AU2002334992B2 - Methods for the synthesis of substituted purines - Google Patents

Description

WO 03/031405 PCT/US02/32679 METHODS FOR THE SYNTHESIS OF SUBSTITUTED PURINES CROSS-REFERENCES TO RELATED APPLICATIONS This patent application claims the benefit of U.S. Provisional Patent Application No. 60/328,741, filed October 12, 2001, U.S. Provisional Patent Application No. 5 60/346,552, filed January 7, 2002, and U.S. Provisional Patent Application No. 60/347,037, filed January 8, 2002, the teachings of all of which are incorporated herein by reference. This patent application is related to U.S. Provisional Patent Application No. 60/328,763, filed October 12, 2001, U.S. Provisional Patent Application No. 60/331,835, filed November 20, 2001, U.S. Provisional Patent Application No. 60/346,480, filed January 7, 2002, and 10 U.S. Provisional Patent Application No. 60/348,089, filed January 10, 2002, the teaching of all of which are incorporated herein by reference. BACKGROUND OF THE INVENTION Field of the Invention [00011 This invention pertains to the field of methods for preparing libraries 15 of purine compounds. For example, the invention relates to methods of preparing 2, 6, 9 substituted purine compounds on solid phase supports by employing sulfenylpurine intermediates. Also provided are methods for preparing 2,9-substituted purines and 0 6 -aryl and 0 6 -alkyl-substituted purines. Background 20 [0002] The sequencing of the human genome and numerous pathogen genomes has resulted in an explosion of new molecular targets that can be modulated by small molecules. Discovering these modulators and further developing them into new therapeutics presents unprecedented opportunities and challenges for academic and industrial researches. One of the most fruitful applications of combinatorial chemistry has been the 25 design of flexible synthetic schemes that generalize a privileged scaffold from a particular target protein to an entire protein family. 1 WO 03/031405 PCT/US02/32679 [0003] Inhibitors of protein kinases have proven to be invaluable tools in the elucidation of signal transduction networks, as well as promising clinical candidates for the treatment of cancer, cardiovascular disease, inflammatory, and neurological diseases. (McMahon et. al., Current Opinion in Drug Discovery & Development, 1, 131 (1998); 5 Adams et al., Current Opinion in Drug Discovery & Development, 2, 96 (1999); Cohen, P., Current Opinion in Chemical Biology, 3, 459 (1999); Garcia-Echeverria et. al., Med. Res. Rev, 20, 28 (2000); Blume-Jensen et al., Nature, 411, 355 (2001) and references therein) Despite concerns that it would be extremely difficult to design specific ATP competitive inhibitors of kinases, there have been a number of success stories including the p38 Map 10 kinase, tyrosine kinases, and cyclin dependent kinases. (McMahon et. al., Current Opinion in Drug Discovery & Development, 1, 131 (1998); Adams et al., Current Opinion in Drug Discovery & Development, 2, 96 (1999); Cohen, P., Current Opinion in Chemical Biology, 3, 459 (1999); Garcia-Echeverria et. al., Med. Res. Rev, 20, 28 (2000); Blume-Jensen et al., Nature, 411, 355 (2001) and references therein; Druker et. al., Nature Medicine, 2, 561 15 (1996); Taylor et al., Current Opinion in Chemical Biology, 1, 2219 (1997); Schindler et. al., Science, 289, 1938 (2000)) Selective inhibitors of each of these kinases are in various stages of clinical testing. The ability to discriminate between extremely homologous kinases such as CDK1 vs. CDK2 has been demonstrated by the development of novel thio flavopiridol derivatives that display enhanced selectivity for CDK1 relative to CDK2. (Kim 20 et. al., J Med. Chem., 43, 4126 (2000).) [00041 The purine ring system is a key structural element of the substrates and ligands of many biosynthetic, regulatory and signal transduction proteins including cellular kinases, G proteins and polymerases. In particular, inhibitors of protein kinases have proven to be invaluable tools in the elucidation of signal transduction networks as well as 25 promising clinical candidates in a multitude of disease such as cancer, cardiovascular disease, inflammatory disease and neurological disease. For example, various purine analogs have been found to be highly potent therapeutic agents for disease. As such, the purine ring system has been a good starting point in the search for inhibitors of kinases, G proteins and many biomedically significant processes. 30 [0005] The design of synthetic schemes for generating a multitude of structurally diverse compounds (i.e., library of compounds) typically involves creating the 2 WO 03/031405 PCT/US02/32679 libraries in situ in solution phase or on solid support, i.e., solid phase. In solid phase synthesis, the desired compounds are generated while attached to the solid support via a linker prepared on a polymeric solid support material, e.g., polystyrene. [00061 Several solid phase and solution phase approaches for the synthesis of 5 purine analogs have been reported in the literature over the past five years. (For 2-, 6-, 8- 9 substituted purine analogs, see, e.g., Gray et. al., Science, 281, 533 (1998); Chang et. al., Chemistry and Biology, 6, 361 (1999); Lucrezia et. al., J. Comb. Chem., 2, 249 (2000); Nolsoe et. al., Synth. Commun., 28, 4303 (1998); for 7- substituted purine analogs, see, e.g., Dalby et. al., Angew. Chem. Int. Ed. Engl., 32, 1696 (1993); Zaitseva et. al., Bioorganic 10 Med. Lett., 5, 2999 (1995).) Unfortunately, these approaches have limitations. One limitation is that one substituent is held invariant in order to anchor the purine ring to the solid phase (Scheme 1). To avoid this limitation, a "traceless" strategy would be desirable that would be compatible with production scale library synthesis in spatially separate or divide-recombine formats. 15 Scheme 1. Previously described combinatorial strategies for 2,6,9-trisubstituted purines HN I17/z 1. amination at C6 (R 1

NH

2 ) or_ I and/or or H alkylation at N9 (R 2 OH) -RI C1 H and/or HN - N amination at C2 (R 3

NH

2 ) 1N - N7 HF N N 2. H. R 3 'N N 4 N )Eor H H 3 R2 [00071 Another limitation of previous synthetic approaches is the low reactivity of the 2-fluoro group once an amino substituent has been installed at C6. For example, complete displacement at C2 of a 2-fluoro-6-benzylaminopurine in solution 20 requires heating at over 100 "C for twelve hours using n-butanol as solvent. Complete aromatic substitution of 2-fluoro or 2-chloro purine compounds on solid support requires even higher temperatures and often results in significant side reactions. This limits the range of functional groups that can be installed at C2 and also creates difficulties in library production. 3 WO 03/031405 PCT/US02/32679 [00081 Subsequently, it was found that 6-amino-2-fluoro-9-alkylpurines react with primary amines in methanol at room temperature. With slightly more forcing conditions (in refluxing methanol) sterically hindered amines such as the alpha-amino group of arginine could be successfully introduced at the C2 position (Scheme 2) with good yields. 5 Unfortunately, these conditions failed to translate to solid support, presumably due to resin swelling problems in methanol. Despite testing a range of solvent systems (NMP, DMF, dioxane, DMSO, THF and their combinations such as DMFv/MeOHv: 1/1), no solvent was found that allowed complete substitution below 100 "C. Scheme 2. Couple amino acids to purine C2 position - R HN CI HO NH 2 HN cl R=i-butyl N Rt-butyl N CN ' R N \ N1-hydroxylethyl l '> DiEA, CH 3 oH, reflux HO 3-guanidinylpropyl F N NN N 4-aminobenzyl 10 0 H [00091 Since a number of purine compounds have been shown to possess diverse phannacological properties and biological activities in a number of therapeutic areas, the generation of a substituted purine compound library would be useful as a screening tool to identify the structures of compounds that possess the desired biological activity. In 15 addition, the generation of substituted purine library would permit alteration of the structures of the identified compounds to identify derivatives of these compounds which exhibit the best biological activity and which also have ideal pharmacological and pharmacokinetic properties. Accordingly, the development of an efficient, rapid in situ method for the generation of a multitude of highly substituted purine compounds that would overcome 20 limitations of known procedures would be highly desirable. [00101 Moreover, to date a variety of heterocyclic scaffolds including pyrimidines, indolines, pyrrolopyrimidines, indirubins, purines, quinazolines, trisubstituted imidazoles, pyrazolopyrimidines, have been developed as kinase inhibitors. ((a) McMahon et. al., Current Opinion in Drug Discovery & Development, 1, 131 (1998); (b) Adams et al., 25 Current Opinion in Drug Discovery & Development, 2, 96 (1999); (c) Cohen, P., Current Opinion in Chemical Biology, 3, 459 (1999); (d) Garcia-Echeverria et. al:, Med. Res. Rev, 4 WO 03/031405 PCT/US02/32679 20, 28 (2000); (e) Blume-Jensen et al., Nature, 411, 355 (2001) and references therein; (f) Druker et. al., Nature Medicine, 2, 561 (1996); (g) Taylor et al., Current Opinion in Chemical Biology, 1, 2219 (1997); (h) Schindler et. al., Science, 289, 1938 (2000).) As each scaffold presents unique opportunities for the presentation of functional groups to the 5 kinase active site, there is a need for efficient and flexible methods for preparing libraries of each of these inhibitor classes. The present invention fulfills these and other needs. SUMMARY OF THE INVENTION [00111 The present invention is directed to methods of preparing diverse 2,6-, 2,9, 2,6,9-, 0 6 -alkyl- and 0 6 -aryl-substituted purine compounds on solid support phase. In 10 some embodiments, the methods involve using a novel intermediate compound, a 2-halo-6 sulfenyl purine. The invention also encompasses these sulfenylpurine intermediates and methods for their preparation. [0012] As such, in one aspect, the present invention provides a method of preparing a 2,6,9-substituted purine compounds having the Formula I:

NR

3

R

4 NN NN HN N 15 R1 the method comprising: a) oxidizing a resin-bound compound of Formula II: <D_(CH2)nS N/ \ N~N NN N1 N N Q1iN\2 to provide a resin-bound compound of Formula III: 5 (CH2)nO N N N N N b) reacting the compound of Formula III with an amine of Formula IV

HNR

3

R

4 IV, to provide a resin-bound compound of Formula V

NR

3

R

4 N N N in' R2 5 R and c) cleaving the resin-bound compound of Formula V from the resin to provide the substituted purine compounds of Formula I. [0013] In Formula I, R 1 and R 2 are independently selected from alkyl, substituted alkyl, cycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl and 10 heterocyclyl; R 3 is hydrogen; and R 4 is selected from alkyl, substituted alkyl, cycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl and heterocyclyl; or, R 3 and R 4 when taken together with the nitrogen atom to which they are respectively attached form an optionally substituted saturated heterocyclic ring. [00141 In Formulae II and III, R 1 and R 2 are as defined above and n is 0 or 1. 15 In Formula IV, R 3 and R 4 are as defined above. In Formula V, R 1 , R 2 , R 3 and R4 are as defined above. [00151 In a preferred embodiment, the compound is a compound of Formula II, and can be prepared using a method comprising: a) alkylating a compound of Formula VI: 6 WO 03/031405 PCT/US02/32679 0(CH2)n--S N N __ N X N H to provide a compound of Formula VII: /\ (CH 2 )n-S N / N )K Vi' X N N

R

2 ;and b) capturing the compound of Formula VII with a resin-bound amine to 5 provide the resin-bound compound of Formula II. [0016] In the above method, X, in Formula VI, is fluoro, chloro or bromo, and n is as defined above. In Formula VII, R 2 , X and n are as defined above. [00171 In another preferred embodiment, the compound of Formula II can be prepared using a method comprising: 10 a) capturing a compound of Formula VI 0-(CH2)n-S / \ '~~N X N with a resin-bound amine to provide a resin-bound compound of Formula VIII:

(CH

2 )n-S N / N VIII N N NH and 7 WO 03/031405 PCT/US02/32679 b) alkylating the resin-bound compound of Formula VIII to provide the resin-bound compound of Formula II. [0018] In Formula VI, X and n are as defined above. In Formula VII, R 1 and n are as defined above. 5 [00191 In a preferred embodiment, the compound of Formula VI is prepared by reacting 2-halo-6-halo-purine with a compound of Formula IX: / \ (CH 2 )nSH I to provide the compound of Formula VI, wherein n is as defined above. In a preferred embodiment, the halo groups of the 2-halo-6-halo-purine compound are fluoro, chloro, 10 bromo or iodo and, preferably chloro. [00201 In a preferred embodiment of the above method, step (a) is carried out with m-chloroperbenzoic acid in a buffered solution. In a preferred embodiment of the above method, step (c) is carried out in the presence of trifluoroacetic acid. In a preferred embodiment, alkylation of the compound of Formula VI is carried out in the presence of an 15 inert solvent and a phosphine (e.g., triphenylphosphine, tricyclohexylphosphine, etc.). In a preferred embodiment, the resin is 4-formyl-3,5-dimethyloxyphenoxymethyl functional polystyrene resin. [00211 The present invention also provides a combinatorial library or array comprising a plurality of 2, 6, 9-substituted purine compounds prepared by the above 20 method. [00221 In another aspect, the present invention provides a compound of Formula VI: &(CH2)n--S N \N N --- ) VI N X N H wherein X is fluoro, chloro, or bromo, and n is 0 to 1. 8 WO 03/031405 PCT/US02/32679 [0023] In another aspect, the present invention provides a method of preparing 2, 9-substituted purine compounds having the Formula X: 0 N HN N I R2 R1 the method comprising: 5 a) oxidizing a resin-bound compound of Formula II 0 (CH2)n--S / \N N 1NR2 to provide a resin-bound compound of Formula III (cH2)n---==o / \ N____ N NNN R2 9 b) hydrolyzing the sulfonyl group at the 6-position of the resin-bound compound of Formula III; and cleaving the hydrolyzed resin-bound compound from the resin to provide the substituted purine compounds of Formula X [0024] In the above method, R 1 and R 2 are independently selected from alkyl, 5 substituted alkyl, cycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl and heterocyclyl; and n is 0 or 1. [00251 In the above method for 2,9-substituted purine compounds, the resin bound compound of Formula II is prepared using one of the methods set forth above for preparing such compound. 10 [00261 The present invention also provides a combinatorial library or array comprising a plurality of 2,9-substituted purine compounds prepared by the above method. [0027] In another aspect, the present invention provides a method for preparing 06 -alkyl-purine compounds of Formula XI:

