Review pubs.acs.org/CR

Biologically Driven Synthesis of Pyrazolo[3,4‑d]pyrimidines As Protein Kinase Inhibitors: An Old Scaffold As a New Tool for Medicinal Chemistry and Chemical Biology Studies Silvia Schenone,*,† Marco Radi,‡ Francesca Musumeci,† Chiara Brullo,† and Maurizio Botta§,⊥ †

Dipartimento di Farmacia, Università degli Studi di Genova Viale Benedetto XV, 3, 16132 Genova, Italy Dipartimento di Farmacia, Università degli Studi di Parma Viale delle Scienze, 27/A, 43124 Parma, Italy § Dipartimento di Biotecnologie, Chimica e Farmacia, Università degli Studi di Siena Via Aldo Moro, 2, 53100 Siena, Italy ⊥ Sbarro Institute for Cancer Research and Molecular Medicine, Center for Biotechnology, College of Science and Technology, Temple University, BioLife Science Building, Suite 333, 1900 N 12th Street, Philadelphia, Pennsylvania 19122, United States ‡

4.6.1. Cyclin-Dependent Kinase (CDK) Inhibitors 4.6.2. Aurora Kinase Inhibitors 5. 1H-Pyrazolo[3,4-d]pyrimidines As Receptor Tyrosine Kinase (RTK) Inhibitors 5.1. Epidermal Growth Factor Receptor (EGFR) Inhibitors 5.1.1. Synthesis and Biological Properties 5.2. Insulin-Like Growth Factor 1 Receptor (IGF1R) Inhibitors 5.2.1. Synthesis and Biological Properties 6. 1H-Pyrazolo[3,4-d]pyrimidines As Cytoplasmic Tyrosine Kinase Inhibitors 6.1. Src Family Kinase Inhibitors 6.1.1. Synthesis and Biological Properties of Src Inhibitors 6.1.2. Synthesis and Biological Properties of Src-Engineered Kinase Inhibitors 6.1.3. Synthesis and Biological Properties of Other Src and Lck Inhibitors 6.2. Dual Src/Abl Tyrosine Kinase Inhibitors 6.2.1. Synthesis and Biological Properties of Dual Src/Abl Inhibitors 6.2.2. Molecular Modeling Studies and Optimization of the Substituents 6.3. Type II (DGF-out Binding) Dual Src/Abl Inhibitors 7. 1H-Pyrazolo[3,4-d]pyrimidines As Inhibitors of Other Kinases 8. Multitargeted Inhibitors 9. Conclusions Author Information Corresponding Author Notes Biographies Acknowledgments References

CONTENTS 1. Introduction 2. Synthesis of 1H-Pyrazolo[3,4-d]pyrimidines 2.1. Synthesis Starting from the Pyrazole Ring 2.1.1. Synthesis of 1-Substituted/1-Unsubstituted Compounds 2.1.2. Synthesis of 3-Substituted and 1,3Disubstituted Compounds 2.1.3. Synthesis of 6-Substituted Compounds 2.2. Synthesis Starting from the Pyrimidine Ring 3. Kinases as Target Enzymes for Anticancer Therapy 4. 1H-Pyrazolo[3,4-d]pyrimidines As Serine-Threonine Kinase Inhibitors 4.1. mTOR Inhibitors 4.1.1. Synthesis and Biological Properties of 4Morpholino Derivatives 4.1.2. Molecular Modeling Studies and Substitution Optimization 4.1.3. Other mTOR Inhibitors 4.2. 70-kDa Ribosomal Protein S6 Kinase (p70S6K) Inhibitors 4.3. PI3K Inhibitors and Dual PI3K/mTOR Kinase Inhibitors 4.3.1. Synthesis and Biological Properties 4.4. Mitogen Activated Protein Kinase (MAPK) Inhibitors 4.4.1. Raf Inhibitors 4.4.2. p38 MAPK Inhibitors 4.5. Glycogen Synthase Kinase-3 (GSK-3) Inhibitors 4.5.1. Synthesis and Biological Properties 4.6. Other Serine-Threonine Inhibitors © 2014 American Chemical Society

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Received: May 16, 2013 Published: May 29, 2014 7189

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1. INTRODUCTION Nitrogen-containing heterocycles are widely distributed in nature and essential for life, playing a vital role in the metabolism of all living cells. Among the many nitrogencontaining heterocycles, the pyrazolo[3,4-d]pyrimidine nucleus (Figure 1) is an important drug-like scaffold that is present in

and initiated their biological evaluation, particularly as antitumor agents. Reaction of ethoxymethylenemalononitrile 1 with hydrazine monohydrate without solvent or with different substituted hydrazines in refluxing ethanol produced 5-amino-1H-pyrazole4-carbonitriles 2a−d and similar derivatives, which were then hydrolyzed with concentrated sulfuric acid to the corresponding amides 3a−d. These amides were subsequently cyclized into the corresponding 1H-pyrazolo[3,4-d]pyrimidin-4-ols 4a− d by treatment with boiling formamide. C4 chlorination of the latter compounds with POCl3 gave 5a−d, which underwent nucleophilic displacement of the chlorine atom by primary or secondary amines in refluxing alcoholic solution to afford the 4amino derivatives 6 (Scheme 1).2,3

Figure 1. 1H-Pyrazolo[3,4-d]pyrimidine.

Scheme 1. Synthesis of 4-Amino-Substituted 1HPyrazolo[3,4-d]pyrimidines

many pharmacologically active compounds. This structure is an isostere of adenine, which is fundamental for every aspect of cell life as a constituent of DNA and RNA. Due to continuing interest in pyrazolo[3,4-d]pyrimidine derivatives as drugs with the potential to modulate purine activity or metabolism and purinergic receptor activation, researchers have dedicated much effort to investigating new approaches for the synthesis of these derivatives. The antitumor activity of pyrazolo[3,4-d]pyrimidines was initially reported many years ago,1 but it was only recently shown that this effect is primarily dependent on tyrosine or serine/threonine kinase inhibition. Due to the large role of kinases in several diseases (especially cancer), chemical modification of the pyrazolo-pyrimidine scaffold to improve the biological activity and kinase selectivity of these compounds has been extensively studied. The most common methods for the synthesis of pyrazolo[3,4-d]pyrimidines are described in the first section of this review, beginning with the work of Robins, which occurred when the understanding of the biological behavior of these compounds was in an embryonic state, and extending to present day. Classic approaches and alternative strategies, starting from either the pyrimidine or the pyrazole nucleus, are reviewed. The body of the article is focused on pyrazolo[3,4d]pyrimidines as serine/threonine and tyrosine kinase inhibitors. Information on the biological targets is also reported to provide useful insight into the synthesis of novel drug candidates and to suggest specific functionalities that could be introduced onto the scaffold to improve biological activity. Examples of this target-based synthesis of specific pyrazolo[3,4d]pyrimidines are given along with a description of how the evolution of chemical methodologies, chemical biology, pharmacology and medicine have led to many new drug candidates. Many pyrazolo-pyrimidine inhibitors have been synthesized, and it is almost impossible to exhaustively report the known derivatives. Most of the compounds described here were reported in the most important medicinal chemistry journals, and some representative examples from the patent literature are also included.

