The utility of pyrimidine derivatives in the construction of azolo[d]pyrimidine: part II-multi-nitrogen azole systems with potential biological applications

Abstract

In this review (part II), we continue our compilation of studies highlighting the use of the pyrimidine ring as a key core precursor for the construction of azolo[d]pyrimidine scaffolds containing more than one nitrogen atom in the azole moiety. Fusion of the azole ring owing to the presence of multiple nitrogen atoms onto the pyrimidine nucleus affords structurally diverse regioisomeric systems, including pyrazolo[3,4-d]pyrimidines, pyrazolo[4,3-d]pyrimidines, imidazolo[4,5-d]pyrimidines (purines) and [1,2,3]triazolo[4,5-d]pyrimidines (8-azapurines), depending on the position of the nitrogen atom within the fused heterocycle. This review provides a comprehensive overview of recent advances during the last decade (2015–2026) in the synthesis and biological applications of fused azolo[d]pyrimidine scaffolds (Part II). In addition, the results presented in this review highlight the importance of various azolo[d]pyrimidine derivatives as promising scaffolds for the development of new therapeutic agents.

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Introduction

Building upon our previous study (Part I), which explored pyrimidine nucleus as a key building block for the synthesis of azolo[d]pyrimidine frameworks bearing mono-nitrogen azole rings,¹ this work extends that approach to the construction of azolo[d]pyrimidine scaffolds incorporating azole moieties containing more than one nitrogen atom, utilizing pyrimidine derivatives as versatile precursors. No doubt that the pyrimidine ring represents a fundamental heterocyclic scaffold in organic and medicinal chemistry owing to its presence in numerous biologically essential molecules, including nucleic acid bases, vitamins, and pharmaceuticals.^2–18 The electron-deficient aromatic character of pyrimidine, together with its versatile substitution patterns, renders it a valuable core precursor for the construction of fused-pyrimidine scaffolds.¹⁹ In fact, the diversity of pyrimidine derivatives is further expanded through the fusion of the pyrimidine ring with other heterocyclic moieties leading to the formation of more bioactive molecules compared to the individual separated moieties. Among them, azolo[d]pyrimidines constitute an important class of bicyclic heterocyclic frameworks formed through the annulation of azole rings onto the pyrimidine nucleus (Fig. 1). The fusion of nitrogen-rich five-membered heterocycles, such as pyrazole, imidazole, and triazole, onto pyrimidine scaffolds has resulted in structurally diverse compounds exhibiting a broad spectrum of biological activities.^20–40

Fig. 1 Azolo[d]pyrimidines generated by combining pyrimidines at the d position with different heterocyclic rings such as pyrazole, imidazole, and triazole.

Specifically, fusion of a pyrazole ring onto the pyrimidine nucleus affords structurally diverse regioisomers, including pyrazolo[3,4-d]pyrimidines and pyrazolo[4,3-d]pyrimidines, which have been extensively studied due to their structural resemblance to purine analogues and their notable bioactivities, particularly in kinase inhibition and anticancer drug discovery.^41–44

Several FDA-approved drugs contain the pyrazolo-pyrimidine nucleus, reflecting its pharmaceutical importance (Fig. 2). For example, allopurinol, a pyrazolo[3,4-d]pyrimidine, was the first xanthine oxidase inhibitor approved by the U.S. Food and Drug Administration for the treatment of chronic gout and hyperuricemia.⁴⁵ Additionally, ibrutinib, a covalent Bruton's tyrosine kinase (BTK) inhibitor bearing a pyrazolo[3,4-d]pyrimidine core, has been approved for the treatment of several B-cell malignancies.⁴⁶ One of the most significant pharmacological applications of pyrazolo[4,3-d]pyrimidine derivatives is exemplified by sildenafil (Viagra®), a selective phosphodiesterase type 5 (PDE5) inhibitor widely used as an oral therapeutic agent for the treatment of male erectile dysfunction.⁴⁷ Consequently, a series of sildenafil analogues (R = Me and Et; R₁ = Me, Et, and –CH₂CH₂OH) was synthesized, and their inhibitory activities were evaluated. The results demonstrated enhanced inhibitory potency along with improved selectivity toward PDE5.⁴⁸

Fig. 2 FDA approved-drug containing a pyrazolopyrimidine moiety.

Similarly, fusion of an imidazole ring with pyrimidine yields purine frameworks, key heterocycles that constitute the core of nucleosides, nucleotides, and numerous therapeutic agents (Fig. 3), such as roscovitine, a cyclin-dependent kinase (CDK) inhibitor with anticancer potential,⁴⁹ acyclovir (an antiviral nucleoside analogue),⁵⁰ and mercaptopurine (an antileukemic agent).⁵¹

Fig. 3 Therapeutic agents containing a purine moiety.

Furthermore, the incorporation of a triazole ring into the pyrimidine nucleus affords [1,2,3]triazolo[4,5-d]pyrimidines (8-azapurines), a class of pharmacologically active heterocycles with diverse therapeutic potential.^34–41 Notable examples include zaprinast, a [1,2,3]triazolo[4,5-d]pyrimidine derivative investigated as a phosphodiesterase inhibitor that contributed to the development of subsequent PDE-targeting drugs,⁵² and 8-azaguanine, an early purine analog exhibiting antineoplastic activity (Fig. 4).⁵³ Beyond anticancer applications, triazolopyrimidine frameworks have been broadly reviewed for their wide spectrum of biological activities, including antimicrobial,⁵⁴ antiviral,^35,55,56 antiplatelet,⁵⁷ antimalarial,⁵⁸ antiepileptic,⁵⁹ and anti-Alzheimer's activities,⁶⁰ emphasizing their versatility as therapeutic scaffolds.

Fig. 4 Therapeutic agents containing a [1,2,3]triazolo[4,5-d]pyrimidine moiety.

The present review summarizes synthetic strategies leading to these frameworks and emphasizes their importance in heterocyclic and medicinal chemistry during the last decade (2015–2026).

Pyrazolo[d]pyrimidines

Pyrazolo[d]pyrimidines are bicyclic heterocycles formed by the fusion of a pyrazole ring with a pyrimidine nucleus, resulting in a rigid, planar framework. Depending on the mode of annulation, two regioisomeric systems can be obtained: pyrazolo[3,4-d]pyrimidine and pyrazolo[4,3-d]pyrimidine. Numerous synthetic methodologies have been developed for the preparation of pyrazolo[4,3-d]pyrimidines; however, both classical and contemporary approaches predominantly rely on pre-substituted pyrazole derivatives as key starting materials.

