AU2004200192B2 - Purine inhibitors of cyclin dependent kinase 2 and 1k-Aa - Google Patents

Description

P/00/01Il Regulation 3.2 AuSTRALIA Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Invention Title: Purine inhibitors of cyclin dependent kinase 2 and lk-Aa The following statement is a full description of this invention, including the best method of performing it known to us: Ireenills carter ~mitn I~eadIe Melbourne\004421 518 Printed 16 January 2004 (13:47) page 2 Freehills Carter Smith Beadle Melbourne\004421818 Printed 16 January 2004 (13:47) page 2 Title: PURINE INHIBITORS OF CYCLIN DEPENDENT KINASE 2 and IrB-a BACKGROUND OF THE INVENTION Field of the Invention This invention concerns 2,6,9-trisubstituted purines that have been discovered to be selective inhibitors of cell cycle kinases and, as such, the compounds are inhibitors of cell proliferation. The 2,6,9-trisubstituted purines are useful in for example in treating autoimmune diseases, e.g. rheumatoid arthritis, lupus, type I diabetes, multiple sclerosis, etc., in treating cancer, cardiovascular disease, such as restenosis, host vs graft disease, gout, polycystic kidney disease and other proliferative diseases whose pathogenesis involves abnormal cell proliferation.

This invention also concerns 2,6,9-trisubstituted purines that have been discovered to be potent and specific inhibitors of IKB-at kinase which prevents signal induced NF-KB activation and cytokine synthesis in vitro and in vivo. Such inhibitors are expected to inhibit the synthesis of cytokines and adhesion proteins whose synthesis is transcriptionally regulated by NF-KB. Proinflammatory cytokines such as IL-1, 1L-6, TNF and adhesion proteins (e.g.

ICAM, VCAM and selections) belong to this class of molecules and have been implicated in the pathogenesis of inflammatory diseases. Thus a potent inhibitor of IVcB-cz kinase is useful in the clinical management of diseases where NF-icB activation is required for disease induction.

Description of the Art In the past few years, advances in molecular and cellular biology have contributed to our understanding of the mechanisms of cell proliferation and of specific events that occur during progression of cells through mitosis. "Progress in Cell Cycle Research" Vol 1, Eds. L.

Meijer, S. Guidet and H.Y.L. Tung; Plenum Press, New York, 1995. These studies have shown that progression through the cell cycle is controlled by a family of serine/threonine kinases called cyclin dependent kinases. These enzymes contain a catalytic protein called cyclin dependent kinase (CDK) that uses ATP as a substrate and a regulatory protein called cyclin. Different cyclin-CDK combinations control events such as growth, DNA replication and cell division. One key member of the CDK famnily of enzymes is CDK2.

CDK2 activity has been shown to be essential for mamnmalian cell cycle progression at the Gl/S boundary. Microinjection of antibodies directed against CDK2 blocks the progression of human diploid fibroblasts into the S phase of the cell cycle. Expression of a CDK2 dominant negative mutant in human osteosarcoma cells has a similar effect. Together, these studies indicate that inhibition of cellular CDK2 activity will prevent progression of cells through the mitotic cycle and induce growth arrest prior to the S phase. Consistent with this view, in vitro studies 'with olomnoucine (2-(hydroxye'hylaniino)-6benzylamino-9-methylpurine), have shown that it is a specific inhibitor of CDK2 with an IC 50 of approximately 2.1 pgg/ml J. Vesely, et al.; Eur. J.Biochem 224, 771-786 (1994), L. Meijer "Chemical Inhibitors of Cyclin-Dependent Kinases" pp 351-356 in "Progress in Cell Cycle Research Vol 1, Eds. L. Meijer, S. Guidet and H.Y.L. Tung; Plenum Press, New York, 1995.

In vivo studies using mammalian cells in culture have shown that olomoucine inhibits cell proliferation at an approximate concentration of 50 gml.

In this invention, we have developed several compounds whose biological activity is considerably more potent than olomnoucine. In vivo studies using mammalian cells indicate that some of the disclosed compounds inhibit cell proliferation at concentrations that are significantly lower than olomoucine.

Recently an 1KB-ct kinase activity has been described in the cytoplasm of stimulated human umbilical vein endothelial cells (Bennett et al (1996) J. Biol.Chem 271, 19680- 19688). Some of the compounds of this invention have been identified as potent and specific inhibitors of IKB-ct kinase which prevents signal induced NF-icB activation and cytokine synthesis in vitro and in vivo. The activation of the heterodimeric transcription factor NF-rB is a complex process. In unstimulated cells, the NF-xB (p50/p65) heterodimer is located in the cytosol where it is complexed with an inhibitory subunit IxBax, 1KB-ct, binds to NF-KB thus masking its nuclear localization signal and preventing translocation to the nucleus. Upon stimulation of cells with a variety of signals (e.g.

lipopolysaceharide) 1KB-ct is rapidly phosphorylated, uniquitinated and degraded by the proteasome. Degradation of 1KB-ct, allows the translocation of NF-KB- to the nucleus where it activates transcription of a number of inflammatory response genes.

These observations suggest that 1KB-ct kinase is an attractive target for the identification of inhibitors that may be useful in the treatment of inflammatory diseases where NF-KB activation is required for disease induction.

Reference to any prior art in the specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in Australia or any other jurisdiction.

004889492 4 SUMMARY OF THE INVENTION

C.)

This invention seeks to provide 2,6,9-trisubstituted purine compounds, which inhibit the cyclin dependent kinase 2.

This invention also seeks to provide 2,6,9-tri substituted purine compounds which are S 5 useful for inhibiting cell proliferation.

This invention also constitutes a pharmaceutical composition, which comprises a 2,6,9-trisubstituted purine compound and a pharmaceutically acceptable carrier.

This invention further constitutes a method for inhibiting cell proliferation, which comprises administering to a mamnmal in need thereof an effective amount of a 2,6,9trisubstituted purine compound.

This invention also constitutes the use of a 2,6,9-trisubstituted purine compound for the preparation of a medicament for inhibiting cell proliferation in a mamnmal.

In a first aspect of this invention is a compound selected from the group consisting o f: {2-[(2-aminoethyl)amino] -9-(methylethyl)purin-6-yl chlorophenyl)methyl]amine; f 2- [(2-aminopropyl)amino] -9-(methylethyl)purin-6-yl [(4-chlorophenyl)methyl] amine; 2- [(2-aminoethyl)(6- f [(4-chlorophenyl)methyl] amino)} -9-(methylethyl)purin-2yl)aminolethan-1-ol; and 2-[(2-hydroxyethyl)(6- [(4-chlorophenyl)methyl] amino)} -9-(methylethyl)purin-2yl)amino]ethan-lI-ol.

In another embodiment, this invention is A 2,6,9-trisubstituted purine composition of matter having the following formula: 004889492

I

N

R

3 N N\ N

N

R

3

NN

(I)

wherein R, is halogen or R' 1 -X wherein X NH, 0, S, S In the composition, is alkyl, heterocyclyl, aryl, heteroaryl, aralkyl, heteroarylalkyl, alkenyl, and alkynyl, each having one to 20 carbon 'atoms, which alkyl, heterocyclyl, aryl, heteroaryl, aralkyl, heteroarylalkyl, alkenyl, and alkynyl, are optionally substituted with from 1 to 3 substituents independently selected from the group consisting of halo, aryl, CF 3 heteroaryl, heterocyclyl, R, SR 20

S(O)R

1 S0 2

R

21

SO

2

NR

2

R

23 S0 2

NR

20 C0R 1

SO.,NR'

0 00NR 20 RY3,

SONR

20 00 2

R

2 1, NR 20 R23, NR 20 C0R 1

NR

20 C0 2

R

1

NR

20

CONR

2 3, N(R' 0

)C(NR

2

)NHIRY,

NR

20 SO.R", OR 20

OCONR

2 0

R

23 000NR 20 S0 2

R

1 000NR 2

R

23 ON, C0 2

R

2 0 CONR 2 3, C0NR 2 1S0 2 ,R and C0R 2 1. Also in the composition, R 2 is a hydrogen or hydrocarbon selected from the group alkyl, heterocyclyl, aryl, heteroaryl, aralkyl, heteroarylalkyl, alkenyl,and alkynyl, each having one to 20 carbon atoms, which alkyl, heterocyclyl, aryl, heteroaryl, aralkyl, heteroarylalkyl, alkenyl,and alkvnyl,are optionally substituted with from 1 to 3 sub stituents independently selected from the group consisting of halo, aryl, heteroaryl, heterocyclyl, R 22

SR

20

S(O)R

1 S0 2

R

2 1 S0 2

NR

2 0R 2 1, S0 2

NR

20 C0R 2 1 SO2NR 20

CONR

20 R.2, SO2NR 2 1 CO 2 R 2 1

NR

2 0RnI, NR 20 00R 21

NR

2 'C0 2

R

2

WROCONR&R

2

N(R

20 )C(NR 20

)NHR

3

NR

20 S0 2

R

21 OR 20 000NR 0

R

2 3 000NR 20 S0 2

R

2 1

OCONR

20 R23, ON, CO0R0 0

CONR

20 R21, CONR 20 SO,R 2 1 and C0R 20 Further, in the compositions, R, is a halogen, hydroxyl, thio, ailkoxy, alkylthio, alkyl, -NRR, or a component having the formula: 6 where m=1-3, o=1,3, y=carbonyl, -NRR,, hydroxyl, thiol, alkoxy, alcyithiol; R, and R, are each independently hydrogen, OR 2 0

NR

2

,R

23 or a hydrocarbon selected from the group including alkyl, acyl, heterocyclyl, aryl, heteroaryl, aralkyl, heteroarylalkyl, alkenyl,and alkynyl, each having one to 20 carbon atoms, which ailkyl, acyl, heterocyclyl, aryl, heteroaryi, aralkyl, heteroarylalcyl, alkenyl,and allcynyl,are optionally substituted with from 1 to 3 substituents independently selected from the group consisting of halo, aryl, heteroaryl, heterocyclyl, R 1 2

SR

20

S(O)R

21 SORL, S02 NR 0

R

2 3 So, NR 20

COR

1 S 2

N

20

ON~R

3

,SON

2 0R

N

R20 RR, NR 0 0 21

NR

2 0 C 21

NR

20 C0NR 20 R 23

N(R

20 )C(NjR 20

)NER_

23

R

0 0R' OR 20 0C0NR 20

R

23

OCONR

20 SOR 1, OCONR 2 0pR 23

,CN

C0 2

R

20 C0NR 2

R

23

CON

20 SO,R 2 1 and C0R 20 R 20 is a member selected from the group consisting of H, ailcyl, C 2 5 alkeny, C 2 alkynyl, heterocyclyl, aryl, and heteroaryl, which ailkyl, alkenyl, alkcynyl, heterocyclyl, aryl, and heteroaryl are optionally substituted with 1 to 3 substituents independently selected from halo, alkyl, mono- or dialkylamino, alkyl or aryl or heteroaryl amiide, CN, 0-C.

6 ailkyl, CF 3 aryl, and heteroaryl; R 2 1 is a member selected from the group consisting of C,4, 5 alkyl, 5 alkenyl, C., alcynyl, heterocyclyl, aryl, and heteroaryl, which alkyl, alkenyl, alkynyl, aryl, heterocyclyl, and heteroaryl are optionally substituted with 1 to 3 substi tuents independently selected from the group of halo, heterocyclyl, aryl, heteroaryl, CF 3 CN, OR 20 SR 2 1, N(R 0 2

S(O)R

22

SO,R

22

SO

2

N(R

0

SO,NR

20 C0R 2 S0 2

NV

20 C0 2

R

2 I SO 2

NR

2

CONR

2 2

N(R

20

NR

20 C0R 2 2

NR?

0 C0 2

R

22

NR

2

CON(R

20 2

NR

2 0

C(NR

20

)NHR

2 3 C0R 20 C0 2

R

20

CON(R

20 2

CONR

20 SOR 2 NR 2 0

SOR

22 S0 2

NR

2 3 C0 2 R1 2

OR

20 0C0NR 0 S0 2

R?

2

OC(O)R

0

C(O)OCH.,OC(O)R

20 and OCON(R 20 2 and each optional heteroaryl, aryl, and heterocyclyl 7 substituent is optionally substituted with halo, alkyl, CF 3 amino, mono- or di- alkylarnino, alkyl or aryl or heteroaryl amide, NCOR 2

NR

20 S0 2

R

2

COR

0 CO,R 20 CON(R 2 0 2

NR

20 00N(R 0 2

OC(O)R

20

OC(O)N(R

0 2

SR

20 S(0)R 1 2 S0 2

R

22

SO,N(R

20 2 ON, or OR 2 0 R 22 is a member selected from the group consisting of 01.15 alkyl, C2.15 alkenyl, 213 allkynyl, heterocyclyl, aryl, and heteroaryl, which alkyl, alkenyl, alkynyl, heterocyclyl, aryl, and heteroaryl are optionally substituted with 1 to 3 substituents independently selected from halo, alkyl, mono- or dialkylamino, alkcyl or'aryl or heteroaryl amide, CN, alkyl, CF 3 aryl, and heteroaryl; and R' is W 21 or H.

There are some limitations to the scope of the compositions of this invention. When Y is carbonyl, Y and R' 4 together may be a single oxygen atom, R 4 and together may be a single oxygen atom, R 4 and may together be a single oxygen atom. When R 3 is 2hydroxyethylarnino and R 2 is methyl, R, is not amino, 3-methyl-2-butenylanuino, benzylamino, or m-hydroxybenzylamino when R 3 is not 2-hydroxyethylamino. When R, is isopropyl, is not benzylamino, m-hydroxybenzylamino, or 3-methylbutylamino. When

R

3 is 2-hydroxyethylamino and R, is 2-hydroxyethyl, R 1 is not benzylamino and when R 3 is selected from the group consisting of 2-methyl-2-hydroxypropylainino, and 2dimethylaxninoethylamino, and when R, is methyl, then is not benzylamino In another embodiment, this invention is a method for inhibiting cell proliferation in mammals comprising administering a therapeutically effective amount of the composition of claim 1 to the marnmal. The method is useful for treating cell proliferation disorders such as rheumatoid arthritis, lupus, type I diabetes, multiple sclerosis, cancer, restenosis, host graft disease, and gout.

8 In yet another embodiment, this invention is a pharmaceutical composition of matter comprising the composition above in an admixture with one or more pharmaceutical excipients.

In still another embodiment, this invention is a composition useful for treating fungal infections (fungi) in humans, animals, and in plants.

DESCRIPTION OF THE FIGURE Figure 1 is a plot of the mean neointimal area of a rat carotid artery treated with a saline vehicle and treated with compound 3 prepared according to Example 2 wherein the unshaded bar represents the untreated section of the carotid artery and the shaded bar represents the treated section of the carotid artery.

It will be understood that the term "comprises" or its grammatical variants as used herein is equivalent to the term "includes" and is not to be taken as excluding the presence of other elements or features.

DESCRIPTON OF THE CURRENT EMBODIMN The present invention relates to a 2,6,9-trisubstituted purine compound having the following formulas: Rl-.% where: R, is halogen or wherein X NH, 0, S, S(0 2

R'

1 is alkyl, heterocyclyl, aryl, heteroaryl, arailkyl, heteroarylalkyl, alkenyl, and alkynyl, each having one to 20 carbon atoms, which alkyl, heterocyclyl, aryl, heteroaryl, arailkyl, heteroarylalkyl, alkenyl, and alkynyl, are optionally substituted with from I to 3 substituents independently selected from the group consisting of halo, ar-yl, CF 3 heteroaryl, heterocyclyl, R 22

SR

20

S(O)R

21 S02R 21

SONR

0

R

3 ,I So NR 20 C0R 2 1

SONR

20 C0NR 2

OR

2 1,

SO,NR

2 0

,R

2

NR

20

R

23

NR

20 00R 21

NR

20 C0 2

R

2

NR

2 0 C0NR 2

"R

3

N(R

2 0 )c(NR 20

)NHR

2

NR

20 S0 2

R

21 OR 2 0

OCONR

20 R23, 0C0NR 20 S0 2

R

1 0C0N 20

R

2 3 CN, C0 2 R 2 1, C0NR 2

R

23

CONR

2 0 1SOR 2 1 and C0R 0 R, is a hydrogen or hydrocarbon selected from the group alkyl, heterocyclyl, aryl, heteroaryl, aralkyl, heteroarylalkyl, alkenyl,and alkynyl, each having one to 20 carbon atoms, which alkyl, heterocyclyl, aryl, heteroaryl, aralkyl, heteroarylalcyl, alkenyl,and alkynyl,are optionally substituted with from 1 to 3 substituents independently selected from the group consisting of halo, aryl, heteroaryl, heterocyclyl., SR 20

S(O)R

2 1 S0 2

R

21

SO

2

NR

0

R

22 S &,NR 20 C0R 1

SO

2

NR

2

OCONR

2

&R

23

SON

2

C

2 2

,NR

20 R21, NR 20

CO

21

NR

2 C0R,

NR

20 C0NR 20 R 23

N(R

20

)C(NR

20

)NIR

2 3

NR

20 SOR 21

OR

20 0C0NR 2

R

23 0C0NR 2 1S0 2 R 21 0C0NR 2

R

23 CN, C0 2 R 20 C0NR 2

R

23

CONR

20

SO'R

2 and C0R 2 1

R

3 is a halogen, hydroxyl, thio, alcoxy, alkylthio, alkyl, -NRR., or a component having the formula: Yj n where m=1-3, n=1-3, o=1,3, y=carbonyl, -NR 4

R.