Notably, the reaction of monosubstituted hydrazines R-NHNH2 with 1,3-difunctional compounds (α,β-unsaturated ketones, esters, acids and nitriles) can lead to different pyrazole regioisomers depending on the nature of the hydrazine, the 1,3difunctional compound, and the reaction medium. The regioselectivity of this reaction has been extensively investigated.4 By reacting α,β-unsaturated nitriles with monosubstituted hydrazines, the 5-amino-pyrazolo 7 or the 3-aminopyrazolo derivatives 8 (Figure 2) can be obtained.5 However, as reported in several examples in this article, the 5-amino derivatives are usually obtained as the main products under neutral conditions. The fusion of the N1-unsubstituted derivative 3a with urea or thiourea afforded 1H-pyrazolo[3,4-d]pyrimidin-4,6-diol 9a or the corresponding 6-mercapto derivative 10, respectively. Treatment of the latter with methyl iodide yielded 6methylthio-derivative 11, which was subjected to the chlorination/amination protocol previously described to afford 4-amino-substituted 6-methylthio derivatives 13 (Scheme 2).2

2. SYNTHESIS OF 1H-PYRAZOLO[3,4-d]PYRIMIDINES 2.1. Synthesis Starting from the Pyrazole Ring

2.1.1. Synthesis of 1-Substituted/1-Unsubstituted Compounds. In the mid-1950s, Robins performed the synthesis of different substituted pyrazolo[3,4-d]pyrimidines 7190

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Scheme 3. Synthesis of 1H-Pyrazolo[3,4-d]pyrimidin-4amine 14, 4-Amino-1H-pyrazolo[3,4-d]pyrimidin-6-ol 15, and 4-Amino-1H-pyrazolo[3,4-d]pyrimidin-6-thiol 16

Figure 2. Structures of 5-amino-pyrazolo derivatives 7 and of 3-aminopyrazolo derivatives 8.

Reaction of 5-amino-1H-pyrazole-4-carbonitrile 2a with formamide, urea or thiourea produced the following corresponding 4-NH2 derivatives: adenine isostere 1H-pyrazolo[3,4d]pyrimidin-4-amine 14 (4-APP), 4-amino-1H-pyrazolo[3,4d]pyrimidin-6-ol 15 or 4-amino-1H-pyrazolo[3,4-d]pyrimidin6-thiol 16, respectively (Scheme 3).2 Chlorination of 9, accomplished using POCl3 and N,Ndiethylaniline, afforded 4,6-dichloro-pyrazolo[3,4-d]pyrimidines 17 in good yield (Scheme 4). Selective replacement of the chlorine atoms with amines under carefully controlled conditions gave a variety of 4-amino- and 4,6-diaminosubstituted derivatives.6,7 Generally, treatment of 17 with ethanolic solutions of primary or secondary amines with steam bath heating for 15 min afforded the corresponding 4-amino-6chloro derivatives 18, whereas longer reaction times in the presence of a large excess of amines usually produced the disubstituted derivatives 19. Biological studies with the pyrazolopyrimidines synthesized by Robins showed that the compounds possessed interesting activity, including inhibition of xanthine oxidase 8 and antiproliferative activity in adenocarcinoma and leukemia cell lines and in vivo tumor models.9,10 Following a similar synthetic approach, Schmidt and Druey reported the synthesis of several pyrazolo[3,4-d]pyrimidines starting from the 5-aminopyrazole-4-carboxylates 21a,c, which were obtained from ethyl(ethoxymethylene)cyanoacetate (20) and the appropriate hydrazine.11 The 4-hydroxy-derivatives 4a and 4c were then directly obtained by reacting 21 with boiling formamide (Scheme 5). The replacement of the C4 cyano group with an ester moiety within the pyrazole structure allowed the sometimes tricky CN group hydrolysis to be avoided. This shortening of the procedure by one step is particularly useful when an NH2 at C4 is not required.

Chlorination of 4 and subsequent substitution of the chlorine atom with amines led to products 6. In a different synthetic approach, Nagahara and Robins reported the synthesis of compounds 4b−e by reaction of the corresponding pyrazolo-amides 3b−e with ethoxymethylenemalononitrile (1) in refluxing DMF, thus affording the desired products in satisfactory yields (Scheme 6).12 The pyrazolo[3,4-d]pyrimidine ring can also be synthesized by reacting pyrazole derivatives with isocyanates or isothiocyanates, which react in a manner similar to other cyclic enaminonitriles and ortho-aminonitriles.13 The reaction of ethyl 5-amino-1-phenyl-1H-pyrazole-4-carboxylate (21c) with chlorosulfonyl isocyanate and subsequent treatment with aqueous KOH produced 22 (Scheme 7).14 4-Amino-1phenyl-5H-pyrazolo[3,4-d]pyrimidin-6-one 23 was synthesized in 68% yield by Quinn and colleagues in a one-pot reaction involving the condensation of 5-amino-1-phenyl-1H-pyrazole4-carbonitrile (2c) with benzoyl isocyanate followed by annulation with ammonia (Scheme 7).15 Taylor and Patel utilized an aza-Wittig-mediated pyrimidine annulation reaction to synthesize 6-phenylamino-substituted pyrazolo[3,4-d]pyrimidines and other fused-pyrimidine deriva-