In contrast, strategies that construct the pyrazolo[4,3-d]pyrimidine scaffold directly from pyrimidine substrates remain relatively limited.^61–63 This limitation is largely attributed to the inherent challenges associated with the regioselective activation of the electron-deficient pyrimidine ring, as well as the controlled formation of the N–N bond during pyrazole annulation.

Accordingly, this section focuses exclusively on synthetic approaches that utilize the pyrimidine ring as the principal core precursor for the construction of pyrazolo[3,4-d]pyrimidine frameworks.

Synthesis of pyrazolo[3,4-d]pyrimidine derivatives

6-Chloropyrimidine-5-carbaldehyde (1) has been widely employed in the literature for the preparation of 1H-pyrazolo[3,4-d]pyrimidine derivatives 2. Thus, condensation of 1 with various hydrazine derivatives in the presence of triethylamine afforded the corresponding pyrazolo[3,4-d]pyrimidine derivatives 2 bearing different substituents (Scheme 1).^64–70

Scheme 1 Preparation of pyrazolo[3,4-d]pyrimidines from 6-chloropyrimidine-5-carbaldehyde.

According to Ofitserova et al., protection of the aldehyde group in 4,6-dichloropyrimidine-5-carbaldehydes alters the selectivity of their reaction with arylhydrazines. This protection is presumably attributed to steric hindrance and a change in reaction sequence between the reacting centers. Thus, treatment of 4,6-dichloropyrimidine-5-carbaldehyde derivatives 3 with 1,3-butylene glycol in the presence of p-toluenesulfonic acid monohydrate afforded 4,6-dichloro-5-(4-methyl-1,3-dioxan-2-yl)pyrimidines 4. The latter compounds were treated with hydrazine derivatives to give the corresponding 4-chloro-5-(4-methyl-1,3-dioxan-2-yl)-6-(2-arylhydrazinyl)pyrimidines 5, which were cyclized using p-toluenesulfonic acid, leading to the formation of the desired 2-arylpyrazolo[3,4-d]pyrimidines 6 (Scheme 2).⁷¹

Scheme 2 Preparation of pyrazolo[3,4-d]pyrimidine from protected 4,6-dichloropyrimidine-5-carbaldehydes.

On the other hand, Ebrahimpour et al. reported that the microwave-assisted transformation of ethyl 4-chloropyrimidine-5-carboxylates 7 provides direct access to the corresponding pyrazolo[3,4-d]pyrimidines 8 through reaction with hydrazine, methylhydrazine, or phenylhydrazine. In contrast, the classical synthesis of compounds 8 was achieved by heating the reactants in ethanol in the presence of sodium ethoxide as a base (Scheme 3).⁷²

Scheme 3 Preparation of pyrazolo[3,4-d]pyrimidine from ethyl 4-chloropyrimidine-5-carboxylates.

Wang et al. reported that the treatment of 4-amino-6-chloropyrimidine-5-carbonitrile 9 with isopropylhydrazine hydrochloride afforded the corresponding pyrazolo[3,4-d]pyrimidine derivatives 10 (Scheme 4).⁷³

Scheme 4 Preparation of pyrazolo[3,4-d]pyrimidine from 6-chloropyrimidine-5-carbonitrile.

Ragab et al. described the acid-mediated intramolecular cyclization of 6-hydrazinylpyrimidine-5-carbonitrile derivatives 11a–d, where treatment with HCl promoted ring closure to afford the corresponding 1H-pyrazolo[3,4-d]pyrimidin-3-amine derivatives 12a–d (Scheme 5).⁷⁴

Scheme 5 Preparation of pyrazolo[3,4-d]pyrimidine from 6-hydrazinylpyrimidine-5-carbonitriles.

El-Kalyoubi et al. described the Michael-type addition of 6-hydrazinyluracils 13a–c to benzylidene malononitrile or benzylidene ethyl cyanoacetate. Refluxing the reaction mixture in DMF in the presence of triethylamine promoted cyclization, followed by the elimination of malononitrile or ethyl cyanoacetate to furnish the corresponding 3-substituted-7-propyl- and/or 5,7-dipropyl-1H-pyrazolo[3,4-d]pyrimidine-4,6(5H,7H)-diones 14a–f (Scheme 6).⁷⁵

Scheme 6 Preparation of pyrazolo[3,4-d]pyrimidine from 6-hydrazinyluracils.

In another study, Komkov et al. reported that treatment of 5-acetyl-4,6-di(methylsulfanyl)-2-phenylpyrimidine (15) with hydrazine hydrate promoted cyclization to afford 4-hydrazino-3-methyl-6-phenyl-1H-pyrazolo[3,4-d]pyrimidine (16) (Scheme 7).⁷⁶

Scheme 7 Preparation of pyrazolo[3,4-d]pyrimidine from a methylsulfanylpyrimidine derivative.

El-Mekabaty reported that the treatment of enaminones 17 with hydrazine hydrate or phenyl hydrazine in refluxing DMF containing triethylamine furnished the corresponding pyrazolo[3,4-d]pyrimidinones 19 through the non-isolable intermediates 18 (Scheme 8).⁷⁷

Scheme 8 Preparation of pyrazolo[3,4-d]pyrimidine from enaminones.

Synthesis of imidazolo[4,5-d]pyrimidines (purines)

Various synthetic strategies have been developed for the construction of the imidazolo[4,5-d]pyrimidine fused heterocyclic system. The most common approaches involve the cyclization of appropriate substituted pyrimidine derivatives, particularly 4,5-diaminopyrimidines with suitable reagents such as aldehydes, ketones, carboxylic acids, carboxylic anhydrides, amides, ortho-esters, cyanoacetimidate, malononitrile, isothiocyanates, carbon disulfide or primary alcohols. These reactions typically lead to the formation of an imidazole ring fused to the pyrimidine nucleus, producing the desired framework. Consequently, the synthesis of substituted purine derivatives has attracted considerable attention, and a variety of strategies have been developed using different catalysts, such as acetic anhydride, ethanesulfonic acid, NaH, FeCl₃–SiO₂, polyphosphoric acid (PPA), Pd(OAc)₂–CuI, Cs₂CO₃, H₂S/base, H₂S/S₈, Pd₂(dba)₃, cellulose sulfuric acid, and pyridine, as well as microwave-assisted conditions. However, many of these methods suffer from several drawbacks, including the use of toxic organic solvents, tedious work-up procedures, prolonged reaction times, and the requirement for expensive reagents or catalysts.