OR

3 N HN N N RI R2 15 the method comprising: a) oxidizing a resin-bound compound of Formula II

(CH

2 )n- S N N\ N /N' N O R2 to provide a resin-bound compound of Formula III 10

(CH

2 )n-- N N N ON R2 b) hydrolyzing the sulfonyl group at the 6-position of the resin-bound compound of Formula III to form a resin-bound compound of Formula XII 0 HN XII O H N N

R

2 ; and 5 c) alkylating the resin-bound compound of Formula XII and cleaving the resin bound alkylated compound from the resin to provide a compound of Formula XI [00281 In the above method, R 1 and R 2 are independently selected from alkyl, substituted alkyl, cycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl and heterocyclyl; R 3 is selected from alkyl and substituted alkyl; and n is 0 or 1. 10 [00291 In the above method for preparing 0 6 -alkyl-purine compounds, the resin-bound compound of Formula II is prepared using one of the methods set forth above for preparing such compound. [00301 The present invention also provides a combinatorial library or array comprising a plurality of 0 6 -alkyl-purine compounds prepared by the above method. 15 [0031] In another aspect, the present invention provides a method for preparing 0 6 -aryl purine compounds of Formula XIII: 11

OR

3 N XIII HN HN N N

R

2 the method comprising: a) oxidizing a resin-bound compound of Formula II (CH2)n-S N \ N N R2 5 to provide a resin-bound compound of Formula III

(CH

2 )n O N N N N I R2 Ri ;and b) reacting the resin-bound compound of Formula III with a phenolic aryl compound followed by cleavage of the resin-bound compound to provide a compound of Formula XIII [0032] In the above method, R 1 and R 2 are independently selected from alkyl, 10 substituted alkyl, cycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl and heterocyclyl; R 3 is selected from aryl and substituted aryl; and n is 0 or 1. 12 [0033] In the above method for preparing 06 -aryl-purine compounds, the resin-bound compound of Formula II is prepared using one of the methods set forth above for preparing such compound. [00341 The present invention also provides a combinatorial library or array 5 comprising a plurality of 0 6 -aryl-purine compounds prepared by the above method. 100351 In another aspect, the present invention provides a method for preparing a compound of Formula VI: 0(CH2)n---S N X N H the method comprising: 10 reacting a 2-halo-6-chloro-purine with a compound of Formula IX / \ (CH 2 )nSH IX to provide the compound of Formula VI. [0036] In the above method, X is fluoro, chloro or bromo; and n is 0 or 1. [0037] In another embodiment, the present invention provides a method for 15 the for synthesis of a C2-substituted purine, the method comprising reacting a C6 sulfenylpurine with a nucleophile (e.g.: an amine), wherein the nucleophile is substituted at the C2 position of the purine, and the C6-sulfenylpurine comprises a solid support-bound compound of Formula XIV: Q(C

H

2 )n S N XIV H 13 wherein X is a halogen and n is 1. In another embodiment, the method further comprises oxidizing a thioether linkage between the purine and the sulfenyl moiety to a sulfone of Formula XV: O- (CH 2 )n 0 S=O HN NX NIN H 5 wherein R 1 is alkyl, substituted alkyl, cycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl or heterocyclyl, and substituting a different group at the C6 position of the purine. In a preferred embodiment, the C6 sulfenylpurine is a 2-fluoro-6-phenylsulfenyl or a 2 fluoro-6-benzylsulfenyl. [0038] Other features, objects and advantages of the invention and its preferred 10 embodiments will become apparent from the detailed description which follows. BRIEF DESCRIPTION OF THE DRAWINGS [00391 Figure 1 shows examples of substituents that have been introduced at the C2 position of purines using the methods of the invention as shown in Scheme 6. These 15 substituents were introduced by palladium catalyzed cross-coupling reactions using Pd 2 (dba) 3 , carbene or phosphine ligand, corresponding boronic acid and Cs 2

CO

3 for C-C bond formation, aniline and KO'Bu for C-N bond formation, phenol and K 3 P0 4 for C-O bond formation. These substituents can also be introduced by reacting with corresponding aniline/phenol and KO t Bu in THF at 70 'C. 20 [00401 Figure 2 shows validated substituents at the C2 position of purine introduced by nucleophilic aromatic substitution with primary and secondary amines as shown in Scheme 11. [0041] Figure 3 shows validated substituents at the C6 position of purine introduced by nucleophilic aromatic substitution with primary and secondary amines as 25 shown in Scheme 11. 14 DETAILED DESCRIPTION OF THE INVENTION AND PREFERRED EMBODIMENTS Definitions [0042] Unless defined otherwise, all technical and scientific terms used 5 herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures for organic and analytical chemistry are those well known and commonly employed in the art. [0043] As used herein, the term "leaving group" refers to a portion of a 10 substrate that is cleaved from the substrate in a reaction. [0044] "Protecting group," as used herein refers to a portion of a substrate that is substantially stable under a particular reaction condition, but which is cleaved from the substrate under a different reaction condition. A protecting group can also be selected such that it participates in the direct oxidation of the aromatic ring component of the 15 compounds of the invention. For examples of useful protecting groups, see, for example, Greene et al., Protective Groups In Organic Synthesis, John Wiley & Sons, New York (1991). [0045] The term "alkyl," by itself or as part of another substituent, means, unless otherwise stated, a straight or branched chain, hydrocarbon radical having the number 20 of carbon atoms designated (i.e. C 1 -Cio means one to ten carbons). Examples of saturated hydrocarbon radicals include groups such as methyl, ethyl, n-propyl, isopropyl, n-butyl, t butyl, isobutyl, sec-butyl, cyclohexyl, (cyclohexyl)methyl, cyclopropylmethyl, homologs and isomers of, for example, n-pentyl, n-hexyl, n-heptyl, and n-octyl The terms "alkenyl" and "alkynyl" refer to unsaturated hydrocarbon groups having one or more double bonds or 25 triple bonds, respectively. Examples of suitable unsaturated hydrocarbon groups include vinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl, 3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and the higher homologs and isomers. 15 WO 03/031405 PCT/US02/32679 [0046] The terms "alkoxy," "alkylamino" and "alkylthio" (or thioalkoxy) are used in their conventional sense, and refer to those alkyl groups attached to the remainder of the molecule via an oxygen atom, an amino group, or a sulfur atom, respectively. [0047] The term "heteroalkyl," by itself or in combination with another term, 5 means, unless otherwise stated, a stable straight or branched chain, or cyclic hydrocarbon radical, or combinations thereof, consisting of the stated number of carbon atoms and from one to three heteroatoms selected from the group consisting of 0, N, Si and S, and wherein the nitrogen and sulfur atoms may optionally be oxidized and the nitrogen heteroatom may optionally be quaternized. The heteroatom(s) 0, N and S may be placed at any interior 10 position of the heteroalkyl group. The heteroatom Si may be placed at any position of the heteroalkyl group, including the position at which the alkyl group is attached to the remainder of the molecule. Examples include -CH 2

-CH

2 -0-CH 3 , -CH 2

-CH

2

-NH-CH

3 , -CH 2 CH 2

-N(CH

3

)-CH

3 , -CH 2

-S-CH

2

-CH

3 , -CH 2

-CH

2

,-S(O)-CH

3 , -CH 2

-CH

2 -S(0) 2

-CH

3 , CH=CH-0-CH 3 , -Si(CH 3

)

3 , -CH 2

-CH=N-OCH

3 , and -CH=CH-N(CH 3

)-CH

3 . Up to two 15 heteroatoms may be consecutive, such as, for example, -CH 2

-NH-OCH

3 and -CH 2 -0 Si(CH 3

)

3 . Similarly, the term "heteroalkylene" by itself or as part of another substituent means a divalent radical derived from heteroalkyl, as exemplified by -CH 2

-CH

2

-S-CH

2

CH

2 and -CH 2

-S-CH

2

-CH

2

-NH-CH

2 -. For heteroalkylene groups, heteroatoms can also occupy either or both of the chain termini (e.g., alkyleneoxy, alkylenedioxy, alkyleneamino, 20 alkylenediamino, and the like). Still further, for alkylene and heteroalkylene linking groups, no orientation of the linking group is implied. [0048] The terms "cycloalkyl" and "heterocycloalkyl", by themselves or in combination with other terms, represent, unless otherwise stated, cyclic versions of "alkyl" and "heteroalkyl", respectively. Additionally, for heterocycloalkyl, a heteroatom can occupy 25 the position at which the heterocycle is attached to the remainder of the molecule. Examples of cycloalkyl include cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, cycloheptyl, and the like. Examples of heterocycloalkyl include 1 -(1,2,5,6-tetrahydropyridyl), 1 piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-morpholinyl, 3-morpholinyl, tetrahydrofuran-2-yl, tetrahydrofuran-3-yl, tetrahydrothien-2-yl, tetrahydrothien-3-yl, 1 -piperazinyl, 2 30 piperazinyl, and the like. 16 [00491 The terms "halo" or "halogen," by themselves or as part of another substituent, mean, unless otherwise stated, a fluorine, chlorine, bromine, or iodine atom. Additionally, terms such as "haloalkyl," are meant to include monohaloalkyl and polyhaloalkyl. For example, the term "halo(C1-C 4 )alkyl" is mean to include trifluoromethyl, 5 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl, and the like. [00501 The term "aryl" means, unless otherwise stated, an aromatic, hydrocarbon substituent, which can be a single ring or multiple rings (up to three rings), which are fused together or linked covalently. The term "heteroaryl" refers to aryl groups (or rings) that contain from zero to four heteroatoms selected from N, 0, and S, wherein the 10 nitrogen and sulfur atoms are optionally oxidized, and the nitrogen atom(s) are optionally quaternized. A heteroaryl group can be attached to the remainder of the molecule through a heteroatom. Non-limiting examples of aryl and heteroaryl groups include phenyl, 1 naphthyl, 2-naphthyl, 4-biphenyl, 1-pyrrolyl, 2-pyrrolyl, 3-pyrrolyl, 3-pyrazolyl, 2 imidazolyl, 4-imidazolyl, pyrazinyl, 2-oxazolyl, 4-oxazolyl, 2-phenyl-4-oxazolyl, 5 15 oxazolyl, 3-isoxazolyl, 4-isoxazolyl, 5-isoxazolyl, 2-thiazolyl, 4-thiazolyl, 5-thiazolyl, 2 furyl, 3-furyl, 2-thienyl, 3-thienyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5-benzothiazolyl, purinyl, 2-benzimidazolyl, 5-indolyl, 1-isoquinolyl, 5-isoquinolyl, 2 quinoxalinyl, 5-quinoxalinyl, 3-quinolyl, and 6-quinolyl. Substituents for each of the above noted aryl and heteroaryl ring systems are selected from the group of acceptable substituents 20 described below. [00511 For brevity, the term "aryl" when used in combination with other terms (e.g., aryloxy, arylthioxy, arylalkyl) includes both aryl and heteroaryl rings as defined above. Thus, the term "arylalkyl" is meant to include those radicals in which an aryl group is attached to an alkyl group (e.g., benzyl, phenethyl, pyridylmethyl and the like) including 25 those alkyl groups in which a carbon atom (e.g., a methylene group) has been replaced by, for example, an oxygen atom (e.g., phenoxymethyl, 2-pyridyloxymethyl, 3-(1 naphthyloxy)propyl, and the like). [0052] Each of the above terms (e.g., "alkyl," "heteroalkyl," "aryl" and "heteroaryl") are meant to include both substituted and unsubstituted forms of the indicated 30 radical. Preferred substituents for each type of radical are provided below. 17 WO 03/031405 PCT/US02/32679 [0053] Each of the above terms (e.g., "alkyl," "heteroalkyl," "aryl" and "heteroaryl") is meant to include both substituted and unsubstituted forms of the indicated radical. Preferred substituents for each type of radical are provided below. [0054] Substituents for the alkyl and heteroalkyl radicals (including those 5 groups often referred to as alkylene, alkenyl, heteroalkylene, heteroalkenyl, alkynyl, cycloalkyl, heterocycloalkyl, cycloalkenyl, and heterocycloalkenyl) can be one or more of a variety of groups selected from, but not limited to: -OR', =0, =NR', =N-OR', -NR'R", -SR', -halogen, -SiR'R"R"', -OC(O)R', -C(O)R', -CO 2 R', -CONR'R", -OC(O)NR'R", NR"C(O)R', -NR'-C(O)NR"R"', -NR"C(O) 2 R', -NR-C(NR'R"R'")=NR"", 10 -NR-C(NR'R")=NR', -S(O)R', -S(O) 2 R', -S(O) 2 NR'R", -NRSO 2 R', -CN and -NO 2 in a number ranging from zero to (2m'+1), where m' is the total number of carbon atoms in such radical. R', R", R"' and R"" each preferably independently refer to hydrogen, substituted or unsubstituted heteroalkyl, substituted or unsubstituted aryl, e.g., aryl substituted with 1-3 halogens, substituted or unsubstituted alkyl, alkoxy or thioalkoxy groups, or arylalkyl 15 groups. When a compound of the invention includes more than one R group, for example, each of the R groups is independently selected as are each R', R", R"' and R"" groups when more than one of these groups is present. When R' and R" are attached to the same nitrogen atom, they can be combined with the nitrogen atom to form a 5-, 6-, or 7-membered ring. For example, -NR'R" is meant to include, but not be limited to, 1-pyrrolidinyl and 4 20 morpholinyl. From the above discussion of substituents, one of skill in the art will understand that the term "alkyl" is meant to include groups including carbon atoms bound to groups other than hydrogen groups, such as haloalkyl (e.g., -CF 3 and -CH 2