Scheme 2. Synthesis of 6-Thiomethyl-Substituted 1H-Pyrazolo[3,4-d]pyrimidines

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35b,c by treatment with ethanol saturated with ammonia (Scheme 10). (4-Amino-6-mercapto-1H-pyrazolo[3,4-d]pyrimidin-3yl)acetonitrile (36) was synthesized by reacting pyrazole derivative 33a with benzoyl isothiocyanate followed by acidification (Scheme 10).19 The synthesis of 3-cyano-derivatives was performed starting with tetracyanoethylene (37), which reacted easily with monosubstituted hydrazines to give 5-amino-3,4-dicyanopyrazoles 38.20 Pyrazoles 38 were converted to 1-substituted-3cyano-pyrazolo[3,4-d]pyrimidines 39 with the protocol used for the synthesis of 35b,c (Scheme 11).21 Tominaga and colleagues synthesized a variety of pyrazolo[3,4-d]pyrimidines starting from ketene dithioacetals, particularly from bis(methylthio)methylene malononitrile 40a and bis(methylthio)methylene cyanoacetamide 40b and from anilino(methylthio)methylene malononitriles 41a and the corresponding amides 41b.22 These last two derivatives, 41a and 41b, were prepared by reacting 40a and 40b with suitable anilines. The reaction of 40a,b and 41a,b with substituted hydrazines under reflux gave the corresponding 5-aminopyrazoles 42a,b and 44a,b, which were subsequently treated with formamide to give the corresponding 3-substituted pyrazolo[3,4-d]pyrimidine derivatives 43a,b and 45a,b. The 4-amino-pyrazolo-pyrimidines were obtained from 4-cyanopyrazoles 42a and 44a, and the 4-hydroxy-pyrazolo-pyrimidines were obtained from the pyrazolo-4-carboxamides 42b and 44b (Scheme 12). Tominaga also reported the synthesis of other 3-substituted derivatives.22 The N1 phenyl pyrazolo-pyrimidines 46 and 47 were prepared by reacting the N1 phenyl-substituted compounds of the 42a series with guanidine carbonate or urea (Scheme 13). Similarly, starting from the pyrazolocarboxamide of the 42b series, the 4,6-dihydroxy derivatives 48 were synthesized, with yields higher than 90%. Moreover, the reaction of the aminocarboxamides 42b with carbon disulfide in the presence of potassium hydroxide afforded the 6methylthio analogues 49 in reasonable yield (Scheme 13). Treatment of the 4-hydroxy derivatives 48 and 49 with phosphorus oxychloride gave 4-chloro-pyrazolo[3,4-d]pyrimidines, which are key intermediates for the synthesis of the 4-amino substituted derivatives. The synthesis of highly functionalized dicyanoethylenes 52 and their application to the preparation of C3-substituted pyrazolo[3,4-d]pyrimidines were reported in a different article from the same authors.23 The reaction of 50 with tetracyanoethylene oxide (51) gave the corresponding dicyanoethylene compounds 52, which were subsequently reacted with hydrazine or phenyl hydrazine to obtain 5-aminopyrazolo-4carbonitriles 53 (Scheme 14). The latter compounds were then

Scheme 4. Synthesis of 4,6-Disubstituted 1H-Pyrazolo[3,4d]pyrimidines

tives starting from aminonitriles and aminoesters.16 The reaction of pyrazolo-nitrile 2b or pyrazolo-ester 21b with dibromotriphenylphosphorane, generated in situ by the slow addition of bromine to a cold solution of triphenylphosphine (PPh3) in DCM, afforded the corresponding iminophosphoranes 24 and 25, which undergo aza-Wittig reaction with phenyl isocyanate to produce the corresponding carbodiimides 26 and 27 in good yields. Treatment of the latter with ammonia followed by heating in methanol or ethanol gave, via the guanidine intermediates, the final cyclized derivatives 28 and 29. In the case of 29, the cyclization was greatly facilitated by the addition of sodium ethoxide (Scheme 8). 2.1.2. Synthesis of 3-Substituted and 1,3-Disubstituted Compounds. Several procedures have been reported for the synthesis of pyrazolo[3,4-d]pyrimidines bearing different substituents at the 3 position of the pyrazole ring. Robins and colleagues reported the synthesis of 3-methyl-1H-pyrazolo[3,4-d]pyrimidin-4-amine 32 through reaction of (1-ethoxyethylidene)-malononitrile 30 and hydrazine monohydrate followed by treatment of the formed cyanopyrazole 31 with boiling formamide (Scheme 9).3 Taylor and colleagues prepared a number of new 4-aminopyrazolo[3,4-d]pyrimidines bearing a C3 cyanomethyl or cyano group.17,18 An example of the synthesis of a 3-cyanomethyl derivative is the reaction of 5-amino-3-(cyanomethyl)-1-phenyl1H-pyrazole-4-carbonitriles 33b,c with ethyl orthoformate to give the intermediates 34b,c. The latter were finally cyclized to

Scheme 5. Synthesis of 1H-Pyrazolo[3,4-d]pyrimidin-4-ol Derivatives

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Scheme 6. Different Synthesis of 1H-Pyrazolo[3,4-d]pyrimidin-4-ol Derivatives

Scheme 7. Synthesis of the N1-Phenyl Derivatives 22 and 23

Scheme 9. Synthesis of 3-Methyl-1H-pyrazolo[3,4d]pyrimidin-4-amine 32

The authors also prepared a number of 3-substituted 4,6diamino-pyrazolo[3,4-d]pyrimidines 59 by modifying a procedure previously reported by Davoll and Kerridge for the onestep synthesis of 4,6-diamino-pyrazolo[3,4-d]pyrimidine.25 In the modified method, 1- and/or 3-substituted-5-amino-4cyanopyrazoles 57 were maintained under reduced pressure during fusion with guanidine carbonate to reduce exposure of the mixture to atmospheric oxygen and remove the water formed in the reaction. The reaction temperature was kept 10− 30 °C above the melting point of the pyrazole derivative for 2− 3 h. In a few cases, the authors used triethanolamine as the reaction medium (Scheme 15).24 Similar 1,3-disubstituted pyrazolo[3,4-d]pyrimidines have been synthesized from 1-alkyl-5-amino-3-aryl-4-cyanopyrazoles 60 in a one-pot synthesis. To a THF solution of malononitrile 54 sodium hydride, acyl chloride, dimethylsulfate and finally alkylhydrazine dihydrochloride in the presence of TEA were added in succession to give the expected intermediates in 21− 58% yield.26 The structure of 60 was confirmed by NOE experiments, as cyclization could have also resulted in the corresponding 1-alkyl-3-amino-5-aryl-4-cyanopyrazoles. The aminonitriles 60 were converted into pyrazolo-pyrimidines 61

cyclized with formamide to afford the corresponding C3substituted 4-amino-pyrazolo[3,4-d]pyrimidines. Southwich and Dhawan prepared 3-aryl- or 3-benzylpyrazolo[3,4-d]pyrimidines 58.24 In their synthesis, malononitrile 54 was reacted with the appropriate acyl chloride and TEA in benzene or, alternatively, with sodium ethoxide and acyl chlorides to give compounds 55. These intermediates were transformed into the corresponding methoxy derivatives 56 by methylation with dimethylsulfate and sodium bicarbonate. The aryl- or benzyl-substituted methoxymethylenemalononitriles 56 reacted with a suitable hydrazine to provide pyrazole intermediates 57, which were then cyclized to pyrazolopyrimidines 58. Scheme 8. Synthesis of 4,6-Disubstituted Derivatives 28 and 29