5 hydroxyl, thiol, alkoxy, alkyithiol; R, and R, are each independently hydrogen, OR 20

NR

20 R23, or a hydrocarbon selected fromn the group including alkyl, acyl, heterocyclyl, aryl, heteroaryl, aralkyl, heteroarylalkyl, alkenyl,and alkcynyl, each having one to 20 carbon atoms, which alkyl, acyl, heterocyclyl, aryl, heteroaryl, aralkyl, heteroarylalkyl, alkenyl,and allcynyl,are optionally substituted with from I to 3 substituents independently selected from the group consisting of halo,. aryl, 12 heteroaryl, heterocyclyl, R" 2

SR

20

SOW)R

2 S0 2

R

21 S0NlR S0N 2 0R S0 2

NR'

0 C0NR 20

R

3 S0 2

NR

20 C0 2

R

21

NR

2

&R

23

NR

20 COR 21

NR

20 C0 2

R

21

NR

20 CoiR2OR 23

N(R

20

)C(NR

20 )NH;lR 23

NR

2 0 S0 2

R

2 1 OR 20

OCONR

20 R OOONR 20 SO2R 2 1 OCONR 20 'R 2

CN,

C0 2

R

20

CONR

20 R13, C0NR 20

SO

2 R 2 and C0R 2 0 with the proviso that when Y is carbonyl, Y and together may be a single oxygen atom, R 4 and R.

5 together may be a single oxygen atom, and may together be a single oxygen atom, and wherein when R 3 is 2hydxoxyethyl amino and R, is methyl, R 1 is not amino, 3-methyl-2-butenylamino, benzylamnino, or m-hydroxybenzylamino, when R 3 is not 2-hydroxyethylamino, when R, is isopropyl, is not benzylamino, m-hydroxybenzylamino, or 3-methylbutylamino, when

R

3 is 2-hydroxyethylamino and is 2-hydroxyethyl, R, is not benzylamino and when R 3 is selected from the group consisting of 2-methyl-2-hydroxypropylamino, and 2dimethylaminoethylamino, and when R 2 is methyl, then R 1 is not benzylamino.

In the compositions, R" 0 is a member selected from the group consisting of H, alkyl, alkenyl, alkcynyl, heterocyclyl, aryl, and heteroaryl, which ailcyl, alkenyl,, alkynyl, heterocyclyl, aryl, and heteroaryl are optionally substituted with 1 to 3 substituents independently selected from halo, alkyl, mono- or dialkylamino, alkyl or aryl or heteroaryl amide, ON, 0-01. alkyl, OF 3 aryl, and heteroaryl.

Also in the compositions, R 1 is a member selected from the group consisting of alkyl, 02.,5 alkenyl, C2.1, alkcynyl, heterocyclyl, aryl, and heteroaryl, which alkyl, alkenyl, alkcynyl, aryl, heterocyclyl, and heteroaryl are optionally substituted with 1 to 3 substituents independently selected from the group of halo, heterocyclyl, aryl, heteroaryl, OF 3 ON, OR" 0

N(R

20 2

S(O)R

22

SO

2 R22, SOXNR 2 S0 2

NR

20 00R 2 2 S0 2

NR

2 0 00 2 R 2

SO

2

NR

20 C0N(R 2 0 2

N(R

20 2

NR

2 0 00R 22

NR

20

CO

2 R22, NR? 0 00N(R 20 2

NR

2 0

C(NR

2 O)NKM3, 13 CopR 2 1, C0R 0

O(R

2 0

CONTR

2 0 1SO,R 22

NR

20

SO

3

R

22 SON 2

OR

22

OR

20

OCONR

2 0

SO,R

22 OC(O)R 20

C(O)OCHOO(O)R

0 and OCON(R 2 and each optional heteroaryl, aryl, and heterocyclyl substituent is optionally substituted with halo, alkyl, OF 3 amino, mono- or di- alkylamino, alkyl or aryl or heteroaryl amide, NCOR 22

NR

20 SO,R 22

COR'

0

COR

20

CON(R

2

NR

20 C0NR 0 2

OC(O)R

0

OC(O)N(R

20 2

SR

20

S(O)R

2 S0 2 R 22

SO

2 N(R 2 0 2 ON, or OR 20 In the compositions, R 2 2 is a member selected from the group consisting of alkyl, 02.15 alkenyl, alkynyl, heterocyclyl, aryl, and heteroaryl, which alkyl, alkenyl, alkynyl, heterocyclyl, aryl, and heteroaryl are optionally substituted with 1 to 3 substituents independently selected from halo, alkyl, mono- or dialkylamino, alkyl or aryl or heteroaryl amide, ON, alkyl, OF3, aryl, and heteroaryl, and is R or H.

is preferably an aryl, substituted aryl, each having 6 carbon atoms wherein substitution includes optional substitution with from 1 to 2 substituents independently selected from the group consisting of halo, OF3, aryl, R22, Nl 0 R?3 NR 20 C0R 21 OR 20 ON; an aralkyl, substituted arailcyl, each having 6-8 carbon atoms wherein substitution includes optional substitution with from 1 to 2 substituents independently selected from the group consisting of halo, OF3, aryl, R 22

NR?

0

R

2 3

NR

20 C0R 21

OR

20 ON; -CH,-phenyl wherein the phenyl ring is optionally substituted with from I to 2 substituents independently selected from the group consisting of halo, OF3, R 22

OR"

0 ON; substituted pyridylalkyl; unsubstituted pyridylalkyl; pyraidyl; substituted pyridyl; and benzyl substituted with a halogen, alkoxy, phenyl, pyridyl or nitro group. In some compositions, is preferably OH 2 aryl or 0112 substituted aryl. Most preferably, is selected from 3-methylthiophenyl, 4methylthiophenyl, 4-phenylbenzyl, 4-methoxybenzyl, 4-biphenyl, 3-methoxybenzyl, 4-(2thienyl)benzyl, 4-(4-methyl)phenylbenzyl, 4-(4-trifluoromethyl)phenylbenzyl, 4-(4nitrilo)phenylbenzyl, 4-(2-pyridinyl)benzyl, piperonyl, 3-methoxbenzyl, 4-chlorobenzyl, and 4-nitrobenzyl.

R

2 is preferably hydrogen or a hydrocarbon selected from the group substituted lower alkyl, cycloallcyl, substituted cycloalcyl each having one to 6 carbon atoms wherein substitution includes optional substitution with from 1 to 2 substituents independently selected from the group consisting of halo, R 22 NR'cOR 2

OR

20 substitUted alkyl, cycloalkyl, substituted cycloalkyl each having one to 6 carbon atoms wherein substitution includes optional substitution with from 1 to 2 substituents independently selected from the group i0 consisting of halo, R 22

NR

2 and OR 20 More preferably, R2 is isopropyl.

R

3 is preferably -NR 4 R, wherein R, and R, are each selected from the group consisting of hydrogen, ailkyl, heterocyclyl, acyl, aryl, heteroaryl, aralkyl, heteroaralkyl, alkyl alkenyl, alkyl alkynyl, each having one to 20 carbon atoms, which ailkyl, acyl, heterocyclyl, aryl, heteroaryl, aralkyl, heteroarvlalkyl,are optionally substituted with from 1 to 3 substituients independently selected from the group consisting of halo, aryl, heteroaryl,heterocyclyl, R 22

SR

20

S(O)R

2 1

SOR

21

SONR

0

R

2 3 Nle&R2 NR 20 C0R 1

NR

20 C0 2

R

1

NR

20

CONR

20 R21,

NR'

0

SO,R

1

OR

20 CN, CO,R 0

CONR

20 R'3, and COR 2 Q. In some compositions, and R, are preferably each H, -CH 2

CH

2 OH -CHR'CHOH, or -CH,CHR' OH, -CH.,CH,NH,,

CHR'CH.NH

2

-CH,CHR'NR

2 wherein R' is hydrogen or alkyl having from I to 6 carbon atoms.

R"

0 is preferably a member selected from the group consisting of H, C,.

8 alkyl.

R

2 is preferably a member selected from the group consisting of C, 3 alkyl, which alkyl is optionally substituted with 1 to 2 substituents independently selected from the group of halo,

OF

3 ON, OR 20

SR

20

N(R

20 2

R

22 is preferably a member selected from the group consisting of 0 1 3 alkyl, aryl, heteroaryl which alkyl, aryl, and heteroaryl are optionally substituted with 1 to 3 substituents independently selected from halo, ailkyl, mono- or dialkylamino, alkyl or aryl, ON, O-CIalkyl, and CF 3 Preferred 2,6,9-trisubstuted purine compositions of this invention include hydroxyethyl)[9-(methylethyl)-6-( {[4-(trifluoromethyl)phenyi~methyl I amino)purin-2yl]aniino ethan- 1 -ol, {((2S)oxolan-2-yl)methyl](6- [(4-fluorophenyl)methyl] amino)} -9- (methylethyl)purin-2-yl)anmjne, [((2R)oxolan-2-yl)methyl)(6- fluorophenyl)methyl] amino)} -9-(methylethyl)purin-2-yI)amine, (2-aminoethyl)(6- dichlorophenyl)methyflamino -9-(methylethyl)purin-2-yl)amine, (2-aminoethyl)[6-( chloro-3-(trifluoromethyl)phenyl~methyl) arnino)-9-(methylethyl)purin-2-yljamine, chlorophenyl)methyl]amnino -9-(methylethyl)purin-2-yl) amino] -3 -methylbutanamide, (2amino-2-methylpropyl)(6- [(4-chlorophenyl)methyllamino -9-(methylethyl)purin-2yl)an-ine, 3 -(2-[bis(2-hydroxyethyl)aminoj-6- f [4-chlorophenyl)methyl] amino) purin-9yl)butan-2 -one, [(4-chlorophenyl)methyl]axnino -9-(methylethyl)purin-2-yl)amino]-3-methylbutan- 1 ol, {2-[(2-aminoethyl)amino]-9-(methylethyl)purih-6yl) am aino)methyl]benzenesulfonamide, 2-[(2-hydroxyethyl)(6- rnethoxyphenyl)methyl] amino)} -9-(methylethyl)purin-2-yl)aznino]ethan- I -ol, 2(2 hydroxyethyl) methylethyl)-6-[(4-phenylphenyl)anino]puin-2-yl} amino)ethan-l1-ol, 16 [(2-amino-2-propyl)amino] -9-(methylethyl)purin-6-yl} [(4-hlorophenyl)methyl]amine, {2-[(2-aminoethyl)amino]-9-( methylethyl)purin-6-yl }[(4-chlorophenyl)methyl]amine, [(2-aminopropyl)arnino]-9-( methylethyl)purin-6-yl }[(4-chlorophenyl)methyl~amine and 2-[(2-aminoethyl)(6- [(4-chlorophenyl)methyl]amino -9-(methylethyl)purin-2yl)am-inojethan- 1 -ol.

There are some limitations to the scope of R 1 R, and R 3 As mentioned above, when R 3 is 2-hydroxyethylamino and R 2 is methyl, R 1 cannot be amino, 3 -methyl-2-butenyl amino, benzylamino, or m-hydroxybenzyl-amino. When R, is 2-hydroxyethylamino and R 2 is isopropyl, R 1 cannot be benzylamino, mhydroxybenzylamino, or 3-methylbutylamino. When R 3 is 2-hydroxyethylamnino and R, is 2-hydroxyetbyl, R 1 cannot be benzylamino. When R 3 is 2-propanol-2-methylamino or 2-dimethylaminoethylamino and R2 is methyl, R 1 cannot be benzylaxnino.

The following are definitions for certain terms used herein.

"Halo" or "Halogen" alone or in combination means all halogens, that is, chioro (Cl), fluoro bromo iodo "Hydroxyl" refers to the group -OH.

"Thiol" or "mercapto" refers to the group -SH.

"Alkyl" alone or in combination means an alkane-derived radical containing from I to preferably 1 to 15, carbon atoms (unless specifically defined). It is a straight chain alkyl, branched alkyl or cycloalkyl. Preferably, straight or branched alkyl groups containing from 1 more preferably 1 to 8, even more preferably 1-6, yet more preferably 1-4 and most preferably 1-2, carbon atoms, such as methyl, dthyl, propyl, isopropyl, butyl, t-butyl and the like. The term "lower ailkyl" is used herein to describe the straight chain ailkyl groups described immediately above. Preferably, cycloalkyl groups are monocyclic, bicyclic or tricyclic ring systems of 3-8, more preferably 3-6, ring members per ring, such as cyclopropyl, cyclopentyl, cyclohexyl, adamantyl and the like. Ailkyl also includes a straight chain or branched alkyl group that contains or is interrupted by a cycloalkyl portion. The straight chain or branched alkyl group is attached at any available point to produce a stable compound.

Examples of this include, but are not limited to, 4-(isopropyl)-cyclohexylethyl or 2-methylcyclopropylpentyl. A substituted alkyl is a straight chain alkyl, branched alkyl, or cycloaikyl group defined previously, independently substituted with 1 to 3 groups or substituents of halo, hydroxy, alkoxy, allcylthio, alkylsulfinyl, alkylsulfonyl, acyloxy, aryloxy, heteroaryloxy, amino optionally mono- or di-substituted with ailkyl, aryl or heteroaryl groups, amidino, urea optionally substi tuted with alkyl, aryl, heteroaryl or heterocyclyl groups, amninosulfonyl optionally N-mono- or N,N-di-substituted with alkyl, aryl or heteroaryl groups, alkylsulfonylamino, arylsulfonylaxnino, heteroarylsulfonylarnino, alkylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, or the like.

"Alkenyl" alone or in combination means a straight branched, or cyclic hydrocarbon containing 2-20, preferably 2-17, more preferably 2-10, even more preferably 2-8, most preferably 2-4, carbon atoms and at least one, preferably 1-3, more preferably 1-2, most preferably one, carbon to carbon double bond. In the case of a cycloalkyl group, conjugation of more than one carbon to carbon double bond is not such as to confer aromaticity to the ring.

Carbon to carbon double bonds may be either contained within a cycloalkyl portion, with the exception of cyclopropyl, or within a straight chain or branched portion. Examples of alkenyl groups include ethenyl, propenyl, isopropenyl, butenyl, cyclohexenyl, cyclohexenylalkyl and the like. A substituted alkenyl is the straight chain alkenyl, branched alkenyl or cycloalkenyl 18 group defined previously, independently substituted with 1 to 3 groups or substituents of halo, hydroxy, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, acyloxy, aryloxy, heteroaryloxy, amino optionally mono- or di-substituted with alkyl, aryl or heteroaryl groups, amidino, urea optionally substituted with alkyl, aryl, heteroaryl or heterocyclyl groups, aminosulfonyl optionally N-mono- or N,N-di-substituted with alkyl, aryl or heteroaryl groups, alkylsulfonylamino, arylsulfonylamino, heteroarylsulfonylamino, allcylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino,' carboxy, alkoxycarbonyl, aryloxycarbonyl, heteroaryloxycarbonyl, or the like attached at any available point to produce a stable compound.

"Alkynyl" alone or in combination means a straight or branched hydrocarbon containing 2-20, preferably 2-17, more preferably 2-10, even more preferably 2-8, most preferably 2-4, carbon atoms containing at least one, preferably one, carbon to carbon triple bond. Examples of alkynyl groups include ethynyl, propynyl, butynyl and the like. A substituted alkynyl refers to the straight chain aikynyl or branched alkenyl defined previously, independently substituted with I to 3 groups or substituents of halo, hydroxy, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, acyloxy, aryloxy, heteroaryloxy, amino optionally mono- or disubstituted with alkyl, aryl or heteroaryl groups, amidino, urea optionally substituted with alkyl, aryl, heteroaryl or heterocyclyl groups, amninosulfonyl optionally N-mono- or N,N-disubstituted with alkyl, aryl or heteroaryl groups, alkylsulfonylanuno, arylsulfonylamino, heteroarylsulfonylamino, alkylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, or the like attached at any available point to produce a stable compound.

"Alkyl alkenyl" refers to a group R"7, where R is lower alkyl, or substituted lower alkyl, R""may independently be hydrogen, halogen, lower alkyl, 19 substituted lower alkyl, acyl, aryl, substituted aryl, hetaryl, or substituted hetaryl as defined below.

",Alkyl alkynyl" refers to a groups -RC=-CR' where R is lower alkyl or substituted lower alkyl, R' is hydrogen, lower alkyl, substituted lower alkyl, acyl, aryl, substituted aryl, hetaryl, or substituted hetaryl as defined below.

"Alkoxy" denotes the group -OR, where R is lower alkyl, substituted lower ailkyl, acyl, aryl, substituted aryl, aralkyl, substituted aralkyl, heteroalkyl, heteroarylalkyl, cycloalkyl, substituted cycloalkyl, cycloheteroalcyl, or substituted cycloheteroalkyl as defined.