CF

3 ) and acyl (e.g., -C(O)CH 3 , -C(O)CF 3 , -C(O)CH 2 0CH 3 , and the like). [0055] Similar to the substituents described for the alkyl radical, substituents 25 for the aryl and heteroaryl groups are varied and are selected from, for example: halogen, -OR', =0, =NR', =N-OR', -NR'R", -SR', -halogen, -SiR'R"R"', -OC(O)R', -C(O)R',

-CO

2 R', -CONR'R", -OC(O)NR'R", -NR"C(O)R', -NR'-C(O)NR"R"', -NR"C(O) 2 R', -NR-C(NR'R")=NR'", -S(O)R', -S(O) 2 R', -S(O) 2 NR'R", -NRSO 2 R', -CN and -NO 2 , -R', N 3 , -CH(Ph) 2 , fluoro(C1-C 4 )alkoxy, and fluoro(C 1

-C

4 )alkyl, in a number ranging from zero 30 to the total number of open valences on the aromatic ring system; and where R', R", R"' and R"" are preferably independently selected from hydrogen, (C 1 -Cs)alkyl and heteroalkyl, 18 WO 03/031405 PCT/US02/32679 unsubstituted aryl and heteroaryl, (unsubstituted aryl)-(CI-C 4 )alkyl, and (unsubstituted aryl)oxy-(Ci-C 4 )alkyl. When a compound of the invention includes more than one R group, for example, each of the R groups is independently selected as are each R', R", R"' and R"" groups when more than one of these groups is present. 5 [00561 The term "independently selected" is used herein to indicate that the R groups, e.g., R1 and R2, can be identical or different (e.g., R.1 and R2 may all be substituted alkyls or R1 may be a substituted alkyl and R2 may be an aryl, etc.). A named R group will generally have the structure which is recognized in the art as corresponding to R groups having that name. For the purposes of illustration, representative R groups as enumerated 10 above are defined herein. These definitions are intended to supplement and illustrate, not preclude, the definitions known to those of skill in the art. [00571 Two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -T-C(O)-(CRR')q-U-, wherein T and U are independently -NR-, -0-, -CRR'- or a single bond, and q is an integer 15 of from 0 to 3. Alternatively, two of the substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -A-(CH 2 )rB-, wherein A and B are independently -- CRR'-, -0-, -NR-, -S-, -S(O)-, -S(0)2-, -S(0) 2 NR'- or a single bond, and r is an integer of from 1 to 4. One of the single bonds of the new ring so formed may optionally be replaced with a double bond. Alternatively, two of the 20 substituents on adjacent atoms of the aryl or heteroaryl ring may optionally be replaced with a substituent of the formula -(CRR')s-X-(CR"R')d-, where s and d are independently integers of from 0 to 3, and X is -0-, -NR'-, -S-, -S(O)-, -S(0) 2 -, or -S(0) 2 NR'-. The substituents R, R', R" and R"' are preferably independently selected from hydrogen or substituted or unsubstituted (CI-C 6 )alkyl. 25 [00581 As used herein, the term "heterocycle," refers to both heterocycloalkyl and heteroaryl groups. [00591 As used herein, the term "heteroatom" is meant to include oxygen (0), nitrogen (N), sulfur (S) and silicon (Si). [00601 The term "pharmaceutically acceptable salts" is meant to include salts 30 of the active compounds which are prepared with relatively nontoxic acids or bases, depending on the particular substituents found on the compounds described herein. When 19 WO 03/031405 PCT/US02/32679 compounds of the present invention contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired base, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, 5 ammonium, organic amino, or magnesium salt, or a similar salt. When compounds of the present invention contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such compounds with a sufficient amount of the desired acid, either neat or in a suitable inert solvent. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids like hydrochloric, hydrobromic, 10 nitric, carbonic, monohydrogencarbonic, phosphoric, monohydrogenphosphoric, dihydrogenphosphoric, sulfuric, monohydrogensulfuric, hydriodic, or phosphorous acids and the like, as well as the salts derived from relatively nontoxic organic acids like acetic, propionic, isobutyric, maleic, malonic, benzoic, succinic, suberic, fumaric, lactic, mandelic, phthalic, benzenesulfonic, p-tolylsulfonic, citric, tartaric, methanesulfonic, and the like. 15 Also included are salts of amino acids such as arginate and the like, and salts of organic acids like glucuronic or galactunoric acids and the like (see, for example, Berge et al., Journal ofPharmaceutical Science, 66, 1-19 (1977)). Certain specific compounds of the present invention contain both basic and acidic functionalities that allow the compounds to be converted into either base or acid addition salts. 20 [00611 The neutral forms of the compounds are preferably regenerated by contacting the salt with a base or acid and isolating the parent compound in the conventional manner. The parent form of the compound differs from the various salt forms in certain physical properties, such as solubility in polar solvents, but otherwise the salts are equivalent to the parent form of the compound for the purposes of the present invention. 25 [00621 In addition to salt forms, the present invention provides compounds, which are in a prodrug form. Prodrugs of the compounds described herein are those compounds that readily undergo chemical changes under physiological conditions to provide the compounds of the present invention. Additionally, prodrugs can be converted to the compounds of the present invention by chemical or biochemical methods in an ex vivo 30 environment. For example, prodrugs can be slowly converted to the compounds of the 20 WO 03/031405 PCT/US02/32679 present invention when placed in a transdermal patch reservoir with a suitable enzyme or chemical reagent. [0063] Certain compounds of the present invention can exist in unsolvated forms as well as solvated forms, including hydrated forms. In general, the solvated forms 5 are equivalent to unsolvated forms and are intended to be encompassed within the scope of the present invention. Certain compounds of the present invention may exist in multiple crystalline or amorphous forms. In general, all physical forms are equivalent for the uses contemplated by the present invention and are intended to be within the scope of the present invention. 10 [0064] Certain compounds of the present invention possess asymmetric carbon atoms (optical centers) or double bonds; the racemates, diastereomers, geometric isomers and individual isomers are encompassed within the scope of the present invention. [0065] The compounds of the present invention may also contain unnatural proportions of atomic isotopes at one or more of the atoms that constitute such compounds. ,15 For example, the compounds may be radiolabeled with radioactive isotopes, such as for example tritium (H), iodine-125 ( 1 25 I) or carbon-14 ( 14 C). All isotopic variations of the compounds of the present invention, whether radioactive or not, are intended to be encompassed within the scope of the present invention. Description of the Preferred Embodiments 20 [0066] The present invention provides solid phase methods of preparing diverse purine compounds. For example, the present invention provides methods for producing 2,6-, 2,6,9-, 2,9-, 0 6 -alkyl-, and 06 aryl-substituted purine compounds as well as novel substituted purine compounds. In some embodiments, the methods use novel C-6 sulfenylpurine compounds as intermediates. Methods of preparing the intermediates are also 25 encompassed by the present invention. [0067] The methods of the present invention overcome limitations described above in preparing 2,6 and 2,6,9-substituted purines by, for example, employing 1) an arylsulfenyl group as a "protective" group at the C6 position of the purine ring which when subsequently oxidized to an arylsulfonyl group allows the quantitative and selective 30 substitution by an amine to the C2 position prior to substitution at the C6 position; and 2) in 21 preferred embodiments, a "traceless" amine linkage to solid support, wherein the amine linker is incorporated into the final purine compound. [0068] The methods described herein are useful not only for substitution of purines, but also of other heterocycles. For example, the methods are suitable for use with a 5 combinatorial scaffold approach towards heterocycle libraries, as described in U.S. Provisional Patent Application No. 60/331,835, which is entitled "Kinase Inhibitor Scaffolds," and which was filed on November 20, 2001, in U.S. Provisional Patent Application No. 60/346,480, which is entitled "Kinase Inhibitor Scaffolds," and which was filed on January 7, 2002, and in U.S. Provisional Patent Application No. 60/348,089, which is entitled "Kinase Inhibitor Scaffolds," and 10 which was filed on January 10, 2002. The methods are also suitable for use in conjunction with the methods described in U.S. Provisional Patent Application No. 60/328,763, which is entitled "Expanding the Diversity of Purine Libraries," and which was filed on October 12, 2001. Priority from all four of these U.S. applications is claimed in international application WO 03/031406. Preparation of 2,6,9-substituted purines 15 [0069] Accordingly, in one aspect, the present invention provides a method of preparing a 2,6,9-substituted purine compounds having the Formula I

NR

3

R

4 HN N NN HN N I R2 R, the method comprising: a) oxidizing a resin-bound compound of Formula II:

(CH

2 )n-S N N NN 20 N R2 to provide a resin-bound compound of Formula III: 22 (CH2)n- 0 N N N N R1 NR 2 b) reacting the compound of Formula III with an amine of Formula IV

HNR

3 R4 IV, to provide a resin-bound compound of Formula V

NR

3

R

4 N N N N N 5 ;1, N R2 and c) cleaving the resin-bound compound of Formula V from the resin to provide the substituted purine compounds of Formula I. [00701 In Formula I, R 1 and R 2 are independently selected from alkyl, substituted alkyl, cycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl and 10 heterocyclyl; R 3 is hydrogen; and R 4 is selected from alkyl, substituted alkyl, cycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl and heterocyclyl; or, R 3 and R 4 when taken together with the nitrogen atom to which they are respectively attached form an optionally substituted saturated heterocyclic ring. [00711 In Formulae II and III, R 1 and R 2 are as defined above and n is 0 or 1. 15 In Formula IV, R 3 and R 4 are as defined above. In Formula V, R 1 , R 2 , R 3 and R4 are as defined above. [00721 The resin utilized to bind the compound of Formula II in step (a) refers to a polymeric resin, including, but not limited to, polystyrene, polypropylene, polyethylene glycol, polyacrylamide, cellulose and the like which has been chemically 20 23 WO 03/031405 PCT/US02/32679 modified by an amino group as is described in more detail below. The amino group is eventually incorporated into the purine compound as described in more detail below. [00731 Oxidation of the resin-bound compound of Formula II is generally carried out at room temperature utilizing a suitable oxidizing reagent and a solvent. Suitable 5 oxidizing agents include, but not limited to, perbenzoic acid, m-chloroperbenzoic acid, performic acid, peracetic acid, perphthalic acid, and the like. Preferably, the oxidizing agent is m-chloroperbenzoic acid. Suitable solvents include, but are not limited to, water, alcohol (e.g., methanol, ethanol, isopropyl alcohol, etc.), tetrahydrofuran, dioxane, dichloromethane, ethylene dichloride, chloroform, NN-dimethylformamide, N,N-dimethylacetamide, or any 10 other organic solvent which does not adversely affect the reaction. [00741 Preferably, the reaction is also carried out in the presence of a base to neutralize the oxidizing agent, thereby providing a buffered solution. Suitable bases include, but are not limited to, inorganic bases such as alkali metal hydroxides, e.g., sodium hydroxide, potassium hydroxide, etc., and alkaline earth metal hydroxides such as 15 magnesium hydroxide and calcium hydroxide. In a typical protocol, a solution comprising an oxidizing agent and a solvent is cooled down to about 0"C, followed by the addition of NaOH and the resin-bound compound of Formula II. The suspension is then shaken or mixed at room temperature, and the resultant resin-bound oxidized compound of Formula III is then washed with a solvent, such as methanol and dichloromethane, and dried under 20 vacuum. [00751 The amine employed in step (b) to displace the sulfone group of the compound of Formula III includes, but is not limited to, primary amines, cyclic secondary amines (such as piperazines) and electron-rich anilines (see, e.g., Table 1-"C6-substituent" column). The reaction is conveniently effected by suspending the compound of Formula III 25 in an inert solvent, such as 1,4-dioxane, followed by the addition of the amine. The resin bound compound is then washed in a solvent such as methanol and dichloromethane, and dried under vacuum to provide the resin-bound compound of Formula V. [0076] Cleavage of the resin-bound compound of Formula V and liberation of the desired compound of Formula I from the resin as described in step (c) of the method is 30 typically carried in the presence of an acid. Suitable acids include, but are not limited to, an organic acid such as formic acid, acetic acid, propionic acid, trichloroacetic acid, 24 WO 03/031405 PCT/US02/32679 trifluoroacetic acid and the like, and inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, hydrogen chloride, etc., or the like. The reaction is usually carried out in a solvent such as water, an alcohol such as methanol, ethanol, 1,4, dioxane, methylene chloride, tetrahydrofuran, a mixture thereof or any other solvent which does not adversely 5 influence the reaction. [0077] Once the desired purine compound is freed from the resin by cleavage, it can be isolated by simply removing the solvent, e.g., by filtration. [00781 In a preferred embodiment, the compound is a compound of Formula II, and can be prepared using a method comprising: 10 [0079] a) alkylating a compound of Formula VI: 0-(cH2)n--S N N N X N H to provide a compound of Formula VII: 0-(cH2)n-S N \N VII N~N '7 X N N