In recent years, heterogeneous catalysts have gained increasing importance in heterocyclic chemistry due to their significant advantages, such as simple work-up procedures, environmental compatibility, low cost, low toxicity, and ease of separation from the reaction mixtures.

In this concept, Maddila et al. developed a convenient, simple, and efficient catalytic procedure for the synthesis of 6-chloro-8-substituted-9H-purine derivatives 21. This method involves the one-pot condensation of 6-chloropyrimidine-4,5-diamine (20) with various aldehydes using silver supported on silica (Ag/SiO₂) or cellulose sulfuric acid as a heterogeneous catalyst. The target compounds were obtained in excellent yields within short reaction times under environmentally friendly conditions (Scheme 9).^78,79

Scheme 9 Preparation of purine derivative via the reaction of 6-chloro-pyrimidine-4,5-diamine with substituted benzaldehyde.

Similarly, Polat et al. reported the use of another catalyst for the preparation of 6-chloro-N-cyclopentylpyrimidine-4,5-diamine 23 via the one-pot condensation of 6-chloro-N⁴-cyclopentylpyrimidine-4,5-diamine (22) with substituted benzaldehydes in the presence of p-toluenesulfonic acid (p-TSA) as a catalyst (Scheme 10).⁸⁰

Scheme 10 Preparation of purine derivative via the reaction of 6-chloro-N-cyclopentylpyrimidine-4,5-diamine with substituted benzaldehyde.

Doganc et al. described a two-step synthesis of 2-chloro-8-(4-fluorophenyl)-9H-purine 26. In the first step, Schiff base 25 was formed by the reaction of 2-chloropyrimidine-4,5-diamine (24) with 4-fluorobenzaldehyde under reflux conditions. In the second step, the resulting Schiff base 25 was treated with N-bromosuccinimide (NBS) in chloroform under reflux, yielding the N9 isomer 26 and not the N7 isomer 27. Interestingly, they also reported that the reaction of 4,5-diaminopyrimidine 24 with 4-fluorobenzoyl chloride produced 6-chloro-8-(4-fluorophenyl)-3H-purine (28) as the sole product (Scheme 11).⁸¹

Scheme 11 Preparation of 9H-purines via the reaction of a Schiff base with NBS, and 3H-purine via the reaction of 4,5-diaminopyrimidine with 4-fluorobenzoyl chloride.

Similarly, other reports have described the use of thionyl chloride as a cyclizing agent in the two-step synthesis of 8-substituted-xanthine 31 via treatment of 5,6-diaminouracils 29 with various substituted aldehydes in a MeOH : AcOH (4 : 1) mixture at room temperature to afford the corresponding Schiff bases 30. Subsequent oxidative cyclization of these benzylidene derivatives using thionyl chloride furnished the desired 8-substituted-xanthine 31 in good yields (Scheme 12).^82–84

Scheme 12 Preparation of xanthine derivatives from 5,6-diaminouracils via reaction with substituted aldehydes followed by thionyl chloride.

However, El-Kalyoubi et al. reported that 8-aryl xanthine derivatives 31 could be obtained directly via the condensation of 5,6-diaminouracils 29 with various aromatic aldehydes in DMF under reflux (Scheme 13).⁸⁵

Scheme 13 Preparation of 8-aryl xanthine derivatives from 5,6-diaminouracil with aromatic aldehydes.

Furthermore, El-Kalyoubi et al. reported a simple procedure for the synthesis of the substituted purine derivative 32 in high yield by heating 1,3-dimethyl-5,6-diaminouracil 29 with p-bromoacetophenone in DMF under a light torch flame, producing 8-(4-bromophenyl)-1,3,8-trimethyl-3,8-dihydro-1H-purine-2,6-dione (32) (Scheme 14).⁸⁶

Scheme 14 Synthesis of 8-(4-bromophenyl)-1H-purine-2,6-dione from 1,3-dimethyl-5,6-diaminouracil and p-bromoacetophenone.

In another report, El-Kalyoubi et al. heated diaminouracils 29 in DMF with 2,7-dibromo-9H-fluoren-9-one, resulted in an intramolecular Aza-Michael addition, furnishing 2,7-dibromo-dihydrospiro[fluorene-9,8′-purine] derivatives 33 in moderate yields (Scheme 15).⁸⁷

Scheme 15 Preparation of 2,7-dibromo-dihydrospiro[fluorene-9,8′-purine] derivatives from 5,6-diaminouracil with 2,7-dibromo-9H-fluoren-9-one.

The synthesis of substituted purine derivatives was reported by several authors via the reaction of 5,6-diaminouracil derivatives 29 with appropriate carboxylic acids, such as 4-phenylbutanoic acid, phenylpropanoic acid, phenoxyacetic acid, phenyl carboxylic acid, isoxazole carboxylic acid, pyrazole-3-carboxylic acids, and pyrazole-4-carboxylic in the presence of the coupling/dehydrating reagent N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDAC) at room temperature. The resulting intermediates, 1,3-dialkyl-6-amino-5-carboxamidouracils 34, were subsequently treated with aqueous NaOH to afford the corresponding 3,7-dihydro-1H-purine-2,6-dione derivatives 31 (Scheme 16).^88–91

Scheme 16 Synthesis of purine derivatives from 5,6-diaminouracil using carboxylic acids.

Furthermore, 3-(2-chlorobenzyl)-8-methyl-3,9-dihydro-1H-purine-2,6-dione (36) was efficiently synthesized in higher yield and shorter reaction time, without the use of toxic reagents, by refluxing 5,6-diamino-1-(2-chlorobenzyl)pyrimidine-2,4(1H,3H)-dione (35) with acetic anhydride in acetic acid (Scheme 17).⁹²

Scheme 17 Synthesis of 3-(2-chlorobenzyl)-8-methyl-3,9-dihydro-1H-purine-2,6-dione from a 5,6-diaminouracil derivative with acetic anhydride.