"Allcylthio" denotes the group -SR, 2 where R is lower alkyl, substituted lower alkyl, aryl, substituted aryl, aralkyl or substituted aralkyl as defined herein.

"Acyl" denotes groups where R is hydrogen, lower alkyl substituted lower ailkyl, aryl, substituted aryl and the like as defined herein.

"Axyioxy" denotes groups -OAr, where Ar is an aryl, substituted aryl, heteroaryl, or substituted heteroaryl group as defined herein.

"Amino" denotes the group NRR', where R and R' may independently by hydrogen, lower alkyl, substituted lower alkyl, aryl, substituted aryl, hetaryl, or substituted hetaryl as defined herein or acyl.

"Amido" denotes the group -C(O)NRR', where R and R' may independently by hydrogen, lower alkyl, substituted lower alkyl, aryl, substituted aryl, hetaryl, substituted hetaryl as defined herein.

"Carboxyl" denotes the group -C(O)OR, where R is hydrogen, lower ailkyl, substituted lower alkyl, aryl, substituted aryl, hetaryl, and substituted hetaryl as defined herein.

"Aryl" alone or in combination means phenyl or naphthyl optionally carbocyclic fused with a cycloalkyl of preferably 5-7, more preferably 5-6, ring members and/or optionally substituted with 1 to 3 groups or substituents; of halo, hydroxy, alkoxy, allcylthio, alkylsulfinyl, alkylsulfonyl, acyloxy, aryloxy, heteroaryloxy, amino optionally mono- or disubstituted with alkyl, aryl or heteroaryl groups, amidino, urea optionally substituted with alkyl, aryl, heteroaryl or heterocyclyl groups, aminosulfonyl optionally N-mono- or N,N-disubstituted with alkyl, aryl or heteroaryl groups, alkylsulfonylamino, arylsulfonylamino, hetero arylIsulfonyl amino, alkylcarbonylamino, arylcarbonylaunino, heteroarylcarbonylaniino, or the like.

"Substituted aryl" refers to aryl optionally substituted with one or more functional groups, halogen, lower alkyl, lower alkoxy, alkylthio, acetylene, amino, amido, carboxyl, hydroxyl, aryl, aryloxy, heterocycle, hetaryl, substituted hetaryl, nitro, cyano, thiol, sulfamido and the like.

"Heterocycle" refers to a saturated, unsaturated, or aromatic carbocyclic group having a single ring morpholino, pyridyl or furyl) or multiple condensed rings naphthpyridyl, quinoxalyl, quinolinyl, indolizinyl or benzo[b]thienyl) and having at least one hetero atom, such as N, 0 or S, within the ring, which can optionally be unsubstituted or substituted with, halogen, lower alkyl, lower alkoxy, alkylthio, acetylene, amino, aunido, carboxyl, hydroxyl, aryl, aryloxy, heterocycle, hetaryl, substituted hetaryl, nitro, cyano, thiol, sulfamido and the like.

"Heteroaryl" alone or in combination means a monocyclic aromatic ring structure containing 5 or 6 ring atoms, or a bicyclic aromatic group having 8 to 10 atoms, containing one or more, preferably 1-4, more preferably 1-3, even more preferably 1-2, heteroatoms 21 independently selected from the group 0, S, and N, and optionally substituted with 1 to 3 groups or substituents of halo, hydroxy, alkoxy, alkylthio, alkylsulfinyl, alkylsulfonyl, acyloxy, aryloxy, heteroaryloxy, amino optionally mono- or di-substituted with ailcyl, aryl or heteroaryl groups, aniidino, urea optionally substituted with alkyl, aryl, heteroaryl or heterocyclyl groups, aniinosulfonyl optionally N-mono- or N,N-di-substituted with ailkyl, aryl or heteroaryl groups, alkylsulfonylamino, arylsulfonylamino, heteroarylsulfoflylamlflo, allcylcarbonylamino, arylcarbonylamino, heteroarylcarbonylamino, or the like. Heteroaryl is also intended to include oxidized S or N, such as sulfinyl, sulfonyl and N-oxide of a tertiary ring nitrogen. A carbon or nitrogen atom is the point of attachment of the heteroaryl ring structure such that a stable aromatic ring is retained. Examples of heteroaryl groups are pyridinyl, pyridazinyl, pyrazinyl, quinazolinyl, purmnyl, indolyl, quinolinyl, pyrimidinyl, pyrrolyl, oxazolyl, thiazolyl, thienyl, isoxazolyl, oxathiadiazolyl, isotbiazolyl, tetrazolyl, imidazolyl, triazinyl, furanyl, benzofuiryl, indolyl and the like. A substituted heteroaryl contains a substituent attached at an available carbon or nitrogen to produce a stable compound.

"Heterocyclyl" alone or in combination means a non-aromatic cycloalkyl group having from 5 to 10 atoms in which from I to 3 carbon atoms in the ring are replaced by heteroatoms of 0, S or N, and are optionally benzo fused or fused heteroaryl of 5-6 ring members and/or are optionally substituted as in the case of cycloalkyl. Heterocycyl is also intended to include oxidized S or N, such as sulfinyl, sulfonyl and N-oxide of a tertiary ring nitrogen. The point of attachment is at a carbon or nitrogen atom. Examples of heterocyclyl groups are tetrahydrofuranyt, dihydropyridinyl, piperidinyl, pyrrolidinyl, piperazinyl, dihydroberizofuryl, dihydroindolyl, and the like. A substituted hetercyclyl contains a substituent nitrogen attached at an available carbon or nitrogen to produce a stable compound.

"Substituted heteroaryl" refers to a heterocycle optionally mono or poly substituted with one or more functional groups, halogen, lower alkyl, lower alkoxy, alkylthio, acetylene, amino, anido, carboxyl, hydroxyl, aryl, aryloxy, heterocycle, substituted heterocycle, hetaryl, substituted hetaryl, nitro, cyano, thiol, sulfamido and the like.

"Aralkyl" refers to the group -R-Ar where Ar is an aryl group and R is lower alkcyl or substituted lower alkyl group. Aryl groups can optionally be unsubstituted or substituted with, halogen, lower alkyl, alkoxy, alkylthio, acetylene, amino, amido, carboxyl, hydroxyl, aryl, aryloxy, heterocycle, substituted heterocycle, hetaryl, substituted hetaryl, nitro, cyano, thiol, sulfamido and the like.

"Heteroalkyl" refers to the group -R-Het where Het is a heterocycle group and R is a lower alkyl group. Heteroalkyl groups can optionally be unsubstituted or substituted with halogen, lower alkyl, lower alkoxy, allcylthio, acetylene, amino, amido, carboxyl, aryl, aryloxy, heterocycle, substituted heterocycle, hetaryl, substituted hetaryl, nitro, cyano, thiol, sulfarnido and the like.

"Heteroarylalkyl" refers to the group -R-HetAr where HetAr is an heteroaryl group and R lower alkyl or substituted lower alkyl. Heteroarylalkyl groups can optionally be unsubstituted or substituted with, halogen, lower alkyl, substituted lower alkyl, alkoxy, alkylthio, acetylene, aryl, aryloxy, heterocycle, substituted heterocycle, hetaryl, substituted hetaryl, nitro, cyano, thiol, sulfamido and the lie.

"Cycloalkyl" refers to a divalent cyclic or polycyclic alkyl group containing 3 to carbon atoms.

"Substituted cycloalkyl" refers to a cycloalkyl group comprising one or more substituents with, halogen, lower alkyl, substituted lower alkyl, alkoxy, allcylthio, acetylene, aryl, aryloxy, heterocycle, substituted heterocycle, hetaryl, substituted hetaryl, nitro, cyano, thiol, sulfamido and the like.

"Cycloheteroalkyl" refers to a cycloalkyl group wherein one or more of the ring carbon atoms is replaced with a heteroatom N, 0, S or P).

"Substituted cycloheteroalkyl" refers to a cycloheteroalkyl group as herein defined which contains one or more substituents, such as halogen, lower alkyl, lower alkoxy, alkylthio, acetylene, amino, amido, carboxyl, hydroxyl, aryl, aryloxy, heterocycle, substituted heterocycle, hetaryl, substituted hetaryl, nitro, cyano, thiol, sulfamido and the like.

"Alkyl cycloalkyl" denotes the group -R-cycloalkyl where cycloalkyl is a cycloalkyl group and R is a lower alkyl or substituted lower alkyl. Cycloalkyl groups can optionally be unsubstituted or substituted with e.g. halogen, lower alkyl, lower alkoxy, alkylthio, acetylene, amnino, amido, carboxyl, hydroxyl, aryl, aryloxy, heterocycle, -substituted heterocycle, hetaryl, substituted hetaryl, itro, cyano, thiol, sulfamido and the like.

"Alkyl cycloheteroalkyl" denotes the group -R-cycloheteroalcyl where R is a lower alkyl or substituted lower alkyl. Cycloheteroalkyl groups can optionally be unsubstituted orsubstituted with e.g. halogen, lower alkyl, lower alkoxy, alkylthio, amino, amido, carboxyl, acetylene, hydroxyl, aryl, aryloxy, heterocycle, substituted heterocycle, hetaryl, substituted hetaryl, itro, cyano, thiol, sulfamido and the like.

"Pharmacologically acceptable salt" a salt prepared by conventional means, and are well known by those skilled in the art. The "pharmacologically acceptable salts" include basic salts of inorganic and organic acids, including but not limited to hydrochloric acid, 24 hydrobromic acid, sulphuric acid, phosphoric acid, methanesuiphonic acid, ethanesulfonic acid, malic acid, acetic acid, oxalic acid, tartaric acid, citric acid, lactic acid, fumaric acid, succinic acid, maleic acid, salicylic acid, benzoic acid, phenylacetic acid, mandelic acid and the like. When compounds of formula I-II include an acidic function such as a carboxy group, then suitable pharmaceutically acceptable cation pairs for the carboxy group are well kniown to those skilled in the art and include alkaline, alkaline earth, ammnonium, quaternary ammonium cations and the like. For additional examples of the types of compounds that are "pharmacologically acceptable salts," see Ber-ge et al, Pharm. Sci. 66, 1 (1977).

If the final 2,6,9-trisubstituted purine compound of this invention contains a basic group, then an acid addition salt of the composition may be prepared. Acid addition salts of the compounds of this invention are prepared in a standard manner in a suitable solvent from the parent compound and an excess of acid, such as, but not limited to, hydrochloric, hydrobroinic, sulfuric, phosphoric, acetic, maleic, succinic, citric or methanesulfonic. The hydrochloric salt form is especially useful.

is If the final 2,6,9-trisubstituted purine compound contains an acidic group, then cationic salts of the composition may be prepared. Typically the acidic parent compound is treated with an excess of an alkaline reagent, such as, but not limited to, hydroxide, carbonate or alkoxide, containing the appropriate cation such as but not limited to, Nat, Ca" 2 and NHl' Certain of the compounds form inner salts or zwitterions which are also acceptable.

The compounds of this invention are useful in inhibiting cell proliferation in mammals including humans. The 2,6,9-trisubstituted purines are useful in for example in treating autoimmune diseases, e.g. rheumatoid arthritis, lupus, type I diabetes, multiple sclerosis, cancer, cardiovascular disease such as restenosis following balloon angioplasty and atherectomy, restensosis following vascular modifying surgical procedures, host vs graft disease, gout, polycystic kidney disease and other proliferative diseases whose pathogenesis involves abnormal cell proliferation. In particular, the compositions of this invention are useful in treating cancers including cancers including lymphoyd neoplasm, cancer of the colon, breast cancer, ovarian cancer, pancreatic cancer, and cancers derived of endotherical cells.

The method of treatment comprises the administration parenterally, and orally, of an effective quantity of the chosen compound of this invention, preferably dispersed in a pharmaceutical carrier. Therapeutically useful amounts of the composition of this invention will generally range from about 0.01 to about 100 mg/kg, but will be readily determined by one skilled in the art depending upon the route of administration, and the age and condition of the patient. Therapeutically useful amounts of the composition of this invention may be administered from one to ten times daily or more for acute or chronic disease. No unacceptable toxicological effects are expected when compounds of the invention are administered in accordance with the present invention.

The compounds of this invention are also useful as antiinflammatory and antifungal agents. As such, the compositions of this invention are useful for treating antiinflammatory and fungal infections in humans, animals, and fungal infections in plants.

Pharmaceutical compositions including the compounds of this invention, and/or 26 derivatives thereof, may be formulated as solutions or lyophilized powders for parenteral administration. Powders may be reconstituted by addition of a suitable diluent or other pharmaceutically acceptable carrier prior to use. If used in liquid form the compositions of this invention are preferably incorporated into a buffered, isotonic, aqueous solution.

Examples of suitable diluents are normal isotonic saline solution, standard 5% dextrose in water and buffered sodium or ammonium acetate solution. Such liquid formulations are suitable for parenteral administration, but may also be used for oral administration.

It may be desirable to add excipients such as polyvinylpyrrolidinone, gelatin, hydroxycellulose, acaia, polyethylene glycol, mannitol, sodium chloride, sodium citrate or any other excipient known to one of skill in the art to pharmaceutical compositions including compounds of this invention. Alternatively, the pharmaceutical compounds may be encapsulated, tableted or prepared in an emulsion or syrup for oral administration.

Pharmaceutically acceptable solid or liquid carriers may be added to enhance or stabilize the composition, or to facilitate preparation of the composition. Liquid carriers include, but are not limited to syrup, peanut oil, olive oil, glycerin, saline, alcohols and water. Solid carriers include, but are not limited to, starch, lactose, calcium sulfate, dihydrate, teffa alba, magnesium stearate or stearic acid, talc, pectin, acacia, agar or gelatin. The carrier may also include a sustained release material such as, but not limited to, glyceryl monostearate or glyceryl distearate, alone or with a wax. The amount of solid carrier varies but, preferably, will be between about 20 mg to about 1 gram per dosage unit.

The pharmaceutical dosages are made using conventional techniques such as, but not limited to, milling, mixing, granulation, and compressing, when necessary, for tablet forms; or milling, mixing and filling for hard gelatin capsule forms. When a liquid carrier is used, 27 the preparation will be in the form of a syrup, elixir, emulsion or an aqueous or non-aqueous suspension. Such a liquid formulation may be administered directly or filled into a soft gelatin capsule.

The Examples which follow serve to illustrate this invention. The Examples are intended to in no way limit the scope of this invention, but are provided to show how to make and use the compounds of this invention. In the Examples, all temperatures are in degrees Centigrade. RT indicates room temperature.

EXAMPLE 1 The compounds of this invention are prepared by conventional methods of organic chemistry. The reaction sequence outlined in the synthesis scheme below is a general method useful for the synthesis of compounds of this invention. 2,6-dichloropurine is dissolved in butanol and the appropriate R, amine is added. After heating for several hours, the reaction mixture is cooled, and the compound 1 is obtained. To compound 1, is added, sodium hydride followed by and compound 2 is isolated. To compound 2, R 3 is added in solution with N-methylpyrrolidinone. The mixture is heated for an appropriate period followed by purification leading to the desired compound.

Ci

R-

C, N butanol, A c NaH R, N N-methylpyrrolidinone cl-, N r

N

R

2 R2 3 2 The following compound was prepared according to the method above.

Preparation of 2-chloro-6-(4-methoxybenzylamino) purine The 2,6-dichloropurine (4.06 g, 21.5 mmol) was suspended in n-butanol (150 ml) and the 4-methoxybenzylamine was added (3.4 ml, 26 mmol). The solution turned clear and then cloudy a few minutes later. The solution was heated at 120°C for 2 hr and then cooled. The n-butanol was evaporated followed by suspension of the residue in water and diethyl ether mixture. A solution of 2N NaOH (1 .3m1, 26 mniol) wasi added and the solution stirred for min before filtration. The filtered precipitate was washed with water and a small portion of ether and then dried under vacuum. The residual liquor was left overnight and more crystals were collected the next day and washed with diethyl ether.

Preparation of 2-chloro-6-(4-methoxybenzylamin o)-9-isopropylp urinle (2) 2-chloro-6-(4-methoxybenzylamino) purine was suspended in dry DMF (5 ml) and treated with sodium hydride, 60% dispersion (82 mg, 2.06 mmol). The suspension was stirred for 30 min over which time it became a clear yellow/green solution. 2-Iodopropane (0.280 mL, 1.7 eq.) was added over 5 min and the resultant solution stirred for 2 days. Water was added and the solution and extracted with ethyl acetate. The organic layer was evaporated to give the product isopropyl purine.

Preparation of 2-diethanolamino-6-(4-methoxybenzylamino)-9-isopropylpurile, The purine (1.65g, 4.98 mmol) was dissolved in DMSO (12 mL) and diethanolamine (4 mL) and then heated at 140*C for 2-3 days and then at 1601C for 1 day. The solution was cooled and water saturated butanol was added (100 mL). The solution was then washed with water (3 x 50 mL), before being evaporated to give a brown oil. The residue was chrornato graphed over silica gel eluting with ethyl acetate, followed by 3% methanol in ethyl acetate to give the product. 'H-NNM( CDC13): 7.29(br s 1H), 7.25(d, 2H), 6.94(br s. IH), 6.83(d. 2H), 5.43(br 4.63(br s. 2H), 4.53(m 1H), 3.86(t. 4H), 3.76(m, 7H), 1.47(d 6H).