R

2 and [0080] b) capturing the compound of Formula VII with a resin-bound amine 15 to provide the resin-bound compound of Formula II. [0081] In the above method, X, in Formula VI, is fluoro, chloro or bromo, and n is as defined above. In Formula VII, R 2 , X and n are as defined above. [0082] Alkylation of the N9 position of the compound of Formula VI as described in step (a) of preparing the compound of Formula II can be effected under known 20 conditions, e.g., Mitsunobu conditions as described, e.g., in Tsunoda et al., Tetrahedron Letters, 34, 1639 (1993). In a typical Mitsunobu reaction, the compound of Formula VI is 25 WO 03/031405 PCT/US02/32679 mixed with an alcohol, e.g., a primary, secondary or tertiary alcohol, in approximately equimolar amounts or with an excess of either compound, in an inert solvent, such as tetrahydrofuran, chloroform or dichloromethane. A slight molar excess of an azodicarboxylate, e.g., diisopropyl azodicarboxylate, and a phosphine, e.g., 5 triphenylphosphine or tributylphosphine, are added, and the reaction is stirred at a temperature, e.g., room temperature, for a sufficient amount of time to obtain the compound of Formula VII. [0083] Alternatively, the compound of Formula II can be prepared using a method comprising: 10 a) capturing a compound of Formula VI 0-(CH2)n-S / \ N X N HN with a resin-bound amine to provide a resin-bound compound of Formula VIII: (CH2)n-S N/ N N and b) alkylating the resin-bound compound of Formula VIII to provide the 15 resin-bound compound of Formula II. [0084] In Formula VI, X and n are as defined above. In Formula VII, R 1 and n are as defined above. [0085] In order to adapt the methods set forth above so that they are useful in a combinatorial scheme, the C6 position can be substituted following oxidation of the 20 thioether to the sulfone, as demonstrated in the synthesis of 2,4-diaminopyridine (Gayo et al., Tetrahedron Lett., 38, 211 (1997)). The 2-fluoro-6-thiophenylpurine can be prepared by 26 WO 03/031405 PCT/US02/32679 reacting excess thiophenol with 2-fluoro-6-chloropurine in methanol at 0 *C and purified by recrystallization. Scheme 3. Sulfenyl group protecting purine C6 position R R F N a N R=

H

3 CO H Ys a. 2 equiv of 4-methoxybenzylamine, 3 equiv of DIEA, BuOH, 80 *C 5 [0086] It has previously been demonstrated that the N9 position can be alkylated on a solid support under Mitsunobu conditions with a variety of alcohols. However, performing the N9-modification reaction on a solid support had several drawbacks including incomplete alkylation with secondary alcohols, consumption of large excesses of reagent, and inconvenient handling of reaction in a 96-well format. To circumvent this 10 problem, a method has now been developed, whereby a resin-bound amine is used to capture a C6-phenylsulfenyl-N9-alkylpurine directly from the crude reaction mixture. This allows the moisture sensitive Mitsunobu reaction to be performed as the first combinatorial step in solution making the overall scheme more convergent. [0087] To achieve a "traceless" linkage to solid support, primary amines are 15 coupled by reductive amination using, e.g., sodium triacetoxyborohydride to a 4-formyl-3,5 dimethoxyphenoxymethyl functionalized polystyrene resin (PAL). (Albericio et. al., J. Org. Chem., 55, 3730 (1990); Boojamra et. al., J. Org. Chem., 62, 1240 (1997); Jin et. al., J. Comb. Chem., 3, 97 (2001).) The purine ring can then, for example, be captured at the C2 position by reacting the PAL-amine resin with 1.5 equivalents of the crude N9-alkylated 2 20 fluoro-6-phenylsulfenylpurine and 3 equivalents of di-isopropylethylamine in n-butanol at 80 "C. The C6 position can then be substituted following oxidation-activation of the thioether to the sulfone (see, Scheme 2). The use of m-chloroperbenzoic acid to oxidize the thioether linkage can result in the premature cleavage from solid support presumably as a result of acid and oxidant sensitivity of the benzylic PAL-amine linkage. This problem can 25 be overcome, for example, by performing the oxidation in buffered solution with m 27 WO 03/031405 PCT/US02/32679 chloroperbenzoic acid that had been neutralized with a stoichiometric amount of sodium hydroxide. [00881 This combinatorial scheme of the present invention has the following merits: (1) the difficulty of C2 substitution is overcome by "directing" the first substitution 5 to C2 by using phenylsulfenyl group as a "protective" group at the C6 position; (2) N9 modification is accomplished in solution and purified by resin-capture; (3) the PAL linker allows traceless cleavage; and (4) construction of focused C6 purine libraries can readily be accomplished because this is the last step in the combinatorial synthesis scheme. As such, the method of the present invention has numerous advantages over any previously used 10 method. Scheme 4. Traceless combinatorial approach toward 2,6,9-trisubstituted purine library

-OCH

3 a -OCH 3 O CHO a Ho -R 1

OCH

3

OCH

3 1 Zt S ' S S= Fb c d F-NI -NN N N N N 1' 2H 3 R2 R5 R2 R1 5 Crude Reaction Mixture HN'R3 HN'R3 N QI 1 HN N N 7 R6 R a. 5 equiv of R 1

-NH

2 , 3 equiv of NaBH(OAc) 3 , 1% HOAc, THF; b. 1.5 equiv of R 2 OH, 1.8 equiv of PPhs, 1.3 equiv of DiAD, THF, RT; c. 0.5 equiv of 1, 1.5 equiv DiEA, BuOH, 80 00; d. 10 equiv of m-CPBA/NaOH (1:1), 1,4-dioxane with 1 0%H 2 0; e. 2 equiv of R 3

-NH

2 , anhydrous dioxane, 80 *C; f. CH 2 Cl 2 :TFA:Me 2

S:H

2 0/45:45:5:5 15 [00891 The scope of the chemistry has been validated for a wide range of substituents. Most primary and secondary alcohols lacking additional acidic hydrogens work well in the Mitsunobu reaction at N9 (see, Table 1). As the Mitsunobu reaction is performed on the C6-phenylsulfenylpurine, there is very little detectable contamination from 28 WO 03/031405 PCT/US02/32679 the N7 regioisomer. Alkylation can also be performed with active alkylbromides (such as t butyl-bromoacetate) or N9 can be temporally protected with, for example, the THP group to provide additional diversity. C2 substituents are preferably limited to primary amines (e.g., Table 1) due to the need for a reaction site to attach the purine to solid support. In some 5 embodiments, it has been found that immobilized anilines only result in partial capture of the purine from solution. Because m-chloroperbenzoic acid is used in preferred embodiments to convert the C6 thioether to a sulfone in the final activation step, substituents having functional groups prone to oxidation cannot be readily used in the first two derivatization steps. C6 displacement of the sulfone works for primary amines, cyclic secondary amines 10 (such as piperazines) and electron-rich anilines (see, Table 1). 29 WO 03/031405 PCT/US02/32679 Table 1. N-9 substituent (R 2 ) introduced by alkylation; C-2 substituent (R 1 ) introduced through resin-bound amines; C-6 substituent (R 3 ) introduced by displacement with amines N9 substituent (R 2 ) C2 substituent (R 1 ) C6 substituent (R 3 ) N N-NCI N N F -- H N N HN N OH H HNN N -'N -N N -NH

CH

3 --N OCH 3 HO 0 N N -N N O H \ 'b 0 N 0 ~ -NH - N a 0 H NBoc H N H" '-"NN 0 H 0 b H 0 ,N HHV c H O bN N OH -NH a

OCH

3 Except c, which was made by reacting purine with 3 equiv of DHP, cat. PPTS, and b, which was made by alkylation with 3 equiv of t-bytyl-bromoacetate all other substituents (R 2 ) were introduced by Mitsunobu alkylation. It should be noted that after final TFA cleavage, a, b and c gave arise to corresponding deprected forms. 30 WO 03/031405 PCT/US02/32679 [0090] In summary, the methods of the present invention provide a concise and traceless linker strategy that is extremely useful for preparing 2,6,9-trisubstituted combinatorial purine libraries. Resin-bound amines are used to capture a C6 phenylmecapto-9-alkylated purine directly from the crude Mitsunobu alkylation reaction 5 mixture. The C6 position is then substituted following oxidation-activation of the thioether to the sulfone. This strategy overcomes the difficulty of C2 substitution by "directing" the first substitution to C2 by using a phenylsulfenyl group to protect the C6 position. A thousand compound combinatorial purine library has been synthesized using this approach in 96-well format. Detailed procedures, and results showing the validation of the methods for 10 synthesis of substituted purines having a wide variety of different substituents are shown in Example 1. [00911 It will be readily apparent that the foregoing discussions regarding oxidizing agents, solvents, bases, alkylating agents and resins as well as the foregoing discussions regarding the capturing step, the alkylating step, the cleaving step, the 15 purification step, etc. are fully applicable to all of the methods disclosed and claimed herein, including the preferred embodiments. Preparation of 2,9-disubstituted and 0 -aryl- and 06 -alkyl-substituted purines [00921 Screening purine libraries has resulted in the identification of diverse molecules that inhibit mitosis, alter cellular morphology, and induce apoptosis. The guanine 20 ring also serves as a key recognition determinant in biological systems and guanine derivatives have been developed as GTPase and phosphodiesterase inhibitors as well as antiviral agents. This aspect of the invention provides diverse solid phase approaches useful for the synthesis of combinatorial guanine and other purine libraries.

R

2 'N' R 2 ' 0 N R R3~ N 'N 'N'N:)NI %>

R

1

R

3 f Purine Scaffold Guanine Scaffold 31 WO 03/031405 PCT/US02/32679 [0093] Initially, the synthesize 2,9-disubstituted guanines was attempted by Mitsunobu alkylation of 2-bromohypoxanthine in solution with an alcohol followed by nucleophilic modification of C2 by a resin-bound amine (see, Scheme 5)

OCH

3 OCH, O CHO - 0 N-R 1

OCH

3

OCH

3 1 O(P) O(P) O(P) 0 HN b BH c H d HN N Br-'N 1 N Br N N ~N N N N~ H R R 2 (P) = suitable protecting group a. R 1

-NH

2 , NaBH(OAc) 3 , 1% HOAc, THF; b. R 2 0H, PPh 3 , DiAD, THF; c.1, DiEA, BuOH, 80 *C; d. CH 2

C[

2 :TFA:Me 2

S:H

2 0/45:45:5:5 5 Scheme 5. 2,9-Disubstituted guanines from protected 2-bromohypoxanthine [0094] Unfortunately, alkylation under Mitsunobu conditions resulted in a mixture of the N9/C6 regioisomers presumably resulting from the presence of the C6 phenol tautomer. Instead of exploring approaches that involve cumbersome protecting group manipulation at C6 and N9 positions, reaction conditions were determined that would 10 discriminate between these positions. This was accomplished, for example, by capturing an N9-alkylated 2,6-dichloropurine onto Wang resin, which serves as both linker and a protective group for phenol, followed by functionalization of C2 by amination or palladium catalyzed cross-coupling chemistry (see, Scheme 6). Modification of N9 was achieved by regioselective alkylation of 2,6-dichloropurine under Mitsunobu conditions as previously 15 reported. The purified N9-alkylpurine could be captured onto Wang resin by treatment with 1.2 equivalents of potassium t-butoxide in THF for 8 hours. The quantitative C2 amination was accomplished with non-hindered primary and secondary amines at 100 *C in DMSO for 12 hours. In order to derivatize the C2 position with anilines, palladium-catalyzed cross coupling conditions were required to drive the reaction to completion. The typical 20 conditions involved reaction of the 2-chloropurine substrate with 5 equivalents of aniline in the presence of Pd 2 (dba) 3 (7 mol%), carbene ligand 1 (14 mol%), and 6 equivalents of KO'Bu in anhydrous 1,4-dioxane under argon. The reactions are typically complete after stirring at 80 'C for 12 hours. Under similar palladium catalyzed coupling condition, the 2 chloro can be reacted with arylboronic acids to form direct C-C bond connections and with 32 phenols to form C-O connections. Although the reaction time and the amount of coupling reagents used could be optimized for each type of reaction and substrate, the general coupling protocol described above was found to be most general for achieving quantitative conversion of the starting material (the chloro-group at the C2 position of purine) with 5 different substrates on the solid support. This approach is ideally suited for focusing diversity at the C2 position of guanine. With R 1 fixed as isopropyl (which can be readily modified with a variety of alcohols by Mitsunobu alkylation), a variety of substituents (R 2 ) have been introduced into the C2 position (see, Figure 1). ci ci O >N b N1 N / -o ci N N c N b C1 N N H R, R1 carbene igand phosphine ligand 0 0 0 0 HN j N,> ~ HN!),N, H N N>N N> R2, R2 3 R2- N N N-N NN R R 2 -1 l a. R 1 0H, PPh 3 , DiAD, THF; b. Wang resin, KOtBu, THF, 0 *C; c. 5 equiv of R 2

R

3 NH, 7.5 equiv of DiEA, DMSO, 100 *C; d. 5 equiv of anilines, 7% of Pd 2 (dba) 3 , 14% of carbene ligand, 6 equiv of KOtBu, dioxanes, 80 *C; e. 5 equiv of boronic acids, 7% of Pd 2 (dba) 3 , 14% of carbene ligand, 6 equiv of Cs2CO3, dioxanes, 80 *C;f. 5 equiv of phenols, 7% of Pd 2 (dba) 3 , 28% of phosphine ligand, 7 equiv of K 3

PO

4 , toluene, 80 *C; g. CH 2 Cl 2 :TFA:Me 2

S:H

2 0/45:45:5:5 10 Scheme 6 [00951 In addition to the foregoing method, which is described in international patent application WO 03/031406, U.S. Provisional Patent Application No. 60/328,763, filed October 12, 2001, U.S. Provisional Patent Application No. 60/331,835, filed November 20, 2001, U.S. Provisional Patent Application No. 60/346,480, filed January 15 7, 2002, U.S. Provisional Patent Application No. 60/348,089, filed January 10, 2002, and U.S. Patent Application 2003/0191312, filed on October 12, 2002, the teaching of all of which are incorporated herein by reference, the present invention provides an alternative method in which a 6-phenylmercaptopurine is alkylated at N9, followed by capture at C2 with a resin 20 33 WO 03/031405 PCT/US02/32679 bound amine and subsequently converted to the guanine derivative by oxidation of the sulfenyl group to sulfonyl group and its subsequent hydrolysis (Scheme 7). This scheme has also been developed to prepare 2,6,9-trisubstituted purines.