Moreover, El-Kalyoubi et al. reported the synthesis of N-substituted purine derivatives 37 through cyclocondensation of suitable 5,6-diaminouracil derivatives 29 with an azlactone (oxazolone) derivative in acetic acid (Scheme 18).⁹³

Scheme 18 Synthesis of N-substituted-8-purine derivatives via the cyclo-condensation of 5,6-diaminouracils with an oxazolone derivative.

Pretze et al. reported that the treatment of 5,6-diaminopyrimidine-2,4(1H,3H)-diones 29 with triethyl orthoformate (TEOF) in the presence of p-toluenesulfonic acid (p-TsOH) afforded the corresponding 3-substituted-1H-purine-2,6(3H,7H)-dione derivatives 38 (Scheme 19).⁹⁴

Scheme 19 Synthesis of 3-substituted-1H-purine-2,6(3H,7H)-dione via the reaction of 5,6-diaminopyrimidine with triethyl orthoformate (TEOF).

In the same manner, Kucukdumlu et al. designed and synthesized novel purine analogs, by the multistep reactions, through condensation of 4,6-dichloro-5-nitropyrimidines 39a and 39b with triethyl orthoformate in the presence of p-toluenesulfonic acid, affording 9-substituted purine derivatives 40a and 40b, respectively (Scheme 20).⁹⁵

Scheme 20 Synthesis of 3-substituted-1H-purine-2,6(3H,7H)-dione via the reaction of 5,6-diaminopyrimidine with triethyl orthoformate (TEOF).

Also, Orduña et al. illustrated the synthesis of new substituted purine derivatives using low-power microwave irradiation. The treatment of 5,6-diaminopyrimidine derivative 39c with trimethyl orthoformate (TMOF), in the presence of HCl, afforded the corresponding 9-substituted purine derivative 40c in good yield (Scheme 21).⁹⁶

Scheme 21 Synthesis of 3-substituted-1H-purine-2,6(3H,7H)-dione via the reaction of 5,6-diaminopyrimidine with trimethyl orthoformate (TMOF).

Verma et al. described a new strategy for the synthesis of 1,3-dimethyl-2,6-dioxo-1H-purine derivatives 31 containing an acetonitrile group. The reaction was carried out by treating 1,3-dimethyl-5,6-diaminouracil 29 with ethyl 2-cyanoacetimidate in the presence of hydrochloric acid under reflux conditions (Scheme 22).⁹⁷

Scheme 22 Synthesis of 1,3-dimethyl-2,6-dioxo-1H-purine derivatives from diaminopyrimidine.

Moreover, El-Kalyoubi et al. developed a novel method for access to numerous functionalized purine scaffolds 31 in less time and improvement in yield by refluxing of 5,6-diaminouracil 29 with malononitrile under solvent-free condition (Scheme 23).⁹²

Scheme 23 Synthesis of purine derivatives from 5,6-diaminouracil with malononitrile.

Lei et al. treated compound 41 with phenyl isothiocyanate or 4-fluorophenyl isothiocyanate, followed by trifluoroacetic acid in dichloromethane, to generate 9-heterocyclyl-substituted 9H-purine derivatives 42 (Scheme 24).⁹⁸

Scheme 24 Synthesis of 9-heterocyclyl-substituted 9H-purine derivatives.

Lorente-Macías et al. explored that the 6-alkoxy purine derivatives were prepared via the reaction compound 43 with phenyl isothiocyanate. It was noticed that using dimethylformamide (DMF) facilitated the synthesis of 6,9-disubstituted purines 44 with monosubstituted C8; however, the use of dimethylbenzamide (DMB) resulted in the synthesis of tri-substituted purines 45 with fragments at both C6 and C8 (Scheme 25).⁹⁹

Scheme 25 Synthesis of 6,9-disubstituted purines from diaminopyrimidine.

Hisham et al. reported a direct cyclization of 5,6-diamino-1,3-dimethyluracil 29 using carbon disulfide in DMF or in an ethanolic solution of KOH under reflux, affording 8-mercapto-1,3-dimethyl-1,2,3,7-tetrahydro-6H-purin-6-one 31 (Scheme 26).¹⁰⁰

Scheme 26 Synthesis of 8-mercapto-purines from diaminopyrimidine.

Also, compound 46 was obtained by heating substituted diamino-pyrimidine-2,4-one derivative 29 with potassium ethylxanthate in DMF (Scheme 27).¹⁰¹

Scheme 27 Synthesis of 8-mercapto-purines from diaminopyrimidine.

Chakraborty et al. explored the synthesis of a variety of 8-substituted purines 48 and 8,9-disubstituted purines 49 via the dehydrogenative coupling reaction of different substituted benzyl alcohol derivatives with 4,5-diaminopyrimidine 47 using a nickel catalyst [Ni(MeTAA)] for a prolonged reaction time and under an argon atmosphere. The ratio between the reactants significantly affected the type and yield of the obtained products (Scheme 28).¹⁰²

Scheme 28 Synthesis of 6-substituted purines from diaminopyrimidine using primary alcohol.

Synthesis of [1,2,3]triazolo[4,5-d]pyrimidines (azapurine derivatives)

Some reports have described the design and synthesis of this scaffold via a multistep transformation starting from 6-amino-2-alkylsulfanylpyrimidine analogues 50. Initially, nitrosation of compounds 50 in the presence of acetic acid affords the corresponding 5-nitrosopyrimidine derivatives 51. Subsequently, a Zinin reduction of the nitroso group using ammonium sulfide provides the corresponding 5,6-diaminopyrimidine intermediates. Due to their high susceptibility to oxidation, these intermediates are not isolated but are immediately subjected to diazotization in aqueous acetic acid, leading to intramolecular cyclization and affording the desired 1,2,3-triazolo[4,5-d]pyrimidine derivatives 52 (Scheme 29).^103,104

Scheme 29 Preparation of [1,2,3]triazolo[4,5-d]pyrimidine from 6-amino-2-alkylsulfanylpyrimidine.