Table 1 identifies compounds of this invention that were prepared according to the synthesis method set forth in this Example.

ITABLE 1 Cornpoun s Prepared By The Method of Example 1

R

1 X (4-methoxyphenyl)methylamino 3-cyanopropyl Cl (4-methoxyphenyl)methylamino 3-chloropropyl c I (4-methoxyphenyl)methylamino benz1 ClI (4-methoxyphenyl)rnethylamino (4-methylcarboxyphenyl)methyl Cl (4-methoxyphenyl)methylamino 2-(N-phtyhaloyl)ethyl Cl (4-rnethoxyphenyl)methylamino isopropyl Ethanolan-dne (4-methoxyphenyl)methylamino isopropyl Diethanolammne (4-methoxyphenyl)rnethylarnino 3-inethylbutyl clI (4-methoxyphenyl)methylamino 2-methylbutyl Cl (4-methoxyphenyl)methylaniino cyclopentyl c I (4-methoxyphenyl)methylamino (3-nitrophenyl)methyl clI (4-methoxyphenyl)methylamino (4-nitrophenyl)methyl Cl (4-methoxyphenyi)methylamino ethyl Cl (4-methoxyphenyl)methylainino propyl

I

(4-methoxyphenyl)rnethylamino (3-methylphenyl)methyl c 1 (4-methoxyphenyi)methylamino (4-methylphenyl)rnethyl c heptylamino H ClI N-benzyl-N-hydroxylamino H Cl propylamino H clI noradarnantylammo H ClI cyclobutylamino H clI 3-methoxypropylamino H Cl 2-methoxyethylamino H ClI cyclapentylaxnzno H ClI I -hydroxy-2-methyl-2- H ClI propylamiuo I-benzylpipenidinyl)-4-annno H ClI heptylamino Methyl ClI

R

1 X R, R,21 N-benzyi-N-hydroxyl mmo Methyl Cl propylaniino Methyl Cl noradamantylamuno Methyl Cl cyclobutylammno Methyl Cl 3-methoxypropylainino Methyl Cl 2-rnethoxyethylamino Methyl Cl cyclopentylamino Methyl Cl I -hydroxy-2-methyl-2- Methyl Cl propylaino I -benzylpipenidinyl)-4-ammno Methyl Cl Methyl Cl diinethoxyphenvl)methylarnino________________ (2-methoxyphenyl)methylamino H Cl (2-pryidinyl)methylamino H Cl (3,4-dimethoxyphenyl)ethylamino H Cl (3-pyridinyl)methylamino H Cl (4-pyridinyl)methylamrino H Cl 6-bydroxy- I-hexylammno H C I phenethylamino H ClI (2.benzothiazolyl)amino H Cl H Cl dirnethoxyphenyl)methyalrnino (2-metboxyphenyl)rnethylamino Methyl ClI (2-pyridinyl)methylaxmno Methyl Cl (3,4-ditnethoxyphenyl)ethylarniino Methyl clI WO 00/44750 RI R2R (4-methoxyphenyl)xuethylamino Methyl Cl (3-pyridinyl)methylarmno isopropyl 2-aiaoethylamino H Cl l-hydroxy-6-hexylamino H ClI phenethylamino H clI (2-benzothiazolyl)qmino H clI (4-methoxyphenyl)methyaihno H c I 3-phenyl- I -propylaino isopropyl 3-hydroxypyrrolidino (2-indanyl)amino H ClI (4-methoxyphenyl)ethylano H C I (4-nitrophenyl)methylamino H C I (2,6-difluorophenyi)methylamino H ClI 3-phenyl-lI-propylaxnino H Cl (2-indanyl)aino Methyl ClI (4-methoxyphenyl)ethylammio Methyl ClI (4-nitrophenyl)methylamino Methyl C I (2,6-difluorophenyl)methylamino Methyl c I cyclopropyimethylamino Methyl clI 4-(1,2-methylenedioxyphenyl) H Cl1 nmthylamno H ClI aminosulfonylphenyl)methylammno (cyclohexanol)- I-methylamino H Cl (2-benzimidazoly)methylamino H Cl cyclohexylmnethylarnino H Cl1 (4-methoxyphenyl)methylamino H Cl R,-X 1, (4-methoxyphenyl)methylamino isopropyl (2-hydroxy- I hydroxyrnethyl)ethylanaino cyclopropyimethylarnino isopr opyl 3-amino-2-hydroxypropylamilo 4-(l 2-rnethylene- Methyl cl dioxyphenyl)rnethylaiio (4-axninosulfonyl- Methyl Cl phenyl)methylamino________________ (cyclohexanol)- 1 -methylamino Methyl c I (2-benziiniidazolyl)methylamfino Methyl c cyclohexyiniethylamino Methyl (3-pyridinyl)nmethylamnlfo Methyl

I

(4-pyridinyl)methylamino 2-methyipropyl 1 6-hydroxyhexylamino cyclopentyl ClI phenethylamino propyl c 1 (2-benzothiazolyl)axnino ethyl clI 3-phenyl- 1 -propylainino isopropyl Cl (2-indanyl)axnino 2-metbylpropyl Cl 2-(4-methoxyphenyl)ethylamino cyclopentyl Cl (4-nitropbenyl)methylaxniino propyl Cl (2,6-difluorophenyl)methylaxnmo ethyl Cl (4-methoxyphenyl)methylanino isopropyl c I 3-phenyl- 1 -propylamino isopropyl 4-hydroxypiperidino (2-indanyl)amino H Cl 2-(4-metboxyphenyl)ethylarnino H Cl (4-nitrophenyl)methylarnimo H Cl (2,6-difluorophenyl)methylamno H Cl

[,R

(4-methoxyphenyl)methylainino H Cl (4-methoxyphenyl)methylamino isopropyl N-(2-cyanoethyl)-N-benzlano 3-phyenyl-l1-propylam~ino isopropyl 1 -(RS)-hydroxyrnethyl-3methylbutylamnino (2-indanyl)aniino isopropyl Cl 2-(4-Methoxyphenylethylaxnino isopropyl Cl (4-aitrophenyl)methylanaino isopropyl Cl (2,6-difluorophenyl)methylamino isopropyl Cl (4-methoxyphenyl)methylaznino isopropyl Cl (4-mcthoxyphenyl)mnethylamino isopropyl Pipenidino (4-methoxyphenyl)methylaniino isopropyl 3-hydroxypiperidino 3-phenyl- I -propylamnimo isopropyl I -(S)-hydroxymethyl-2-(4'imidazolyl)etbylanmino (2-indanyl)amino isopropyl diethanolainino (4-methyoxyphenyi)methylamino isopropyl diethanolamino (4-methyoxyphenyl)methylamino isopropyl 2-(S)-hydroxymetbylpyrrolidino (4-rnethyoxyphenyl)methylamino isopropyl diethnolanino (4-methyoxyphenyl)methylamino benzyl morpholino (4-methyoxyphenyl)methylamino 3-methylbutyl diethanolainino (4-rnethyoxyphenyl)methylaniino 2-metbylbutyl diethanolainino (4-methyoxyphenyl)methylamino cyclopentyl diethanolainino (4-methyoxyphenyl)methylamino (3-aitrophenyi)methylamino diethanolamino (4-methyoxyphenyl)methylamino (4-nitrophenyl)methylamino diethanolarnino (4-rnethyoxyphenyl)methylamino ethyl diethanolamuino (4-methyoxyphenyl)methylaznino propyl diethanol amino (4-methyoxyphenyl)methylaxnino (3-methylphenyl)methylamino diethanolamino ,heptylarnino, (4-methylphenyl)mnethylaxnino diethanolamino R,-X N-benzyl-N-hydroxyamino Methyl diedhzolammo propylamino Methyl diethanolamino noradamantylamino Methyl diethanolanuno cyciobutylamino Methyl diethanolamino 3-methoxypropylamino Methyl diethanolamino 2-methoxyethylamino Methyl diethanolamino cyclopentylainino Methyl diethanolannno I -hydroxy-2-methyl-2- Methyl diethanolamino propylamino 4-(lI -benzylpiperidinyl)anino Methyl diethanolannno (4-metboxyphenyl)rnethylaxnino Methyl diethanolamino (4-methoxyphenyl)methylamino isopropyl diethanolammno (2,4-dimethoxy-phenyl) isopropyl 3-hydroxypyrrolidino methylanmno (2-methoxyphenyl)methylamino Methyl 2-(3'indolyl)etbylamino (2-pyridinyl)methylaniino Methyl diethanolamino Methyl diethanolamino ditnethoxyphenyl)ethylamino (3-pyridinyl)methylamino Methyl diethaolamino (4-pyridinyl)methylamino Methyl diethanolamino 6-hyclroxy- I -hexylamino Methyl dietbanolzmino phenethylamaino Methyl diethanolamino (2-benzotbiazolyl)arnino Methyl diethanolamino 3-phenyl- I -propylamino Methyl diethanolamino (2-indanyl)amino Methyl diethaolainino 2-(4-methoxyphenyl)ethylamino Methyl diethanolamino (4-nitrophenvl)rnethylaxnino Methyl diethanolamino (2,6-difluorophenyl)methylamino Methyl diedhaoLamino R,-X R,

R,

cyclopropylmethylamino Methyl diethanolamino 1,2-rnethylenedioxy- Methyl diethanolamino phenyl)methylamino (4-aminosulfonylphenyl)- Methyl diethanolarnino methylamino (cyclohexanol)- 1 -methylammuo Methyl diethaolamino (2-benzimidazolyl)methylaniino Methyl diethanolamino cyclohexylmethylaimino Methyl diethanolamiAno (3-pyridyl)methylamino Methyl diethanolainino (4-pyridyl,)methylamino 2-methylpropyl diethanolamino 6-hydroxy- I -hexylanuno cyclopentyl diethanolamino 2-phenethylamino propyl diethanolanino (2-benzothiazolyl)amino ethyl diethanolammno 3-phenyl- I -propyiamino isopropyl diethanolamino (2-indanyl)arnino 2-methyipropyl diethanolamino 2-(4-methoxyphenyl)ethylainino cyclopentyl diethanolarnino (4-nitropbenyl)methylamno propyl diethanoiaino (2,6-difluorophenyl)methylanino ethyl diethaoLamino (4-methyoxyphenylmethylamiino isopropyl diethanolammno (4-methyoxyphenyl)rnethylamino isopropyl 1-hydroxyrnethylcyclopenrylamino (4-methyoxyphenyl)methylamino isopropyl 2-(R.S)-hydroxymethylpiperidino cyclopropylxnethylanaino isopropyl 2,3-dihydroxy- 1 -propylamino 1,2-methylenedioxyphenyl) isopropyl cl1 methylamino (4-aminosulfonylphenyl) isopropyl ClI methylammlo (cyclohexanol)- I -rnethylarnino isopropyl IC 1 R,-X R2 R3 (2-benzimidazolyl)amino isopropyl cl cyclohexyimethylaino isopropyl Cl 3-phenyl- I -propylamino isopropyl Cl cyclopropylmethylamino cyclopentyl Cl 1,2-metbylenedioxyphenyl) isopropyl diethanolammno methylarnio (4-methoxyphenyl)methylamino isopropyl diethanolammo (4-methoxyphenyl)metbylanmmo isopropyl diisopropylamino (4-methoxyphenyl)methylamiino isopropyl (trans-2-hydroxycyclohexyl)immno (4-methoxyphenyl)methylamino isopropyl 1-hydroxy-3- (4-methoxyphenyl)metbylanino isopropyl 5-(S).(2,2-dimethyl-4(S)phenyldioxalanyl)amino (4-methoxyphenyl)methylarnino isopropyl I -imidazolyl)propyianiino (4-methoxyphenyl)methylamino isopropyl 4-hydroxyl-4-phenylpiperidino (4-methoxyphenyl)methylamino isopropyl (2-benzylthio-1 hydroxymethyl)ethylamino (4-methoxyphenyl)methylamino isopropyl N-methyl-N-(2-hydroxy-2-(3,4dihydroxyphenyl)ethyl)amino (4-methoxyphenyl)methylamino isopropyl diallylanino (4-methoxyphenyl)methylaniio isopropyl Piperazmo (4-methoxyphenyl)methylaruno isopropyl (+/-)N-methyl-N-(2-hydroxy-2phenylethyl)aniina (4-methoxyphenyl)methylarnino isopropyl (anilinomethyl)pyrrolidino (4-mttioyphnyi~ethianino soprpyl(+/-)N-(2-propenyl)-N-2-(4- (4-mthoyphnyl~ethlamno ioprpylhydroxy-2-methylpentyl)amiino (4-methoxyphenyl)methylaniino isopropyl N-(2-hydroxyethyl)-N-(3hydroxypropyl)amino R2 R (4-methoxyphenyl)methylamino isopropyl Di-N- 1-(2-hydroxy-2- (4-methoxyphenvl)methylainino isopropyl Di-N-2-(3-hydroxybutyl)amino Example 2 This example describes a method for preparing compounds of this invention according to the following general synthesis scheme:

R

1

X

c N Ethanol, Reflux CHN K2CO3

DMF

RR3H

R

3 N N NMP, 140 o C C R R, R2 Preparation of {2-chloropurin-6-yl} [(4-chlorophenyl)methyl]amine: To a suspension of 15 g (0.0794 mol) of 2,6-dichloropurine in 250 mL of absolute ethanol was added 12.7 mL (0.0873 mol) of triethylamine and 10.62 mL (0.0873 mol) of 4chlorobenzylamine. The mixture was refluxed at 80 °C for 16h (the formation of creamy white precipitate was observed). The reaction mixture was cooled and the precipitated product was removed by filtration. The precipitate was washed with ethanol (350 mL) and dried in high vacuum for 24h. Product was characterized by 'H-NMR.

Preparation of {2-chloro-9-(methylethyl)purin-6-yl} [4-chlorophenyl)methyl] amine: To a solution of 6 g (0.020 imol) of 2-chloro-6-(4-chlorophenyl)methylaminopurine in 41 mL of anhydrous DMF was added 5.64 g (0.041mol) anhydrous. potassium carbonate and 3.41 mL,(0.035 mol of 2-iodopropane) and stirred at room temperature for 16h. To the mixture 500 mL of water was added and stirred for Ih. The precipitate was filtered, washed with water (350 mL), and dried in vacuum oven at 50 'C for 16h. The product was obtained as an off white solid and characterized by 'H-N MR.

Preparation of {2-[(2-amiooethyl)aminol-9-(methylethyl)purin-6-y} 1(4chlorophenyl)methyl] amin e: To a solution of 3.36 g (0.01 mi) of {2-chloro-9-(nethylethyl)purin-6-yl} [4chlorophenyl)methyllamine in 13 mL of anhy. 1-methyl-2-pyrroldinone was added 4.68 mL (0.70 mol) of 2-aininoethylamine and the mixture was heated at 140 0 C for 24h. The compound was subjected to variable gradient chromatography on silica gel with dichloromethane/methanol mixtures and yielded 1-methyl-2-pyrrolidinone. The mixture was dissolved in dichioromethane and extracted with water (520 mL). The organic layer was dried over anhydrous. Sodium sulfate and evaporated to an off white solid. The product was characterized by 'H-NMR and purity checked by RP-HIPL.C (YMC C-i18 column; 50X4.4 mmn; S -5 120 A' 0. 1% TFA-water/ 0. 1% TFA-acetonitrile).

Table 2, below identifies compounds of this invention that were prepared according to the general synthesis method set forth in this Example. In Table 2, MS Mass Spectrum and mass of parent molecular ion plus one hydrogen atom.

