Patent Application
20070167466
This invention relates to new pyrazolo[4,3-d]pyrimidine derivatives and to their use in suitable utilities, especially in cancer therapy and agricultural practice.
The cell division cycle is an evolutionarily conserved process in all eukaryotic cells to control growth and division. The cell cycle consists of four distinct stages illustrated in
Proliferative disorders such as cancer are recognised as diseases of the cell cycle. It has been found that in tumour cells, the mechanisms that normally function to restrain cell division are defective, whilst those that promote division become more active. The genes responsible for these changes in growth and proliferation are generally named “tumour suppressors” and “oncogenes”. Cell-cycle regulatory compounds are pivotal in the modulation of abnormal cellular proliferation as they provide ideal targets for therapy for a range of proliferative disorders.
A series of 3-,7-disubstituted pyrazolo[4,3-d]pyrimidines are useful for inhibition of cyclin-dependent kinases (preferably p34cdc2/cyclin B). Hence they can be used as antimitotic and apoptotic drugs, particularly as anticancer drugs and herbicides. Likewise, the compounds can be used as anti-fungal agents, which may have high value in the treatment of aspergillosis, penicilliosis, actinomycosis and the like. Difference in homology of insect CDK genes permit selection of compounds of this invention which discriminate between insect/mammalian CDK enzymes and thus leads to insecticides.
It is an object of this invention to provide antimitotic, anticancer, herbicidal, fungicidal and insecticidal compounds having improved selectivity and efficiency index, i.e. that are less toxic yet more efficacious than analogues known heretofore.
It is an object of this invention to provide 3,7-disubstituted pyrazolo[4,3-d]pyrimidines, which inhibit the cdks, cell proliferation or block cytokinin receptors.
A further object of this invention is to provide a pharmaceutical composition, which comprises a 3,7-disubstituted pyrazolo[4,3-d]pyrimidine, and a pharmaceutically acceptable carrier.
A further object of this invention to provide a method for inhibiting cell proliferation and/or inducing apoptosis to a mammal or plant in need of an effective amount of 3,7-disubstituted pyrazolo[4,3-d]pyrimidines.
This invention further constitutes a method for inhibiting cell proliferation to a plant in need of an effective amount 3,7-disubstituted pyrazolo[4,3-d]pyrimidines.
The solution of this object are 3-,7-disubstituted pyrazolo[4,3-d]pyrimidines represented by the general formula I
and pharmaceutically acceptable salts thereof, wherein
R3 is selected from the group consisting of
alkyl, cycloalkyl, cycloalkyl alkyl, cycloheteroalkyl alkyl, cycloheteroalkyl, aryl, heterocycle, heteroaryl, arylalkyl, heteroarylalkyl, and heteroalkyl, wherein each of the groups may optionally be substituted,
R7 is selected from the group consisting of halogen, hydroxyl, hydroxylamino, amino, carboxyl, cyano, nitro, amido, sulfo, sulfamido, carbamino, unsubstituted or substituted alkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted arylalkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted heteroarylalkyl, substituted or unsubstituted cycloalkyl alkyl, substituted or unsubstituted cycloheteroalkyl alkyl;
R7′-X— wherein X is an —NH—, —N(alkyl)-, —O— or —S— moiety and
R7′ is selected from the group consisting of H, alkyl, cycloalkyl, aryl, alkylcycloalkyl, arylalkyl, heterocycle, heterocycloalkyl, substituted alkyl, substituted cycloalkyl, substituted aryl, substituted arylalkyl, substituted heterocycle, substituted heteroaryl, substituted heteroarylalkyl, substituted heteroalkyl, substituted cycloalkyl alkyl and substituted cycloheteroalkyl alkyl.
If the above groups are substituted, they are preferably substituted by halogen, hydroxyl, amino, mercapto, carboxyl, cyano, nitro, amido, sulfo, sulfamido, carbamino, alkyl, alkoxy, and/or substituted alkyl group, in particular by more than one of the above substituents, preferably by 1 to 3 substituents.
In another embodiment, this invention is a method for inhibiting cdks and cell proliferation and/or for inducing apoptosis in plants, comprising administering an effective amount of a composition comprising one or more compounds according to claim 1 to the plant. The cdk inhibiting molecules are useful for treating disorders, some of them involving cell proliferation, and thus are useful as herbicides.
In yet another embodiment, this invention is a pharmaceutical composition comprising one or more compounds according to claim 1 in an admixture with one or more pharmaceutical excipients.
In still another embodiment, this invention is a composition comprising one or more compounds according to claim 1 useful for treating fungal infections (fungi) in plants.
In another embodiment, this invention is a composition comprising one or more compounds according to claim 1 useful for treating insects and yeasts on plants.
3,7-disubstituted pyrazolo[4,3-d]pyrimidines result in the acquisition of extremely high potency against plant viruses on the part of the defined compounds. As used herein, and unless modified by the immediate context:
“Halogen” preferably refers to fluorine, bromine, chlorine and iodine atoms.
“Hydroxy” refers to the group —OH.
“Mercapto” refers to group —SH.
“Alkyl” preferably refers to branched or unbranched C1-C8 alkyl chain which is saturated or unsaturated. Thus, the term “alkyl” when used herein encompasses alkyl alkenyl and alkinyl groups. Alkenyl groups preferably have 2 to 8 carbon atoms, alkinyl groups preferably have 3 to 8 carbon atoms. Such groups as methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, tert-butyl, allyl, ethinyl, propargyl, and the like can exemplify this term.
“Substituted alkyl” preferably refers to alkyl as described above including one to six, in particular 1 to 3 substituents such as hydroxyl, mercapto, alkylthio, halogen, alkoxy, acyloxy, amino, acylamino, hydrazino, carbamoyl, amido, carboxyl, sulfo, acyl, guanidino and the like. These groups may be attached to any carbon atom of the alkyl moiety.
“Alkoxy” denotes the group —OR, where R is preferably alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, cycloalkyl, substituted cycloalkyl, cycloheteroalkyl or substituted cycloheteroalkyl as defined herein.
“Alkylthio” denotes the group —SR, where R is preferably as defined for “alkoxy” group.
“Sulfo” denotes the group —SO3R, where R is preferably H, alkyl or substituted alkyl as defined above.
“Sulfamido” denotes to the group SO2NRR″ where R and R″ is preferably H, alkyl or substituted alkyl as defined above.
“Acyl” denotes groups —C(O)R, where R preferably is alkyl, substituted alkyl, aryl, substituted aryl, arylalkyl, substituted arylalkyl, cycloalkyl, substituted cycloalkyl as defined herein.
“Aryloxy” denotes groups —OAr, where Ar is preferably an aryl, substituted aryl, heteroaryl or substituted heteroaryl group as defined herein.
“Alkylamino” denotes the group —NRR′, where R and R′ may independently be hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl or substituted heteroaryl as defined herein.
“Amido” denotes the group —C(O)NRR′, where R and R′ may independently be hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl as defined herein.
“Carboxyl” denotes the group —C(O)OR, where R is preferably hydrogen, alkyl, substituted alkyl, aryl, substituted aryl, hetaryl or substituted hetaryl as defined herein.
“Acylamino denotes the group —NHCOR, where R may be alkyl, substituted alkyl, heterocycle, aryl, substituted aryl, heteroaryl and substituted heteroaryl as defined herein.
Carbamoylamino denotes the group NHCOOR, where R is preferably alkyl or aryl.