Alternatively, many reports stated that the reduction of nitrosopyrimidine derivatives 53 using iron powder in methanol/acetic acid afforded the corresponding aminopyrimidine derivatives 54. Subsequently, heating of compounds 54 with the appropriate amine produces the intermediates 55. Without further purification, upon treatment of 55 with sodium nitrite in the presence of acetic acid, it undergoes intramolecular cyclization to furnish the corresponding [1,2,3]triazolo[4,5-d]pyrimidine derivatives 56 (Scheme 30).^105–109

Scheme 30 Preparation of [1,2,3]triazolo[4,5-d]pyrimidines from nitrosopyrimidine derivatives.

Moreover, diazotization of tetraaminopyrimidine (57) using sodium nitrite in sulfuric acid, followed by intramolecular cyclization, leads to the formation of the fused [1,2,3]triazolo[4,5-d]pyrimidine ring system 58. In addition, nitration of compound 57 using nitric acid as the nitrating agent and upon quenching the reaction mixture with ice-water, the fused heterocyclic compound N-(7-oxo-6,7-dihydro-2H-[1,2,3]triazolo[4,5-d]pyrimidin-5-yl)nitramide (59) was isolated in good yield (Scheme 31).¹¹⁰

Scheme 31 Preparation of [1,2,3]triazolo[4,5-d]pyrimidine from tetraaminopyrimidine.

Similarly, triazolo[4,5-d]pyrimidine 60 was synthesized in good yield via cyclocondensation of the 4,5-diaminopyrimidine precursor 35 with in situ generated nitrous acid (HNO₃) at ambient temperature (Scheme 32).⁹²

Scheme 32 Preparation of [1,2,3]triazolo[4,5-d]pyrimidine from the 4,5-diaminopyrimidine derivative.

Recently, another important approach utilizes pyrimidine-5-carboxaldehyde derivatives as key precursors for the construction of the [1,2,3]triazolo[4,5-d]pyrimidine framework. For instance, in 2026, Omanakuttan et al. reported the synthesis of triazolo[4,5-d]pyrimidine analogues via nucleophilic aromatic substitution of pyrimidine-5-carboxaldehyde (61) with (R)-alaninol, affording the corresponding pyrimidine derivative 62. Subsequent condensation of compound 62 with hydrazine hydrate promoted intramolecular cyclization, leading to the formation of the fused [1,2,3]triazolo[4,5-d]pyrimidine derivative 63 in good yield (Scheme 33).¹⁰⁴

Scheme 33 Preparation of [1,2,3]triazolo[4,5-d]pyrimidine from 4,6-dichloropyrimidine-5-carboxaldehyde.

In addition, hydrazino-substituted pyrimidines have been employed as versatile building blocks for the construction of [1,2,3]triazolo[4,5-d]pyrimidine derivatives, where oxidative or nitrosative cyclization promotes the formation of the fused triazole ring. In this context, Debnath et al. explored a method for converting 1,3-dimethyl-5-(arylazo)-6-aminouracil derivatives 64 into novel 1,3-dimethyl-8-arylazapurin-2,6-dione derivatives 65 through an oxidative transformation in the presence of copper ions under alkaline conditions. The reaction proceeds efficiently in a dimethylformamide–water medium, affording the desired fused heterocyclic products in good yields (Scheme 34).¹¹¹

Scheme 34 Preparation of [1,2,3]triazolo[4,5-d]pyrimidine from (arylazo)-6-aminouracil derivatives in the presence of copper ions.

Subsequently, Alexander K. Eltyshev and co-workers investigated several oxidizing systems for the conversion of 5-(arylazo)-6-aminouracil derivatives 66 into 8-arylazapurine derivatives 67. A variety of oxidants, including oxygen, potassium persulfate, sodium perborate, and N-bromosuccinimide, were evaluated for this transformation. Among the tested systems, the combined use of copper(II) acetate in pyridine was found to be the most effective oxidizing system, providing the desired azapurine derivatives with efficiency under the optimized reaction conditions (Scheme 35).^112–114

Scheme 35 Preparation of [1,2,3]triazolo[4,5-d]pyrimidine from (arylazo)-6-aminouracil derivatives using copper(II) acetate in pyridine.

Biological activity

Anticancer

Salem et al. and Nassar et al. reported the design and synthesis of novel pyrazolo[3,4-d]pyrimidine derivatives as structural analogues of classical antifolate agents, including methotrexate (MTX), pralatrexate, and pemetrexed (PMX). In vitro biological evaluation demonstrated that compounds 68 and 69 exhibit potent inhibitory activity against dihydrofolate reductase (DHFR), along with significant antiproliferative effects across a panel of human cancer cell lines compared to the reference compound sorafenib (Scheme 36).^115–117

Scheme 36 Pyrazolo[3,4-d]pyrimidine derivatives as anticancer agents.

In addition, numerous studies have focused on the design, synthesis, and biological evaluation of diverse pyrazolo[3,4-d]pyrimidine derivatives for their anticancer potential. These compounds have been screened against a panel of human cancer cell lines, including HCT-116 (colon, EGFR mutant), HT-29 (colon, EGFR wild-type), MCF-7 and MDA-MB-231 (breast), HepG2 (hepatocellular), HGC-27 (gastric), and A549 (lung). Several derivatives, namely 70a–d, 71a, 71b, 72a, 72b, 73, 74, and 75, exhibited significant antiproliferative activity against selected cell lines, in some cases comparable to or exceeding that of standard reference drugs such as sorafenib, erlotinib, and doxorubicin (Scheme 37).^43,118–122

Scheme 37 Pyrazolo[3,4-d]pyrimidine derivatives as anticancer agents.

Moreover, Nemr et al. reported that compounds 76 and 77 exhibit pronounced inhibitory activity against cyclin-dependent kinase 2 (CDK2) and related isoforms. Molecular interaction studies revealed that these derivatives bind efficiently within the CDK active site, adopting binding modes comparable to those of endogenous substrates or well-established CDK inhibitors (Scheme 38).¹²³

Scheme 38 Pyrazolo[3,4-d]pyrimidine derivatives as anticancer agents.

Lei et al. designed and synthesized a series of novel 9-heterocyclyl-substituted 9H-purine derivatives as potent inhibitors targeting the mutant EGFR^{L858R/T790M/C797S} tyrosine kinase. Among the synthesized compounds, compound 78 exhibited remarkable antiproliferative activity against the HCC827 and H1975 cell lines and demonstrated strong inhibitory activity against EGFR^{L858R/T790M/C797S} (Scheme 39).⁹⁸

Scheme 39 Substituted-purine derivatives as anticancer agents.