TABLE 2 COMPOUNDS PREPARED BY METHOD OF EXAMPLE 2 R,X R, R, T ms(mH+) (4-MethvlphenvI)methylaniino Isopropyl 2-Aminoethylamino 340 (2.4-Dichlorophenylmethylainino Isopropyl Diethanolainino 439 (2,4-Dichiarophenyl)rnethylaxnino Isopropyl 2-Aminoethylanfino 394 (3-Methylphenyi)methylaniino Isopropyl j Dietbanolaino 385 (3-Methyiphenyl)mtuthylarnino Isolpropyl I 2-Aminoethylamino 340 (3-Methylphenyi,)methylamino Isopropyl 2-(N2-Dimnethylamino)-N 1- 458 benzylethylamino (4-Trifluoromethvlphenyl)methylamino Isopropyl 2-Aminoethylamino 394 (4-Trifluoromethylphenyl)methylamino Isopropyl 1 .Hydroxymethyl-2- 437 methylpropylamino Isopropyl Diethanolamino 507 trifluoromethylphenyl)methylanmino Isopropyl 2-Aminoethylamino 462 trfluoromethylphenyl)methvlamino RX RRms

M+)

Isopropyl I -Hydroxyrnethyl-2- 505 trifluorornethvlphenvl)rnethviamino methyl ropylaino' (3-Chlorophenyl)methylamina Isopropyl 1 -Hvdroxymethvlethvlamino 375 (2-Trifluoromethyl-phenyl)methvianhino Isopropyl 1 -Hydroxymethylethylaxnino 409 (4-Chloro-3-trifluoromethyl- Isopropyl 1 -Hydroxymethylethyian-dno 443 phenyl)inethylammno (3-Chlorophenvl)rnethylamino Isopropyl 2-Hvdroxyethylamino 361 (2-Trifluromethvl-phenvlmethylamino Isopropyl 2-Hydroxyethylamino 395 (4-Chloro-3-trifluoromethyl- Isopropyl 2-Hydroxyethyiammno 429 phenyl)rnethvlamino (3-Chlorophenyl)mcthylamino Isopropyl (I,2S)-2-Hydroxy- 1- 451 methyl-2:phenylethylanino (2-Chlorophe vflmethylan-ino Isopropyl Diethanolaino 450 p I Isopropyl Diethanolamino 407 (1 -Naphthyl)methylammno Isopropyl Diethanolamino 421 (2-Chlorophenvl)methvlamino Isopropyl 2-Aminoethlammno 360 I Isopropyl 2-Arninoethvlaino 362 (I -Naphthyl)methvlarrmino Isopropyl 2-Aminoethylamino 376 (2-Chlorophenyl)methylarnino Isopropyl 1 -Hydroxyrnethyl-2- 403 methviethviamino Isopropyl 1 -Hydroxymethyl-2- 405 methylethvlamino (1 -Naphthyl)methylaxnino Isopropyl 1 -Hydroxymethyl-2- 419 methvlethylainino (3-Methvlphenvl)methylaniino Isopropyl 2-Aniinopropylamino 354 (2-Chlorophenvl)methvlanmino Isopropyl 2-Axninovropylamlilo 374 (3-Chlorophenyl)methylaxruno Isorpropyl 2-Aminopropylarnino 374 (2,S.Difluorophenyl)methylaxn'no Isopropyl 2-Axminopropvlamino 376 (1 -Naphthyl)rnethvlamimo Isopropyl 2-Aniinopropylaxnino 390 Isopropyl Diethanolnmino 473 t-ifluoromethvlphenvl)methylanaino (3-Chlorophenyl)methylamino Isopropyl 1-Hydroxymethyl-2- 403 methyipropylaiino (2-Trifiuoromethylphenyl)methylamino Isopropyl 1 .Hydroxymethyl-2- 437 methyipropyLmnino (3-Methylphenyl)methylamino 1 Isopropyl 2-(2-Hydroxyethylaniino)- 384 (2-Chlorophenyl)methylamino Isopropyl 2-(2-Hydroxyethylamlino)- 404 ethylamino (3-Chlorophenyl)methylamino j Isopropyl 2-(2-Hydroxyethylarnino)- f 404 Isopropyl 2-(2-Hydroxyethylanhino)- 406 (1 -Naphthyl)methylamino Isopropyl 2-(2-Hydroxyethylamino)- 420 _________ethvlamino 1 I]sopropyl 2-(2-Hydroxyethylammno)- 506 p~henvl)methviamino J________ethylamidno (4-Isopropylphenyl)methylamino Isopropyl 2-(2-Hydroxyethylamnio)- 412 ethylanuno (2-Trifluoroxnethylphenyl)methylan-Lino Isopropyl 2-(2-Hydroxyethylammno)- 438 ethylamino (4-Methylphenyl)methylanxno Isopropyl 2-(2-Hydroxyethylaxnino)- 384 ethvlanmunoII R,X

MS(MHF+)

(4-Chloro-3- Isopropyl 2-(2-Hydroxyethylamino)- 472 trifuorometb 1 henvl')methylamino ethylamino Isopropyl 2-(2-Hydroxyethylarmino)- 472 triflucromethylphenyl)methvlamiino ________ethviaxnino (3 ,5-Dichiorophenyl)methylammno Isopropyl 2-(2-Hydroxyethylaxnino)- 438 ethylanuno (1 R)-(4-Methyiphenyl)ethyamilo Isopropyl 2-(2-Hydroxyethylainfo)- 398 ethvianimo________ (IR)-(2-Naphthyl)ethylamino isopropyl 2-(2-Hydroxyetylamfilo)- 434 ethvlaxnmo (2,4-Dichloro-6- Isopropyl 2-(2-Hydroxyethylamfilo)- I 452 methvlphenyl)methylamino ________ethylamino (4-Trifluoromethypheny1)methylamiflo Isopropyl 2-(2.Hydroxyethylamino)- 438 (3-Metbviphenvl)methylamilo Isopropyl (2S)-pyrrolomethylalnino 380 (2-Chlorophenvl)methvlamino Isopropyl (2S-prOlomethvLnio 400 (3-Chlorophenyl)methylanio isopropyl (2S)-pyrrolomethylaniino henyl)methvianino Isopropyl (2S)-pyrro omethvlamino 402 (I -Naphthyl)methylanuno ISOpropyl (2S)-pyrrolomethylanmlno 416 (3 ,5-Bistuifluoromethyl- Isopropyl (2S)-pyrrolomethylamiflo 502 phenyl)methylamino (4-Isopropylphenvl)methvamiDo Isopropyl (2S)-pvrrolomethvlamino 408 (2-Trifluoromethylphenv)methylaliino Isopropyl (2S)-pmrolomethylanino 434 (4-Methylphenyl)methviamilo Isopropyl (2S)-pyrrolomethviamino 380 (4-Chloro-3- Isopropyl (2S)-pyrrolamethylamino 468 trifluoromethylphenvl)nethvanmino Isopropyl (2S)-pyrrolometbylanino 468 trifluoromethylphenyl)methviamino 43 Isopropyl (S-vrlmtvann 3 (I R -4-Methy1 henyP)ethylamino j Isopropyl -(2S)-pyrrolomethylamino 394 (IR)-2-Naphthy1)ethylamino Isopropyl -(2S)-pyrrolomethylaino 430 (2,4-Dichloro-6- Isopropyl (2S)-pyrrolomethyla O 448 methylphenyl)mnethylaniinoI (4-Trifluoromethylphenl)methvano Isopropyl -(2S)-pvrrolomethylammno 434 (3-Methylphenyl)methylamino Isopropyl 2-Hydroxy- 355 methylethvlanmlno (2-Chlorophenyl)methylaniino Isopropyl 2-Hydroxy-l1-(S)- 375 methylethylaino isopropyl 2-Hydroxy- 377 methylethylaino (1 -Naphtbyl)rnethylamino Isopropyl 2-Hydroxy- 391 methylethylamino Isopropyl 2-Hydroxy- 1 477 phenyl)methylamino methylethylamino (4-Isopropylphenyl)methyi n1 n Isopropyl 2-Hydroxy- 383 methylethylamino (4-Methylphenyl)rnethyinmino Isopropyl 2-Hydroxy- 355 methylethylamino Isopropyl 2-Hydroxy-l-(S)- 443 trifluoromethylphenyl)rethvlamino methylethylainiino (5-Fluoro-2- Isopropyl 2-Hydroxy-l-{S)- 427 tnflucromethylphenylhmethvlaniino methylethylamino I j Isopropyl 2-royliar) 409 R,X R R MS(ME+) I -(4-Methylphenyl)ethylamino Isopropyl 2-Hydroxy-l1-(S)- 369 1-(2-Naphthyl)etbylamino Isopropyl 2-Hydroxy-l-(S)- 405 methyl thylarnino (2 ,4-Dichloropheny)nethyimmno Isopropyl 2-Hydroxy- 1 409 methylethviarnino (2,4-DichLoro-6- Isopropyl 2-Hydroxy-l1-(5)- 423 methylphenvl)methylamino methylethylamino (4-Trifluoromethyiphenyl)methylamino Isopropyl 2-H-ydroxy-l1-(5)- 409 rnetbvlethylarnino (3-Methviphenyl)methylamino Isopropyl 2-Hvdroxyethylaxnino34 (2-Chlorophenvl)methylanhno Isopropyl 2-Hydroxvethylaniino 361 Isopropyl 2-Hydr xvethylamino 363 (1 -Naphthvl)methylarmino Isopropyl 2-Hvdroxvethylamino 377 (3 ,5-Bistrifluoromethyl- Isopropyl 2-Hydroxyethylaniino 463 phenyl)methviamino (4-Isop~rop~vlpbenvi)methviaminmo Isopropyl 2-Hydroxyethviamino 369 (4-Methvlphenvl~methvlan-ino Isopropyl 2-Hydroxvethvianmino 341 T Isopropyl 2-Hydroxyethylamino 429 triflucoromethvlphenflmethylamino..

(5-Fluoro.2- Isopropyl 2-Hydroxyethylaxnhno 413 uifluoromethylphenyi)methylamino (3 .5-Dichlorophenvl)rnethvl:mino T Isopropyl 2-Hvdroxvethylainino 395 (lR)-(4-Methvlphenv1)ethylamino Isopropyl 2-Hydroxvethylamino 355 (IR)-(2-Naphthyl)ethylamino Isopropyl 2-Hydroxvethvlaniino 391 (2,4-Dichlorophenyl~methvlamino Isopropyl 2-Hvdroxyethylaxnino (2,4-Dichloro-6- Isopropyl 2-Hydroxyethylamino40 methylphenvl)metbvlamino- (4-Trifluoromethylphenyl~methylaxnino Isopropyl 2-Hvdroxvethvlamino 395 (3-Methylphenyl)methylamino Isopropyl 1- 369 Hvdroxymethylgropylaniino (2-Cblorophenyl)methylanmino Isopropyl 1- 389 Hvdroxymethylpropylaxnino (3-Chlorophenyl)methyammno Isopropyl 1-389 Hydroxvmethylpropylamino Isopropyl 1- 391 Hvdroxvmethvlpropylamino (1 -Naphthyl)methylarnino Isopropyl 1- 405 ____________Hvdroxvinethvipropylamuno Isopropyl 1- 491 pbenyl)methylaxnino ________Hydroxymethylpropylamino (4-Isopropyiphenyl)rnethylamino Isopropyl 1- 397 Hvdroxymethylpropylainino (2-Trifluoromethylphenyl)methylamino Isopropyl 1-423 ydroxymethylpropyLamino (4-Methylphenyl)methylamino Isopropyl 1- 1 369 Hydroxymethvipropylamino (4-Chloro-3 Isopropyl 1- 457 trfluoromethvlphenyl)methylanmino j _______Hydroxyvmetbvlpropylaniino Isopropyl 1- 457 trifhioromethylphenyl)rnethylamino ________Hvdroxvmnethylpropylamino (S-Fluoro-2- 1- 441 trffluoromcthylphenyl)methvlarnino jHvdroxymrethyipropylaminoI R,X J_ R, MS(M]H+) (3 ,5-Dichlorophenyl)methylamino Isopropyl 1- 423 _____________Hydroxyniethvipropvlamino (1 R)-(4-Methylphenyl)ethylamino Isopropyl 1 383 (1 R)-(2-Naphthyl)ethylamino Isopropyl I1- 419 Hydroxymethylpropylarino (2,4-Dichlorophenyl)rnethylamino Isopropyl 1- 423 1HydroxymethylpropylarnioI_______ (2,4-Dichloro-6- Isopropyl 1- 437 metbvlphenvl)methylamino Hydroxytnethylpropylam (4-Trifluorometbylphenyl)methylamino Isopropyl 1- 423 1 Hydroxymethylpropylarnino (3 ,5-Dichloropbenyl)methylamino Isopropyl 12-(2-Hydroxyethylamino)- 438 I ethylainino (3 ,5-Dichlorophenyl)metbvlanmmo Isopropyl I(2S)-pyrrolomethviamino 434 (4-Chlorophenyl)methylaino Isopropyl 2-Aminocyclohexyl iino 414 (4-Chlorophenyi)methylarxmo Isop~rotpvl 3-Amiinocyclohexylaminn 414 (3-Fluoro-6-trifluoromethyl- Isopropyl Diethanolqmino phenyl)methvlamino Isopropyl 2-Aminoethylamino 428 Isopropyl 2-Anxinopropylamino I 476 phenyl)methylaino____________ (2-Trifluoromethyl-pbenvl)metaylamino Isopropvl 2-Aminopropylamino 408 (4-methyiphenyl)methvlamino Isopropyl I 2-Arninopropylarniino j 354 (4-Chloro-3-trifluoromethyl- Isopropyl 2-Aminopropylamino 442 phenyl)rnethylaxnino_____________ Isopropyl 2-Aminopropylamino 442 p env mre FRl-I -(4-meth lphenyl)ethylamn Isopropyl 2-aminoethylamin2 354 1 -(2-Naphtbvl')ethylamnio Isopropyl 2-Aminoethylamino 390 Isopropyl I -Hydroxymethyl-2- 471 Phenvl)rnethylamino methyl ropylarnino (3-fluoro-6-trifluoromethyl- Isopropyl 1 -Hydroxymethyl-2- 455 phenvl)methylamino methylpropylainino FRi- I -(4-methylphenyl)ethvlamino Isopropvl 2-Ammnopropylamino 368 [RI- I -(2-Naiphthvl)ethvlaxnino Isopropyl 2-Aniinopropylamimo F 404 (4-Trifluoromethvl-phenyl)methvlainino J Isopropyl f 2-ArninopropyLamino 408 (3-Methylphenyl)methylaznino Isopropyl 2-Amnino-2- 368 (2-Chlorophcnyl)methylamino Isopropyl 2-Amnino-2- 388 methylpropylanmino (3-Chiorophenyl)methylami o Isopropyl 2-Amino-2- 388 methylpropylamino Isopropyl 2-Amino-2- 390 methvlpropylamino (I -Naphthyl)methylamino Isopropyl 2-Amino-2- 404 methyipropylarnino (3 ,5-Bis-trffluoromethyl- T Isopropyl 2-Amino-2- 490 phenyl)methylamino tmethylpropylamino (2-Trifluorometbyl-phenyl)methylamino Isopropyl 2-Amino-2- 422 methylpropylamino (4-Methylphenyl)methylaniino T Isopropyl 2-Amino-2- 368 T methylpropylamino R,X R, ,MS(Nm+) (4-Cbloro-3-trifluoromethyl- Isopropyl 2-Amino-2- 456 phenylfmethylamino methylpropylamino (2-Chloro-S-trifluoromethyl- Isopropyl 2-Anmino-2- 456 phenyl)methylarmino methvlpropylamino (3-Fluoro-6-triffluorometbyl- Isopropyl 2-Ainino-2- 440 phenvI)methylarmino methylpropvlamino 1 -(4-Methylphenyl)ethylamino Isopropyl 2-Aniino-2- 382 methylpropylamino 1 -(2-Naphthyl)ethylaxmno Isopropyl 2-Amino-2- 418 methylpropylarmuno (4-Trifluoronaethyl-phenyl)methylamino Isopropyl 2-Arnino-2- 422 1 methylpropylamino EXAMPLE 3 This Example describes a method for preparing compounds of this invention. The synthesis method disclosed in this Example is only slightly modified from that disclosed in Example 1.

C1

R

1 butariol, A

N>

jNaH N

N

R

3

N

6

R

3 A

N

N-methytpyrrolidinone cr"A?"N N The following compound was prepared according to the method above.

Preparation of 2,6-dichloro-9-isopropylpurine To a solution of 0.67g of 2,6.-dichloropurine in 5mnL of dry DMF at room temperature was added 0. l6gms 1 eq.) of 50% sodium hydride/oil powder. Upon cessation of hydrogen evolution, a large excess (2 mL) of isopropyl iodide was added to the anionic solution. This reaction solution was stirred for three days at ambient temperature. The reaction was quenched with 30 m.L of water and extracted with ethyl acetate (350 mL). The organic extracts were combined and back washed with 350 mL of water followed by 20 mL of brine. The ethyl acetate solution was dried over anhydrous magnesium sulfate and evaporated. The compound was subjected to variable gradient flash chromatography on silica gel with hexane/ethyl acetate mixtures and yielded the N-9 product and the N-7 isomer.

Preparation of 2-chloro-6-anilino-9-isopropylpurine 2,6-dichloro-9-isopropylpurine (0.019 g, 0.081 mmol) was dissolved in butanol ml) and aniline (0.044 ml, 0.244 mmol) was added. The reaction mixture was heated to 1 20'C for 10 hr, cooled, diluted with EtOAc and washed 3 times with water. The mixture was dried over MgSO, and concentrated to an off white solid.

Preparation of 2-diethanolamino-6-(4-phenylanilino)-9-isopropylpurine A solution of 67mgs of 2,6-dichloro-N-9-isopropylpurine and 1 O0mgs of 4-phenylaniline in 1 mnL of n-octanol was heated to 80'C for 24 hours. The n-octanol was removed in vacuo and then replaced with 1 mL of 40% diethanolamine in DMSO. The solution was heated at 130'C for 48 hours. The reaction was cooled to ambient temperature then diluted with 10 m1L of water and sub.sequently extracted with ethyl acetate (3X30 mL). The organic extracts were combined and back washed with 3X20 mL of water followed by 10 mL of brine. The ethyl acetate solution was dried over anhydrous magnesium sulfate and filtered and the solvent was evaporated. The 65mgs of crude product was crystallized from Tiff-ether solution.

Table 3 below identifies compounds of this invention that were prepared according to the general synthesis method set forth in this Example.