OCH

3

OCH

3 O CHO O

OHN-R

1

OCH

3

OCH

3 Oos N b N \ cN FN F N N N N H R 2 R1 R2 crude reaction mixture d O 1) N f d N' HN N NN d N N R1 k2 R2RR 2 2 3 2 a. R 1

-NH

2 , NaBH(OAc) 3 , 1% HOAc, THF; b. 1.5 equiv of R 2 OH, 2 equiv of PPh 3 , 1.3 equiv of DiAD, THF, RT; c. 0.5 equiv of 1, 1.5 equiv DiEA, BuOH, 80 *C; d. 10 equiv of m-CPBA/NaOH (1:1), 1,4-dioxane with 10%H 2 0; e. 5 equiv of NaOH, 1,4-dioxane with I0%H 2 0, 80 *C; f. CH 2

CI

2 :TFA:Me 2

S:H

2 0/45:45:5:5 5 Scheme 7 [00961 The Wang resin capture strategy allows focused diversity at the C2 position with versatile reactions such as palladium catalyzed cross-coupling reactions, but requires purification of the Mitsunobu product in the first step and high temperatures for chloride displacement conditions. The second approach offers the opportunity of focusing 10 diversity at the N9 position without the need for purification of the Mitsunobu product and effects the C2 functionalization under mild conditions, but limits the C2 substituents to primary amines. [0097] In another aspect, the present invention provides methods to synthesize 0 6 -aryl- and 0 6 -alkyl- purines, which are closely related to guanine analogs and 15 have exhibited interesting biological activities. For example, 06-cyclohexylmethylguanine has been shown to be a competitive inhibitor of cyclin-dependent kinase 1 and 2 (CDK1 and 2) and exhibits an unique binding mode to the ATP-binding site (see, Arris et. al., J. Med. 34 WO 03/031405 PCT/US02/32679 Chem., 43, 2797 (2000)). Resin-bound guanine 3 (from scheme 7) offers direct access to 06 alkyl- purines by Mitsunobu alkylation. Alternatively, resin-bound 6-benzenesolfonylpurine 2 could serve as a versatile intermediate to 0 6 -aryl-, 0 6 -alkyl- purines through DABCO/DBU mediated SNAr displacement reaction (Scheme 8). As shown in Table 2, a 5 variety of phenols were validated using the methods of the present invention and all of them gave satisfactory results. O O'R3 O'R 3 H N N N, a.. N >N N N b HR3=Alkyl Group 4a N NN N HN N N ,

R

2 R1 R 2 3 0 S=OO'R3 O-Ra N N N > R3=Aryl Group 4b > 1 R2 R1 R2 R1 R2 2 a. R 3 0H, PPh 3 , DiAD, THF; b. CH 2

CI

2 :TFA:Me 2

S:H

2 0/45:45:5:5 c. DABCO, DBU, R 3 -OH, molecular sieves, 1,2-DME, 60*C Scheme 8 [00981 When alcohols were used as substrates for displacing the 10 benzenesulfonyl group (resin 2), only the guanine analog was obtained presumably as a result of hydrolysis. To circumvent this problem, the use of a more active sulfonate leaving group was explored, which had been demonstrated displaced by alcohols successfully in solution phase. (Lakshman et. al., Org. Lett., 2, 927 (2000).) To adapt this chemistry to solid phase, 2-bromohypoxanthine was loaded on PAL-amine resin at C2 position without 15 protection of the N9. After activation of the C6 position by reaction with mesitylenesulfonyl chloride, a variety of phenols and alcohols can be coupled to the purine (Scheme 9), providing a more general route to 0 6 -aryl-, 0 6 -alkyl- purines (Table 2). 35 WO 03/031405 PCT/US02/32679 0 0 0 HN a HN b N Br- -N N N )-N IN N )IN N H H H Ic

O-R

2 O-R2 N d N HN N N ONNA N N' R1 H H a. 1, DIEA, BuOH, 80 *C; b. TsCI, DMAP; c. DABCO, DBU, R 2 -OH, molecular sieves, 1,2-DME, 60 *C; d. CH 2

CI

2 :TFA:Me 2

S:H

2 0/45:45:5:5 Scheme 9 [00991 In summary, the methods of the present invention provide general and versatile solid phase strategies for the synthesis of combinatorial guanine and 0 6 -aryl- and 5 0 6 -alkyl- purine libraries. 2,9-Disubstituted guanine analogs and 0 6 -aryl/alkyl purine analogs can be constructed from commercially available purine or hypoxanthine in a traceless fashion, thus allowing ease access to compound libraries. 36 Table 2. Entry C6 substituent (OR) Purity (%) Entry C6 substituent (OR) Purity (%) 83 O K 90 87 O 89 O OCF 3 86 O O"C 6

H

13 82 O OCH 3 86 O 87 00 H 3 4K OCH3 90 0 82 O F 83 OK 83 0 87 O CI 84 Resin-Capture-Release Method for Preparation of 2,6,9-substituted purines 101001 In another aspect, the present invention provides a resin-capture-release 5 strategy for making combinatorial 2,6,9-trisubstituted purine libraries by capturing N9 derivatized purines at the C6 position with a thio-modified polymer. The C2 halo (e.g., fluoro) group is subsequently substituted with primary and secondary amines, followed by thioether oxidation and release by C6 substitution with amines and anilines. This approach complements the strategy discussed above in which a 6-phenylsulfenylpurine scaffold was captured at the C2 position with a 10 resin-bound amine (see also, Sheng and Gray, US Provisional Patent Appl. No. 60/328,741, entitled "A Concise and Traceless Linker Strategy Towards Combinatorial Libraries of 2,6,9-substituted Purines," which was filed on October 12, 2001 and from which priority is claimed). The C2 capture approach, which is shown in Scheme 4, is ideally suited for the traceless synthesis of focused C6 purine libraries. However, it has two limitations for some 15 37 WO 03/031405 PCT/US02/32679 applications: (1) because the C2 amino substitutent is tethered to the solid support prior to being covalently attached to the scaffold, only primary amines can be installed at the C2 position; and (2) it is inefficient to prepare C2 focused libraries as this substitution reaction is the first combinatorial step. To address and overcome these limitations, the present 5 invention provides a complementary approach in which resin capture can be performed at the C6 position, resulting in a resin-bound purine that can be derivatized in a final step at C2. [0101] It is known that the 2-sulfonyl group of 4-aminosubstituted pyrimidines can be displaced by various amines (see, Gayo et aL, Tetrahedron Lett., 38, 211 (1997)). Because 2,4-dichloropyrimidine has similar amination reactivity as 2,6 10 dichloropurines under basic conditions (the 4 position of pyrimidine and the 6 position of purines undergo selective nucleophilic substitution first; complete substitution of the 2 position afterward requires elevated temperature and high concentrations of amine nucleophile), the same can be true for the purine system. Accordingly, the synthetic approach shown in scheme 10 was devised. 15 Scheme 10. C2 sulfone group cannot be displaced by amines. 0 0 CI Ci HN NH N b N N c N Br~< N aS-)N N ~ S N N N H H H R 2 3 Crude reaction mixture d HN' R3 0 N'R3 N'R2 N'R2

R

4

R

4 > .. N N4 N N N N /S N NS N N R3 R2 R h 2 O%1R 0 10 R, R a). 2 equiv of thiophenol, 3 equiv of DIEA, MeOH, 80 *C; b). 6 equiv of POCl 3 , 2 equiv of Bu 4 NCI, 1 equiv of N,N-dimethylaniline, anhydrous CH 3 CN, reflux; c). 1.5 equiv of R 1 0H, 1.8 equiv of PPh 3 , 1.3 equiv of DiAD, THF, RT; d). 0.5 equiv of 1, BuOH, 1 equiv of D!EA, 80 0C; e). 10 equiv of m-CPBA/NaOH (1:1), dioxane; f). 5 equiv of R 3

R

4 NH, 1,4-dioxane, 80 "C; g). CH 2 Cl 2 :TFA:Me 2

S:H

2 0/45:45:5:5 [0102] The starting scaffold, 2-thiophenyl-6-chloropurine, can be obtained by thiophenol displacement of commercially available 2-bromohypoxanthine followed by chlorination with POCl 3 in 90% overall yield. Mitsunobu reaction can first be used to carry 38 WO 03/031405 PCT/US02/32679 out alkylation reactions at the N9 position. Resin-bound amine 1, which is obtained by reductive amination of a 4-formyl-3,5-dimethoxyphenoxymethyl functionalized polystyrene resin (PAL), is used to capture the N9 alkylated purine from the crude Mitsunobu reaction mixture by nucleophilic substitution at C6. Unfortunately, C2 oxidation to the sulfone and 5 subsequent displacement with amines under a variety of conditions resulted in only the 2 phenylsulfonyl-6-aminosubstituted purine product, consistent with the observation that nucleophilic displacement of C2 on a C6 amino-substituted purine is difficult. [0103] To avoid substitution at the C6 position with an amine prior to derivatization of the C2 position, the present invention provides a method in which the 10 purine is first linked to solid support at the C6 via a thioether. The thioether linked purine is obtained by resin-capture of the crude N9 Mitsunobu alkylation product at C6 using a methylmercapto resin. The C2 position is subsequently derivatized by a nucleophilic substitution reaction with amines. The C6 substituent is then introduced by displacement of the sulfonyl group with amines after oxidative activation of thioether linkage and the final 15 product is released into the reaction solution (Scheme 11). This approach offers, inter alia, the following advantages: (1) secondary amines can be introduced at the C2 position; (2) only the activated polymer-bound purine intermediate can be released; (3) the activated sulfone linker allows traceless cleavage; and (4) construction of focused C2 purine libraries can readily be accomplished by coupling different amines at the C2, and using a single 20 amine for the final displacement. As such, this method of the present invention provides an advantage relative to scheme 4 due to the technical difficulty in preparing Pal-amine resins by reductive amination in an array format. [0104] This method of the present invention has been validated for a broad range of substituents on the purine ring. Most primary and secondary alcohols lacking 25 additional acidic hydrogens work well in the N9 Mitsunobu reaction (as described herein). Quantitative alkylation of the starting purine can be achieved by using excess alkylating reagent (1.5 equivalents of alcohol, 1.6 equivalents of diisopropyl azodicarboxylate and 2.0 equivalents of triphenylphosphine). The Mitsunobu alkylation step can also be performed on solid support after the resin-capture of 2-fluoro-6-chloropurine. This procedure resulted in 30 very small quantities of the minor N7 Mitsunobu regioisomer, but required considerably more alkylating reagents. 39 WO 03/031405 PCT/US02/32679 CI CI S? S? N a F N N N cN F-'-N N F-- F'-N NR3 NN 4 R1 5 R1 R2 6 R1 Crude reaction mixture d R N , R2N R3N

R

3

R

2 R2 R 1 8 7 a).1.5 equiv of ROH, 1.8 equiv of PPh 3 , 1.3 equiv of DiAD, THF, RT; b). 0.5 equiv of mercaptomethy polystyrene resin, BuOH, 1 equiv of DiEA, 80 *C. c). 3 equiv of R2R 3 NH, 4 equiv of DiEA, BuOH, 80 C. d). 10 equiv of m-CPBA/NaOH (1:1), dioxane. e). 0.9 equiv of R 4