“Aryl” or “Ar” refers to an aromatic carbocyclic group having at least one aromatic ring (e.g., phenyl or biphenyl) or multiple condensed rings in which at least one ring is aromatic (e.g., 1,2,3,4tetrahydronaphthyl, naphthyl, anthryl, or phenanthryl).
Preferably, the aryl group has more than six, in particular 6 to 10 carbon atoms.
“Substituted aryl” refers to aryl as described above which is optionally substituted with one or more functional groups, in particular 1 to 3 substituents, such as halogen, alkyl, hydroxy, amino, acylamino, carbamoylamino, hydrazino, mercapto, alkoxy, alkylthio, alkylamino, amido, carboxyl, nitro, sulfo and the like as defined herein.
“Heterocycle” refers to a unsaturated or aromatic carbocyclic group preferably having 1 to 3 rings and having at least one, preferably 1 to 3 and in particular 1 or 2 hetero atoms, such as N, O or S, within the ring; the ring can be single (e.g. pyranyl, pyridyl or furyl) or multiple condensed (e.g., quinazolinyl, purinyl, quinolinyl or benzofuranyl) which can optionally be unsubstituted or substituted with, e.g., halogen, amino, hydroxy, cyano, nitro, mercapto, alkoxy, alkylamino, acylamino, carbamoylamino, acyloxy, dialkylamino, alkylthio carboxyl, amido, sulfo, sulfamido, and the like as defined above. The heterocycle groups preferably has 5 to 10 ring atoms, which are either carbon atoms or hetero atoms as defined above.
“Heteroaryl” refers to a heterocycle in which at least one heterocyclic ring is aromatic.
“Substituted heteroaryl” refers to a heterocycle optionally mono or poly substituted with one or more functional groups, preferably 1 to 6, in particular 1 to 3 substituents, e.g., halogen, amino, hydroxy, cyano, nitro, mercapto, alkoxy, alkylamino, acylamino, carbamoylamino, acyloxy, dialkylamino, alkylthio carboxyl, amido, sulfo, sulfamido, and the like.
“Arylalkyl” refers to the group —R—Ar where Ar is an aryl group and R is alkyl or substituted alkyl group as defined above. The aryl groups can optionally be unsubstituted or substituted as defined above with, e.g., halogen, amino, acylamino, carbamoylamino, hydrazino, acyloxy, alkyl, hydroxyl, alkoxy, alkylthio, alkylamino, amido, carboxyl, hydroxy, aryl, nitro, mercapto, sulfo and the like.
“Heteroalkyl” refers to the group —R-Het where Het is a heterocycle group and R is a alkyl group as defined above. Heteroalkyl groups can optionally be unsubstituted or substituted as defined above with e.g., halogen, amino, hydroxy, cyano, nitro, mercapto, alkoxy, alkylamino, acylamino, carbamoylamino, acyloxy, dialkylamino, alkylthio, carboxyl, amido, sulfo, sulfamido, and the like.
“Heteroarylalkyl” refers to the group —R-HetAr where HetAr is an heteroaryl group and R is alkyl or substituted alkyl as defined above. Heteroarylalkyl groups can optionally be unsubstituted or substituted as defined above with, e.g., halogen, alkyl, substituted alkyl, alkoxy, alkylthio, nitro, mercapto, sulfo and the like.
“Cycloalkyl” refers to a divalent cyclic or polycyclic alkyl group containing preferably 3 to 15 carbon atoms.
“Substituted cycloalkyl” refers to a cycloalkyl group comprising one or more substituents as defined above with, e.g., halogen, amino, hydroxy, cyano, nitro, mercapto, alkoxy, alkylamino, acylamino, carbamoylamino, acyloxy, dialkylamino, alkylthio, carboxyl, amido, sulfo, sulfamido, and the like.
“Cycloheteroalkyl” refers to a cycloalkyl group as defined above wherein one or more, preferably 1 to 3, of the ring methylene group is replaced with a heteroatom (e.g., NH, O, S)
“Substituted cycloheteroalkyl” refers to a cycloheteroalkyl group as herein defined which contains one or more substituents as defined above, such as halogen, amino, hydroxy, cyano, nitro, mercapto, alkoxy, alkylamino, acylamino, carbamoylamino, acyloxy, dialkylamino, alkylthio, carboxyl, amido, sulfo, sulfamido and the like.
“Cycloalkyl alkyl” denotes the group —R-cycloalkyl where cycloalkyl is a cycloalkyl group as defined above and R is an alkyl or substituted alkyl as defined above. Cycloalkyl groups can optionally be unsubstituted or substituted as defined above with e.g., halogen, amino, hydroxy, cyano, nitro, mercapto, alkoxy, alkylamino, acylamino, carbamoylamino, acyloxy, dialkylamino, alkylthio, carboxyl, amido, sulfo, sulfamido and the like.
“Cycloheteroalkyl alkyl” denotes he group —R-cycloheteroalkyl where R is a alkyl or substituted alkyl as defined above and cycloheteroalkyl as defined above. Cycloheteroalkyl groups can optionally be unsubstituted or substituted as defined above with e.g. halogen, amino, hydroxy, cyano, nitro, . mercapto, alkoxy, alkylamino, acylamino, carbamoylamino, acyloxy, dialkylamino, alkylthio, carboxyl, amido, sulfo, sulfamido, and the like.