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Scheme 10. Synthesis of 3-Cyanomethyl-1H-pyrazolo[3,4-d]pyrimidines

Scheme 11. Synthesis of 3-Cyano-1H-pyrazolo[3,4-d]pyrimidines

Scheme 12. Synthesis of 3-Methylthio or 3-AminoSubstituted 1H-Pyrazolo[3,4-d]pyrimidines

Scheme 13. Synthesis of the 3-Methylthio-1-phenylSubstituted 1H-Pyrazolo[3,4-d]pyrimidines 46-49

in high yield (82−98%) by treatment with formamide at reflux (Scheme 16). The synthesis of 3-(p-toluenesulfonylamino)-1H-pyrazolo[3,4-d]pyrimidines was performed by Tominaga27 using Nbis(methylthio)methylene-p-toluenesulfonamide 62 as the starting material. Reaction with malononitrile 54 or cyanoacetamide 63 in the presence of potassium carbonate in DMSO afforded the corresponding displacement products 64a or 64b in yields exceeding 90%. The reaction of 64 with various hydrazines afforded the 5-aminopyrazole derivatives 65, which were reacted with formamide to give the desired pyrazolopyrimidines 66 (Scheme 17). A different approach to C3-substituted pyrazolo[3,4-d]pyrimidines has been reported by Schultz and Ding.28 The

synthesis starts with the 3-bromo derivative 67, which was obtained from Robins compound 5a by treatment with Nbromosuccinimide (NBS) under microwave irradiation. The application of microwaves gave high yields of the desired product (more than 95%), provided only a small amount of 7194

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Iodination of 14 was achieved by treatment with Niodosuccinimide (NIS) under microwave irradiation. Compound 74 was alkylated at N1 using Mitsunobu chemistry, involving treatment with an alcohol in the presence of PPh3 and diisopropyl azodicarboxylate (DIAD), thus providing 75 in good yield. However, the need for repeated chromatography to remove triphenylphosphine oxide encouraged the development of an alternate alkylation method, which involved treating 74 with K2CO3 and bromobutane or isobutyl bromide. It should be noted that these two approaches were only suitable for the introduction of primary alkyl groups at N1.30 Arylation at C3 was achieved via Suzuki cross-coupling using a Pd catalytic system including the 1,3,5,7-tetramethyl-2,4,8trioxa-6-phenyl-6-phosphaadamantane (PA-Ph) ligand developed by the same authors. Microwave irradiation of 75 with a variety of boronic acids in the presence of tris(dibenzylideneacetone)dipalladium(0) [Pd2(dba)3] and the PA-Ph ligand afforded the desired N1- and C3-substituted pyrazolo[3,4-d]pyrimidines 76. The same authors also synthesized two N1-tert-butyl derivatives 80a,b and the corresponding N1-unsubstituted derivatives 81 starting from N1-substituted compound 77 using microwave-assisted procedures. In this case, the C3 halogenation was performed using bromine in water (Scheme 21).30 Recently, other authors used MAOS (microwave-assisted organic synthesis) for the preparation of pyrazolo[3,4-d]pyrimidine compounds in high yield. Heravi and colleagues reported the synthesis of pyrazolo[3,4-d]pyrimidines 58 using Keggins heteropolyacids, such as phosphotungstic acid (H3PW12O40) or molybdophosphoric acid (H3PMo12O40), and microwave irradiation starting from the usual 5-amino-4cyanopyrazoles 57 and formamide.31 The microwave-solid acid combination resulted in a convenient catalytic synthesis with yields higher than 61% and reduced reaction times compared to traditional methods (Scheme 22). 2.1.3. Synthesis of 6-Substituted Compounds. Although the synthesis of a few C6 substituted compounds has been described above, this section focuses on the synthetic approaches generally used to obtain C6 alkyl or aryl derivatives. One of the first examples of the synthesis of 6-alkylsubstituted (particularly, 6-methyl and 6-ethyl) pyrazolo[3,4-

Scheme 14. Synthesis of Functionalized Pyrazoles Starting from Tetracyanoethylene Oxide

side products, and required short reaction times. Compound 67 was then subjected to a one-pot, two-step process involving sequential SNAr displacement of the C4 chlorine atom with different amines under mild acidic conditions (acetic acid) followed by a Suzuki coupling with different boronic acids to afford the C3-substituted derivatives 68 (Scheme 18). Interestingly, this approach permitted straightforward functionalization of the C3 position in the presence of relatively reactive functional groups (amine, amide, alcohol) that would have required protection from the strong acidic or basic conditions used in the previously reported procedures. Recently, the synthesis of 6-oxo or 6-thio-substituted derivatives bearing a phenyl ring at C3 has been reported.29 Ethyl benzoylacetate (69) was reacted with trichloroacetonitrile in ethanol in the presence of sodium acetate to afford acrylate intermediate 70. The reaction of 70 with hydrazine hydrate in refluxing ethanol afforded 5-amino-3-phenyl-1H-pyrazole-4carboxylic acid ethyl ester 71. The reaction of 71 with benzoyl isothiocyanate afforded intermediate 72, which was treated with ethanolic sodium ethoxide to afford 3-phenyl-6-thioxo-1,5,6,7tetrahydro-4H-pyrazolo[3,4-d]pyrimidin-4-one (73b). The fusion of 71 with urea or thiourea afforded pyrazolopyrimidinones 73a and 73b in good yield (Scheme 19). The pyrazolo-pyrimidinones thus obtained represent advanced intermediates for further functionalization. Todorovic and colleagues reported the microwave-assisted synthesis of 1,3-disubstituted-pyrazolo[3,4-d]pyrimidine derivatives, including compounds 76, starting from 1H-pyrazolo[3,4d]pyrimidin-4-amine 14 (Scheme 20).30

Scheme 15. Synthesis of 1,3-Disubstituted 1H-Pyrazolo[3,4-d]pyrimidin-4-amines 58 and of 1,3-Disubstituted 1H-Pyrazolo[3,4d]pyrimidin-4,6-diamines 59