R

5 NH, anhydrous 1,4-dioxane, 80 *C. Scheme 11. Resin-capture-release strategy towards combinatorial libraries of 2,6,9 trisubstituted purines [0105] The C2 position can be substituted with variety of primary amines, 5 such as.sterically hindered 2-amino-3-methyl-butanol, and cyclic/acyclic secondary amines (see, Figure 2). Normally five equivalents of amines are used at a concentration of 2M in butanol to ensure quantitative substitution of 2-chloro-purine. Finally, the C6 displacement of the sulfonyl group can be carried out with diverse primary and secondary amines and electron-rich anilines (see, Figure 3). Rather than use excess amine to quantitatively release 10 resin-bound purine, which requires follow-up purification by solid supported liquid extraction (SLE) (Johnson et al., Tetrahedron Lett. 54: 4097 (1998)), a limited amount of amine (0.8 equivalent) can be used. This procedure essentially gave the pure purine product without the need of SLE purification. It should be noted that the final derivatization step should preferably be carried under anhydrous conditions, since water can also react with the 15 sulfonyl purine to release guanine as a by-product. [0106] Detailed reaction conditions useful in these methods are described in Example 2. [0107] In summary, this aspect of the invention provides an alternative approach or method for making combinatorial 2,6,9-trisubstituted purine libraries by 20 capturing an N9 substituted 2-fluoro-6-chloropurine at the C6 position via a thioether linkage and subsequently modifying the polymer-bound purine intermediate at the C2 position, followed by substitution at the C6 position with concomitant release. 40 WO 03/031405 PCT/US02/32679 Pharmaceutical Formulations [0108] In another preferred embodiment, the present invention provides a pharmaceutical formulation comprising a compound of the invention and a pharmaceutically acceptable carrier. 5 [0109] The compounds described herein, or pharmaceutically acceptable addition salts or hydrates thereof, can be delivered to a patient using a wide variety of routes or modes of administration. Suitable routes of administration include, but are not limited to, inhalation, transdermal, oral, rectal, transmucosal, intestinal and parenteral administration, including intramuscular, subcutaneous and intravenous injections. 10 [0110] The compounds described herein, or pharmaceutically acceptable salts and/or hydrates thereof, may be administered singly, in combination with other compounds of the invention, and/or in cocktails combined with other therapeutic agents. Of course, the choice of therapeutic agents that can be co-administered with the compounds of the invention will depend, in part, on the condition being treated. 15 [0111] For example, when administered to a patient undergoing cancer treatment, the compounds may be administered in cocktails containing anti-cancer agents and/or supplementary potentiating agents. The compounds may also be administered in cocktails containing agents that treat the side-effects of radiation therapy, such as anti-emetics, radiation protectants, etc. 20 [0112] Supplementary potentiating agents that can be co-administered with the compounds of the invention include, e.g., tricyclic anti-depressant drugs (e.g., imipramine, desipramine, amitriptyline, clomipramine, trimipramine, doxepin, nortriptyline, protriptyline, amoxapine and maprotiline); non-tricyclic and anti-depressant drugs (e.g., sertraline, trazodone and citalopram); Ca 2 antagonists (e.g., verapamil, nifedipine, 25 nitrendipine and caroverine); amphotericin; triparanol analogues (e.g., tamoxifen); antiarrhythmic drugs (e.g., quinidine); antihypertensive drugs (e.g., reserpine); thiol depleters (e.g., buthionine and sulfoximine); and calcium leucovorin. [0113] The active compound(s) of the invention are administered per se or in the form of a pharmaceutical composition wherein the active compound(s) is in admixture 30 with one or more pharmaceutically acceptable carriers, excipients or diluents. Pharmaceutical compositions for use in accordance with the present invention are typically formulated in a conventional manner using one or more physiologically acceptable carriers 41 WO 03/031405 PCT/US02/32679 comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen. [0114] For injection, the agents of the invention may be formulated in 5 aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiological saline buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. [0115] For oral administration, the compounds can be formulated readily by 10 combining the active compound(s) with pharmaceutically acceptable carriers well known in the art. Such carriers enable the compounds of the invention to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated. Pharmaceutical preparations for oral use can be obtained solid excipient, optionally grinding a resulting mixture, and processing the mixture of 15 granules, after adding suitable auxiliaries, if desired. to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxyniethylcellulose, and/or 20 polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. [0116] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, 25 polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses. [01171 Pharmaceutical preparations, which can be used orally, include 30 push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or 42 WO 03/031405 PCT/US02/32679 lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for such administration. 5 [0118] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner. [01191 For administration by inhalation, the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, e.g., 10 dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In the case of a pressurized aerosol the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of e.g., gelatin for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch. 15 [0120] The compounds may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multi-dose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, 20 stabilizing and/or dispersing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. [01211 Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable 25 lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents, which increase the solubility of the compounds to allow for the 30 preparation of highly, concentrated solutions. [01221 Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen-free water, before use. 43 WO 03/031405 PCT/US02/32679 [0123] The compounds may also be formulated in rectal compositions such as suppositories or retention enemas, e.g., containing conventional suppository bases such as cocoa butter or other glycerides. [0124] In addition to the formulations described previously, the compounds 5 may also be formulated as a depot preparation. Such long acting formulations may be administered by implantation or transcutaneous delivery (e.g., subcutaneously or intramuscularly), intramuscular injection or a transdermal patch. Thus, for example, the compounds may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for 10 example, as a sparingly soluble salt. [0125] The pharmaceutical compositions also may comprise suitable solid or gel phase carriers or excipients. Examples of such carriers or excipients include but are not limited to calcium carbonate, calcium phosphate, various sugars, starches, cellulose derivatives, gelatin, and polymers such as polyethylene glycols. 15 EXAMPLES [0126] The following examples are offered to illustrate, but not to limit the present invention. General [01271 N9 Mitsunobu alkylation reactions and C6 sulfone displacement 20 reactions are carried out under anhydrous conditions under argon atmosphere. C2 resin capture reactions are carried out in 4 mL scintillation glass vials unless otherwise noted. Anhydrous tetrahydrofuran, and 1,4-dioxane are obtained by passing them through commercially available alumina columns. All other reagents, resins, and solvents are purchased at highest commercial quality and used without further purification. Purity of 25 compounds are assessed by reverse-phase liquid chromatography / mass spectrometer (Agilent Series 1100 LC-MS; elution method: starting from 5% to 95% acetonitrile in water with 0.5% acetic acid in 8 minutes and finishing at 10 minute with 95% acetonitrile using a Phenomenex Luna 50*2.00mm 5pt C18 column) with an UV detector at k = 255 nm (reference at 360 nm) and an API-ES ionization source. NMR spectra are recorded on 30 Bruker-400MHz instrument and calibrated using residual undeuterated solvent as an internal 44 WO 03/031405 PCT/US02/32679 reference. The following abbreviations are used to designate the multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet. EXAMPLE 1 General Procedure for the Combinatorial Synthesis of 2,6,9-Trisubstituted Purines 5 Reductive Amination for Synthesis of PAL-Resin-Bound Amine (1) [0128] To a suspension of 4-formyl-3,5-dimethoxyphenoxymethyl functionalized polystyrene resin (PAL) (10.0 g, 11.3 mmol) in DMF (350 mL) is added a primary amine (56.5 mmol), followed by addition of sodium triacetoxyborohydride (7.18 g, 33.9 mmol) and acetic acid (6.52 mL, 113 mmol). The mixture is shaken gently at room 10 temperature for 12 hours and then washed with methanol (300 mL x 4) and dichloromethane (300 mL x 4) and dried under vacuum. The complete conversion of PAL aldehyde to resin bound amine is confirmed by disappearance of the aldehyde stretch. 2-Fluoro-6-phenylsulfenylpurine (2) [01291 To a solution of 2-fluoro-6-chloropurine (10.0 g, 57.9 mmol) in 15 methanol (200 mL) is added diisopropylethylamine (25.2 mL, 144.7 mmol). The mixture is cooled to O "C and followed by slow addition of thiophenol (11.9 mL, 115.8 mmol) via an addition funnel over one hour. The reaction is stirred at 0 *C for 12 hours. The solvent is then removed under reduced pressure and the solid is collected by filtration and washed twice with hexanes. The collected solid is further purified by re-crystallization from methanol to 20 afford desired product (11.8 g, 83% yield). 1 H NMR (400 MHz, (CD 3

)

2 SO) 8 7.54 (in, 3H), 7.66(m, 2H), 8.49(s, 1H); MS CnH 7

FN

4 S [MWf] 246.04, found: 247.05 2-Fluoro-6-phenylsulfenyl-9-alkylpurine (3) [0130] To a flame-dried round bottom flask (500 mL) was added 2-fluoro-6 phenylsulfenylpurine (10.0 g, 40.6 mmol), triphenylphosphine (19.2 g, 73.1 mmol) and 25 alcohol (52.8 mmol), followed by dissolving them in THF (anhydrous, 350 mL). The solution was cooled down to -30 "C and diisopropyl azodicarboxylate (12.0 mL, 60.9mmol) was added dropwise. The reaction was allowed to warm up to room temperature and stirred under argon. After overnight, the solvent was removed under reduced pressure and the crude material was directly used in the next step without further purification. 45 WO 03/031405 PCT/US02/32679 Resin Capture of 3 at C2 From Crude Mitsunobu Reaction (4) [0131] To a solution of crude 2-fluoro-6-phenylsulfenyl-9-alkylpurine (0.15 mmol) in n-butanol (1.0 mL) is added PAL-resin-bound amine 1 (0.10 mmol), followed by addition of diisopropylethylamine (0.30 mmol). The suspension is heated to 80 *C under 5 argon. After 12 hours, the resin is washed with methanol (3 mL x 4) and dichloromethane (3 mL x 4) and dried under vacuum. The complete conversion of secondary amine (PAL amine) to tertiary amine is confirmed using the bromophenol blue test. ((a) KrchAk et. al., Int. J. Petp. Protein Res., 32, 415-416 (1988); (b) Krchfkk et. al., Collect. Czech. Chem. Commun., 53, 2542-2548 (1988).) 10 Activation of C6 by Oxidation of Thioether to Sulfone (5) [01321 To a solution of m-CPBA (0.23 g, 75%, 1.0 mmol) in 1,4-dioxane (9 mL) cooled to 0 *C is added NaOH (1 mL, 1M, 1.0 mmol) aqueous solution, followed by addition of resin 4 (0.10 mmol). The suspension is shaken gently at room temperature. After 8 hours the resin is washed with methanol (3 mL x 4) and dichloromethane (3 mL x 4) and 15 dried under vacuum. C6 Displacement with Amines (6) and Product Cleavage (7) [01331 The resin 5 (0.05 mmol) is suspended in anhydrous 1,4-dioxane (0.6 mL), followed by addition of an amine (0.1 mmol). After overnight shaking at 80 "C, the resin is washed with methanol (1 mL x 4) and dichloromethane (1 mL x 4) and dried under 20 vacuum to afford resin 6. Resin 6 is subsequently cleaved using

CH

2 Cl 2 :TFA:Me 2

S:H

2 0/45:45:5:5/v:v:v:v (0.5 mL) to afford desired product 7 (in average >85% HPLC purity, 80% purified yield). Validation of Methods Using Various Substituents [01341 The following Tables 3 through 5 provide retention times, and 25 calculated and observed molecular weights for compounds having various substituents at the N9, C2, and C6 positions of purines. These results demonstrate that the methods of the invention are applicable for a wide variety of different substituents. 46 Table 3. Validation of N9 substituents. a Mitsunobu alkylation with t-Butyl N-(2 hydroxyethyl)-carbamate. b Alkylation with t-Butyl-bromoacetate (2 equiv) in the presence of K 2

CO

3 (3 equiv) in DMF at RT. ' N9 protected with THP. a,bc For these three entries, the protecting groups were all removed during final TFA cleavage. 1 16 minutes elution method 5 was used, except for entry 9 which 10 minutes elution method was used. NH N \ > N N N H H2

H

3 CO Retention calculated Observed HPLC Isolated Entry N9 substituent (R 2 ) Time (min)' [MI] [MH*] purity (%) yield (%) O 1 4.79 482.25 483.3 91 83 2 3.96 485.29 486.3 84 76 3 / -7.09 514.25 515.2 86 81 4 -CH 3 4.88 388.20 389.2 93 86 5 / 6.31 464.23 465.2 90 85 6 -N O 3.92 487.27 488.3 85 80 7N a 296 417.23 418.2 86 79 8 oHOb 3.77 43219 433.2 89 77 9 -H 4.65 374.19 375.2 86 79 47 WO 03/031405 PCT/US02/32679 Table 4. Validation of C2 substituents. 2 10 minutes elution method was used. N

R

1 N N Retention Calculated Observed HPLC Isolated Entry C2 substituent (R 1 ) Time (min) 2 [M] [MH*] purity (%) yield (%) 1 -N / C 4.87 386.16 387.2 92 85 2 HN 5.62 344.23 345.2 88 78 3 HN -H 2.93 306.18 307.20 87 80 4 -NOCH 3 4.42 382.21 383.3 93 84

FNH

5 3.33 387.24 388.3 90 82 H 0 6 N 5.07 416.23 417.2 83 77 H 4.38 396.19 397.2 91 85 7 yN 0~o H 8 N NH 4.12 405.23 406.3 86 80 H 9 OH 3.63 348.23 349.3 85 80 48 WO 03/031405 PCT/US02/32679 Table 5. Validation of C6 substituents. ' 10 minutes elution method was used. R3 N N N 1-,N N H Retention Calculated Observed HPLC Isolated Entry C6 substituent (R 3 ) Time (min) 3 [M] [MH*] Purity (%) yield (%) 1 --N N F 5.11 459.25 460.3 91 85 2 -NH -N 2 / N3.52 387.22 388.2 86 79 H 3 N -N-N, N 3.01 404.24 405.3 85 80 -- NH 4 HO 4.27 428.23 429.2 86 80 0 5 -N 'N o 3.86 451.27 452.3 90 83 6 HN 7.05 372.21 373.2 83 75 H 7 N? 3.70 421.26 422.3 89 81 0 H 8 N 3.44 423.31 424.3 88 82 / OCH 3 4.54 416.23 417.20 90 84 9 -NH4 49 WO 03/031405 PCT/US02/32679 HN NO N N H [0135] 1H NMR (400 MHz, CDC1 3 ) 5: 1.52 (d, 6H, 6.8 Hz), 3.59-3.63 (m, 2H), 3.84-3.87 (m, 2H), 4.57-4.65 (m, 1H), 5.51-5.53 (m, 1H), 7.03-7.07 (m, 1H), 7.27-7.33 (m, 2H), 7.59 (s, 1H), 7.75-7.77 (m, 2H), 8.16 (s, 1H); HRMS calc'd for [MH*] C 16

H

21

N

6 0 313.1777, 5 found: 313.1778. 0 HN N -~ N> H [0136] 1H NMR (400 MHz, CDC1 3 ) 5: 1.54 (d, 6H, 6.8 Hz), 1.94 (m, 4H), 2.39 (m, 2H), 2.94 (t, 2H, J=7.3 Hz), 3.41-3.49 (m, 8H), 3.66 (br, 2H), 4.67 (m, 1H, 6.8 Hz), 7.23 (m, 3H), 7.31 (m, 2H), 7.62 (s, 1H); MS (ES) calc'd for [MH+] C 23

H

32

N

7 0 422.27, found 422.30 HN N N \ > N N N 10 [0137] 1H NMR (400 MHz, CDC1 3 ) 3: 1.57 (d, 6H, J=6.8 Hz), 2.96 (t, 2H, J=7.6 Hz), 3.73 (m, 2H), 4.68 (m, 1H), 5.02 (br, 1H), 4.67 (m, 1H, J=6.8 Hz), 7.05 (m, 1H), 7.21-7.34 (m, 6H), 7.58 (s, 1H), 7.79 (m, 2H); MS (ES) calc'd for [MH*] C 2 2

H

25

N

6 373.21, found 373.20 50 WO 03/031405 PCT/US02/32679 -N N N cN 01" N N N H [01381 1H NMR (400 MHz, CDC1 3 ) 8: 1.55 (d, 6H, J=6.8 Hz), 2.90 (t, 2H, J=7.2 Hz), 3.65 (m, 2H), 4.65 (m, 1H, J=6.8 Hz), 4.81 (d, 2H, J=5.1 Hz), 4.87 (br, 1H), 6.06 (br, 1H), 7.05 (m, 1H), 7.21-7.31 (m, 6H), 7.50 (s, 1H), 7.69 (m, 1H), 8.50 (dd, 1H, J=1.3 Hz), 8.64 (d, 1H, 5 J=1.5 Hz); MS (ES) calc'd for [MH*] C 22