In a preferred embodiment the invention relates to 3-,7-disubstituted pyrazolo[4,3-d]pyrimidines, which inhibit the cyclin-dependent and MAP kinases have formula I
and the pharmaceutically acceptable acid salts thereof, wherein
R3 is
selected from an alkyl, cycloalkyl, cycloalkyl alkyl, cycloheteroalkyl, cycloheteroalkylalkyl, cycloalkyl alkyl, aryl, arylalkyl, heteroaryl, heteroarylalkyl, wherein each of the groups may be optionally be substituted by a halogen;
R7 is selected from the group consisting of halogen, hydroxyl, hydroxylamino, amino, hydrazino, carboxyl, cyano, nitro, amido, sulfo, sulfamido, carbamino, NHCONH2, NHC(═NH)NH2, alkyl, cycloalkyl, cycloalkylalkyl, aryl, arylalkyl, heteroalkyl, heteroaryl, heteroarylalkyl, cycloheteroalkyl, cycloheteroalkylalkyl, which is substituted independently at each occurrence with 0-5 substituents selected from the group halogen, hydroxy, alkoxy, amino, hydrazo, mercapto, carboxyl, cyano, nitro, amido, sulfo, sulfamido, acylamino, acyloxy, alkylamino, dialkylamino, alkylthio and carbamoyl group;
The following derivatives are particularly preferred, namely: 7-(2-hydroxy-3-chlorobenzyl)amino-3-(methyl, ethyl, isopropyl, cyclopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-hydroxy-4-chlorobenzyl)amino-3-(methyl, ethyl, isopropyl, cyclopropyl, 3-pentyl benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-hydroxy-5-chlorobenzyl)amino-3-(methyl, ethyl, isopropyl, cyclopropyl, 3-pentyl. benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-hydroxy-6-chlorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-hydroxy-3-iodobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-hydroxy-4-iodobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-hydroxy-5-iodobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, . 7-(2-hydroxy-6-iodobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-hydroxy-3-bromobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 3-pentyl, 7-(2-hydroxy-4-bromobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-hydroxy-5-bromobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-hydroxy-6-bromobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-hydroxy-3-fluorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-hydroxy-4-fluorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-hydroxy-5-fluorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 6-(2-hydroxy-6-fluorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,3-dihydroxy-4-methoxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,5-dihydroxy-4-methoxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,6-dihydroxy-3-methoxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,3-dihydroxy-3-methoxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,5-dihydroxy-3-methoxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,6-dihydroxy-4-methoxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,3-dihydroxy-4-chlorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,3-dihydroxy-4-chlorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,5-dihydroxy-4-chlorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,6-dihydroxy-4-chlorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,6-dihydroxy-4-bromoxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,6-dihydroxy-4-iodobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,6-dihydroxy-3-chlorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,6-dihydroxy-3-bromobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,6-dihydroxy-3-iodobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,6-dihydroxy-3-fluorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,6-dihydroxy-3,5-dichlorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,6-dihydroxy-3,5-dibromobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,6-dihydroxy-3,5-diiodobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,6-dihydroxy-3,5-difluorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-fluorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-fluorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-fluorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-bromobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-bromobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-bromobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-iodobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-iodobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-iodobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-chlorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-chlorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-chlorobenzylamino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine 7-(4-chlorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-acetylbenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-acetylbenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-acetylbenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-karboxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-karboxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-acetoxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-acetoxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-acetoxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-nitrobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-nitrobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-nitrobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-cyanobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-cyanobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-cyanobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(5-nitro-2-methylbenzyl)amino-3-(methyl, ethyl, isopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-methylbenzyl)amino-3-(methyl, ethyl, isopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-methylbenzyl)amino-3-(methyl, ethyl, isopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-methylbenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-methylaminobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-methoxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-methoxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-methoxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-hydroxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-hydroxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-hydroxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-hexylbenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-hexyloxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-ethoxybenzyl)amino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-ethoxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-ethoxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-ethylbenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-penthylbenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-penthyloxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-fenoxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-fenylbenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-propylbenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-propyloxybenzyl)aminopurin, 7-(4-oktylbenzylamino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-octyloxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-ethyloxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3,4-diacetoxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3,5-diacetoxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 67-(2,5-diaminobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3,5-dibromobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3,5-dibromo-4-methoxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,3-dichlorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,4-dichlorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,5-dichlorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,6-dichlorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3,4-dichlorobenzyl)amino 3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3,5-dichlorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,3,4,5-tetrafluorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-chloro-3,6-difluorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(5-chloro-2-fluorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,3,4-trifluorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,3,5-trifluorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,4,5-trifluorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3,4,5-trifluorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,3,6-trifluorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-chloro-2,6-difluorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-chloro-6-fluorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,6difluorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,4-difluorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3,4-difluorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,5-difluorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3,5-difluorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-[5-fluoro-2-(trifluoromethyl)benzyl]amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-[4-fluoro-2-(trifluoromethyl)benzyl]amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-chloro-5-(trifluoromethyl)benzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-(difluoromethoxy)benzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-(difluoromethoxy)benzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-(difluoromethoxy)benzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-[2-fluoro-5-(trifluoromethyl)benzyl]amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-[3-fluoro-4-(trifluoromethyl)benzyl]amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-[2-fluoro-4-(trifluoromethyl)benzyl]amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-(trifluoromethylthio)benzylamino)purin, 7-[2-fluoro-3-trifluoromethyl)benzy]lamino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-chloro-6-fluoro-3-methylbenzylamino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(6-chloro-2-fluoro-3-methylbenzylamino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-[3-chloro-2-fluoro-5-(trifluoromethyl)benzyl]amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-[3-chloro-2-fluoro-6-(trifluoromethyl)benzyl]amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,3-difluoro-4methylbenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,6-difluoro-3-methylbenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-fluoro-6-(trifluoromethyl)benzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-chloro-2,6-difluorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-[3-(trifluoromethylthio)benzy]lamino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-fluoro-4-methylbenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-[4-fluoro-3-methylbenzyl]amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(5-fluoro-2-methylbenzylamino)-3-(methyl, ethyl, isopropyl, 3-pentyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-chloro-3,6-difluorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-[4-trifluoromethylthio)benzyl]amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-fluoro-5-(trifluoromethyl)benzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-chloro-4-fluorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-(trifluoromethoxy)benzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-(trifluoromethyl)benzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-[2-(trifluoromethyl)benzyl]amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-trifluoromethyl)benzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-[4-chloro-3-(trifluoromethyl)benzyl]amino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-fluoro-3-(trifluoromethyl)benzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-[3,5-di(trifluoromethyl)benzyl]amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-[3-(trifluoromethoxy)benzyl]amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-[4-(trifluoromethoxy)benzyl]amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-(trifluoromethyl)benzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-[4-(diethylamino)benzyl]amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3,4-dihydroxybenzyl]amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3,5-dihydroxybenzyl]amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3,4-dihydroxybenzyl]amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,3-ethylenedioxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,4-dihydroxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,5-dihydroxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, isopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,6-dihydroxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3,4-dimethoxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3,4-dimethoxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3,5-dimethoxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,3-dimethoxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,4-dimethoxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,5-dimethoxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,6-dimethoxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-hydroxy-4-methoxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-hydroxy-3-methoxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-hydroxy-3-methoxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-hydroxy-4-methoxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-hydroxy-2-methoxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-hydroxy-5-methoxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-hydroxy-4-methoxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-hydroxy-3-methoxybenzylamino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-hydroxy-6-methoxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-hydroxy-5-methoxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4,5-dimethoxy-2-nitrobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3,4-dimethylbenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,3-dimethylbenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,4-dimethylbenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,6-dimethylbenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,6-dimethyl-4-hydroxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3,5-dimethyl-4-hydroxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-fluoro-4-hydroxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-fluoro-4-methylbenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3,4-dinitrobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3,5-dinitrobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-methyl-5-nitrobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-methyl-4-nitrobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3,4-diiodo-4-hydroxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-chloro-3,4-dimethoxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-chloro-3,5-dinitrobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-chloro-4-fluorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-chloro-4-fluorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-chloro-6-methylbenzyl)amino-3-(methyl, ethyl, isopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-chloro-2-methylbenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-chloro-4-methylbenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(5-chloro-2-methoxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-chloro-4-fluorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-chloromethylbenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-chloro-5-nitrobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-chloro-6-nitrobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-chloro-3-nitrobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(5-chloro-2-nitrobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-bromo-4-hydroxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3,5-dibromo-4-hydroxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-bromo-4-methoxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-butoxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-butoxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-/t-butyl/benzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-t-butyl-2,6-dimethylbenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-aminobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-aminobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-aminobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-amino-6-chlorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-amino-4-chlorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-amino-3-chlorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-amino-4-chlorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-amino-6-chlorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,6-diamino-3-chlorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,6-diamino-4-chlorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-amino-3-chlorobenzyl)amino-3-(methyl, ethyl, isopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-amino-5-dichlorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(5-amino-2-methylbenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-amino-3-nitrobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-amino-3-nitrobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-benzyloxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-acetylbenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-acetylbenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,3,4-trimethoxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,4,5-trimethoxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,4,6-trimethoxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3,4,5-trimethoxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,4,6-trimethoxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,3,4-trihydroxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,4,6-trihydroxybenzyl)lamino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,3,4-trihydroxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3,4,5-trihydroxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,4,6-trihydroxybenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,4,5-trichlorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,4,5-trichlorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,4,6-trichlorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,3,4-trichlorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,3,5-trichlorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,3,6-trichlorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,5,6-trichlorobenzyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-anilino-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-[2,4-bis(trifluoromethyl)anilino]-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-[2,5-bis(trifluoromethyl)anilino]-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-[2,4-bis(trifluoromethyl)anilino]-3-(methyl, ethyl, isopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3,5-bis(trifluoromethyl)anilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-bromoanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-bromoanilino)-3-(methyl, ethyl, isopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-bromoanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-bromo-2-chloroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-bromo-3-chloroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-[2-bromo-6-chloro-4-(trifluoromethyl)anilino]-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-bromo-5,6-difluoroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-bromo-4,6-difluoroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-bromo-2,6-difluoroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-bromo-2-fluoroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-bromo-4-fluoroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-bromo-4-methylanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-bromo-2-methylanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 6-(4-bromo-3-methylanilino))-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-[2-bromo-4-(trifluoromethoxy)anilino]-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-[3-bromo-4-(trifluoromethoxy)anilino]-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-[4-bromo-2-(trifluoromethoxy)anilino]-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-bromo-4,5,6-trifluoroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,4-dibromoanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,5-dibromoanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,4-dibromo-3,6-dichloroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,6-dibromo-4-fluoroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-[2,6-dibromo-4-(trifluoromethoxy)anilino]-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-[2,4-dibromo-6-(trifluoromethyl)anilino]-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-[2,6-dibromo-4-(trifluoromethyl)anilino]-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,3-dichloroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,4-dichloroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,5-dichloroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,6-dichlorooanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3,4-dichloroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3,5-dichloroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-[2,6-dichloro-4-(trifluoromethoxy)anilino]-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-[2,4-dichloro-6-(trifluoromethyl)anilino]-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-[2,6-dichloro-4-(trifluoromethyl)anilino]-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,3-difluoroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,4-difluoroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,5-difluoroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,6-difluoroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3,4-difluoroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3,5-difluoroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-difluoromethoxyanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-difluoromethoxy-5-nitroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,3-difluoro-6-nitroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,4-difluoro-6-nitroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,4-dijodoanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,3-dimethylanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,4-dimethylanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,3-dimethyl-6-nitroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,4-dimethoxyanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,3-dimethoxyanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,3-dinitro-6-methylanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-hydroxy-2-methylanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-chloroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-chloroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-chloroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, (7-chloro-2,6-dibromo-4-fluoroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-chloro-4-fluoroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-chloro-5-fluoroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-chloro-6-fluoroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-chloro-2-fluoroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-chloro-4-fluoroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-chloro-2-fluoroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(5-chloro-2-fluoroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-chloro-4-fluoro-5-methylanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(5-chloro-4-fluoro-2-nitroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(5-chloro-2-hydroxyanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-chloro-2-iodoanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-chloro-4-iodoanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-chloro-6-methylanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-chloro-2-methylanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-[3-chloro-4-(trifluoromethoxy)anilino]-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-[4-chloro-2-(trifluoromethoxy)anilino]-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-fluoroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-fluoroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-fluoroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-fluoro-4-iodoanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-fluoro-5-nitroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-fluoro-4-methylanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-fluoro-2-methylanilino)-3-(methyl, ethyl, isopropyl, 3-cyklopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-fluoro-4-methylanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-fluoro-2-methylanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-fluoro-4-methylanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(5-fluoro-2-methylanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-fluoro-2-nitroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-fluoro-3-nitroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-jodoanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-[2-fluoro-4-(trifluoromethyl)anilino]-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-iodo-2-methylanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-methoxyanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-methoxyanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-methoxyanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-methoxy-5-methylanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-methoxy-6-methylanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-methoxy-2-methylanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(5-methoxy-2-methylanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 6-[4-methoxy-3-(trifluoromethyl)anilino]-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-methylanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-methylanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-methylanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-[2-methyl-3-(trifluoromethoxy)anilino]-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-[2-methyl-4-(trifluoromethoxy)anilino]-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-[2-(methylthio)anilino]-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-[4-(methylthio)anilino]-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-nitroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-nitroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-nitroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-nitro-4,5,6-trifluoroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-[2-nitro-4-trifluoromethoxy)anilino]-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-nitrotetrafluoroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,3,4,5,6-pentabromoanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 6-(2,3,4,5,6-pentafluoroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,3,4,5-tetrachloroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,3,5,6-tetrachloroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-(1,1,2,2-tetrafluoroethoxy)anilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,3,4,5,-tetrafluoroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,3,4,6,-tetrafluoroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,3,5,6,-tetrafluoroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-[2,3,5,6-tetrafluoro-4-(trifluoromethyl)anilino]-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,4,6-tribromoanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,4,6-tribromo-3,5-dijodoanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,3,4-trichloroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,4,5-trichloroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,4,6-trichloroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,4,5-trifluoroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,3,5-trifluoroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,3,6-trifluoroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,3,4-trifluoroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-trifluoromethoxyanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-trifluoromethoxyanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-trifluoromethoxyanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,3,4-trifluoro-6-nitroanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,4,5-trimethylanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2,4,6-trimethylanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-chloro-4-carboxyanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-carboxy-4-hydroxyanilino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-cyclohexylamino-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-cyclopentylamino-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-cyclobutylamino-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-allylamino-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-diallylamino-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine 7-isopentylamino-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3,3-dimethylallylamino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-hydroxymethyl-3-methylallyl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-propargylamino-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-furfurylamino-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(oxazol-4-yl)amino-3-(methyl, ethyl, isopropyl, 3-pentyl, cyclopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(2-pyridylamino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(3-pyridylamino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-pyridylamino)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(4-morfolinyl)-3-(methyl, ethyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(1-chinuklidinyl)-3-(methyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(1-etyleniminyl)-3-(methyl, ethyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(1-propyleniminyl)-3-(methyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(1-pyrolidinyl)-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(1-piperidinyl)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(1-piperazinyl)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-pyrazol-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-imidazol-3-(methyl, ethyl, isopropyl, 3-pentyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(1-imidazolinyl)-3-(methyl, ethyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine, 7-(1-pyrazolinyl)-3-(methyl, ethyl, isopropyl, 3-pentyl, cyklopropyl, benzyl)pyrazolo[4,3-d]pyrimidine.
The novel compounds of this invention per se or as intermediates in the preparation of novel compound having a wide variety of industrial utilities.
The compounds of the formula I and their pharmaceutically acceptable salts inhibit selectively the enzyme p34cdc2/cyclin B kinase and related cdks (cdk1, cdk2, cdk5, cdk7, MAP kinases).
In another embodiment, this invention is a method for inhibiting cdks and cell proliferation and/or for inducing apoptosis in plants comprising administering an effective amount of the composition of claim 1 to the plant.
In still another embodiment, this invention is a composition useful for treating fungal infections (fungi) in humans, animals and plants.
Disubstituted pyrazolo[4,3-d]pyrimidine derivatives result in the acquisition of extremely high potency against viruses on the part of the defined compounds. An important aspect of the present invention is a method for inhibiting proliferation of a DNA virus dependent upon events associated with cell proliferation for replication. The DNA virus includes any of the retrovirus family. The effective amount is that sufficient to inhibit cellular CDK activity to extent impending viral replication.
In addition to other CDK1-related kinases, this kinase controls certain steps of cell division cycles, in particular the transition from G1 phase into the S phase and in particular the transition from the G2 phase into the M-phase. Out the basis of this findings, it can be expected that the compounds of the formula I and their acceptable salts can be used as antimitotic compounds and for treatment of proliferative diseases.