TABLE 3 Compounds Prepared By The Method Of Example 3 R,X (8-guinolinvI)amino Isopropyl CI (6-guinolinyl~aniino Isopropyl C1 (3-gwinolinyl)amino ISOPrOPYl CI amihno Isopropyl CI I Isopropyl C1 4-butvianilino Isopropvl C1 (8-guinolinyl)amino Isopropyl Diethanolamino (6-guinolinyl)armno Isopropyl Diethanolammlo (3-quinolinyl)amino ISOPTOPVI Diethanoiamnino anilmo Isopropyl Diethanolamino Isopropyl Diethanolamino 4-butviaihno Isopropyl Diethanolaniino (6-ethoxy-2-benzothiazoivl)amino Isopropyl C1 4-morpholino-2-methylamino Isopropyl CI (4-anuinosulf'onyl-p henvflmethviamino Isopropyl CI 4-bromoanilino Isopropyl diethanolamino 3,4-dichloroanilino Isopropyl diethanolamino 2-(2-(l1 -methyl)pyrrolidinvI)ethyaxnino Isopropyl diethaolarnino 3-bromoanilino Isopropyl CI 4-methoxvanilino Isopropyl diethanolamnio 4-iodoaniluao Isopropyl C1 3-iodoanilino Isopropyl CI 3-methoxyanilbno Isopropyl C1 I -piperidinyl)ethylamino ISOPropyl diethanolamino I -pyrrofidinvIethvlamino Isopropyl diethanolamino (I -indanyl)amino Isopropyl diethanolarnino 2-(6-ethoxybenzothiazolylamino Isopropyl diethanolaxnino 4-morpholino-2-methylan-ino Isopropyl diethanolamino (4-aminosulfonyl-phenyl)methvlamino Isopropyl diethaolarmno 4-bromoanilino Isopropyl diethanolamino 3 .4-dichioroanilino Isopropyl diethanoiamino 2 -methyl)pyrrolidinvl~ethylamino Isopropyl diethanolamino 3-brmoanlinoIsopopyldiethanolamino 4-methvoxvanilino Isopropyl diethanolammno 4-iodoanilino Isopropyl diethanolainuno 3-iodoanilino Isopropyl diethaolamino 3-methoxvanilino Iorpldiethanolamino 1-piperidinyl)ethylamino Isopropyl diethanolano 1-pyrrohiyl)ethylamj no Isopropyl diethanolamino (I -indanyl')aniino Isopropyl diethanolamino 3-iodonflino Isopropyl diethanolamino RAX

R,

3-phenoxyanilino Isopi-opyl diethanolarnino 4-iodoanilino Isopropyl diethanolamino 4-phenoxvanilino Isopropyl diethanolamino 3-phenoxvanilino Isopropyl diethanolamino 2-fluorenvlammno Isoipropyl diethanolarnino 1 -fluorenylamino Isopropyl diethanolammno 2-anthracenviarnino Isopropyl diethanolamino 1 -anthracenviamino Isopropyl diethanolano 2-(6-ethoxybenzotiazolyl)amino Isopropyl diethanolamino (1 -indanvl)ammno j Isopropyl diethanolamino 2-(6-ethoxybenzothiazoivl)axpino j Isopropyl diethanolamino 4-inorpholino-2-metbviamino Isopropyl diethanolamino (4amnoulonl-hny~mthlaiio Isopropyl diethanolamino 4-bromoanilino Isopropyl diethanolamino 3,4-dichloroanilino Isopropvl diethanolaino I -methyl)pyrrolidinyl)ethylan-ino Isopropyl diethanolamnio 3-bromoanilino Isopropyl diethanolamino 4-methoxyanjiimo Isopropyl diethanolaniino 4-iodoanilino Isopropyl diethanolamino 3-iodoanilino j Isopropyl diethanolainino 3-methoxyanilino Isopropyl diethanolamino 241 -piperidinyl)ethyiainino Isopropyl diethanolarnino 241 -pyrrolidinyl)ethviarnino Isopropyl diethanolamino (1 -indanyl)amino Isopropyl diethaolamino 3-iodoanilino Isopropyl diethanolamino 3-pheoxvanilino Isopropyl diethanolamino 4-iodornilno Isopropyl diethanoiamino 4-phenoxyanilino Isoprcypyl diethanolainino 3-phenoxvanilino Isopropyl diethanolamino 2-fluorenvlamo -Isopropyl diethanolarnino I -fluorenviaiio Isopropyl diethaolamino 2-anthracenylarnino Isopropyl diethanolamino 1 -anthracenylaxrno Isopropyl dehnlmn 2-(6-ethoxvbenzothiazolyI amino Isopropyl diethanolamino (2-biphenyi)methylamino Isopropyl diethaolamino (4-biphenyl)methylaniino Isopropyl diethanolamino 2-naphthylmethyLimino Isopropyl diethanolamino 1 -naphthvimethvlamino Isopropyl diethanolainino (4-Chlorophenyl)methylamino Isopropvl Diethanolamino (4-Fluorophenyl)methylamino Isopropyl Diethanolamino (4-Methoxyphenyl methylainino Isopropyl 4 -Trifluoromethylphenyl)methyiarnino Isobutvi Diethanolamino (4-TrifluoromethylphenyI meth lamino Isopropyl Diethanoamno 4 -Chlorophenyl)rnethylamino Isopropyl (S)-2-An uno-3phenylpropylamino (4-Fluorophenyl)methvlaniino Isopropyl 2-Axninoethylaniino (4-Fluorophenyl)methylamino Isopropyl 1-Hydroxymnethyl-2methyl-propylamino (4-Fluorophenyl)methyl ni o Isopropyl I -Hydroxymethyl-2rnethylmpropvlamino (4-Chlorophenyl)methylamino Isopropyl (D)-l1-Hydroxymethyl-2- I methyl-propylarnino RX RR (4-Cblorophenyl)niethylaqnino Isopropyl 1-Hydroxymethyl-2- (4-Cb~orophnyl~mthylamno Ispropy 2 -Hyroy-2-henyl (4-Chlorophenyl)methylaniino Isopropyl -yrx2phnl hyroxethletbvlmin (4-Chlorophenyl)methylaxnino Isopropyl 2-Amino-N2-(2bvdroxyethyl)ethylamino (4.-Chlorophenyl)metliylaniino Isopropyl (S)-2-Phenyl- 1 -carboxamida- ___________ethylantino (4-Chlorophenyl)methylami o lsopropyl 2-Anmino-N2-(2hydroxyethyl)-N I (hvdroxvethyl)ethvlamino (4-Sulfonarnidophenylmethvlamino Isopropyl 2-Aminoetbylamino (4-Fluorophenyl)methylamino 2-Oxo-3-buryl Diethanolamino (4-Fiuoropheiivl)methylamino 2-Oxo-3-butvl 2-Aminoethylamino (4-Chlorophenvl)methylamino 2-Oxo-3-butvl Diethanolamino (4-Chlorophenvl)methvianiino 2-Qxo-3-butvl 2-Aminoethylamino (4-Methylphenyl)methylamino Isopropyi 1 -Hydroxymetbyl-2-methylpylaninmo (3-Methylphenyl)methylamino Isopropyl 1 -Hydroxyrnethyl-2-methyl- (4-Chlorophenyl)methyiamino Isopropyl 2-(N2-dimethylamino)-Nl- (4-Chlorophenyl)methylaniino Isopropyl 1 -Carboxamido-2-methyl- (4-Chlorophenyl~methylam~p Ipoyl2micylamino (4-Chlorophenyl)methylaniino Isopropyl 2-AniinopropyLanino (4-Cblorophenvl)methvlaxmno Isopropy) (4-Cbilorophenyl)methylamino Isopropyl 2-Amino-2-methyl- (4-Chlorophenyl)methylamino Isopropyl 1- (Hydroxymethyl)propylaniin (4-Chlorophenyl)methylamino Isopropyl (Hydroxymethyl)propylarniin 0 (4-Chlorophenyl)methylamino Isopropyl 1- (Hydroxymethvl)ethvlamino (4-Chiorophenyl)methylamino Isopropyl(R(- (Hydroxymethvl)ethylamidno (4-Chlorophenyl)methylainino Isopropyl Hydroxypropylaniino (4-Chlorophenyl)methylamino Isopropyl Hydroxyprorviarino (4-Fluorophenyl)methviarnino Isopropyl 2-Amino-propylaxnino (3-Chloropbenyl)methvlamino Isopropyl Diethanolamino (3-Chlorophenyl)methylaxnino Isopropyl 2-Aminoethvlanhno (2-Trifluoromethylphenvl)methylamino isapro yl Diethaolammno (2-Trifluoromethvlphenyl)methl 0an Isopropyl 2-Anainoethylamino (4-Chloro '-3-trifluoromethyiphenyl) Isopropyl Diethanolamno methvlamo-2Aiotvamino (4-Chloro-3-trifluoromethvlphenvl) Isopropyl 2Aanehlmn

R,XR,,

(3 ,5-Dichlorophenyl)mthvlammno Isopropyl Diethanolaino (3 ,5-Dichlorophenyl)methylamino Isopropyl 2-Amino-ethvlaxnino (2-Trifluoromethyl-phenyl)methylamino Isopropyl 2-(N2-dimethylammno)-N 1benzyl-ethylamino (3-Chlorophenvl)rnethviamimo Isopropyl Diethanolamino (3-Chlorophenyvhmethylarmino ISOPTDPYI 2-Aminocthviammio (2-Trifluoromethvlphenyl)methylamino Isopropyl Diethanoiamino (2-Trifluoromethvlphenyl)methylamino Isopropyl 2-Arninoethylanmino (4-Chloro-3- .Isopropyl Diethanolanino trifluoromethylphenv'~methviamino (4-Chloro-3- Isopropyl 2-Aminoethylamuno trifluoromethylphenyl)methvlamino (3 .5-Dichlorophenyl)methvlamino Isopropyl Dietbanolamiria (3 ,5-Dichlorophenvl)methylan-ino Isopropyl 2-Aminoethvlamino (2-Trifluoromethylphenyl)methylamino Isopropyl 2-(N2-Diniethylamino)-NlI benzyletbviamino (3-ChLorophenyl)methylamino Isopropyl 2-(N2-Dixnethylamino)-N I1benzvlethviamino Isopropyi 2-(N2-Dimethylaamino)-N 1 benzvlethylamino (4-Ghloro-3- Isopropyl 2-(N2-Dimrethylamino)-N I1- U-Mu~oromethvlphenvl)metbvlamino benzylethvianmuno (4-Cblorophenyl)methylamino Isopropyl 2-Amino-2rethylpropylamino (4-Fluorophenyl)methylamino Isopropyl Tetrahydrofuranyl)methylam (4-Fluorophenyl)methylamino Isopropyl Teahydrofuranylmethylam m (4-Methylphenvlmrethylamino Isopropvl Diethanolanino (4-Fluorophenyl)methylan-ino Isopropyl 2-Hydroxy- 1 methvlethvlainino (4-Fluorophenyl)methylaniino Isopropyl (S)-2-Hydroxy-2methviethvlan'uno (4-Fluorophenyl)methylamin Isopropyl (R)-2-Hydroxy-2methylethylammno (4-Fluorophenyl)methylamino Isopropyl I- ___________HvdroxvmethylPTOPYlamn (4-Flurophenyi)methylamino Isopropyl 2-Amino-2- I methvlpropylamin 51 EXAMPLE 4 This Example describes a method for preparing compounds of this invention. The synthesis method disclosed in this Example is only slightly modified from that disclosed in Example 1.

Cl

CI

N N NaH N Cl N N C N N H4 1 bulanol RI R 1 Or N S^ N a. N-msthypyrohicnone N N R3 N N ouplng C N N 6A R2 5A R2 The following compound was prepared according to the method above.

Preparation of 2,6-dichloro-9-isopropylpurine The 2,6-dichloropurine (5.00 g, 26.46 mmol) was suspended in 55 ml of dry DMF at room temperature and treated with sodium hydride, 60% dispersion (1.27 g, 31.75 mmol) added in portions. After stirring for 1 hr, 2-iodopropane (4.5 ml, 44.98 mmol) was added and the reaction stirred for 2 days. The reaction was poured into diethyl ether and washed once with saturated sodium bicarbonate solution and once with water. The mixture was dried over anhydrous sodium sulfate and concentrated in vacuo. The concentrate was chromatographed over silica gel eluting with 10% acetone in dichloromethane solution to give the desired N-9 52 alkylation product as a white solid.

Preparation of 2-chloro-6-(4-methylmercapto) anillno-9-isopropiypurie 2,6-Dichloro-9-isopropylpurine (0.15 g, 0.649 rnmol) was dissolved in n-butanol (4 ml) and 4-(methylrnercapato) aniline (0.089 ml, 0.714 mmol) and triethylamnine (0.20 ml, 1.43 mmol) were added. The reaction mixture was heated at 800 overnight. The cooled reaction was diluted ethyl acetate and washed 1 x IM HCI, 1 x saturated sodium bicarbonate, and I x brine before being dried with anhydrous sodium sulfate and concentrated in vacuo. The residue was chromatographed over silica gel and eluting with 2% methanol in dichloromethane to give the desired product as a white solid.

io Preparation of 2-diethanolamine-6-(4-metbylmercapto) anilino-9-isopropylpurine (6A).

The purine (0.18 g, 539 mmol) was dissolved in N-methylyrrolidinone (3 ml) and diethanolamine (1 ml) and then heated at 120'C overnight. The cooled reaction was poured into diethyl ether and washed three times with water before drying over anhydrous sodium sulfate and concentrating in vacuo. The residue was chromatographed over silica gel eluting with 5% methanol in dichloromethane to give the desired product as an off-white solid. 'H- NMR(6, CDC' 3) 8.08(s,lH), 7.58(d, 2H), 7.47(s,1H), 7.18(d, 2H), 4.95(br s, 4.52(m, IM), 3.94(m, 4H), 3.83(m,4H), 2.43(s, 3H), 1.47(d, 6H).

Preparation of 4-(2-tbienyl) benzonitrile.

Some groups must first be synthesized before reacting with the 2,6-dichloro-9isopropylpurine. These groups can be synthesized through various coupling methods and other synthetic procedures known to those skilled in the art of organic synthesis.

To a pressure tube was added 4-bromobenzonitrile (0.20 g, 1.10 nmnol), tetrakis(triphenylphosphine) palladium (0.127 g, 0.1 eq) and 2-thiopheneboronic acid 53 (0.211 g, 1.65 mmol). The reaction was flushed under vacuum and flushed with dry nitrogen three times. Following flushes, ethyleneglycol dimethyl ether (5.5 ml) and an aqueous solution of sodium carbonate (2.53 ml, IM) were added to the tube. The tube was then sealed and heated at 80'C overnight. The cooled reaction was the diluted with diethyl ether and washed twice with water before drying over sodium sulfate and concentrating in vacuo. The residue was chromatographed over silica gel eluting with 10% ethyl acetate in hexane to' give the desired product as a white solid.

Pre paration of 4-(2-thienyl) benzylamine.

The 4-(2-thienyl)benzonitrile (0.086 g, 0.464 rnmol) was dissolved in dry tetrahydrofuran (1.6 ml) before lithium aluminum hydride (0.46 ml, 0.464 minol, 1 M in THE) was added dropwise. The reaction was allowed to stir at room temperature overnight.

TLC methanol in dichloromethane) still showed starting material remaining. Another I eq of LAH was added. After an additional hour, the reaction was quenched by the Fieser and Fieser method using wager (17.4641l), aqueous sodium hydroxide solution (17.441, soin.), and water (52.37 41) added sequentially to the reaction. The reaction was then diluted with diethyl ether and water and extracted twice with diethyl ether before drying over sodium sulfate and concentrating in vacuo. The residue was carried on crude without any further purification.

Table 3 below identified compounds of this invention that were prepared according to the general synthesis method set forth in this Example.