H

26

N

7 388.22, found 388.20 N N N N N N H [0139] 1H NMR (400 MHz, CDC1 3 ) 5: 1.57 (d, 6H, J=6.8 Hz), 2.97 (t, 2H, J=7.1 Hz), 3.66 (t, 2H, J=7.3Hz), 3.81 (t, 4H, J=4.8Hz), 4.25 (br, 4H), 4.75 (m, 1H, J=6.8 Hz), 7.28 (m, 3H), 7.34 (m, 2H), 7.56 (s, 1H); MS (ES) calc'd for [MH+] C 20

H

27

N

6 0 367.22, found 367.20 HN H O N H N N , N H 10 [0140] 1H NMR (400 MHz, CDC1 3 ) 6: 1.03 (d, 3H, J=6.8 Hz), 1.05 (d, 3H, J=6.8 Hz), 1.98 2.03 (m, 1H), 1.52 (d, 3H, J=6.8 Hz), 1.54 (d, 3H, J=6.8 Hz), 2.01 (m, 1H), 3.71-3.76 (m, 1H), 3.89-3.92 (m, 2H), 4.57 (sept., 1H, 6.8 Hz), 5.03 (br, 1H), 7.07 (t, 1H, J=8.8 Hz), 7.39 (br, IH), 7.57 (s, 1H), 7.89 (s, 1H), 8.02 (br, 1H); MS (ES) calc'd for C 19

H

27

N

6 0 [MH*]: 15 355.22, found 355.20 51 WO 03/031405 PCT/US02/32679 HN \/ OCH3 HO N HO,, N N N H [0141] 1H NMR (500 MHz, d6-DMSO) 5: d 0.85 (d, 6H, 7.1 Hz), 1.44 (d, 6H, 7.1 Hz), 1.93 (m, 1H), 3.31 (s, 2H), 3.46 (d, 2H, 5.3 Hz), 3.69 (s, 3H), 3.79 (m, 1H), 4.49 (m, 3H), 5.75 (s, 1H), 6.83 (d, 2H, J = 8.7), 7.27 (d, 2H, J = 8.7), 7.75 (s, 1H); HRMS calc'd for C 21

H

30

N

6 0 2 5 [MIH*] 399.2508, found: 399.2510. HN \/ OCH 3 O N N N' N N N H [0142] 1H NMR (400 MHz, CDCl 3 ) 6: 1.61 (d, 6H, J=6.8 Hz), 1.92 (m, 2H), 2.10 (m, 2H), 2.52 (t, 2H, J=6.3 Hz), 3.44-3.56 (m, 6H), 3.80 (s, 3H), 4.81 (b, 3H)), 6.88 (d, 2H, J=7.9 Hz), 7.32 (d, 2H), 8.08 (s, 1H); MS (ES) calc'd for [MJH*] C 23

H

32

N

7 0 2 438.26, found 438.30 HN N N> llz N N N

H

3 CO N 10 [0143] 1H NMR (400 MHz, CDCl 3 ) 6: 1.44 (m, 2H), 1.56 (m, 4H), 2.45 (m, 4H), 2.65 (t, 2H, J=13.5 Hz), 2.94 (t, 2H, J=7.1 Hz), 3.78 (s, 3H), 3.84 (m, 2H), 4.14 (t, 2H, J=13.6 Hz), 4.57 (d, 2H, J=5.8 Hz), 6.85 (d, 2H, J=8.7 Hz), 7.21-7.32 (m, 7H), 7.57 (s, 1H); MS (ES) calc'd for [MH*] C 28

H

36

N

7 0 486.30, found 486.30 52 WO 03/031405 PCT/US02/32679 EXAMPLE 2 A Resin-Capture-Release Strategy towards Combinatorial Libraries of 2,6,9 substituted Purines [0144] General. Purity of compounds were assessed by reverse-phase liquid 5 chromatography - mass spectrometer (Agilent Series 1100 LC-MS) with an UV detector at 2 = 255 nm (reference at 360 nm) and an API-ES ionization source. NMR spectra were recorded on Bruker-400MHz and 500MHz instrument and calibrated using residual undeuterated solvent as an internal reference. The following abbreviations were used to designate the multiplicities: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet. LC 10 elution methods (using a Phenomenex Luna 50*2.00mm 5p C18 column): (1) 10 minutes method: starting from 5% solvent A (acetonitrile) in solvent B (water with 0.5% acetic acid) and running the gradient to 95% A in 8 minutes, followed by 2 minutes elution with 95% A. (2) 6 minutes method: starting from 5% solvent A (acetonitrile) in solvent B (water with 0.5% acetic acid) and running the gradient to 95% A in 5 minutes, followed by 1 minutes 15 elution with 95% A. Representative Experimental Procedures: [0145] 2-Phenylsulfenylhypoxanthine (2). To a solution of 2 bromohypoxanthine (10.0 g, 46.5 mmol) in methanol (200 mL) was added thiophenol (9.55 mL, 93.0 mmol) and diisopropylethylamine (20.2 mL, 116.3 mmol). The reaction was stirred 20 at 90 *C for overnight. The solvent was removed under reduced pressure and the solid was collected by filtration and washed with hexanes (100 mL x 2). The collected solid was further purified by re-crystallization from methanol to afford desired product (10.1 g, 89% yield). 'H NMR (400 MHz, (CD 3

)

2 SO) 8 7.50(m, 3H), 7.62 (m, 2H), 7.97 (s, 1H); MS (ES) calc'd for Cn IH 8

N

4 OS [MH+] 245.05, found: 245.05 25 [0146] 2-Phenylsulfenyl-6-chloro-purine (3). To a flame-dried round bottom flask (200 mL) was added 2-Phenylsulfenylhypoxanthine 2 (1.22 g, 5.0 mmol), tetrabutyl-ammonium chloride (anhydrous, 2.78 g, 10.0 mmol) and N,N-dimethylaniline (0.63 mL, 5.0 mmol), followed by dissolving them in acetonitrile (anhydrous, 50 mL). To the solution phosphorus oxychloride (2.8 mL, 30.0 mmol) was added dropwise. The reaction 30 was refluxed under argon. After 2 hours, the reaction was cooled to 0 "C on an ice bath and 53 WO 03/031405 PCT/US02/32679 quenched by addition of saturated sodium bicarbonate solution. The aqueous layer was extracted with ethyl acetate (4 x 100 mL). The combined organic layers were dried over sodium sulfate, and were concentrated on the rotary evaporator. The crude product was further purified by re-crystallization from methanol to afford desired product 3 as white solid 5 (0.94 g, 72%). 1H NMR (400 MHz, (CD 3

)

2 SO) 7.50 (m, 3H), 8 7.65 (m, 2H), 8.53 (s, 1H); MS (ES) calculated for CnIH 7 C1N 4 S [MI] 263.02, found: 263.00 [0147] Solution phase N9 Mitsunobu Alkylation of Purine (4). To a flame dried roundbottom flask (500 mL) was added 2-fluoro-6-chloro-purine (7.0 g, 40.6 mmol), triphenylphosphine (19.2 g, 73.1 mmol) and alcohol (52.8 mmol), followed by dissolving 10 them in THF (anhydrous, 350 mL). The solution was cooled down to -30 "C and diisopropyl azodicarboxylate (12.0 mL, 60.9mmol) was added dropwise. The reaction was allowed to warm up to room temperature and stirred under argon. After overnight, the solvent was removed under reduced pressure and the crude material was directly used in the next step without further purification. 15 [01481 Resin Capture of N9-alkylated Purine Scaffold at C6 (5). To a solution of crude 2-fluoro-6-chloro-9-alkylpurine (15.0 mmol) in n-butanol (200 mL) was added mercaptomethyl polystyrene resin (10.0 mmol, Midwest Biotech), followed by addition of diisopropylethylamine (5.2 mL, 30.0 mmol). The suspension was heated to 80 *C under argon. After overnight, the resin was washed by methanol (200 mL x 4) and 20 dichloromethane (200 mL x 4) and dried under vacuum. [01491 C2 Amination of Captured Purine (6). The resin 5 (0.10 mmol) was suspended in n-butanol (1.0 mL), followed by addition of an amine (0.30 mmol) and diisopropylethylamine (0.40 mmol). After overnight shaking at 80 "C, the resin was washed by methanol (3 mL x 4) and dichloromethane (3 mL x 4) and dried under vacuum. 25 [0150] Activation of C6 by Oxidizing Sulfenyl Group to Sulfonyl Group (7). To a solution of m-CPBA (0.23 g, 75%, 1.0 mmol) in 1,4-dioxane (9 mL) cooled to 0 *C was added NaOH (1 mL, IM, 1.0 mmol) aqueous solution, followed by addition of resin 4 (0.10 mmol). The suspension was shaken gently at room temperature. After 8 hours the resin was washed by methanol (3 mL x 4) and dichloromethane (3 mL x 4) and dried under 30 vacuum. 54 WO 03/031405 PCT/US02/32679 [01511 C6 Displacement with Amines and Product Releasing (8). The resin 7 (0.05 mmol) was suspended in anhydrous 1,4-dioxane (0.6 mL), followed by addition of an amine (0.1 mmol). After overnight shaking at 80 "C, the resin was filtered using a polypropylene cartridge (45pt PTFE frit) and the flow-through solution was collected. The 5 resin was subsequently washed by dichloromethane (0.5 mL x 3) and flow-through was combined and solvent was removed under reduced pressure to afford desired product 8 (in average >85% HPLC purity, 80% purified yield). [0152] The following Tables 6 and 7 show experimental data obtained for compounds prepared using the methods of the invention and having various substituents at 10 the C2 and C6 positions. These results demonstrate the wide applicability of the methods of the invention. 55 WO 03/031405 PCT/US02/32679 Table 6. Validation of C2 substituents. 110 minutes elution method was used. For other entries, 6 minutes elution method was used. 2 Isolated yields are based on the amount of amine (0.8 equiv) used for the final displacement releasing. N

R

3 Entry N9 substituent (R 2 ) Retention Calculated Observed HPLC Isolated Time (min) [M] [MH*] purity (%) yield 2 (%) 1 N N NH 4.57 462.25 463.3 90 81 2 4.38 396.19 397.2 91 86 3 HN 5.621 344.23 345.2 87 81 4 5.781 366.22 367.3 90 82 5 N"O 5.301 332.20 333.2 94 87 6 N O 4.63 476.26 477.3 84 79 6 H VN -'NH 7 4.12 405.23 406.3 84 77 8 5.07 416.23 417.3 85 80 H H 9 \_N OH 3.63 348.23 349.3 83 79 10 N 3.33 387.24 388.3 86 82 0 11 -N'N F 5.22 425.23 426.2 89 83 12 /N OH 2.93 306.18 307.2 90 84 H 56 Table 7. Validation of C6 substituents. 110 minutes elution method was used. For other 2 entries, 6 minutes elution method was used. Isolated yields are based on the amount of amine (0.8 equiv) used for the final displacement releasing. R4, ,RA N N N Entry C6 substituent Retention Calculatedf Observed HPLC Isolated (NR4R 5 ) Time (min) [M] I [MH*] purity (%) yield 2 (%) 1 5.631 366.22 367.2 91 83 2 I-NH N 3.52 387.22 388.2 84 77 3 -- \N 3.01 404.24 405.3 88 82 4 i-N N-Q) F 5.11 459.25 460.3 91 86 5 S OH 4.54 416.23 417.20 90 84 -NH 6 HO 4.27 428.23 429.2 82 77 7 ^N O 3.86 451.27 452.3 87 81 8 HN 7.051 372.21 373.2 86 80 9 N 5.731 378.25 379.3 87 81 OH 10 J-N 3.47 384.23 385.3 84 76 OH 11 N 3.70 421.26 422.3 88 83 0 12 H O 3.04 423.27 424.3 89 82 N ,,N 13 'N- OH 3.87 446.24 447.3 84 76 14 'N 3.44 423.31 424.3 87 81 H 15 -N 4.60 416.23 417.2 92 84 57 WO 03/031405 PCT/US02/32679 0 HN N N N H [01531 1H NMR (400 MHz, CDC1 3 ) 6: 1.54 (d, 6H, 6.8 Hz), 1.94 (m, 4H), 2.39 (m, 2H), 2.94 (t, 2H, J=7.3 Hz), 3.41-3.49 (m, 8H), 3.66 (br, 2H), 4.67 (m, 1H, 6.8 Hz), 7.23 (m, 3H), 7.31 (m, 2H), 7.62 (s, 1H); MS (ES) calc'd for [MH] C 23

H

32

N

7 0 422.27, found 422.30 HN NI N N N,, N 5 H [01541 1H NMR (400 MHz, CDC1 3 ) a: 1.57 (d, 6H, J=6.8 Hz), 2.96 (t, 2H, J=7.6 Hz), 3.73 (m, 2H), 4.68 (m, 1H), 5.02 (br, 1H), 4.67 (m, 1H, J=6.8 Hz), 7.05 (m, 1H), 7.21-7.34 (m, 6H), 7.58 (s, 1H), 7.79 (m, 2H); MS (ES) calc'd for [MH*] C 22

H

25

N

6 373.21, found 373.20 -N HN N N N H 10 [0155] 1H NMR (400 MHz, CDC1 3 ) 5: 1.55 (d, 6H, J=6.8 Hz), 2.90 (t, 2H, J=7.2 Hz), 3.65 (m, 2H), 4.65 (m, 1H, J=6.8 Hz), 4.81 (d, 2H, J=5.1 Hz), 4.87 (br, 1H), 6.06 (br, 1H), 7.05 (m, 1H), 7.21-7.31 (m, 6H), 7.50 (s, 1H), 7.69 (m, 1H), 8.50 (dd, 1H, J=1.3 Hz), 8.64 (d, 1H, J=1.5 Hz); MS (ES) calc'd for [MH*] C 22