In addition to therapeutic applications it will be apparent the subject compounds can be used as a cell culture additive for controlling proliferative and/or differentiation states of cells in vitro, for instance, by controlling the level of activation of a CDK. By preventing the activation of a Go/G1 CDK, the subject inhibitors can prevent mitotic progression and hence provide a means for ensuring an adequately restrictive environment in order to maintain cells at various stages of differentiations, and can be employed, for instance, in cell cultures designed to test the specific activities of trophic factors. Other tissue culture systems which require maintenance of differentiation will be readily apparent to those skilled in the art.
It is likely that inhibition by the compounds, of the invention of the catalytic activity of cyclin-dependent kinases in mediated by interaction of the compounds at the ATP-binding site of the enzyme. Such compounds are particularly desirable for reducing excessive cell growth, since they allow inhibition of the kinase activity regardless of the cause underlying the excessive kinase activity leading to excessive cell proliferation. Thus, the compounds of the invention are active in situations in which the excessive kinase activity results from the kinase being a mutated hyperactive, form of the kinase and situations in which the kinase is present at excessive levels. Such compounds can also block excessive kinase activity in situations in which the cyclin regulating the kinase is present at excessive levels or its binding to the kinase is enhanced. Furthermore, compounds which block kinase activity by interacting with the ATP binding site of the enzyme are also useful for inhibiting kinase activity in situations in which a natural inhibitor of cyclin-kinase complexes is mutated.
It will also be apparent that differential screening assays can be used to select for those compounds of the present invention with specificity for CDK enzymes. Thus, compounds, which act specifically on eukaryotic pathogens, e.g., are anti-fungal or anti-parasitic agents, can be selected from the subject of the inhibitors.
By way of illustration, the assays described in the art can be used to screen for agents which may ultimately be useful for inhibiting at leas one fungus implicated in such mycosis as aspergillosis, blastomycosis, chromoblastomycosis, coccidiomycosis, conidiosporosis, actinomycosis, penicilliosis, monoliasis, or sporotrichosis. For example, if the mycotic infection to which treatment is desired is aspergillosis, an assay as described above or in the appended examples can comprise comparing the relative effectiveness of a test compound on inhibiting a plant CDK enzyme with its effectiveness towards a CDK enzyme from yeast. Likewise, the differential screening assays can be used to identify anti-fungal agents which may have value in the treatment of aspergillosis by making use of the CDK genes cloned from yeast such as Aspergillus fumigatus, Aspergillus flavus, Aspergillus niger, Aspergillus nidulans, or Aspergillus terreus.
In yer another embodiment, certain of the subject CDK inhibitors can be selected on the basis of inhibitory specificity for plant CDK s relative to the mammalian enzyme. For example, a plant CDK can be sidposed in a differential screen with one or more of the human enzymes to select those compounds of greatest selectivity for inhibiting the plant enzyme. Thus, the present invention specifically contemplates formulations of the subject CDK inhibitors for agricultural applications, such as in the form of a defoliant or the like.
In one approach the 3-isopropyl-7-substituted pyrazolo[4,3-d]pyrimidine of the formula I, wherein R7 substituents are defined above for a compound of the formula I, are prepared by reaction of 7-chloro-3-isopropylpyrazolo[4,3-d]pyrimidine with appropriate nucleophile as 1)amine (ammonium hydroxide, hydrazine, hydroxylamine, benzylamine; 2-, 3-, 4-hydroxybenzylamine; dihydroxybenzylamine; 3-chloroaniline, etc.) or 2) lithium (natrium, kalium) salt of alcohole or mercaptane. Preferably, the appropriate nucleophil reactant may be R7′-X—Y, wherein R7′-X— is as defined in claim 1 and Y is H. A nucleophil is reagent is able to attack a place in molecule with absence of electrons. An appropriate alkylating agent is a reagent which is source of carbo cations which attack a place in a molecule with excess of electrons—preferentially free electron couples, usually oxygen, nitrogen and sulfur, such as R7′-Z, wherein R7′ is as defined in claim 1 and Z is selected from halogen, toluensulfonate, and mesylate. 7-Chloro-3-isopropylpyrazolo[4,3-d]pyrimidine is dissolved in chloroform and appropriate R7′—NH2 or R7′—O(Li, Na, K), or R7′—S(Li, Na, K) (5-20 eq.) was added. After heating for several hours, the reaction mixture is cooled and the 7-substituted-3-isopropylpyrazolo[4,3-d]pyrimidine is obtained.
In another approach the 3-ethyl substituted pyrazolo[4,3-d]pyrimidine of the formula I, wherein R7 substituents are defined above for a compound of the formula L are prepared by reaction of 7-chloro-3-ethylpyrazolo[4,3-d]pyrimidine with appropriate nucleophile as 1)amine (ammonium hydroxide, hydrazine, hydroxylamine, benzylamine; 2-, 3-, 4-hydroxybenzylamine; dihydroxybenzylamine; 3-chloroaniline, etc.) or 2) lithium(natrium, kalium) salt of alcohole or mercaptane. 7-Chloro-3-isopropylpyrazolo[4,3-d]pyrimidine is dissolved in chloroform and appropriate R7′—NH2 or R7′—O(Li, Na, K), or R7′—S(Li, Na, K) (5-20 eq.) was added. After heating for several hours, the reaction mixture is cooled and the 7-substituted-3-ethylpyrazolo[4,3-d]pyrimidine is obtained.
In yet another approach the 3-methyl substituted pyrazolo[4,3-d]pyrimidine of the formula I, wherein R7 substituents are defined above for a compound of the formula I, are prepared by reaction of 7-chloro-3-methylpyrazolo[4,3-d]pyrimidine with appropriate nucleophile as 1)amine (ammonium hydroxide, hydrazine, hydroxylamine, benzylamine; 2-,3-,4hydroxybenzylamine; dihydroxybenzylamine; 3-chloroaniline, etc.) or 2) lithium(natrium, kalium) salt of alkohole or mercaptane. 7-Chloro-3-isopropylpyrazolo[4,3-d]pyrimidine is dissolved in chloroform and appropriate R7′—NH2 or R7′—O(Li, Na, K), or R7′—S(Li, Na,K) (5-20 eq.) was added. After heating for several hours, the reaction mixture is cooled and the 7-substituted-3-methylpyrazolo[4,3-d]pyrimidine is obtained.
Further,
pictures a-e—control cells
pictures a′-e′—cells treated with 200 mM A2.16.32 for 12 hr.
picture a—lower magnification showing frequency od mitosis (a), and aberrant mitosis (a′). b,b′—prophase, c,c′—metaphase, d,d′—anaphase, e,e′—telophase.
1. molecular weight markers
2. control I—DNA after 10 h incubation without the drug
3. DNA after 10 h incubation with bohemine
4. control II—DNA after 44 hod inkubation without the drug and
5. DNA pafter 44 hod incubation with bohemine.
The starting material for the compounds of the formula I is available from commercial sources (Sigma, Aldrich, Fluka, etc.). Melting points were determined on a Koffler block and are uncorrected. Evaporations were carried out on a rotary evaporator under vacuum at temperatures below 80° C. The 1H NMR spectra (c, ppm; J, Hz) were measured on Varian VXR-400 (400 MHz) or on Varian Unity 300 (400 MHz) instruments. All spectra were obtained at 25° C. using tetramethylsilane as on a internal standard. Electron impact mass spectra m/z (rel. %, composition, deviation) were measured on a VG 7070E spectrometer (70 eV, 200° C., direct inlet). Quadrupole mass spectra were measured on a Micromass ZMD detector with electrospray ionization. Merck silica gel Kieselgel 60 (230-400 mesh) was used for column chromatography. All compounds gave satisfactory elemental analyses (±0.4%).