In addition, numerous studies have reported the evaluation of purine derivatives against a broad spectrum of cancer cell lines, including HUH7 (liver), MCF7 (breast) and T47D (breast), HCT-116 (colon), A549 (lung), HeLa (cervical), Panc-1 (pancreatic), HCC827 and H1975 (lung). Structure–activity relationship (SAR) analysis revealed that compounds 79–85 and 86a, 86b represent the most potent derivatives, exhibiting significant antiproliferative activity across the tested cell lines. Notably, their IC₅₀ values were comparable to those of established anticancer agents such as camptothecin (CPT), cladribine, fludarabine, fexagratinib (AZD4547), and 5-fluorouracil (5-FU) (Scheme 40).^{80,97,124–127}

Scheme 40 Substituted-purine derivatives as anticancer agents.

Also, a series of novel 9H-purine-2,6-diamine derivatives was designed, synthesized, and biologically evaluated as dual inhibitors targeting Janus kinase 2 (JAK2) and the second bromodomain of BRD4 [BRD4(BD2)]. Both in vitro and in vivo studies demonstrated their potential therapeutic relevance. Among them, compound 87 displayed promising inhibitory activity against both targets, exhibiting favorable IC₅₀ values for BRD4(BD2) and JAK2 (Scheme 41).¹²⁸

Scheme 41 Substituted-purine derivatives as anticancer agents.

Sivakrishna Narra and Nethaji Munirathinam reported the synthesis and biological evaluation of sulfonamide-decorated N6-benzylaminopurine derivatives 88 and 89 for their anticancer potential. In silico studies indicated that compound 89 exhibits a higher binding affinity toward the ATP-binding sites of cyclin-dependent kinases CDK1, CDK2, and CDK4 compared to roscovitine, a well-established CDK inhibitor. Furthermore, cytotoxicity assays demonstrated that compound 89 showed enhanced antiproliferative activity relative to both compound 88 and roscovitine. Collectively, these findings suggest that compound 89 represents a promising lead scaffold for the development of novel anticancer agents (Scheme 42).¹²⁹

Scheme 42 Substituted-purine derivatives as anticancer agents.

Liu et al. reported the design and synthesis of novel [1,2,3]triazolo[4,5-d]pyrimidine derivatives as inhibitors of ubiquitin-specific peptidase 28 (USP28), exhibiting activity in the low micromolar range. Among them, compound 90 demonstrated potent inhibitory activity against USP28 and displayed high selectivity over related targets, including USP7 and lysine-specific demethylase 1 (LSD1), with IC₅₀ values exceeding 100 µmol L⁻¹ (Scheme 43).³⁸

Scheme 43 [1,2,3]Triazolo[4,5-d]pyrimidine derivatives as anticancer agents.

Several authors reported the design and synthesis of novel [1,2,3]triazolo[4,5-d]pyrimidine derivatives, which were subsequently evaluated for their antiproliferative activity against a panel of human cancer cell lines, including MGC-803 (gastric), NCI-H1650 and PC9 (lung), PC-3 (prostate), and EC9706 (esophageal) cells. Among these compounds, derivatives 91–94 displayed the most potent anticancer activity when compared to the effect of the commonly used anticancer agent doxorubicin (Scheme 44).^{34,37,105,106}

Scheme 44 [1,2,3]Triazolo[4,5-d]pyrimidine derivatives as anticancer agents.

Van Hoof et al. and Omanakuttan et al. reported some triazolo[4,5-d]pyrimidine analogues displaying dual antagonistic activity against CCR7 and CXCR2. Among them, lead compounds 95 and 96 revealed promising activity. Structure–activity relationship studies revealed that shortening or elongation of the length of the aliphatic group at the 2-thio-position was detrimental to enhancing the CCR7 antagonistic potency and selectivity, and in both cases, resulted in compounds exhibiting low micromolar activity as CCR7 and CXCR2 antagonists (Scheme 45).^104,130

Scheme 45 [1,2,3]Triazolo[4,5-d]pyrimidine derivatives as anticancer agents.

Antimicrobial activity

Several studies have investigated the antimicrobial potential of pyrazolo[3,4-d]pyrimidine derivatives against both Gram-positive and Gram-negative bacteria. The results demonstrated that compounds 97–99 exhibited nearly complete inhibition of Staphylococcus aureus and Escherichia coli growth, respectively. Additionally, compound 100 displayed the highest antibacterial activity against Bacillus subtilis and Pseudomonas aeruginosa, showing strong efficacy with low minimum inhibitory concentration (MIC) values (Scheme 46).^131–133

Scheme 46 Pyrazolo[3,4-d]pyrimidine derivatives as antimicrobial agents.

Given the critical role of dihydrofolate reductase (DHFR) inhibition in treating bacterial and protozoal infections, Ibrahim et al. evaluated a series of pyrazolo[3,4-d]pyrimidine derivatives for their ability to inhibit bacterial DHFR. These compounds were further assessed both in vitro and in vivo for their antibacterial activity against multiple bacterial strains. Among the tested derivatives, compounds 101 and 102 exhibited potent antibacterial effects, demonstrating low minimum inhibitory concentrations (MICs) against Pseudomonas aeruginosa and Staphylococcus aureus (Scheme 47).¹³⁴

Scheme 47 Pyrazolo[3,4-d]pyrimidine derivatives as antimicrobial agents.

Maddila et al. and Hu et al. investigated the antimicrobial potential of novel purine derivatives against both Gram-positive and Gram-negative bacteria. In vitro studies revealed that compounds 103 and 104 exhibited superior antibacterial activity against Escherichia coli and Klebsiella pneumoniae compared to the reference drugs norfloxacin and ciprofloxacin, respectively. Additionally, a purine–metronidazole hybrid (compound 105) demonstrated significant inhibitory activity, with an MIC value of 6 µM, approximately fourfold more potent than norfloxacin (MIC of 25 µM) (Scheme 48).^79,135

Scheme 48 Purine derivatives as antimicrobial agents.