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Scheme 16. Synthesis of Other 1,3-Disubstituted 1H-Pyrazolo[3,4-d]pyrimidin-4-amines

propionic anhydride to give the corresponding 5-acylamino-4cyanopyrazoles 82, which were then treated with an alkaline solution of hydrogen peroxide to give 6-alkyl-4-hydroxypyrazolo-pyrimidines 83. Finally, C4 chlorination with phosphorus oxychloride and subsequent nucleophilic displacement with a primary or secondary amine gave the desired compounds 84 (Scheme 23). The Taylor group, during a study of the base-catalyzed condensation of aromatic and heterocyclic ortho-aminonitriles with nitriles, synthesized a series of 4-amino-pyrazolo[3,4d]pyrimidines 85 by the reaction of 4-cyano-5-amino-pyrazoles 2a−c with different nitriles in methanolic ammonia at 200 °C for 20 h in a steel hydrogenation bomb (Scheme 24).33 Subsequently, Baker and Kozma synthesized 6-aryl-substituted derivatives 86 by reaction of 2a with substituted benzamidine hydrochlorides and sodium acetate at 200 °C under solventfree conditions (Scheme 24).34 A method for obtaining 1,6-alkyl/aryl disubstituted 1,5dihydro-pyrazolo[3,4-d]pyrimidin-4-ones 87 is represented by the straightforward reaction of carboxamides 3b,c with different esters in the presence of sodium ethoxide in ethanol. The resulting derivatives 87 can subsequently be chlorinated with POCl3 and reacted with amines, as previously reported, to give the 4-amino analogues (Scheme 25).35 Interestingly, 6-trifluoromethyl derivatives 92 were synthesized by other authors using a similar procedure.36 Cyanopyrazole 2a was reacted with trifluoroacetic anhydride (TFAA) to afford intermediate 88, which was hydrolyzed to the corresponding amide 89. The latter compound was cyclized at 200 °C to give the pyrazolo-pyrimidine 90, which was

Scheme 17. Synthesis of 3-(p-Toluenesulfonylamino)-1Hpyrazolo[3,4-d]pyrimidine Derivatives

Scheme 18. Synthesis of C3-Substituted 1H-Pyrazolo[3,4d]pyrimidines

d]pyrimidines 84 was reported by Robins and colleagues.32 5Amino-4-cyanopyrazoles 2a−d were acylated by acetic or

Scheme 19. Synthesis of 3-Phenyl-Substituted 1H-Pyrazolo[3,4-d]pyrimidin-4-ones

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Scheme 20. Microwave-Assisted Synthesis of 1,3-Disubstituted 1H-Pyrazolo[3,4-d]pyrimidines

Scheme 21. Synthesis of 3-Aryl-1H-pyrazolo[3,4d]pyrimidin-4-amines 80 and 81

Scheme 23. Synthesis of 6-Methyl or 6-Ethyl 1HPyrazolo[3,4-d]pyrimidines

Scheme 22. Synthesis of 1,3-Disubstituted 1H-pyrazolo[3,4d]pyrimidines Using Keggins Heteropolyacids Scheme 24. Synthesis of 6-Aryl and 6-Alkyl-1H-pyrazolo[3,4d]pyrimidin-4-amines

chlorinated to give 91 and underwent subsequent nucleophilic substitution with NH3 or different amines (Scheme 26). The amino-pyrazole carboxamides 3a−c were condensed with different aromatic aldehydes to provide 5-(Narylideneamino)pyrazolo derivatives 93, which were cyclized with HCl or para-toluenesulfonic acid (p-TSA) to give the corresponding 6-substituted-1H-pyrazolo[3,4-d]pyrimidin-4ones 94. These compounds can also be obtained directly by reaction of 3a−c with aromatic carboxylic acids in heated polyphosphoric acid (PPA) or polyphosphate ester (PPE) (Scheme 27).37 Derivatives 94 are useful intermediates for the synthesis of 4-amino-substituted compounds following the procedures mentioned above. Similar compounds, 95, have recently been obtained through a one-pot procedure reacting derivatives 2 with aliphatic acids in the presence of POCl3.38 The latter reagent acted as both a

chlorinating agent, generating the acyl chloride in situ from the corresponding acid, and also as a hydrolyzing agent to convert the cyano group to the corresponding amide. The reaction of the amide with the acyl chloride formed an intermediate that cyclized into target compounds 95 (Scheme 28). 7197

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using MAOS. In a modification of the Taylor preparation,33 5amino-1H-pyrazole-4-carbonitriles 2, aromatic nitriles and tBuOK were irradiated in an open vessel for 10 min in the absence of solvent to give the final derivatives 96 in 40−85% yield (Scheme 29).39

Scheme 25. Synthesis of 1,6-Disubstituted 1H-Pyrazolo[3,4d]pyrimidin-4-ones

Scheme 29. MW-Assisted Synthesis of 1,6-Disubstituted 1HPyrazolo[3,4-d]pyrimidin-4-amines

Scheme 26. Synthesis of 6-Trifluoromethyl-Substituted 1HPyrazolo[3,4-d]pyrimidines

2.2. Synthesis Starting from the Pyrimidine Ring

The pyrazolo[3,4-d]pyrimidine scaffold can be prepared starting from pyrimidines, although this procedure has been used less frequently than cyclizations starting from pyrazolo intermediates. This synthesis generally involves reaction of hydrazine derivatives with pyrimidines possessing a carbonyl function at the C5-position.40 2-Amino-4,6-dichloro-pyrimidine-5-carbaldehyde 97 was cyclized with hydrazine hydrate to give 4-chloro-1H-pyrazolo[3,4d]pyrimidin-6-ylamine 98 in good yield (Scheme 30).41 Scheme 30. Synthesis of 4-Chloro-1H-pyrazolo[3,4d]pyrimidin-6-ylamine 98 Starting from 2-Amino-4,6dichloro-pyrimidine-5-carbaldehyde 97 Scheme 27. Synthesis of 6-Substituted 1H-Pyrazolo[3,4d]pyrimidin-4-ones

Recently, Quiroga and colleagues reacted 97 with amines to obtain N4-substituted-2,4-diamino-6-chloro-5-carbaldehydes 99, which were transformed into the corresponding pyrazolo[3,4-d]pyrimidines 100 by reaction with hydrazine hydrate under microwave irradiation in the absence of solvent.42 The authors performed the reaction of equimolar amounts of aldehyde 97 with a variety of aliphatic, aromatic and heterocyclic amines in a basic medium in refluxing ethanol. When the amination of 97 was performed in the presence of two equivalents of amines and TEA, the disubstituted products 101 were obtained (Scheme 31). 4-Amino-2-dimethylamino-6-chloro-5-cyanopyrimidine 102 was cyclized with methyl hydrazine to produce the corresponding 1-methyl-pyrazolo[3,4-d]pyrimidine 103 in high yield with no trace of the 2-methyl regioisomer (Scheme 32).43 A new synthesis of 1-alkyl or 1-aryl-pyrazolo[3,4-d]pyrimidines has been reported by Waring and colleagues.44 The reaction of 4,6-dichloropyrimidine-5-carbaldehyde 104 with a number of substituted hydrazines provided the final compounds 105 in a single step. The N2-substituted regioisomer has also been isolated from the reaction, but it is readily separable using chromatography (Scheme 33).