Prepared according to: E. Royals: J. Am. Chem. Soc. 67, 1508 (1945)
A solution of methyl 2,4-dioxo-5-methylhexenoate II (31g; 180 mmol) and N2H4.H2O (13 mL, 180 mmol) in ethanol (120 mL ) was refluxed for 2 hours. The mixture was poured into H2O and extracted with chloroform. The chloroform extract was concentrated under reduced pressure to give a yellow liquid. The liquid and aq. 3 M NaOH (120 mL) was stirred overnight at room temperature. The acidification of the yellow solution to pH=2 with conc. HCl affords crystals. Crystals were filtered off and washed with ice-water. Yield 68%, mp=136-140° C.
1H NMR(300 MHz, MeOD): 1.14d(6H, 7.1 Hz), 3.02sept.(1H, 7.1 Hz), 6.59s(1H).; CHN required: C=54.54%; H=6.54%; N=18.17%; found: C=54.58%; H=6.38%; N=18.12%.
To an ice-cooled and stirred solution of 2.9 g (18.8 mmol) 5-isopropylpyrazol-3-carboxylic acid III in fuming sulphuric acid 1 mL (65%,), sulphuric acid 7.6 mL (100%) and the nitric acid 3 mL (65%) was added portionwise. The stirring was continued for 1 h at room temperature and then another 3 h at 104° C. temperature and then poured into ice-water. The white precipitate of product was filtered and crystallized from water; (yield 76%); mp=139-142° C.; 1H NMR(300 MHz, DMSO): 1.22d(6H, 7.1 Hz), 2.94sept(1H, 7.1 Hz), 3.33s(1H), 6.45bs(1H); CHN required: C=42.02%; H=4.56%; N=21.09%; found: C=42.41%; H=4.49%; N=21.01%.
5-Isopropyl-4-nitropyrazole-3-carboxylic acid was added to a 4.5M solution of HCl in absolute methanol. The reaction mixture was heated at 60° C. for 7 hours and then was evaporated to dryness. The title compound was crystallised from ethyl acetate; yield 91%; mp=78-80° C. 1H NMR (300 MHz, CDCl3): 1.39d(6H, J=7.1 Hz); 3.64sept(1- J=7.1 Hz), 3.98s(3H). CHN required: C=45.07%; H=5.20%; N=19.70% found: C=45.21%; H=5.23%; N=19.65%.
Mode A
To a solution of methyl 5-isopropyl-4-nitropyrazol-3-carboxylate (4.34g, 24 mmol) in 20 mL ethanol and 5 mL water was added 1 g RaNi (an activity W5). The mixture was stirred under hydrogen atmosphere (760 Torr) for 12 hours. The RaNi was filtered off and the filtrate was concenrated in vacuo. The residue crystals were washed with cooled ethylacetate; yield 95%; mp=122-123° C. MS(EI, 70 eV, direct inlet): 183(88; C8H13N3O2.+.; −1.0), 168(59), 152(3), 136(100), 108(8), 80(16), 68(20). 1H NMR(400 MHz, CDCl3): 1.31d(6H; J=6.9 Hz), 2.93sept(1H; J=6.9 Hz), 3.9s(3H). IR (KBr, cm−1): 3399, 3296, 1710, 1626, 1584, 1302.
Mode B
To a solution of methyl 4-nitropyrazol-3-carboxylate V (7.34g, 34.4 mmol) in 36 mL n-propanole, 6 mL water and 5.6 mL 10 M HCl was added 0.55 g PtO2. The mixture was stirred under hydrogen atmosphere (760 Torr) for 9 hours. The reaction mixture was filtered and the filtrate was concentrated to dryness in vacuo. The desired amine was liberated by treatment of aq. ammonia during extraction into chloroform. The product crystallised after evaporation; yield 95%; mp=122-123° C.
A mixture of aminoester VI (1.5 g, 8.42 mmol), formamidine acetate (2.47 g, 24 mmol) and triethylamine (5.25 mL ) in 32 mL of 2-ethoxyetanol was heated for 2 hours at 90° C. under argon atmosphere. The excess of triethylamine was evaporated from cellosolve solution in vacuo, the crystallised product was filtered off and washed with chloroform. An analytical sample was obtained by recrystallisation from ethanol. Yield 96%; mp=302-304° C. 1H-NMR (300 MHz, CD3OD): 1.41d(6H, J=7.15 Hz), 3.40sept(1H, J=7.15 Hz), 7.82s(1H). 13C-NMR (400 MHz, DMSO-d6+AcOD): 21.912, 25.985, 141.85, 172.17. CHN required: C=53.92%; H=5.66%; N=31.44; found: C=53.20%; H=5.58%; N=31.39%. MS (EI): 178(35; C8H10N4O.+); 177(8) 163(25;C7H7N4O.+); 150(18;C6H6N4O.+); 54(18); 53(10); 41(11); 28(38); 27(11).
7-Hydroxy-3-isopropylpyrazolo[4,3-d]pyrimidine X (200 mg, 1.122 mmol) was dissolved in the mixture of 0.81 mL (11 mmol) of thionyl chloride, 0.12 mL (1.56 mmol) of dimethylformamide and 5 mL of chloroform. This mixture was heated under reflux for 3 hours. The solution was evaporated to dryness in vacuo and the residue was dissolved in chloroform. This solution was extracted twice with a small portions of water and combined chloroform extract was dried over Na2SO4. This compound VIII was not isolated and was used immediately as chloroform solution in following reaction step.
To chloroform solution of XI (prepared in the subsequent step from 200 mg X) was added 3 mL of benzylamine. This mixture was stirred at room temperature 10 minutes and then evaporated to dryness in vacuo. The crude product was purified by column chromatography on silica gel, the mixture of chloroform/methanol (98.5 /1.5) was used as mobile phase. Yield 82%; mp=153-154° C. 1H-NMR (400 MHz, CDCl3): 1.40d(6H, J=7.02 Hz); 3.41sept(1H, J=7.02 Hz); 4.80s(2H), 7.25m(5H), 6.57s (1H), 8.4s (1H). CHN required: C=67.39%; H=6.41%; N=26.20; found: C=67.33%; H=6.43%; N=26.24%. MS (EI): 267(62;C8H10N4O.+); 266(18) 252(44); 239(6); 106(49); 91(100); 65(21); 43(14); 41(16).
To chloroform solution of XI (prepared in the subsequent step from 200 mg X) was added 1.31 mL (7.7 mmol) N-ethyldiisopropylamine, 3 mL EtOH and 500 mg (4.06 mmol) 2-hydroxybenzylamine. This mixture was heated one hour at 60° C. and then was evaporated to dryness in vacuo. The crude product was purified by chromatography on silica gel in the mixture of chloroform/methanol/AcOH (20:0.4:0.1). Yield 40%; mp=214-217° C. 1H-NMR (400 MHz, DMSO-d6): 1.36d(6H, J=6.96 Hz); 3.28sept(1H, J=6.96 Hz); 4.65s(2H), 6.78-7.26m(4H), 8.21s (1H). CHN required: C=63.59%; H=6.05%; N=24.72; found: C=63.39%; H=6.07%; N=24.62%. MS (ES): [M+H]+=274.3 (100).