Finger et al. and Nadaf et al. synthesized a series of substituted purine derivatives and evaluated their in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Rv, along with their binding interactions with M. tuberculosis enoyl reductase. The results demonstrated that 6H-purin-6-ones (106–108) and alkyl-substituted purines (109) exhibited potent antimycobacterial activity against both drug-sensitive and drug-resistant M. tuberculosis strains, while displaying limited cytotoxicity (Scheme 49).^136,137

Scheme 49 Purine derivatives as antimicrobial agents.

Antiviral

Additionally, pyrazolo[3,4-d]pyrimidine derivative 110 was evaluated for antiviral activity against tobacco mosaic virus (TMV). The results demonstrated that compound 110 exhibited remarkable inactivation potential, with a half-maximal effective concentration (EC₅₀) of 53.65 µg mL⁻¹, significantly more potent than the reference antiviral ribavirin (EC₅₀ = 150.45 µg mL⁻¹) (Scheme 50).¹³⁸

Scheme 50 Pyrazolo[3,4-d]pyrimidine derivative as an antiviral agent.

Alseud et al. designed and synthesized a novel series of pyrazolo[3,4-d]pyrimidines (HCQ-PPs) as potential anti-SARS-CoV-2 agents. Among the synthesized derivatives, compound 111 exhibited the highest antiviral activity, surpassing that of remdesivir. These findings highlight compound 111 as a promising lead for further optimization and development as an anti-SARS-CoV-2 therapeutic (Scheme 51).¹³⁹

Scheme 51 Pyrazolo[3,4-d]pyrimidine derivative as an antiviral agent.

Mohammed et al. synthesized a series of tricyclic penciclovir (PCV) derivatives and evaluated their antiviral activity, particularly against herpes simplex virus (HSV). Among them, derivative 112 demonstrated potent antiviral effects, with EC₅₀ values of 1.5, 0.8, and 0.8 µM against HSV-1, HSV-2, and HSV-1 TK + VZV Oka strains, respectively. These activities are comparable to or better than the reference antivirals cidofovir (EC₅₀ = 2.0, 2.0, and 5.0 µM) and acyclovir (EC₅₀ = 0.9, 0.9, and 100 µM) (Scheme 52).¹⁴⁰

Scheme 52 Purine derivative as an antiviral agent.

A series of purine nucleoside derivatives incorporating sulfa ethylamine moieties was designed, synthesized, and evaluated for antiviral activity against tobacco mosaic virus (TMV), cucumber mosaic virus (CMV), and potato virus Y (PVY). Several derivatives exhibited notable antiviral effects, with compound 113 showing particularly strong protective activity against CMV and PVY, surpassing that of the reference antiviral agent pingyangmycin (Scheme 53).¹⁴¹

Scheme 53 Purine derivative as an antiviral agent.

Furthermore, Deng et al. reported the synthesis of novel purine–quinazoline hybrids and evaluated their antiviral activity against tobacco mosaic virus (TMV). In vivo bioassays revealed that several purine–quinazoline derivatives exhibited higher antiviral efficacy than the commercial antiviral ribavirin. Notably, compound 114, containing an 8-fluoroquinazoline moiety, demonstrated curative and protective activities of 65.2% and 60.2%, respectively, surpassing those of ribavirin (50.1% and 57.2%) (Scheme 54).¹⁴²

Scheme 54 Purine derivative as an antiviral agent.

On the other hand, high-throughput virtual screening identified compound 115 as a promising candidate, supported by molecular dynamics simulations and compliance with Lipinski's rule of five. These results suggest that compound 115 may possess potential as a broad-spectrum antiviral agent against diarrhea-causing viruses (Scheme 55).¹⁴³

Scheme 55 Purine derivative as an antiviral agent.

Gómez-Sanjuan and colleagues identified a series of 3-aryl-[1,2,3]triazolo[4,5-d]pyrimidin-7(6H)-ones as selective inhibitors of chikungunya virus (CHIKV) replication. To enhance their physicochemical properties and antiviral potency, a new series of derivatives was synthesized. Among them, compounds 116, 117, and 118 demonstrated markedly improved inhibitory activity against various CHIKV strains (Scheme 56).^144,145

Scheme 56 [1,2,3]Triazolo[4,5-d]pyrimidine derivative as an antiviral agent.

Anti-inflammatory

Somakala et al. synthesized a series of N-substituted 4-((1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-yl)amino)benzamides and evaluated their in vitro anti-inflammatory activity. Among the derivatives, compound 119 exhibited notable anti-inflammatory potential, achieving 83.73% inhibition compared to the standard drug diclofenac sodium (78.05%). Furthermore, compound 119 demonstrated reduced ulcerogenic liability and decreased lipid peroxidation relative to the standard (Scheme 57).¹⁴⁶

Scheme 57 Pyrazolo[3,4-d]pyrimidine as an anti-inflammatory agent.

Similarly, several studies have reported the synthesis of pyrazolo[3,4-d]pyrimidine derivatives bearing diverse functional groups, which have been evaluated for their anti-inflammatory activity via in vitro COX-1/COX-2 inhibition assays. Preliminary results indicated that derivatives 120–123 and 124a–d exhibited higher COX-1/COX-2 selectivity indexes (SI values) compared to the reference drugs celecoxib and diclofenac sodium (Scheme 58).^147–150

Scheme 58 Pyrazolo[3,4-d]pyrimidines as anti-inflammatory agents.

In other studies, Shi et al. and Wang et al. synthesized a series of novel pyrazolo[4,3-d]pyrimidine analogues and evaluated their anti-inflammatory activity. Structure–activity relationship (SAR) analysis revealed that the introduction of a 3-morpholinopropan-1-amine moiety, a substituted 3,4,5-trimethoxystyryl group at C-5, and an alkylamine at C-7 of the pyrazolo[4,3-d]pyrimidine scaffold significantly enhanced anti-inflammatory effects. Among the synthesized derivatives, compounds 125 and 126 were identified as the most potent candidates (Scheme 59).^151,152

Scheme 59 Pyrazolo[4,3-d]pyrimidines as anti-inflammatory agents.

Afifi et al. evaluated the anti-inflammatory activity of a series of designed purine-pyrazole derivatives against 15-lipoxygenase (15-LOX). All the tested compounds demonstrated potent 15-LOX inhibitory activity, comparable to the reference inhibitors zileuton, quercetin, and meclofenamate sodium. Among them, purine derivative 127 exhibited the highest potency (Scheme 60).¹⁵³

Scheme 60 Purine derivative as an anti-inflammatory agent.

