearrangement. Nucleophilic attack of the amino group in compounds 1a–1d onto the activated carbonyl group of acyl chloride [I], which had been obtained by chlorination of carboxylic acid 2a or 2b with POCl₃, afforded the intermediate [II]. This intermediate undergoes an intramolecular Pinner reaction to give the oxazine intermediate [III]. The subsequent Dimroth rearrangement of the latter intermediate gave the final tricyclic products 3a–3f. Attempts to isolate the proposed intermediates failed even after careful monitoring of the reactions.

Scheme 2:

Plausible mechanism for the formation of compounds 3a–3f.

Finally, we were interested in studying the role of the amount of POCl₃ in the reaction. For this purpose, the model reaction was again tested in the presence of excess POCl₃ (1 mL) under reflux. During monitoring of the reaction mixture by TLC, it was observed that a product, with different R_f’s of that expected for compound 3a, was forming. During workup and identification, it was established that O-acylation of the amidic group in the pyrimidine ring had occurred and the product 5-acetoxy-7-methyl-4-(4-methylphenyl)-3-phenyl-1,4-dihydropyrazolo[4′,3′:5,6]pyrano[2,3-d]pyrimidine 5 was isolated. As is shown in Scheme 3, the formation of compound 5 is assumed to be the result of a cascade reaction by formation of the intermediate 4 followed by aromatic nucleophilic displacement with acetic acid. However, under these conditions, attempts to isolate the intermediate 4 failed. Compound 5 can also be formed through the reaction of previously isolated compound 3a with 2a in the presence of excess POCl₃ under reflux. The ester hydrolysis of compound 5 in H₂O-EtOH containing NaOH under reflux afforded the product 3a in high yield (Scheme 3).

Scheme 3:

Formation of compound 5.

The synthesized compounds 3a–3f and 5 were screened for their antibacterial activity against reference strains of Staphylococcus aureus (S. aureus, American Type Culture Collection 6538), and Staphylococcus epidermidis (S. epidermidis, ATCC 12228) as Gram-positive bacteria and Escherichia coli (E. coli, ATCC 8739) and Pseudomonas aeruginosa (P. aeruginosa, ATCC 9027) as Gram-negative bacteria. Gentamicin was used as the reference compound in the evaluation of antibacterial activity. None of the compounds had an antibacterial effect against Gram-negative bacteria, E. coli, and P. aeruginosa, in the primary screening test. The results revealed that compound 3f has antibacterial activity against S. aureus and S. epidermidis in disks containing 3 mg per disk with inhibition zones of 11.00±0.00 mm, while compound 3e showed an antibacterial effect against only S. aureus bacterium in disk containing 3 mg per disk with the inhibition zone of 13.00±0.00. The minimum inhibitory concentration (MIC) was 3 mg mL⁻¹ for compound 3f against tested Gram-positive bacteria and 2 mg mL⁻¹ for compound 3e against S. aureus. Growth inhibition of S. aureus and S. epidermidis was also observed against gentamicin (10 μg per disk), which was 20.00±0.00 and 22.00±0.00 mm, respectively. Thus, the antibacterial activity of compounds 3e and 3f against tested bacteria was satisfactory but less than those of the standard.

3 Conclusion

In summary, we have reported the synthesis of new 7-alkyl-4,6-dihydropyrazolo[4′,3′:5,6]pyrano[2,3-d]pyrimidin-5(1H)-one derivatives 3a–3f in high yields by the reaction of 6-amino-1,4-dihydropyrano[2,3-c]pyrazole-5-carbonitriles 1a–1d with aliphatic carboxylic acid 2a or 2b in the presence of a limited amount of POCl₃ under reflux. Using excess amount of POCl₃, in a model reaction of 1a with 2a, the corresponding 5-acetoxy derivative 5, instead of 3a, was isolated. Compound 5, which can also be formed through the reaction of 3a with 2a in the presence of excess POCl₃ under reflux, could be converted again to 3a under hydrolysis conditions using NaOH in H₂O-EtOH at reflux temperature. An intramolecular Pinner–Dimroth rearrangement was also suggested for the formation of compounds 3a–3f. All new products were characterized by FT-IR, ¹H, and ¹³C NMR spectra and microanalytical data. Furthermore, the antibacterial assay of the synthesized compounds showed that two of them have satisfactory antibacterial effects on tested Gram-positive bacteria.

4 Experimental section

5 Supplementary information

Copies of the FT-IR, ¹H NMR, and ¹³C NMR spectra as well as pictures of disks with the inhibition zones for the evaluation of the antibacterial activity are given as Supplementary information available online (http://dx.doi.org/DOI: 10.1515/znb-2018-0166).