To chloroform solution of XI (prepared in the subsequent step from 200 mg X) was added 1.5 mL (8.8 mmol) N-ethyldiisopropylamine, 5 mL EtOH and 500 mg (4.06 mmol) 3-hydroxybenzylamine. This mixture was heated one hour at 60° C. and then was evaporated to dryness in vacuo. The crude product was purified by column chromatography on silica gel in the mixture of chloroform/methanol/AcOH (20:0.6:0.1). Yield 48%; mp=220-221° C. 1H-NMR (300 MHz, DMSO-d6): 1.38d(6H, J=7.14 Hz); 3.26sept(1H, J=7.14 Hz); 4.68s(2H), 6.62-7.17m(3H), 8.22s (1H); 9.31s(1H). MS (ES): [M+H]+=274.3 (100).
To chloroform solution of XI (prepared in the subsequent step from 200 mg X) was added 1.5 mL (8.8 mmol) N-ethyldiisopropylamine, 5 mL EtOH and 500 mg (4.06 mmol) 4-hydroxybenzylamine. This mixture was heated one hour at 60° C. and then was evaporated to dryness in vacuo. The crude product was purified by column chromatography on silica gel in the mixture of chloroform/methanol/AcOH (20:1:0.1). Yield 49%; mp=234-236° C. 1H-NMR (300 MHz, DMSO-d6): 1.28d(6H, J=6.59 Hz); 3.58sept(1H, J=6.59 Hz); 4.60s(2H), 6.73-7.21m(4H), 8.20s (1H). MS (ES): [M+H]+=274.3 (100).
To chloroform solution of XI (prepared in the subsequent step from 200 mg X) was added 1.20 ml (11.2 mmol) 3-chloroaniline. This mixture was heated one hour at 60° C., then was cooled at room temperature and crystals were precipitated. These colourless crystals were washed with ether; the analytical sample was obtained by recrystallisation from mixture ethanol-ether. Yield 58%; mp=213-216° C. 1H-NMR (400 MHz, DMSO-d6): 1.40d(6H, J=6.94 Hz); 3.48sept(1H, J=6.94 Hz); 7.30dd(1H), 7.49dd(1H), 7.86dd(1H), 8.20s(1H), 8.78s (1H). CHN(C14H14N5Cl.HCl.H2O) required: C=49.20%; H=5.01%; N=20.48%; Cl=20.48%; found: C=49.43%; H=5.09%; N=20.13%; Cl=20.58%. MS (ES): [M+H]+=288.5 (100), 290.5 (33).
To chloroform solution of XI (prepared in the subsequent step from 200 mg X) was added 3.5 mL (20 mmol) N-ethyldiisopropylamine, 3 mL EtOH and 620 mg (4.7 mmol) isopent-2-en-1-(yl)amino hydrochloride. This mixture was stirred 12 hours at room temperature and then was evaporated to dryness in vacuo. The crude product was purified by column chromatography on silica gel in the mixture of chloroform/methanol/aq. NH4OH (98:2:1). Yield 48%; syrupy. 1H-NMR (400MHz, CDCl3): 1.45d(6H, J=6.96 Hz); 1.65d(1H, J=1.28 Hz), 3.47sept(1H, J=6.96 Hz); 4.18d(2H, J=1.28 Hz), 5.25m(1H, J=1.28 Hz), 6.25s(1H), 8.22s (1H). COSY[1.45d(6H, J=6.96 Hz); 3.47sept(1H, J=6.96 Hz)], COSY[1.65d(6H, J=1.28 Hz); 4.18d(2H, J=1.28 Hz); 5.25m(1H, J=1.28 Hz)], COSY[4.18d(2H); 6.25s(1H)]. MS (ES): [M+H]+=246.5 (100).
To the chloroform solution of XI (prepared in the subsequent step from 150 mg X) was added 2 mL (0.205 mol) furfurylamine. This mixture was stirred 1 hour at 50° C. and then was evaporated to dryness in vacuo. The crude product was purified by column chromatography on silica gel in the mixture of chloroform/methanol/aq. NH4OH (98:2:0.2). Yield 43%; mp=179-182° C. 1H-NMR (500 MHz, MeOD): 1.422d (6H, J=7.0 Hz); 3.455sept. (1H, J=7.0 Hz); 4.802s (2H); 6.373s (2H); 7.468s (1H); 8.273s (1H). MS (ES): [M+H]+=258.3 (100).
To the chloroform solution of XI (prepared in the subsequent step from 150 mg X) was added 0.29 mL (2.53 mmol) 3-pentylamine. This mixture was stirred 1 hour at 50° C. and then was evaporated to dryness in vacuo. The crude product was purified by column chromatography on silica gel. in the mixture of chloroform/methanol (99:1). Yield 25%; mp=73-75° C. 1H-NMR (500 MHz, MeOD): 0.933t (3H, J=7.0 Hz); 1.374bs (2H); 1.388bs (2H); 1.418d (6H, J=6.9 Hz); 1.715pent. (2H, J=7.0 Hz), 3.447hept.(1H, J=6.9 Hz); 3.583t (2H, J=7.0 Hz); 8.207s (1H). MS (ES): [M+H]+=248.2 (100).
To chloroform solution of XI (prepared in the subsequent step from 152 mg X, 0.855 mmol) was added 0.44 mL (2.60 mmol) N-ethyldiisopropylamine, 2 ml methanol and 343 mg (1.54 mmol) 2-bromobenzylamine hydrochloride. This mixture was heated two hours at 60° C. and then was evaporated to dryness in vacuo. The crude product was purified by column chromatography on silica gel in the mixture of chloroform/methanol (98.5:1.5) crystallization from Et2O. Yield 42%; mp=194-196° C. 1H-NMR (300 MHz, CH3OD): 1.44d(6H, J=6.9 Hz); 3.43hept(1H, J=6.9 Hz); 4.89s(2H); 7.20t(1H, J=7.1 Hz); 7.32t(1H, J=7.1 Hz); 7.45bs(1H); 7.62d(1H, J=7.1 Hz); 8.24s (1H). MS (ES): [M+H]+=246.2 (95), 248.2 (100).
To chloroform solution of XI (prepared in the subsequent step from 152 mg X, 0.855 mmol) was added 0.34 mL (2.60 mmol) 4-methoxybenzylamine. This mixture was heated one hour at 52° C. and then was evaporated to dryness in vacuo. The crude product was purified by column chromatography on silica gel in the mixture of chloroform/methanol (98:2), crystallization from Et2O. Yield 42%; mp=143-144° C. 1H-NMR (300 MHz, CDCl3): 1.36d(6H, J=6.9 Hz); 3.46sept. (1H, J=6.9 Hz); 3.69s (3H), 4.86bs (2H); 6.72d (2H, J=8.8 Hz); 7.30d (2H, J=8.8 Hz); 8.34s (1H). MS (ES): [M+H]+=298.3 (100).