Scheme 28. Synthesis of 6-Substituted 1H-Pyrazolo[3,4d]pyrimidin-4-ones

Other pyrazolo[3,4-d]pyrimidines bearing different 6-substitutions, including heterocyclic rings, have been obtained 7198

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Scheme 31. Synthesis of 4,6-Diamino-Substituted 1H-Pyrazolo[3,4-d]pyrimidines

Scheme 32. Synthesis of N6,N6,1-Trimethyl-1Hpyrazolo[3,4-d]pyrimidine-3,4,6-triamine 103

Scheme 34. Synthesis of 1,4,6-Trisubstituted-1Hpyrazolo[3,4-d]pyrimidines Starting from 2,4,6Trichloropyrimidin-5-carbaldehyde 106

Scheme 33. Synthesis of N1-Substituted 4-Chloro-1Hpyrazolo[3,4-d]pyrimidines

Recently, Webb and colleagues45 synthesized a library of 4,6diamino-substituted pyrazolo[3,4-d]pyrimidines 109 starting from 2,4,6-trichloropyrimidin-5-carbaldehyde 106, which reacted with anilines in the presence of catalytic amounts of the phase transfer catalyst tetrabutylammonium iodide (TBAI) to give the intermediates 107 in moderate to high yields. These compounds were subsequently treated with different anilines under the same basic conditions to give the substituted diamino intermediates 108 in a regioselective manner. Finally, reaction of 108 with hydrazines gave pyrazolo[3,4-d]pyrimidines 109 (Scheme 34). After the development of this stepwise protocol, the authors also perfected a two-pot protocol that allowed the preparation of compounds 109 without the need for chromatographic purification of any synthetic intermediates. Compound 107 was prepared first, and different anilines and substituted hydrazines were then added to 107 in sequence. The two-pot protocol allowed the preparation of a large number of compounds in a high-throughput manner, with the aim of obtaining sufficient pure material. The average reaction yield was 35%. The major disadvantage was the unsuccessful results obtained with alkyl

hydrazines, which did not cyclize to form the pyrazolopyrimidine moiety, most likely due to steric interactions.45 Jones and colleagues described an efficient three-step, onepot synthesis of 1-aryl-pyrazolo[3,4-d]pyrimidines under mild 7199

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reaction conditions.46 The nucleophilic substitution of the commercially available 4,6-dichloropyrimidine-5-carboxaldehyde 104 with anilines was performed in acetonitrile at 0 °C. Hydroxylamine-O-sulfonic acid was then added; after dilution with dichloromethane and aqueous 1 M sodium hydroxide solution, a biphasic mixture was obtained. The desired N1-arylpyrazolo-pyrimidines 110 were obtained in yields ranging from 30 to 69% (Scheme 35).

chloro-pyrazolo[3,4-d]pyrimidines bearing aromatic moieties at N1.48 The reaction was performed by reacting 104 with an aromatic hydrazine (as the hydrochloric salt) in acetonitrile or THF, while heating to 200 °C for a short time (1−15 min) or by using microwave irradiation. In this manner, the authors prepared 4-chloropyrazolo[3,4-d]pyrimidines 112.

3. KINASES AS TARGET ENZYMES FOR ANTICANCER THERAPY In one way or another, protein phosphorylation is involved in and regulates every cellular process. Even complex functions such as memory can ultimately be traced to the phosphorylation of a specific protein.49 The family of human protein kinases, of which there are 538 members, represents the third largest and the most important enzyme class; they are estimated to be responsible for modifying one-third of the human proteome.50,51 These enzymes catalyze the transfer of the gamma phosphate group from ATP to specific serine, threonine or tyrosine hydroxy groups on target protein substrates involved in a number of cell signaling pathways. It has been conclusively demonstrated that kinase alterations (especially hyperactivation, hyperproduction, or mutations) leading to the disruption of cell signaling cascades play important roles in several diseases, including cancer, inflammation, neurological disorders and diabetes.52 Thus, kinases represent important targets for drug development. The catalytic domain is highly conserved among kinases and consists of a bilobed structure with Mg-ATP situated in a deep cleft located between the N- and C-terminal lobes. The majority of small-molecule kinase inhibitors developed thus far target the ATP binding site, and the kinase adopts a conformation almost identical to that used to bind ATP (the active conformation; Figure 3A). This is not surprising because, historically, most inhibitors have been discovered using biochemical screening based on activation loop-phosphorylated recombinant kinase catalytic domains (at very low concentrations of ATP), conditions in which hydrophobic compounds are most likely to interact with the active conformation of the ATP cleft. Compounds that target the active state of the enzyme are classified as type I inhibitors and bind to the ATP

Scheme 35. Three-Steps One-Pot Synthesis of 1-Aryl-4chloro-1H-pyrazolo[3,4-d]pyrimidines

Very recently, Moon and colleagues reported the synthesis of 1-substituted 4-chloro-pyrazolo[3,4-d]pyrimidines by the reaction of 4,6-dichloropyrimidine-5-carboxaldehyde 104 with various hydrazines, which were sometimes utilized as their hydrochloride salts.47 For aromatic hydrazines, the reaction was performed in the absence of an external base, which led to the exclusive formation of hydrazone 111. The hydrazones were subsequently cyclized by heating at 200 °C in acetonitrile under MW irradiation or in dioxane with conventional heating to form the desired pyrazolo[3,4-d]pyrimidine products 112. For aliphatic hydrazines, the reaction sequence proceeded in a single step in the presence of an external base to form derivatives 113 (Scheme 36). In further studies of the reactivity of 104 with hydrazines, the same authors optimized a one-pot reaction that provided 4-

Scheme 36. Synthesis of 1-Alkyl or 1-Aryl-4-chloro-1H-pyrazolo[3,4-d]pyrimidines

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Figure 3. A. Binding mode of ATP in the active kinase conformation. B. Binding mode of a type II inhibitor (imatinib). Type II inhibitors bind to both the ATP binding site and an immediately adjacent hydrophobic pocket, an allosteric site, which is created when the DFG motif is in the “out” conformation, thus freezing the kinase in the inactive state.