Table 4 Compounds Prepared By The Method of Example 4 R 1XR2 R3 C! Me cI ethanolaimmo Me ethanolaino cyclopropylmethylaiio isopropyl

CI

cvciopropvLmethyl 1 0n isopov diethanolamino 3-phenoxvanilino isopropyl Cl 4-iodoanilino isopropyl

CI

3-amino uinolino isopropyl Cl isopropyl diethanolamino ciepoxymethyl

CI

4-methoxybenzvIamino 2.3-dihydroxvpropvl diethanoLamino 4-phenylanilino isopropyl diethanolamino 4-phenylbenzvlamino isopropyl

CI

2-naphthalenyimethvlamjno isopropyl Cl I -naphthalenvlmethviamino isopropyl Cl 2-phenylbenzvlamino isopropyl cl 3-guinolinylarruno isopropyl diethanolamino isopropyl diethunolamino 6-guinolinviamino isopropyl diethanolarnino 8-guinciinylamino isopropyl diethanolanino n-butylamino isopropyl Cl 4-(2-thiophenyI)benziarnino isopropyl deithanolamino 4-(2-thiophenyl)benzylamino isopropyl Cl 3-thiomethoxyanilino isoprapyl Cl 4-thiomethoxyanilino isopropyl c 3-thiomethorxyanilino isopropyl diethanoamino 4-thiomethoxvanjlino isopropyl diethanoamino 4-(2-pvridinyl) benzyiamino isopropyl 3-methoxvbenzvlamino isopropyl 3,4-diniethoxybenzvlamino isopropyl 3 ,4,5-nrimnethoxyenzylamino isopropyl c 3-methoxybenzvlanmno isopropyl diethanoiannno 3,4-diimethoxvbenzylarnino Isopropyl diethanolamino 3 ,4,5-trimethoxbenzvianiino IsopropvI diethanolamino 4-(3-thjiohenyI)benzvaiano Isopropyl Cl 4-(4-methoxphenvl) benzylamino Isopropyl

CI

4-(4-brornophenyl) benzylamino Isopropyl diethanolamimo metboxyphenyl) benzvlamino Isopropyi diethanolamnino 4-(4-rnethoxyphenv) benzylamino Isoipropyl diethanolaminio 4-(3-thiophenyl) benzylamino Isopropyl diethanolanino 4-(3-methylpheny) benzylamino Isopropyl Cl 4-(4-methylphenyl) benzylamino Isopropyl Cl 4-(4-trifluoromethylphenyl) Isopropyl Cl 3-(4 nitrilophenyl)anilino Isopropyl CI 3-(4-nitrilophenyl)anilino Isopropyl diethanolainino 4-C 2-pyridinyI)benzvan-dno Isopropyl Cl 4-(2-pryidinyl~benzvlanino Isop~ropyl diethaolamino EXAMPLE This Example describes a method for preparing compounds of this invention. The synthesis method disclosed in this Example is only slightly modified from that disclosed in Example 1.

Cl Cl

HHN

2 A HN 2N 2N N N 7 R2 I R3-C I pyndine CH2CI2 C1 NA

N

R3-, N J N

N

H R2 The following compound was prepared according to the method above.

Preparation of 2 -amino-6-chloro-9-methylpurine The 2-amino-6-chloropurine (1.08 g, 6.4 mmol) was suspended in dry DMF (75 ml) and treated with sodium hydride, 60% dispersion (0.28 g, 7 mmol). The suspension was stirred for 15 min before iodomethane (0.44 ml, 7.06 mmol) was added and the resulting yellow solution stirred for 1 hr 45 min. The solid was filtered and the filtrate evaporated before addition of water for 10 min. The resulting solid was filtered and dried overnight to 56 give the product as a mixture of N-7 and N-9 alkylation products. The residual liquor was left overnight and more crystals were collected the next day and dried.

Preparation of 6-chloro-2-(2-methoxyacetylamino)-9-methyipurine The mixture of isomers from above was dissolved in dlichioromethane and pyridine (2 eq) followed by treatment with rnethoxyacetyl chloride (4 eq). The reaction was stirred at room temperature until complete. The reaction was evaporated and filtered through a plug of silicia gel eluting with 2% methanol in dichioromethane followed by purification on a chomatotron using silica gel and eluting with 2% methanol in dichioromethane to isolate the desired product.

Table 5 identifies compounds of this invention that were prepared according to the synthesis method set forth in this Example.

Table Compounds Prepared By The Method of Example R1 R2 R3 Cl Me H Cl Me 2-methoxyacetylamino EXAMPLE 6 This Example describes a method for preparing compounds of this invention. The synthesis method disclosed in this Example is only slightly modified from that disclosed in Example 1.

Ql Ri P io( n CI i N Ri

R

1 Kl>

I

R

3 N A 3 N H The following compound was prepared according to the method above.

Preparation of 2-chloro-6-(4-phenyl benzylamino) purine The 2,6-dichloropurine (5.0 g, 26.45 mmol) was suspended in n-butanol (50 ml) and the 4-phenylbenzylamine (6.61 g, 29.1 mmol) and triethylamine (4.1 ml, 29.1 mmol) were added. The solution was heated at 120°C overnight then cooled. Filtered off product using excess n-butanol and washed precipitate with 100 ml 1M HCI and 200 ml water.. The solid was dried in vacuum over overnight at 70°C to give the desired product as a pale yellow solid Preparation of 2-diethanolamino-6-(4-phenyl benzylamino) purine 58 The 2-chloro-6-(4-phenyl benzylamino) punine (2.0 g, 5.96 mmol) was added together with diethanolarnine (11.4 ml, 119.2 rnmol) and N-methylpyrrolidinone (10 ml) and heated at 1 20'C overnight. The cooled reaction was poured into dichioromethane and washed twice with water. The organic layer was dried with anhydrous sodium sulfate and concentrated in vacuo to give the desired product as a pale green solid which was further dried in vacuum oven at 70'C for 2 days.

Preparation of 2-diethanolamino-6-(4-phen yl benzylamino)-9-methylpurine (11).

The 2-diethanolamino-6-(4-phenyl benzylamino) purine (0.050 g, 0.124 mmol) was dissolved in dry DMF and treated wit sodium hydride, 60% dispersion (5.5 mgs, 0. 13 6 rmol) for I hr. iodomethane (0.009 ml, 0.148 mmol) was added and the resultant solution stirred at room temperature overnight. Poured reaction into diethyl ether and washed twice with saturated sodium bicarbonate solution before drying over anhydrous sodium sulfate and concentrating in vacuo. The residue was chromatographed over silica gel eluting with methanol in dichloromethane to give the produce as a white solid.

1 H-NMvR(5, CDCI3): 7.55 7.41 (in, 4H) 7.35(m, 4H), 6.41 (br s, IH), 5.10(br s, 4.72 (br s, 2H), 3.86 (in, 4H), 3.74(m, 4H), 3.59(s, 3H).

Table 5 identified compounds of this invention that were prepared according to the synthesis method set forth in this Example.

Table 6 Compounds Prepared By The Method of Example 6 R2 113 4-phenylbenzylamino IMethyl diethanolamino 4-phenylbenzvlainino Cyclopentvl diethanolamio 4-phenvlberizvlamino Alvi diethanolamino 4-phenylbenzylamino Benzvl diethanolaniino 4-phenylbenzylamino 3-methylbutyl dietbanolamino 4-phenylbenzyiamino Isobutvi diethanolamino 4-phenvibenzykammio t-butvaceaxe diethanolamino I 4-phenylbenzyianuno IMethylacetate dietbaoaximo 4-phenyIbenzlammio Cyclobuty1 diethanolamino I 4-phenvibenzylamino Ethyl Idiethanolamino 4-phenvibenzvlamino Propyl diethanolarnino EXAMPLE 7 Composition of this invention were evaluated in the following assays.

CDK2 assays: Compositions of this invention were assayed to determine their CDK2 inhibitory activity. The assay system (total volume of 50 41) contained 50 mM Tris-CI, pH 7.4, 10 MM MgC1 2 5 mM DTT, 1 .tg of histone Hi, 30 jiM ATP (I gCi of gamna 32

P

labeled ATP), 10 jig of BSA and I ng of purified CDK2. After incubation at 30'C for min, the reaction was terminated by the addition of 10 gI of 10% TCA and the samples were blotted onto to nitrocellulose filters. These filters were washed extensively in 10% TCA and assayed for radioactivity. Blanks contained no enzyme.

To ascertain the potency of various compounds of this invention, the compounds were added to the above assay at concentrations ranging from 100 to 0.02 jig/ml. Affer incubation at 30 min., the assay tubes were processed as above. In 'all assays, various concentrations of olomoucine were added and were used as a standard positive control. The IC 5 0 (enzyme) listed in Table 7 is defined as the concentration required to inhibit CDK2 activity by 61 EXAMPLE 8 Cell Proliferation Assays: Early passage rat aortic smooth muscle cells (CV Therapeutics Cell repository) were seeded in 48 well dishes (Falcon, ml/well) at a density of 20,000 cells/ml of DME containing 5% heat inactivated bovine serum. The cells were incubated in a standard tissue culture incubator for 48 hr. The medium was aspirated and the wells were replenished with 0.2 ml of fresh medium. Compounds of this invention were added at concentrations ranging from 100 to 0.37 gg/ml. After 48 hr incubation, the medium was aspirated and the cultures were treated with 0.2 ml of saline 0.25 p of phenozine methosulfate solution containing MTS (Cell Titer 96 Aqueous Non-radioactive cell proliferation assay kit, Catalog G 5430, Promega, 2800 Woods Hollow Road, Madison, WI 53711-5399). The ICs 0 cells listed in Table 6 is defined as the concentration required to inhibit cell proliferation by Olomoucine at various concentrations was added and was used as a standard positive control.

TABLE 7 Bioactivity of Selected Representatives of this Invention R2 R3 ICo (gg/mL) IC, (pg/mL) enzyme cells benzylamino Me Ethanolamino 7 4-methoxybenzylamino H Cl 60 NA 4-methoxybenzylamino Me Cl 6 4-methoxybenzylamino Me Ethanolamino 4 48 4-chlorobenzyloxy H Cl 60 NA 4-chlorobenzyloxy Me Cl 60 NA 4-chlorobenzyloxy trifluoromethyl Cl >60 NA 4-methoxybenzylamino isopropyl Cl 4 77 RI~ R2 R3 IC., (J9/mL) IC, p/ enzyme cells 4-methoxvbenzylaxnino isopropyl Ethanolaznino 4 43 4-methoxybenzylamino Me Diethanoiamino 4 48 4-methoxybenzyiamino 2-methylpropyl cl 60 ethanolamino Me Ethanolamino >60 4-methoxybenzylamino trifluoromethyl C 1 >60 4-methoxybenzylamina benzyl Cl >60 ethanolamino H Benzylamino >60 NA 4-methoxybenzyianiino isopropyl Diethanolamino 0.2 2.! 4-methoxybenzylainino perflUOTOisopropyl C 1 >45 NA 4-methoxybenzylamino perfluoroisopropyl Diethanolamno 40 NA 4-methoxybenzylamino ispropyl 3-pyrroline 1 12.5 4-methox ybenzyl amino hydroxyethyl Diethanolamino 0.5 62 4-methoxybenzylamino isopropyl Serinol 0.4 4-methoxybenzyiamino isopropyl I .3-diamino-2- 0.6 4-methoxybenzylamino 3-cyanopropyl CI >60 NA 4-methoxybenzylarnino 3-chioropTOPY1 CI >60 NA 4-methoxybenzyiamino benzyl Cl >60 NA 4-methoxybenzylamino Methyl 4- CI >60 NA carboxybenzyi 4-methoxybenzyiarnino Naphthaloylethyl cI >60 NA 4-chborobenzylamino Trifluoromethyl Jl 4-methoxybenzylamino isopropyl N-(2-cyaflopropyi)-

NA

N-(3pyridylmethyl)amino 4-methox ybenzyl amino isopropyl 2-(hydroxymethyl).I 3-rnethylbutan-2- NA amino 4-methoxybenzyiarnino isopropyl 3hydroxypipenidino NA cyclohexylmethylarnino isopropyl CI

NA

piperonylarnino isopropyl Diethanolamino 0.8 NA 4-methoxybenzylaniino isopropyl Diisopropanolamin 0.8 NA 0 R2 1 R3 (jig/ML) (pg/mL) enzyme cells aniiino isopropyl C1 I NA 4-rnethoxybenzyiamino isopropyl N-benzyl-N-2- INA hydroxyethylamino 4-phenylanilino isopropyl Diethanolamino 0.6 NA 4-phenylbenzyiamino isopropyl Diethanolamino 0.6 NA 4-phenyibenzylamina isopropyl 3-amino-l,2- 0.6 NA propanediol thiophenyl)benzyiaminoDehanamo0.

4-(4-methylphenyl) isopropyl Diethanolamino 0.6 NA benzylarnino 4-4-iopoylDitanlmio0.6

NA

trifluoromethylphenyl) iorplDehnlmn benzyalmino 4-thiomethoxyaniiino isopropyl C1 0.6 NA 3-(4-nitrilophenyl) isopropyl Diethanolarnino 0.5 NA anilino 3-thiomethoxyanilino isopropyl Diethanolamino 0.1 NA 4-thiomethoxyanilino isopropyl Diethanolarnino 0.07 NA 3-methoxybenzyianiino isopropyl CI 0.9 NA 4-(2-pyridi .nyl) isopropyl Diethanolamino 0.16 N benzylanino

N

3-methoxybenzylarnino isopropyl Diethanolamino 0.5 NA Isopropyl Diethanolamino 0.12 0.3 Cblorophenyl)methyla rnino Isopropyl Diethanolamino 0.15 2.2 Fluorophenyl)methyla miuno Isobutyl Diethanolamino 59 NA Trifluoromethylphenyl) methylamino Isopropyl Diethanolamino 0.56 NA Trifluoromethylphenyl) methviam-ino_______ Isopropyl (S)-2-Aniino-3- 1.07 NA Chlorcphenyl)methyla phenyipropylanimno midno Isopropyl 2- 0.17 1.4 Fluorophenyl)methyla Aminoethylam~ino Isopropyl (D--0.06 2.7 Fluorophenyl)methyla Hydroxymethyl-2wino methylprooviamino________ (4-Fluorophenyl) Isopropyl 0.19 NA methviamino Hydroxymethyl-2-_______ R-XR2 R3 JICso (ji g/ML) 1 ICso (11glML) 1 enzyme cells methylpropylamino Isopropyl I- 0.19 NA Cblorophenyl)methyla Hydroxymethyl.2mino methyl- Isopropyl 0.05 NA Chlorophenyl)methyla Hydroxymethyl.2- Mino methylpropylainino________ Isopropyl 2-Hydroxy-2- 0.08 Chlorophenyl)rnethyla phenyi-ethylamino ndno Isopropyl 2-Axnino-NI-(2- 0.07 0.2 Cblorophenyl)methyla hydrox~yethyl)ethyl m~ino ammno Isopropyl 2-Amino-N2-(2- 2.02 NA Chlorophenyl)methyla hydroxyethyl)ethyl Mino amino Isopropyl (S)-2-Phenyl-l- 1.07 NA Chlorophenyl)methyla carboxaniidorruno ethylamino Isopropyl 2-Amino-N2-(2- 0.43 NA Chlorophenyl)methyla hydroxyethyl)-N I mino (hydroxyethyl)ethy lamino Isopropyl 2- 9 NA Sulfonarmdophenyl)me Aminoethylamino thvlamino 2-Oxo-3-butyl Diethanolamino 1NA Fluorophenyl)methyla Mino 2-Oxo-3-butyl Diethanolamino 37 NA Chlorophenyl)methyla nino Isopropyl 2- 0.35 0.1 Chlorophenyl)methyla Am-inoethylamino ""no Isopropyl 3- 1.0 NA Chlorophenyi)methyla Aminopropylamino mnno Isopropyl 5- 31 NA Chlorophenyl)methyla Aminopentylainino "no0 Isopropyl 2-Amino-2-methyl- 0.05 0.1 Chlorophenyl)methyla ethylamino Isopropyl

NA

Chlorophenyl)methyla (Hydroxymethyl)pr ""DO I ovviamino Isopropyl 1- 0.18 NA Chlorophenyflrnethyla (Hydroxyrnethyl)pr I R2 R3 IC,, (Pg/mL) IC,, (ig/mL) enzyme cells rino opylamino Isopropyl 0.26 NA Cblorophenyl)methyla (Hydroxymethyl)et mino hylamino Isopropyl 0.35 NA Chlorophenyl)methyla (Hydroxymethyl)et mino hvlamino Isopropyl 0.38 NA Chlorophenyl)methyla Hydroxypropylami nino no Isopropyl 0.43 NA Chlorophenyl)methyla Hydroxypropylami Mino no Isopropyl 2-Amino- 0.48 NA Fluorophenyl)methyla propylamino mino.

Isopropyl 0.63 NA Fluorophenyl)methyla Tearahydrofuranyl) rino methvlanino Isopropyl 0.58 NA Fluorophenyl)methyla Tetrahydrofuranyl) Mino methvlamino Isopropyl 2-Hydroxy- 1- 0.18 NA Fluorophenyl)methyla methylethylamino min Isopropyl (S)-2-Hydroxy-2- 0.22 NA Fluorophenyl)methyla methylethylamino mino Isopropyl (R)-2-Hydroxy-2- 0.23 Fluorophe'nyl)methyla methylethylamino mino Isopropyl 1- 0.11 2.4 Fluorophenyl)methyla Hydroxymethyipro mino _pylino The inhibition of cell proliferation properties of the compounds of this invention are demonstrated by their ability to inhibit cell proliferation in the range of about 0.05 pg/mi to 100 g.g/ml, preferably less than 0. 5 pLg/ml.

Similar assays were performed using the following cell lines; P388 mouse lymphoid neoplasm; L 12 10 mouse lymphcytic leukemia; Caco2 human colon adenocarcinoma; MCF7 human breast adenocarcinoina; *PupVSMC rat neonatal aortic smooth muscle cells; Ovcar human ovarian Carcinoma; Pancl human pancreatic adenocarcinomna; and HUVEC human umbilical cord endothelial cells. The inhibitory activity of several compositions of this invention against one or more of the cell lines are reported in Tables 8 and 9 below.