H

26

N

7 388.22, found 388.20 58 WO 03/031405 PCT/US02/32679 N N N H [01561 1H NMR (400 MHz, CDC1 3 ) a: 1.57 (d, 6H, J=6.8 Hz), 2.97 (t, 2H, J=7.1 Hz), 3.66 (t, 2H, J=7.3Hz), 3.81 (t, 4H, J=4.8Hz), 4.25 (br, 4H), 4.75 (m, 1H, J=6.8 Hz), 7.28 (m, 3H), 7.34 (m, 2H), 7.56 (s, 1H); MS (ES) calc'd for [MH+] C 2 0

H

27

N

6 0 367.22, found 367.20 0 0N) N N N \- > N N N 5 F [0157] 1H NMR (400 MHz, CDC1 3 ) a: 1.30 (t, 3H, J=7.1 Hz), 1.59 (d, 6H, J=6.8Hz), 3.52(m, 8H), 3.94 (s, 2H), 4.20 (t, 4H, J=4.5Hz), 4.27 (q, 2H, J=7.lHz), 4.41 (br, 4H), 4.80 (m, 1H, J=6.8 Hz), 7.16 (d, 2H, J=9.lHz), 7.41 (d, 2H, J=9.lHz), 8.11 (s, 1H); MS (ES) calc'd for [MH*] C 26

H

36

FN

8 0 2 511.29, found 511.30 H N O N N N N N 10 [0158] 1H NMR (400 MHz, CDC1 3 ) a: 1.58 (d, 6H, J=6.8 Hz), 3.76 (m, 8H), 4.02 (br, 2H), 4.20 (t, 2H, J=5.9Hz), 4.75 (m, 1H, J=6.8 Hz), 6.93 (m, 3H), 7.26 (m, 2H), 7.84 (s, 1H); MS (ES) calc'd for [MH*] C 2 0

H

27

N

6 0 2 , 383.22, found 383.20 59 WO 03/031405 PCT/US02/32679 EXAMPLE 3 Concise, Traceless Approaches Toward Combinatorial 2, 9-disubstituted Guanines and 06-Aryl-, 06-Alkyl- Purines [0159] Table 8 shows the retention times, as well as calculated and observed 5 molecular weights for purines having various substituents at the C6 and C2 positions that were made using the methods of the invention. These results demonstrate that the methods are applicable to attachment of a wide variety of substituents to purine scaffolds. Table 8 0 > N 1 X-phenethylamino, Y=phenethyl

R

2 N N> 2X= 4-methoxybenzylamino, Y=H X N N C6 substituent (R 2 ) Retention Calculated Observed Tim (mn) [M] [MH]02 substituent (OR) Retention Calculated Observed 5.40 317.10 318.10 Nl 8.54 575.12 576.10 CI PN/-8 .41 33.16 33420 106.45 487.05 488.05 N 4.41 333.16 334.20520.20 H < 0 CF 6.37 431.12 432.10 N OH 2.44 237.10 238.10 8.99 503.27 504.30 N 5.29 275.17 276.20 6.94 415.20 416.20 SOCH3 H 4.84 313.15 314.20 - -6.14 427.16 428.20 -N 0.50 284.14 285.20 N OCH 3 7.41 465.22 466.25 a

OCH

3 5.40 317.10 318.10 5.47 377.15 378.20 a,b HN -OCHa 5.40 317.10 318.10 6.70 491.27 492.30 a~b-11-0a y6.61 403.20 404.20 a,b O / \ OCH 3 5.40 317.10 318.10 Tal 8.49 511.24 512.25 10 0 6.49 423.17 424.20 N8.39 475.24 476.20 /-,0)( :)6.23 387.17 388.20 8.16 485.22 486.20 0 6.04 397.15 398.15 60 WO 03/031405 PCT/US02/32679 -C2 substituent (OR) Retention Calculated Observed Time (min) [M] [MH*] o" C 6

H

13 9.25 535.29 536.30 0 7.38 447.23 448.20 8.28 463.24 464.25 0 6.12 375.17 376.20 7.83 471.19 472.20 5.75 383.12 384.10 F 7.94 449.22 450.20 10 5.73 361.15 362.20 Ci 8.15 469.17 470.20 - / 6.06 381.10 382.10 5 [0160] It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference for all purposes. 61

Claims (26)

1. A method of preparing a 2,6,9-substituted purine compound having the Formula I: NR 3 R 4 N N NN HN N I R2 R1 wherein: R 1 and R 2 are independently selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl and heterocyclyl; R 3 is hydrogen and R 4 is selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl and heterocyclyl or R 3 and R4 when taken together with the nitrogen atom to which they are respectively attached form an optionally substituted saturated heterocyclic ring; the method comprising: a) oxidizing a resin-bound compound of Formula II (CH 2 )n- S N N N N\ R1 R2 wherein: R 1 and R 2 are as defined above and n is 0 or 1; to provide a resin-bound compound of Formula III 62 (CH 2 ) O N N N R wherein R 1 , R 2 and n are as defined above; b) reacting the compound of Formula III with an amine of Formula IV HNR 3 R 4 IV wherein R 3 and R 4 as defined above, to provide a resin-bound compound of Formula V NR 3 R 4 N N\ N N R2\ wherein R 1 , R 2 , R 3 and R 4 are as defined above; and c) cleaving the resin-bound compound of Formula V from the resin to provide the substituted purine compounds of Formula I.

2. The method of claim 1, wherein the compound of Formula II is prepared by a) alkylating a compound of Formula VI (CH2)n-S N N N X N H 63 WO 03/031405 PCT/US02/32679 wherein X is fluoro, chloro or bromo and n is as defined above, to provide a compound of Formula VII \ (CH 2 ) -S NN NN ) VII X N\ R 2 wherein R 2 , X and n are as defined above; and b) capturing the compound of Formula VII with a resin-bound amine to provide the resin-bound compound of Formula II; or a) capturing a compound of Formula VI &(CH2)-S N \N >VI N X N H wherein X and n are as defined above, with a resin-bound amine to provide a resin-bound compound of Formula VIII <D(CH2)n--S / \N N ~N NH wherein R 1 and n are as defined above; and b) alkylating the resin-bound compound of Formula VIII to provide the resin-bound compound of Formula II. 64 WO 03/031405 PCT/US02/32679

3. The method of claim 2, wherein the compound of Formula VI is prepared by reacting 2-halo-6-chloro-purine with a compound of Formula IX: / \ (CH 2 )nSH Ix wherein n is as defined above, to provide the compound of Fonnula VI.

4. The method of claim 3, wherein the halo is fluoro, chloro or bromo.

5. The method of claim 1, wherein step (a) is carried out with m chloroperbenzoic acid in a buffered solution.

6. The method of claim 1, wherein step (c) is carried out in the presence of trifluoroacetic acid.

7. The method of claim 2, wherein alkylating of the compound of Formula VI is carried out in the presence of an inert solvent and a phosphine.

8. The method of claim 1, wherein the resin is 4-formyl-3,5 dimethyloxyphenoxymethyl functionalized polystyrene resin.

9. A chemical library consisting of a plurality of 2, 6, 9 substituted purine compounds prepared by the method of claim 1.

10. A compound of the Formula VI &~(cH2)n-S N/ N NN X N HN wherein X is fluoro, chloro, or bromo and n is 0 to 1. 65 WO 03/031405 PCT/US02/32679

11. A method of preparing 2, 9-substituted purine compounds having the Formula X 0 N HNN HN N R1 R2 wherein R 1 and R 2 are independently selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl and heterocyclyl; the method comprising: a) oxidizing a resin-bound compound of Formula II \ (cH 2 )n-S NN N 1 N R2 wherein R 1 and R 2 are as defined above and n is 0 or 1; to provide a resin-bound compound of Formula III &(CH2)n- ==0 N N N\ 1RR2 wherein R 1 , R 2 and n are as defined above; b) hydrolyzing the sulfonyl group at the 6-position of the resin-bound compound of Formula III and cleaving the hydrolyzed resin-bound compound from the resin to 66 WO 03/031405 PCT/US02/32679 provide the substituted purine compounds of Formula X.

12. The method of claim 11, wherein the resin-bound compound of Formula II is prepared by a) alkylating a compound of Formula VI \ (CH 2 )n-S N X N H wherein X is fluoro, chloro or bromo and n is as defined above, to provide a compound of Formula VII &(CH2)n--S / \N VII X N N\ R2 wherein R 2 , X and n are as defined above; and b) capturing the compound of Formula VII with a resin-bound amine to provide the resin-bound compound of Formula II; or &(CH2)n--S / N N 6 7 67 WO 03/031405 PCT/US02/32679 a) capturing a compound of Formula VI )(CH2)n-S \N N X N H wherein X and n are as defined above, with a resin-bound amine to provide a resin-bound compound of Formula VIII (CH2)n-S N N \N VIII N ~<N H wherein R 1 , X and n are as defined above; and b) alkylating the resin-bound compound of Formula VIII to provide the resin-bound compound of Formula II.

13. A chemical library consisting of a plurality of 2,9-substituted purine compounds prepared by the method of claim 11.

14. A method for preparing 0 6 -alkyl-purine compounds of Formula XI OR 3 N HN XI R1 R2 68 WO 03/031405 PCT/US02/32679 wherein R 1 and R 2 are independently selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl and heterocyclyl; and R 3 is selected from the group consisting of alkyl and substituted alkyl; the method comprising: a) oxidizing a resin-bound compound of Formula II &(CH2)n--S N/\ N N N N 1 wherein R 1 and R 2 are as defined above and n is 0 or 1; to provide a resin-bound compound of Formula III N ~N N N N wherein R 1 , R 2 and n are as defined above; b) hydrolyzing the sulfonyl group at the 6-position of the resin-bound compound of Formula III to form a resin-bound compound of Formula XII 0 HNN X11 HN N N R2 wherein R 1 and R 2 are as defined above; and 69 WO 03/031405 PCT/US02/32679 c) alkylating the resin-bound compound of Formula XII and cleaving the resin bound alkylated compound from the resin to provide a compound of Formula XI.

15. The method of claim 14, wherein the resin-bound compound of Formula II is prepared by a) alkylating a compound of Formula VI 0-(CH2)n--S N \N X N wherein X is fluoro, chloro or bromo and n is as defined above, to provide a compound of Formula VII 0-(CH2)n--S N// N~v X N R 2 wherein R 2 , X and n are as defined above; and b) capturing the compound of Formula VII with a resin-bound amine to provide the resin-bound compound of Formula II; or a) capturing a compound of Formula VI &~(CH2)n-S N X N wherein X and n are as defined above, with a resin-bound amine to provide a resin-bound compound of Formula VIII 70 0_(CH2)-S /N N N N N CH wherein R 1 , X and n are as defined above; and b) alkylating the resin-bound compound of Formula VIII to provide the resin-bound compound of Formula II.

16. A chemical library consisting of a plurality of 0 6 -alkyl-purine compounds prepared by the method of claim 14.

17. A method for preparing 0 6 -aryl purine compounds of Formula XIII OR 3 H N N XIII HN N N I R2 R1 wherein R 1 and R 2 are independently selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl and heterocyclyl; and R 3 is aryl or substituted aryl; the method comprising: a) oxidizing a resin-bound compound of Formula II 0-(CH2)n--S NN N N N\ I R2 R1 wherein R 1 and R 2 are as defined above and n is 0 or 1; 71 to provide a resin-bound compound of Formula III (CH2)n--- O N N N N N JN R2 wherein R 1 , R 2 and n are as defined above; and b) reacting the resin-bound compound of Formula III with a phenolic aryl compound followed by cleavage of the resin-bound compound to provide a compound of Formula XIII.

18. The method of claim 17, wherein the resin-bound compound of Formula II is prepared by a) alkylating a compound of Formula VI \ (CH 2 )n-s N X N H wherein X is fluoro, chloro or bromo and n is as defined above, to provide a compound of Formula VII 72 (CH 2 )n-S N' C N i VI N X N R2 wherein R 2 , X and n are as defined above; and b) capturing the compound of Formula VII with a resin-bound amine to provide the resin-bound compound of Formula II; or a) capturing a compound of Formula VI 0-(CH2)n-S N N NN X N H wherein X and n are as defined above, with a resin-bound amine to provide a resin-bound compound of Formula VIII \ (CH 2 )--S N N N H wherein R 1 , X and n are as defined above; and b) alkylating the resin-bound compound of Formula VIII to provide the resin-bound compound of Formula II.

19. A chemical library consisting of a plurality of 0 6 -aryl-purine compounds prepared by the method of claim 17.

20. A method of preparing a compound of Formula VI 73 0-(cH2)n N N x N H wherein X is fluoro, chloro or bromo and n is 0 or 1; the method comprising: reacting a 2-halo-6-chloro-purine with a compound of Formula IX (CH2)nSH Ix wherein n is as defined above to provide the compound of Formula VI.

21. The method of claim 20, wherein the halo is fluoro, chloro or bromo.

22. A method for synthesis of a C2-substituted purine, the method comprising reacting a C6-sulfenylpurine with a nucleophile, wherein the nucleophile is substituted for X at the C2 position of the C6-substituted purine, and the C6-sulfenylpurine comprises a solid support-bound compound of Formula XIV: O(CHz~n Ns \> Xiv H wherein X is fluoro or chloro and n is 1.

23. The method of claim 22, wherein the nucleophile is an amine.

24. The method of claim 23, wherein the method further comprises: 1) oxidizing the sulfenyl moiety to a sulfone containing C2-substituted purine of Formula XV: 74 O-(CH 2 )n a 6=0 N \> xv HNIN N HH wherein: n is 1 and R, is selected from the group consisting of alkyl, substituted alkyl, cycloalkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl and heterocyclyl; and 2) substituting a different group at the C6 position of the purine.

25. The method of any one of claims 22-24, wherein X is fluoro.

26. The method of any one of claims 22-25, wherein the solid support is a methylmercapto resin. 75