Pournara et al. designed and synthesized 6-piperazinyl-substituted purine analogues incorporating nitric oxide (NO)-donor furoxan and hydrogen sulfide (H₂S)-donor moieties. The anti-inflammatory activity of these derivatives was evaluated by measuring their inhibitory effects on lipopolysaccharide (LPS)-induced IL-1β secretion in human aortic smooth muscle cells (HAoSMCs). The results demonstrated that the compounds bearing [(methylsulfonyl)thio]propanoyl and [(methylsulfonyl)thio]hexanoyl groups (compounds 128 and 129, respectively) effectively suppressed LPS-induced IL-1β release in HAoSMCs (Scheme 61).¹⁵⁴

Scheme 61 Purine derivatives as anti-inflammatory agents.

Anti-diabetic activity

The pyrazolo[3,4-d]pyrimidine derivative 130, featuring a bicyclic moiety and a benzamide functionality, exhibited superior anti-diabetic activity, with an IC₅₀ value of 1.60 ± 0.48 µM, compared to the reference drug acarbose (IC₅₀ = 1.73 ± 0.05 µM) (Scheme 62).¹⁵⁵

Scheme 62 Pyrazolo[3,4-d]pyrimidine as an anti-diabetic agent.

Anti-oxidant activity

Mangasuli et al. synthesized a series of coumarin–purine hybrids using a microwave-assisted method and evaluated their in vitro antioxidant activity. Among the synthesized derivatives, compound 131 exhibited the highest activity, which is attributed to the presence of a hydroxyl (–OH) group, and demonstrated superior antioxidant potential compared to the standard ascorbic acid (Scheme 63).¹⁵⁶

Scheme 63 Purine hybrid as an antioxidant agent.

Anti-Alzheimer's activity

Sharma et al. synthesized three series of caffeine-based triazoles and evaluated their in vitro inhibitory activity against acetylcholinesterase (AChE) and β-site amyloid precursor protein cleaving enzyme-1 (BACE-1) at 10 µM. The results indicated that all the derivatives exhibited moderate to potent inhibition. Notably, purine derivative 132 demonstrated dual inhibitory potential against both AChE and BACE-1. Its activity was comparable to the reference compounds cryptolepine and rivastigmine for AChE, and β-secretase inhibitor IV for BACE-1 (Scheme 64).¹⁵⁷

Scheme 64 Inhibitory activity of the purine derivative against acetylcholinesterase.

Antiplatelet aggregation

A series of novel adenosine derivatives was synthesized, with compound 133 identified as the reference molecule. Based on this lead structure, new derivatives can be rationally designed by introducing favorable substituents at specific positions. In particular, incorporating bulky electron-withdrawing groups at position 6 in region A, such as F, Cl, Br, or –NO₂, and stronger hydrogen-bond-donor groups at the C-5′ position, such as –COOH or –CH₂OH, is predicted to enhance biological activity (Scheme 65).¹⁵⁸

Scheme 65 Pyrazolo[3,4-d]pyrimidine as an antiplatelet agent.

Also, a series of 1,2,3-triazolo[4,5-d]pyrimidines, structurally related to ticagrelor, was synthesized and evaluated as potential antiplatelet agents. Among the derivatives, compound 134 demonstrated notable antiplatelet activity (Scheme 66).¹⁵⁹

Scheme 66 1,2,3-Triazolo[4,5-d]pyrimidine as an antiplatelet agent.

Moreover, a series of 8-azapurine derivatives was synthesized and their antiplatelet activity was evaluated both in vitro and in vivo using ticagrelor as a reference. Structure–activity relationship (SAR) analysis and molecular docking studies were also performed. Among the synthesized compounds, derivative 135 exhibited the highest activity (IC₅₀ = 0.20 µM), approximately fourfold more potent than ticagrelor (IC₅₀ = 0.74 µM). Furthermore, treatment with compound 135 resulted in shorter bleeding times, reduced blood loss, and lower acute toxicity compared to ticagrelor (Scheme 67).⁵⁷

Scheme 67 Novel 8-azapurine derivative as potent antiplatelet agent compared with ticagrelor.

Conclusions

Building on the foundation established in Part I, this review (Part II) highlights the expansion of azolo[d]pyrimidine scaffolds through the incorporation of multi-nitrogen azole moieties. The fusion of these azole rings onto the pyrimidine core affords structurally diverse regioisomeric systems, including pyrazolo[3,4-d]pyrimidines, pyrazolo[4,3-d]pyrimidines, imidazolo[4,5-d]pyrimidines (purines), and [1,2,3]triazolo[4,5-d]pyrimidines (8-azapurines). Over the past decade, extensive studies have demonstrated their broad biological potential, including anticancer, antimicrobial, antiviral, anti-inflammatory, antioxidant, neuroprotective, anti-diabetic, and antiplatelet activities. Despite these promising advances, several critical challenges and research gaps remain. First, most reported compounds are evaluated primarily through in vitro assays, with a relative scarcity of in vivo studies and clinical validation, thereby restricting translational potential. Second, mechanistic insights at the molecular level remain limited for many derivatives, hindering rational drug design.

Future research should therefore focus on expanding biological evaluation to include in vivo models and pharmacokinetic studies, improving target selectivity and safety profiles and integrating green chemistry approaches and advanced synthetic strategies to access novel derivatives with higher structural complexity.

In conclusion, azolo[d]pyrimidine derivatives represent highly versatile and privileged scaffolds in medicinal chemistry. Addressing the current limitations and research gaps will be essential to fully exploit their potential and accelerate the development of next-generation therapeutic agents targeting diverse disease pathways.

Author contributions

Aymn E. Rashad: investigation, writing – original draft, review and editing. Ahmed H. Shamroukh: investigation, writing – original draft, review and editing.

Conflicts of interest

The authors declare no conflicts of interest.

Data availability

The authors confirm that no new experimental data were generated in this study. Data presented in tables and figures are derived from previously published studies, all of which are appropriately cited in the reference list. The authors confirm that this study was carried out using publicly available data.

Acknowledgements

The authors of this work wish to thank the authorities of the National Research Centre for their support in carrying out this study.

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