Table 8 (jyg/ml) for Inhibition of Cell Proliferation R,X R, R, P L Cacm MCF Panc I OvC Pup HU 388 120 2 7 ar VS VE

C

Isopropyl Diethanolamina 1.5 2.5 4.5 8.0 10.0 11.0 0.5 rnethoxyphenyl )-methyiamino Isopropyl Diethanolamino 1.0 4.0 0.5 4.0 4.0 7.0 phenylphenyl)a mino- Isopropyl Diethanolamino 1.0 1.0 3.5 1.0 1.3 phenylphenyl)methvlamino Isopropyl Morpholino >5 5.5 methoxyphenyl )-methylamino T- isopropyl Diethanoiamino 1.5 2.0 2.5 phenoxyphenyl '3benzyloxyphen Table 9 RA R R MRC-5 [PuP VSMC (4-methoxyphenyl)methylamino Isopropyl Diethanolarnino 5 0.4 (4-Chlorophenyl)methylaniino Isopropyl 2-Amninoethylamino 1 0.1 (4-Chlorophenyl).methylamino Isopropyl 2-Ainino-2- 1 .0.1 methylethylamino (4-Chlorophenyl)-methylainino Isopropyl 2-Amino.-N1-(2- 1 0.3 hydroxyethyl)ethylamin (4-Chlorophenyl)-methylarmno -Isopropyl j Diethanolamnc 0.3 Human Fibroblast Pup VSMC Rat neonatal aortic smooth muscle cells EXAMPLE 8 Compounda of this invention was evaluated for effectiveness using the Murine Leukemia Model. The Murine Leukemia Model is a standard model used in the evaluation of antitumor agents. CDF1 mice were injected ip with L1210 cells (1xl0 cells/mouse).

Twenty-four hours later, these mice were treated with various doses (ip) of compound 3 of Example I in saline. The dosing regimen used in this study is outlined in Table 10, below.

Mice were dosed with compound 3 daily or on alternate days. Control mice received saline.

After 7 days, dosing was suspended and survival monitored.

Table Treatment N Median T/CxIOO survival time Days Saline control 7 10(9-13) 100 Compound 3 0.5 mg/kg bid 7 11(10-15) 110 mg/kg bid 7 13 (11-13) 130 2 mg/kg bid 7 12(10-14) 120 4 mg/kg days 7 13 (10-15) 130 1,3,5,7 8 mg/kg days 7 13(12-16) 130 1,3,5,7 The results indicate that rats administered compound 3 survived longer than the control rats.

68 EXAMPLE 9 This example measured the effect of an acute local delivery of compound 3 of Example 1 in reducing neointima formation following balloon angioplasty in the rat carotid artery model. In this example, the left common carotid arteries of adult male rats (n=10 per experimental group) were surgically injured using a Fogarty arterial embolectomy catheter.

Immediately after injury, the common carotid artery was bisected with a vascular clamp, thereby establishing an untreated and treated segment. A drug delivery catheter was then inserted into the distal half of the common carotid. After drug delivery, the catheter was removed and excess drug was washed out by removing the vascular clamp and re-establishing blood flow before closing the artery. The animals were allowed to recover for 14 days before harvesting the common carotid artery. The harvested tissue was sectioned and the neointimal area was digitized and measured with a computer planimetery system. For each animal, measurements were averaged for the untreated segment and 15 for the treated. aminoethyl)amino]-9-( methylethyl)purin-6-yl} [(4-chlorophenyl)methyl]amine was administered at a dose of 5 mg/mL reducing the neointimal area about 90% in comparison to the 6% reduction of saline alone.

The results of this Example are found in Figure 1. According to Figure 1, administering compound 3 of Example 1 to a damaged carotid artery reduced the neointimal area about 88% in comparison to the 6% reduction produced by the saline vehicle alone.

nlA 69 EXAMPLE IKB-ct Kinase Assays: Compositions of this invention were assayed to determine their IKB-a kinase inhibitory activity. The human umbilical vein endothelial cell line (HIJVEC) used in these studies was purchased from Clonetics (San Diego, CA) and was maintained in endothelial cell growth medium supplemented with 2% fetal bovine serum, lOng/mi human recombinant epidermal growth factor, 1 Lg/ml hydrocortisone, 50 4.g/ml gentamnicin, 50 ng/ml amphotericin B and 12 .±g/ml bovine brain extract at 3700 in a tissue culture incubator. All growth media and supplements were purchased from Clonetics (San Diego, CA). E. coli io lipopolysacchaide (LPS) serotype 011 I:B4 was purchased from Sigma (Saint Louis, Ml). All other chemicals were of reagent grade.

Preparation of cell Lvsate: Monolayers (75 cm') of HUVEC cells were treated with LPS (100 ng/ml) for 5 minutes after which the cell media was rapidly removed and the monolayer washed three times with ice cold PBS. The cell layer was scraped into 10 ml PBS and the is cells pelleted by centriftigation (3000 rpm, 5 mi, 400). Cell lysate was prepared by incubating the cell pellet in 0.2 ml lysis buffer (20mM =EES, pH7.3, 50mM NaCI, MgCl,, 1mM EDTA, 1mM EGTA, 1mM sodium orthovanadate, 10m.M 1-glycerophospate, 1mM phenylmethylsulfonylfroride, 1mM dithiothreitol, 0.5% Nonidet P-40 for 15 minutes at 37'C for frequent vortexing. Cell debris was removed from the sample by rnicrocentrifugation (1 0,OO0xg, 15 minutes, 400) and the supernatant was "precleared" by the addition of 100 ml of a suspension of sepharose 4B in lysis buffer and mixing gently for I hour at 4'C. The speharose 4B beads were removed by microcentrifiagation and the supernatant aliquotted and stored at 8000.

Solid Phase IicB-ax kinase assay: 1 4±g of GST- IicB-ct, corresponding to full length IKB-a of human origin, (Santa Cruz Biotechnology,) was incubated with 20 41d of a 50% slurry of glutathione S sepharose 4B (Phannacia) in reaction buffer (20mM HEPES, pH7.3, MgCl 2 1 5mM f-glycerophosphate, 0.5mM sodium orthovanadate, 0.5mM EGTA) for minutes at room temperature. The GST- 1KB-bead complex was the washed three times with ml of reaction buffer by resuspension and microcent-ifugation. 104.g of IiUVEC cell lysate protein in 1 Oi of reaction buffer was then added to the GST- 1KB-bead complex and the mixture incubated with gentle mixing at 41C for 1 hour. The bead complex was then washed three times with reaction buffer containing 0.2 M NaCl and once with reaction buffer alone. Finally the bead complex was resuspended in 2041l of reaction buffer containing 54~Ci [y- 32 P]ATP (>5000 ci/mmol, New England Nuclear Corp. Boston, MIA) and incubated at room temperature for 15 minutes. The reaction was terminated by the addition of 1 0j41 of SDS- PAGE sample buffer and boiled for 3 minutes before separation by SDS-PAGE (10-20% gradient Readygel, BioRad). Following electrophoresis the gel was fixed (50% methanol 10% acetic acid) for 15 minutes, washed three times for 5 minutes each with distilled H.0 and treated with 5% glycerol for 15 minutes before drying down and exposing to film for autoradiography (X-OMAT XAR-5 Kodak).

In gel kinase assay: 1KB-a isozyrnes were assayed for activity using a modification of previously published methods (11, 19, 20). Briefly duplicate samples of the IicB-glutathione sepharose 4B bead complex were prepared as described above and were separated by electrophoresis through a 12% SDS-PAGE gel which had been polymerised in the presence of 4.g/ml GST- 1KB-a. Following electrophoresis the gel was washed gently twice for minutes each with 50m.M Tris-HCI pH8.0, 5mM P-mercaptoethanol; 20% isopropanol to remove SDS. Proteins were then denatured within the gel by incubation for 45 minutes in lO0mi 50mM Tris-HCl pHS.0; 5mM P-mercaptoethanol; 0.04% Tween 40. The gel was then cut in half to separate the duplicate samples, one half was incubated in 10 ml reaction buffer alone and the other in 10 ml reaction buffer containing 1 0~.g/ml of 2-diethanolaxnino-6(4phenyl anilino)-9-isopropyl purine (compound 6 of Example 2) for 1 hour at room temperature which 1 0 Cqy_1 2 P]ATP was added and the incubations continued for a further hour at room temperature. The gels were then subjected to multiple 15 minute washes of each 5% trichloroacetic acid containing 1% sodium pyrophosphate until 1 ml of wash solution gave close to background radioactivity. The gels were then dried down and exposed to file for autoradiograhy.

Pre~aration of 2-di ethanol amino-6-(4-n2henvbenzlamino)-9-isonronvI Rurine Epoxy activated Seoharose 6B Affinity Matrix. Freeze dried epoxy activated Sepharose 6B (Pharmacia LKB, Piscataway, NJ) was chosen for the coupling reaction due to its ability to form an ether bond between an hydroxylI-containing ligand and the epoxide group on the sepharose. The gel was swollen according to the manufacturer's instructions, (100mg) of compound 6 of Example 2 was dissolved in Iln coupling solution (1.2:1 v/v dimethylformnamide 0.1N NaOH) and mixed with 0.5m1 of swollen gel at pH 10-1 1 for 726 hours at room temperature with gentle agitation. Excess reactive groups were blocked with IM ethanolamine for 4 hours at and the gel slurry was poured into 1 ml syringe column. The resin was activated with three alternating cycles of twenty column volumes each of ?H 4.0 (0.1M acetate, 0.5M NaCI) and pH 8.0 1M Tris-HCI, 0.5M NaCi) buffers followed by twenty column volumes of reaction buffer (20mM HEPES, pH7.3, 10mM MgCI.,, 15m.M P-glycerophophate, 0.5mM sodium oi-thovanadate, 0.5mM EGTA). The column was stored at V 0 C in reaction buffer containing 72 sodium azide and regenerated prior to each use with alternating cycles of low and high pH as described above.

Activated I{UVEC cell lysate (500jitg protein in Imi. reaction buffer) was passed over the CV T-1545 sepharose matrix sequentially five times and the flow through was saved (unbound material). The matrix was then washed three times with imi of reaction buffer (wash 1-3) then three times each with reaction buffer containing 0.5M NaCI (eluiate 1-3).

Aliquots (2041t from Imi) of each sample were assayed for their ability to phosphorylate at GST- IKB-sepharose bead complex and analyzed by SDS-PAGE as described above.

Assay of affinity enriched IK-B-(x kinase. The bulked 0.5 M NaCl eluates from the affinity matrix were used as the source of enzyme for development of an IicB-ct kinase filter assay.

Each reaction contained affinity enriched IKB-ct kinase (1lpg protein), 1lOng GST IicB-a kinase and 0.5 pCi[y- 32 P]ATP (>5000 Cilmmnol, New England Nuclear Corp, Boston, MA) in reaction buffer. The reaction was incubated for 15 minutes at room temperature and was terminated by the addition of 2 1 0.5M EDTA. Reaction mixtures were blotted onto phosphocellulose disks (Gibco BRL Life Technologies, Gaithersburg, IM) and the filters washed three times with 0.1 5M phosphoric acid with gentle shaking for 15 minutes (up to ten filters were washed with 300 ml of 0. 1 5M phosphoric acid.) Following a third wash the filters were air dried, added to scintillation fluid and assayed by liquid scintillation spectrometry.

Electrophoretic Mobility Shift Assay: Nuclear extracts were prepared using a high-salt buffer extraction procedure. 10 pmol of double stranded NF--cB consenses oligonucleotide AGTTGAGGGGACTTTCCCAGGC-3') )Promega) was 5' end labeled with 5 ci [y- 32

P]ATP

(>5000 Cilinmol, New England Nuclear Corp, Boston, MA) by incubaton with T4 polynucleotide kinase for 1 hr at 371C. Unincorporated nucleotides were removed by pasing the reaction mixture over I ml Sephadex G-5-spin column. Binding assays were performed at room temperature for 1 hr and consisted of 1 04g nuclear extract protein, 14~g salmon sperm DNA, and Sx 10 cpin of 2 P labeled consensus of oligonucleotide in the presence and absence of fifty fold unlabeled oligonucleotide. DNA-protein complexes were resolved by 8% non denaturing polyacrylamide gel electrophoresis, the gels were dried onto filter paper and visualized by autoradiography.

Table 11 Ac'ti'viv of Selectedl R nrapntativPc nf thi TnVPinn En ~v m p R 1XR2 R3 IC5O(4.nM 4-phenylbenzylamino Isopropyl Diethanolarnino 1.1 4-phenylbenzylaznino Isopropyl Diethylamino >2.4 4-phenylbenzylamino Isopropyl Ethanolamino 4-bromoanilino Isopropyl Diethanolarnino 14 4-(3-methoxphenyl) Isopropyl Diethanolamino benzylamino__________ 4-(4-methoxphenyl) Isopropyl Diethanolanuno 11 3-(4-nitrilophenyl) Isopropyl Diethanolanuno 2.2 anilino 4-thiomethoxyanilino Isopropyl Diethanolamino 12.4 4-(2-pyridinyl) Isopropyl Diethanolanmino [benzylamnino

I__

Claims (27)

1. A compound selected from the group consisting of: 0 [(2-aminoethyl)amino] -9-(methylethyl)purin-6-yl chlorophenyl)methyl]amine; [(2-aminopropyl)amino] -9-(methylethyl)purin-6-yl chlorophenyl)methyl] amine;

2- [(2-aminoethyl)(6- [(4-chlorophenyl)methyl] amino)} -9- (methylethyl)purin-2-yl)amino] ethan-lI-ol; and 2-[(2-hydroxyethyl)(6- [(4-chlorophenyl)methyl] amino) -9- (methylethyl)purin-2-yl)amino] ethan-l1-ol. 2. The compound of claim I wherein the compound is aminoethyl)amino] -9-(methylethyl)purin-6-yI [(4-chlorophenyl)methyl] amine.

3. The compound of claim I wherein the compound is aminopropyl)amino] -9-(methylethyl)purin-6-yl [(4-chlorophenyl)methyl] amine.

4. The compound of claim 1 wherein the compound is 2-[(2-aminoethyl)(6- [(4-chlorophenyl)methyl] amino)} -9-(methylethyl)purin-2-yl)amino]ethan- I -ol.

The compound of claim I wherein the compound is 2-[(2-hydroxyethyl)(6- [(4-chlorophenyl)methyl]amino }-9-(methylethyl)purin-2-yI)amino]ethan- I -ol.

6. A cationic salt of a compound of any one of claims I to

7. An acid addition salt of a compound of any one of claims I to

8. A method for inhibiting cell proliferation in mammals comprising administering a therapeutically effective amount of a compound of any one of claims I to to the mammal.

9. The method of claim 8 wherein the therapeutically effective amount ranges from about 0.001 to about 100 mg/kg weight of the mammal. The method of claim 8 or 9 wherein the compound is administered to a mammal suffering from a cell proliferation disorder selected from the group consisting of rheumatoid arthritis, lupus, type I diabetes, multiple sclerosis, cancer, restenosis 004889492 0 following ballon angioplasty or atherectomy, restenosis following vascular modifying C' surgical procedures, host graft disease, and gout.

O

11. The method of claim 10 wherein the cell proliferation disorder is restenosis.

12. The method of claim 10 wherein the cell proliferation is disorder cancer.

13. The method of claim 10 wherein the cell proliferation disorder is polycystic kidney disease.

14. The method of claim 10 wherein the cell proliferation disorder is selected from the group consisting of lymphoyd neoplasm, cancer of the colon, breast cancer, ovarian cancer, pancreatic cancer, and cancers derived from endothelial cells.

The method of any one of claims 8 to 14 wherein the mammal is a human.

16. Use of a compound of any one of claims 1 to 5 for the preparation of a medicament for inhibiting cell proliferation in a mammal.

17. The use of claim 16 wherein the medicament comprises the compound of any one of claims 1 to 5 in an amount ranging from about 0.001 to about 100 mg/kg weight of the mammal.

18. The use of claim 16 or 17 wherein the medicament is to be administered to a mammal suffering from a cell proliferation disorder selected from the group consisting of rheumatoid arthritis, lupus, type I diabetes, multiple sclerosis, cancer, restenosis following ballon angioplasty or arthrectomy, restenosis following vascular modifying surgical procedures, host graft disease, and gout.

19. The use of claim 18 wherein the cell proliferation disorder is restenosis.

The use of claim 18 wherein the cell proliferation is disorder is cancer.

21. The use of claim 18 wherein the cell proliferation disorder is selected from the group consisting of lymphoid neoplasm, cancer of the colon, breast cancer, ovarian cancer, pancreatic cancer, and cancers derived from endothelial cells. 004889492 76 I

22. The use of claim 18 wherein the cell proliferation disorder is polycystic N, kidney disease. O

23. The use of any one of claims 16 to 22 wherein the mammal is a human.

24. A pharmaceutical composition comprising a compound of any one of claims 1 to 5 and one or more pharmaceutical excipients.

An antifungal agent useful for treating fungal infections in humans, and animals comprising a compound of any one of claims 1 to

26. A compound according to claim 1 substantially as hereinbefore described with reference to the examples.

27. A method for inhibiting cell proliferation in mammals according to claim 8 substantially as hereinbefore described with reference to the examples. Dated: 25 October 2006 Freehills Patent Trade Mark Attorneys Patent Attorneys for the Applicant: CV Therapeutics, Inc.