binding site through the formation of hydrogen bonds to the kinase “hinge” residues and through hydrophobic interactions in and around the region occupied by the adenine ring of ATP.53 Serendipity, in combination with structure−activity relationship (SAR)-guided medicinal chemistry, has allowed the identification of type II kinase inhibitors, whose members preferentially bind to the inactive conformation of the kinase, thereby preventing its activation.54 Type II inhibitors bind to the ATP binding cleft and also exploit an adjacent hydrophobic pocket, known as an allosteric site, created by the activation loop (which contains the conserved DFG motif) positioned in an “out” conformation. The binding mode of a type II inhibitor is shown in Figure 3B: the compound shown is imatinib, one of the first type II inhibitors identified and the first kinase inhibitor approved for cancer therapy.55 Type III non-ATP competitive inhibitors exclusively bind to the allosteric site adjacent to the ATP binding region. More recently, type IV inhibitors, which bind to allosteric sites several angstroms away from the nucleotide binding region, have been identified. They act by inducing conformational changes that modulate enzymatic activity. Type III and type IV inhibitors are often referred to as “allosteric inhibitors”.56,57 Finally, type V inhibitors are covalent inhibitors that target the catalytic site of the kinase.58 Approximately half of the current research and development budget of the pharmaceutical industry is spent on kinases and, in particular, on their inhibitors. Synthetic small molecules are the most prominent family of kinase inhibitors. In the last ten years, ten small molecule inhibitors of different kinases have received FDA approval and entered clinical therapy for the treatment of solid and hematological malignancies, starting in 2001 with imatinib for chronic myeloid leukemia (CML). Many other compounds are being tested in preclinical and clinical trials. All of these inhibitors contain at least one nitrogen heterocycle but belong to very different chemical families. The pyrazolo[3,4-d]pyrimidine-based inhibitor ibrutinib was very recently approved by the FDA for the treatment of lymphomas, and a large number of other derivatives have recently shown promising preclinical activity. The following paragraphs provide selected examples of pyrazolo-pyrimidines

as kinase inhibitors as well as a description of some important features of the specific biological targets. Some examples of the most active compounds cited in this section are reported in Table 1.

4. 1H-PYRAZOLO[3,4-d]PYRIMIDINES AS SERINE-THREONINE KINASE INHIBITORS 4.1. mTOR Inhibitors

The serine-threonine kinase mammalian target of rapamycin (mTOR) is a member of the phosphatidylinositol 3-kinaserelated kinase (PIKK) family.59 mTOR is a critical component of the phosphatidylinositol 3-kinase (PI3K) pathway, the protein kinase B (Akt/PKB) pathway and the Ras/extracellular signal regulated kinase (ERK) pathway, all which are fundamental for cell growth, survival, motility, proliferation, protein synthesis, and transcription.60 These signaling cascades are frequently deregulated in human cancer.61,62 Intracellular mTOR is present in two complexes, in which it is associated with different proteins: mTOR complex 1 (mTORC1) and mTOR complex 2 (mTORC2). The first is regulated by growth factors (i.e., IGF1 and IGF2) through the PI3K and Akt/PKB pathways and by nutrients in a complex network of interactions. Specifically, Akt is an upstream enzyme in the signaling cascade that leads to mTORC1 activation.63 In turn, mTORC1 stimulates cell growth and proliferation, and its activity is mediated by two major downstream targets, i.e., the p70 S6 ribosomal kinase 1 (p70S6K1)64 and the eukaryotic initiation factor 4E-binding protein 1 (4E-BP1).65 Moreover, mTORC1 is involved in the regulation of different proteins, including cyclins, cyclin dependent kinases, protein phosphatases and RNA polymerases.66 The second complex, mTORC2, is involved in the regulation of cytoskeleton functions by stimulating actin fibers, paxillin, RhoA, Rac1 and protein kinase Cα (PKCα).67 Importantly, mTORC2 activates Akt, which is upstream of mTOR in the mTORC1 pathway and downstream in the mTORC2 pathway. Similarly to mTORC1, mTORC2 appears to be activated by growth factors, perhaps in an Akt-independent manner. mTOR activity is deregulated in human diseases, particularly in malignancies, in which increased levels and/or the 7201

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Table 1. Examples of Pyrazolo-Pyrimidines, Their Target Kinases, Pharmaceutical Company or Author, and Main Activities target kinase

key compound

research group

mTOR

141a WYE-13282

Wyeth

p70S6K

147 XL41889

Exelixis

PI3K PI3K/mTOR

15297 15598

Genentech Wyeth

Raf p38 MAPK

159110 162a111

Genentech Bristol-Myers Squibb Novartis

169c112

GSK-3

189a130

CDK2

192136

CDK9

198138

GlaxoSmith Kline Bristol-Myers Squibb Exelixis Inc.

Aurora kinase

205141

Biogen

EGFR

222a157

AstraZeneca

IGF-1R

226a, A-928605166

Abbott

Src and other SFK members Engineered Src

230a PP1, and 230b PP2176 233a 1NM-PP1183 244 A-770041193 2633h209,214

Hanke et al.

281a PCI-32765 ibrutinib233

Pharmacyclics Inc.

Lck Abl Btk

Shokat et al. Abbott Schenone et al.

biological activity IC50 of 0.21 nM in enzymatic assays. IC50 of 2 nM in LNCaP cells. antitumor in vivo activity. IC50 of 2 nM in enzymatic assays. IC50 values of 30 and 55 nM in A549 and PC-3 cells, respectively. antitumor in vivo activity. IC50 of 3 nM for PI3Kα in enzymatic assays. IC50 values of 1, 68, and 13 nM against PI3Kα, PI3Kγ and mTOR, respectively, in enzymatic assays. IC50 of 0.9 nM against B-Raf V600E in enzymatic assays. IC50 of 5 nM in enzymatic assays. IC50 of 6 nM for the inhibition of TNFα production in human peripheral blood mononuclear cells. IC50 of 0.6 nM in enzymatic assays. 96% inhibition of LPS-induced TNFα release in mice pIC50 of 8 in enzymatic assays. IC50 of 18 nM in enzymatic assays. IC50 of 16 nM in NCI H460 cells. antitumor in vivo activity. IC50 of 1 nM in enzymatic assays. IC50 values of 4, 5, and 22 nM against Aurora kinase A, Aurora kinase B and CDK1, respectively. IC50 of 10 nM in HCT116 cells. IC50 values of 1 and 5 nM against ErbB2 and EGFR, respectively, in enzymatic assays. IC50 of 17 nM in BT474C cells. IC50 of 37 nM in enzymatic assays. IC50 of 90 nM in A431 cells. antitumor in vivo activity. IC50 values in the nanomolar range in enzymatic assays. antiproliferative activity in several cell lines. IC50 of 1.5 nM against engineered Src (I338G v-Src). IC50 of 147 nM in enzymatic assays. Ki of 80 nM in enzymatic assays. Antiproliferative activity on CML cell lines. IC50 of 0.5 nM in enzymatic assays. approved November 2013 for the treatment of mantle cell lymphomas.

