Three-component microwave-assisted synthesis of 3,5-disubstituted pyrazolo[3,4-d]pyrimidin-4-ones

A practical three-component method for the synthesis of pyrazolo[3,4-d]pyrimidin-4-ones was developed. The reaction was performed in a one-pot manner under controlled microwave irradiation using easily accessible methyl 5-aminopyrazole-4-carboxylates, trimethyl orthoformate, and primary amines. Under the optimized conditions, challenging substrates, such as N-1 unsubstituted 5-aminopyrazole-4-carboxylates with another substituted amino group in position 3, reacted selectively affording 5-substituted 3-arylamino-1,5-dihydro-4H-pyrazolo[3,4-d]pyrimidin-4-ones. The reaction tolerated a range of primary amines, including anilines. The advantages of the developed protocol include short reaction time, pot- and step-economy, and convenient chromatography-free product isolation. The structural features of representative products were explored by X-ray crystallography.


Introduction
The pyrazolo [3,4-d]pyrimidin-4-one scaffold has been utilized for bioactive molecule construction for a long time. Without other substituents, this bioisostere of hypoxanthine is known as the anti-gout drug allopurinol (Fig. 1). However, the pharmacological prole of substituted pyrazolo [3,4-d]pyrimidin-4-ones is more diverse. For example, pyrazolo [3,4-d]pyrimidin-4-ones substituted in position 5 were reported to possess antiviral, 1 antimicrobial, 2-4 and anticancer [5][6][7] properties. This substitution pattern is shared by highly specic ubiquitin-specic protease 7 inhibitors FT827 and FT671, which were found to be active against cancer in vitro and in vivo. 8,9 Recently, ASN7186636 was reported as an inhibitor of another anticancer target, tropomyosin receptor kinase A. 10 Compound 1 possessing 3,4dichlorobenzyl substitution in position 5 of the pyrazolo [3,4-d] pyrimidin-4-one system was identied as an inhibitor of prometastatic protein fascin and served as a starting point for the development of highly potent inhibitors. 11 Anti-inammatory and analgesic effects were demonstrated by a potent dual inhibitor cyclooxygenase 2 and inducible nitric oxide synthase, compound 2. 12 The synthesis of 5-substituted pyrazolo [3,4-d]pyrimidin-4ones typically involves the pyrimidinone annulation followed by the N-alkylation in position 5. Developed in 1956, 13 this approach was based on the conversion of 5-amino-1methylpyrazole-4-nitrile to the corresponding amide and the subsequent pyrimidinone ring closure upon heating in formamide (Scheme 1, pathway 1). Many modications of this ring closure have been reported. Instead of formamide, formic acid or triethyl orthoformate were used as one-carbon inserting reagents in the cyclization step. 5,14 Pyrazolo[3,4-d]pyrimidin-4ones were also prepared by heating 5-aminopyrazole-4-nitriles in formic acid 1,6,7,12 or alkyl 5-aminopyrazole-4-carboxylates in formamide. 15,16 However, the main drawbacks of this approach remain (1) lack of selectivity in the alkylation of pyrazolo [3,4-d] pyrimidin-4-ones unsubstituted at N-1 (ref. 17 and 18) and (2) limitation of groups introduced at N-3 to alkyls.
These limitations were overcome by the preparation of substituted 5-aminopyrazole-4-carboxamides, for example by amidation of esters (pathway 2), followed by the cyclization in the reaction with formic acid. 19 The pyrimidinone ring formation was also achieved by the treatment of substituted 5aminopyrazole-4-carboxamides with triethyl orthoformate 20 or N,N-dimethylformamide dimethyl acetal (DMF-DMA). 21,22 The reaction with DMF-DMA is typically performed in two steps with isolation of the corresponding formamidines as intermediates. 2,3,5,23 Two synthetic approaches to 5-substituted pyrazolo[3,4-d] pyrimidin-4-ones were developed by Finlander and Pedersen. 24 The reaction of ethyl 5-amino-1-methylpyrazole-4-carboxylate with triethyl orthoformate resulted in the formation of the formimidate, which upon the treatment with anisidine transformed to the corresponding formamidine (pathway 3). The thermal cyclization of this formamidine afforded the desired pyrazolo [3,4-d]pyrimidin-4-one. This approach, however, was unsuccessful with the N-1 unsubstituted analogue. A more general approach utilizes a different reaction sequence: preparation of formimidate from anisidine and triethyl orthoformate in the rst step, followed by the reaction with 5-aminopyrazole-4-carboxylates (pathway 4). In pathway 3, DMF-DMA was also used instead of triethyl orthoformate. 25 Pathways 2, 3, and 4 are based on similar types of reagents: 5-aminopyrazole-4-carboxylate, primary amines, and triethyl orthoformate (or its synthetic equivalents). However, these pathways are different in the order of steps combining the reagents. Since the outcome of these pathways does not depend on the sequence of their individual reactions, we decided to develop a three-component one-pot methodology introducing the reagents to the reaction mixture together (pathway 5). Multicomponent reactions involving 5-aminopyrazoles and orthoformates oen benet from microwave irradiation. [26][27][28][29][30] Moreover, it has been reported 31 that 5-aminopyrazoles react with orthoformates and secondary amines under microwave irradiation affording N-pyrazolylformamidines, which resemble intermediates for the synthesis of pyrazolo [3,4-d]pyrimidin-4-ones. Therefore, we applied microwave-assisted methodology for the development of our three-component protocol.