To chloroform solution of XI (prepared in the subsequent step from 152 mg X, 0.855 mmol) was added 0.44 mL (2.60 mmol) N-ethyldiisopropylamine, 7 mL methanol and 236 mg (1.54 mmol) 3-hydroxy-4-methoxybenzylamine. This mixture was heated one hour at 52° C. and then was evaporated to dryness in vacuo. The crude product was purified by column chromatography on silica gel in the mixture of chloroform/methanol/NH4OH (93.5:6.5:0.1) crystallization from chloroform/Et2O. Yield 62%; mp=197-199° C. 1H-NMR (300 MHz, CH3OD): 1.43d(6H, J=7.15 Hz); 4.45hept(1H, J=7.15 Hz); 3.83s(3H); 4.67s(2H), 6.82-6.90m (3H), 8.24s (1H). MS (ES): [M+H]+=314.3 (100).
a) solution: MeOH p.a. + HCOOH
b) solution: MeOH p.a. + H2O +NH3
a) solution: MeOH p.a. + HCOOH
b) solution: MeOH p.a. + H2O + NH3
a) solution: MeOH p.a. + HCOOH
b) solution: MeOH p.a. + H2O + NH3
Proteins
Cyclin-dependent kinases (p34cdc2, p33cdk2) and cyclins (cyclin B, E) are produced in Sf9 insect cells coinfected with appropriate baculoviral constructs. The cells are harvested 68-72 hrs post infection in lysis buffer for 30 min on ice and the soluble fraction is recovered by centrifugation at 14.000 g for 10 min. The protein extract is stored at −80° C.
Lysis buffer: 50mM Tris pH 7.4, 150mM NaCl, 5 mM EDTA, 20 mM NaF, 1% Tween, 1 mM DTT, 0.1 mM PMSF, leupeptine, aprotonine.
Enzyme Inhibition Assays
To carry out experiments on kinetics under linear conditions, the final point test system for kinase activity measurement is used. The kinase is added to reaction mixture in such a way as to obtain linear activity with respect to the concentration of enzyme and with respect to time.
The p34cdc2 and p33cdk2 kinase inhibition determination involves the use of 1 mg/ml histone H1 (Sigma, type III-S) in the presence of 15 μM [γ-33P]ATP (500-100 cpm/pmol) (ICN) in a final volume of 10 μl. Kinase activity is determined at 30° C. in the kinase buffer.
Tested compounds are usually dissolved to 100 mM solutions in DMSO, final concentration of DMSO in reaction mixture never exceeds 1%. The controls contain suitable dilutions of DMSO.
After 10 min, addition 3×SDS sample buffer stops the incubations. Phosphorylated proteins are separated electrophoretically using 10% SDS polyacrylamide gel. The measurement of kinase activity is done using digital image analysis.
The kinase activity is expressed as a percentage of maximum activity, the apparent inhibition constants are determined by graphic analysis.
Kinase buffer: 50 mM Hepes pH 7.4, 10 mM MgCl2, 5 mM EGTA, 10 mM
2-glycerolphosphate, 1 mM NaF, 1 mM DTT
Table 4 shows the results of inhibitory activity of novel compounds against CDK1 and CDK2 in comparison with the data on a prototype compound (trisubstituted purine olomoucine). Most of the 3,7-disubstituted pyrazolo[4,3-d]pyrimidine derivatives showed marked inhibitory activity in in vitro kinase assays
Protein extraction and purification of pant CDK by binding to p13suc1-beads or immunopurification with an antibody specific to the cdc2a-MS protein was carried out as described previously (Bögre et al. 1997, Plant Physiol. 113, 1997, 841-852). The MMK1 protein kinase was purified with a specific antibody from Vicia faba extracts as described by Bögre et al. 1997a, Plant Cell 9, 75-83). Protein kinase activity was measured as described above in Example 19. The quantification of radioactivity incorporated into histone H1 or myelin basic protein was undertaken using Phosphoimager (original gel images shown on
Table 5 shows the results of inhibitory activity of novel compounds against plant in comparison with the data on the prototype compounds (disubstituted purines olomoucine, roscovitine and purvalanol A). Most of the 3,7-disubstituted pyrazolo[4,3-d]pyrimidine derivatives showed marked inhibitory activity in in vitro plant kinase assays.
We have been using the following cell lines: HELA (human cervical carcinoma), MCF7 (human breast adenocarcinoma), NIH 3T3 (mouse fibroblasts), HOS (human osteogenic sarcoma), HL 60 (human promyelocytic leukemia), G 361 (human malignant melanoma), K562 (human chronic myeloblastic leukemia), CEM (human lymphoblastoid leukaemia). Tested drugs were added to the cell cultures in six different concentration and kept at 37° C. and 5% CO2 for three days. All cell lines were grown in DMBM medium (Gibco BRL) supplemented with 10% (v/v) fetal bovine serum and
Cytoxicity of novel compounds was tested on panel of cell lines of different histogenetic and species origin (Tab. 6). Higher activities were found in all tumour cell lines tested. Notably, the higher effectiveness of novel derivatives was also found in cell lines bearing various mutations or deletions in cell cycle associated proteins, e.g. HL-60, BT549, Hela, U2OS, MDA-MB231, and Saos2. It indicates that these substances should be equally effective in tumours with various alterations of tumour suppressor genes, namely p53, Rb, etc. Importantly, this observation distinguishes the novel compounds from flavopiridol and related compounds, as their biological activity is dependent on p53 status.
The novel compounds have also been tested in tobacco callus bioassay for cytotoxicity (herbicidal activity) and induction of cell death. The compounds to be tested were dissolved in dimethylsulfoxide (DMSO) and the solution brought up to 10−3 M with distilled water. This tock solution was further diluted in the respective media used for the tobacco bioassay to concentration ranging from 10−8 M to 10−4 M. The final concentration of DMSO in the media did not exceed 0.2%, and therefore did not affect biological activity in the assay system used. Cytokinin-dependent tobacco callus Nicotiana tabacum L. cv. Wisconsins 38 Murashige-Skoog medium, containing per 1 liter: 4 μmol nicotinic acid, 2.4 μmol pyridoxine hydrochloride, 1.2 μmol thiamine, 26.6 μmol glycine, 1.37 μmol glutamine, 1.8 μmol myoinositol, 30 g of sucrose, 8 g of agar, 5.37 μmol α-naphtylacetic acid and 0.5 μmol 6-benzylaminopurine. Subcultivation was carried out every three weeks. Fourteen days before the bioassay, the callus tissue was transferred to the media without 6-benzylaminopurine. Compounds were tested with two different concentrations of 6-benzylaminopurine (10−5 M and 10−6 M). Inhibitory growth activity was determined from the increase in fresh callus weight after four weeks of cultivation. Five replicates were prepared for each concentration tested and the entire test was repeated at least twice. Inhibitory activity was compared with growth response curve of 6-benzylaminopurine in the range from 10−8 M to 10−4 M and IC50 was calculated for each compound for 10−5 M and 10−6 M of 6-benzylaminopurine.
Table 7 shows the results of inhibitory activity of novel compounds on growth of tobacco cells cultivated in vitro in comparison with the data on the prototype compound (trisubstituted purine olomoucine). Most of the 3,7-disubstituted pyrazolo[4,3-d]pyrimidine derivatives showed marked inhibitory activity on in vitro growth. Furthermore, these compounds are able to induce apoptosis in plants cells (are able to kill plant cells) and induce strong antimitotic activities (see
| Number | Date | Country | Kind |
|---|---|---|---|
| 030 10 184.4 | May 2003 | EP | regional |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/EP04/04855 | 5/6/2004 | WO | 8/3/2006 |