antitumor agents; indeed, the overall objective response rates achieved with rapamycin therapy for solid tumors have been modest. In preclinical and clinical settings, the treatment of certain tumors with rapamycin increased PI3K/Akt activity, thus decreasing the therapeutic potential of mTORC1 inhibition.72 The discovery that mTORC2 directly phosphorylates Akt, which is usually hyperactivated in tumors, suggests that mTORC2 inhibitors could be valuable anticancer drugs.67 Furthermore, recent studies in cancer biology have suggested that mTORC2 activity is essential for the survival of several cancer cells but is less necessary in normal cells. For all of these reasons, several small molecule ATP-competitive inhibitors that target the kinase domain of mTOR, in both mTORC1 and mTORC2, have been developed in the past few years with the aim of circumventing the problems associated with allosteric inhibition by rapamycins.68 The inhibition of signaling through mTORC1 is usually detected by a decrease in the phosphorylation of proteins such as 4E-BP1 and p70S6K1, whereas the inhibition of mTORC2 is indicated by decreased Akt Ser473 phosphorylation.

phosphorylation of its downstream targets have been correlated with tumor aggressiveness and poor prognosis.68 The macrolide rapamycin and its derivatives are selective and allosteric inhibitors of mTOR. They form a complex with the ubiquitous intracellular protein FK506-binding protein-12 (FKBP12); this complex binds to the FRB (FKBP-rapamycin binding) domain of mTOR, located near the kinase domain,69 and inhibits enzymatic functions.70 However, rapamycins inhibit the enzyme in the mTORC1 complex, but not in mTORC2, which was originally defined as rapamycin insensitive. It has subsequently been shown that rapamycins suppress mTOR activity in both mTORC1 and mTORC2 complexes but at very different concentrations; indeed, whereas mTORC1 is inhibited by low nanomolar range concentrations of rapamycin, mTORC2 inhibition requires low micromolar concentrations.71 However, low doses of rapamycin have been shown to induce feedback mechanisms that lead to an IGF1 receptor-dependent mTORC2 activation and subsequent increased Akt phosphorylation. The differential inhibitory activity of rapamycins against the two mTOR complexes may limit their effectiveness as 7202

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The largest family of mTOR inhibitors is the pyrazolo[3,4d]derivatives. 4.1.1. Synthesis and Biological Properties of 4Morpholino Derivatives. Zask and colleagues from Wyeth have synthesized several potent mTOR inhibitors, including 117 (WAY-600), 121a (WYE-687), and 123 (WYE-354), by preparing the pyrazolo[3,4-d]pyrimidine scaffold starting from the pyrimidine ring or the pyrazole ring.73 The synthesis of a number of compounds starts with 2,4,6-trichloropyrimidin-5carbaldehyde 106 (Scheme 37).

Scheme 38. Synthesis of the mTOR Inhibitor 121a (WYE687)

Scheme 37. Synthesis of the mTOR Inhibitor 117 (WAY600)

enzymatic assays, and block substrate phosphorylation by mTORC1 and mTORC2 in vitro in response to growth factors, amino acids and PI3K/Akt hyperactivation. These pyrazolopyrimidines inhibited the phosphorylation of p70S6K1 and Akt and showed antiproliferative activity associated with G1 cell cycle arrest, apoptosis induction and the inhibition of protein synthesis in rapamycin-sensitive and rapamycin-resistant cancer cell lines. Importantly, 123a (WYE-354) is also active in vivo, showing antitumor activity when injected into mice bearing PTEN-null U87MG glioma. 4.1.2. Molecular Modeling Studies and Substitution Optimization. Molecular modeling studies have demonstrated the importance of a urea moiety in position C6, showing that this group interacts more favorably with the ATP-binding site of the enzyme than the corresponding carbamoylphenyl group.73−75 Indeed, the same Wyeth researchers built an mTOR homology model, starting from the known X-ray crystal structure of PI3Kγ, which was based on the highly similar sequences of the ATP-binding sites of the two enzymes. The X-ray structure of a pyrazolo-pyrimidine inhibitor bound to PI3Kγ (PDB code 3IBE) was used as the basis for docking studies with the mTOR homology model and showed that the urea group of the inhibitor makes three hydrogen bond contacts within the ATP-binding pocket, two between the urea NHs and Asp2195 and one between the urea carbonyl and Lys2187. The binding mode of these inhibitors showed the importance of the C4 morpholino substituent, which participates in a critical interaction with Val2240 in the hinge region of mTOR. Moreover, the picolylpiperidine tail in the front of the binding site interacts with the region containing Leu2249 and Ala2248 (Figure 4).

The reaction of 106 with the appropriate hydrazine affords 4,6-dichloropyrazolo-pyrimidine 114. The chlorine atom in C4 is selectively displaced by morpholine, leading to 115. Suzuki coupling with the appropriate boronic acid gives compound 116. Then, the benzyl protecting group on the piperidinyl nitrogen is removed by hydrogenation, and the resulting secondary amine is reductively alkylated to give 117 (WAY600). For the synthesis of other derivatives, compound 115 was debenzylated with α-chloroethyl chloroformate (ACE-Cl) to afford the free piperidino derivative 118, which is converted into picolyl-functionalized intermediate 119 by reductive amination. When 119 was subjected to Suzuki-Miyaura coupling conditions, the 6-anilino-derivative 120 was produced, which was treated with triphosgene and TEA and subsequently with a suitable alcohol or amine to afford carbamoylphenyl or ureidophenyl derivatives 121, including 121a (WYE-687; Scheme 38).74 Product 118 was treated with a suitable carbamoyl chloride to afford intermediates 122, which were subjected to the reaction sequence presented for 121 to afford the ureas or carbamates 123, including 123a (WYE-354; Scheme 39).75 Inhibitors 117 (WAY-600), 121a (WYE-687) and 123a (WYE-354) are the most studied compounds.76 These compounds inhibit mTOR with IC50 values in the 5−9 nM range, show significant selectivity (>100-fold) over PI3Ks in 7203

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Scheme 39. Synthesis of the mTOR Inhibitor 123a (WYE-354)

Structure-based drug design suggested the preparation of several 6-arylureidophenyl-1H-pyrazolo[3,4-d]pyrimidines substituted with water-solubilizing groups on the arylureido moiety; compound 126a (Figure 5), with a 4-hydroxymethylphenyl group, showed an IC50 value of 0.08 nM against mTOR, as well as increased water solubility and cellular potency.75

Figure 5. Structures of the mTOR inhibitors 126a and 126b. Figure 4. Schematic interactions between an ureidophenyl pyrazolopyrimidine and mTOR in the homology model of mTOR ATPbinding pocket. Hydrogen bonds are indicated as dotted lines.

Derivatives bearing ethyl, pyrrolidino, morpholino or methylpiperazino groups as solubilizing moieties on the para position of the urea phenyl ring showed mTOR inhibition and unprecedented antiproliferative activity in cell assays; compound 126b (Figure 5) showed an IC50 value of 0.7 nM in an enzymatic assay and an IC50 value