Synthesis
The synthesis of starting 3-substituted 5-aminopyrazole-4carboxylates 3 was performed according to the previously reported method. 32 For the trial reaction and subsequent condition optimization, we used the model reaction of 5aminopyrazole-4-carboxylate 3a, benzylamine, and trimethyl orthoformate under microwave irradiation in a Discover SP reactor (CEM, USA) ( Table 1).
The reaction at 160 C for 35 min resulted in the formation of the desired 5-benzyl-3-phenylaminopyrazolo [3,4-d]pyrimidin-4one (4a), which was isolated by simple ltration. Several solvents, including emerging sustainable solvents 2-methyltetrahydrofuran (2-MeTHF) and eucalyptol, were screened under these conditions and the best results were obtained in EtOH (Table 1, entry 3). Further improvements in the yield were achieved by increasing the reaction time to 55 min (entry 9). An attempt to carry out the reaction at a lower temperature (150 C) resulted in a lower yield (entry 11) while temperatures above 160 C were precluded by an increase of the pressure above the instrument safety limits. The reaction performed using conventional heating under reux in EtOH did not afford the desired product even aer 3 days (entry 12). We also attempted to carry out this reaction using conventional heating in pressurized vessels resembling the conditions of the reaction under microwave irradiation (entry 13). This reaction in the Monowave 50 (Anton Paar) reactor resulted in the isolation of equally pure 4a but in lower yield (27%).
Therefore, for the exploration of the multicomponent reaction scope, we used microwave irradiation at 160 C for 55 min ( Table 1, entry 9) as optimised conditions. Two points of diversity in positions 3 and 5 of pyrazolo [3,4-d]pyrimidin-4-ones 4 were generated by different combinations of 5-aminopyrazole-4-carboxylates 3 and primary amines (Scheme 2). Overall, the multicomponent reaction under microwave irradiation was found to be selective and its scope was rather general. The method allowed selective pyrimidine ring annulation on the N-1 unsubstituted 5-aminopyrazole-4-carboxylates 3 and no reactions at pyrazole ring nitrogen atoms or 3-arylamino group were observed. A variety of 3-arylamino substituents on the pyrazole ring of 3 were equally well tolerated. The method optimized for benzylamine was successfully applied for substituted benzylamines and their analogues affording pyrazolo [3,4-d]pyrimidin-4-ones 4 in 60-85% yields. However, the yields decreased to 21-53% when aromatic amines were used as substrates.
The structure of the prepared pyrazolo [3,4-d]pyrimidin-4ones 4 was conrmed using NMR spectroscopic data. The carbonyl group signal from the constructed pyrimidinone ring appears in the 13 C NMR spectra at 156.8-157.2 ppm. The methine group of this heterocyclic ring gives a signal at 150.7-151.3 ppm in the 13 C NMR spectra and a downeld-shied singlet at 8.06-8.54 ppm in the 1 H NMR spectra. This signal in the 1 H NMR spectra of compounds 4a-k appears $0.3 ppm more towards low eld compared to the signals of 4m-t possessing an aryl substitution at N-5. The shielding effect of the aryl group at N-5 of 4m-t indicates the positioning of the phenyl out-of-plane of the pyrazolo [3,4-d]pyrimidin-4-one skeleton thus resulting in the anisotropic effect of the aryl substituent on H-6 located under the plane of this ring. Such an orientation of the phenyl ring at N-5 was further conrmed by X-ray crystallography and can be explained by the steric hindrance between the phenyl ring and oxygen atom of the carbonyl group.

X-ray crystallography
The molecular structures of two representative derivatives were established by X-ray crystallography, thereby providing further supporting evidence for the structures of the products. The molecular structures of 4d and 4p are illustrated in Fig. 2 and selected geometric parameters are collated in Table 2. For 4d, the 10 atoms comprising the pyrazolo [3,4-d]pyrimidin-4-one core exhibit a root-mean-square (r.m.s.) deviation of 0.0124Å with the maximum deviation from the least-squares plane being 0.0223(10)Å for the C3a atom. The dihedral angles between the central plane and the planes through the N-bound tolyl and phenyl rings are 4.36 (5) and 71.10(4) , respectively, indicating near to co-planar and perpendicular relationships; the dihedral angle between the outer rings is 69.35 (4) . The planarity of the fused-ring system coupled with the systematic variations in bond lengths, Table 2, suggest considerable delocalization of pelectron density in this residue. The most notable elongations are seen in the C3-N2 and C6-N7 bond lengths with commensurate shortening in the N1-N2, C3-C3a and C3a-C4 bonds.
The molecular structure of 4p shows features similar to those exhibited by 4d. The 10-membered core exhibits minor distortions from planarity having a r.m.s. deviation of 0.0319Å with the maximum deviation of 0.0605(13)Å noted for the C3a atom; the dihedral angle between the veand six-membered rings ¼ 4.52 (8) . The dihedral angles between the central plane and the planes through the N-bound bromo-and methoxy-phenyl rings are 13.15 (6) and 58.81(3) , respectively, are indicative of signicant twisting in the molecule; the dihedral angle between the outer rings is 52.82(4) .
In both 4d and 4p, an intramolecular amine-N-H/ O(carbonyl) hydrogen bond is noted (Table 3). Hydrogen bonding features prominently in the supramolecular association evident in the crystals of 4d and 4p, Table 3. Centrosymmetric dimers are formed in the crystal of 4d, being mediated by pyrazolyl-N-H/N(pyrimidyl) hydrogen bonds giving rise to eight-membered {/NCNH} 2 synthons. These are connected into a supramolecular chain with a twisted topology via rather short tolyl-C-H/O(carbonyl) interactions, Fig. 3a. As detailed in ESI Fig. S1, † the chains are connected into a three-

Conclusions
In conclusion, we developed an efficient three-component method for the synthesis of 3,5-disubstituted pyrazolo [3,4-

General method for the synthesis of 5-substituted 3arylaminopyrazolo[3,4-d]pyrimidin-4-ones (4)
Substituted 5-aminopyrazole-4-carboxylates 3 (1 mmol), trimethyl orthoformate (0.33 mL, 3 mmol), and a primary amine (3 mmol) were added to EtOH (2 mL) in a 10 mL seamless pressure vial. The reaction mixture was irradiated in a Discover SP (CEM) microwave reactor operating at a maximal microwave power of 150 W and pressure limit of 435 psi at 160 C for 55 min. Aer cooling, the precipitated product was isolated by vacuum ltration and recrystallised using an appropriate solvent.