Cyclin dependent kinase inhibiting purine derivatives

Method of treating a tumour or other cell proliferation disorder which comprises administering an effective amount of a purine compound which inhibits cyclic dependent kinase activity. Novel purine compounds and pharmaceutical compositions are also disclosed.

FIELD OF THE INVENTION
 The present invention relates to certain purine derivatives which show
 activity in biological systems as cyclin dependent kinase (CDK) inhibitors
 and which are accordingly of interest as potentially useful therapeutic
 agents that may be incorporated in pharmaceutical compositions or
 formulations for use in controlling or inhibiting cell growth or
 proliferation in mammals, for example in connection with antitumour or
 cancer treatment.
 BACKGROUND
 Cyclin dependent kinases (CDK's) are a family of enzymes which form
 complexes with other activating proteins known as cyclins to provide key
 regulatory factors that are involved in the control of growth and division
 in animal cells. More particularly, the progression of animal cells
 through the cell division cycle (G1, S, G2 and M phases) is regulated by
 the sequential formation, activation and subsequent inactivation of a
 series of CDK/cyclin dimer complexes which control passage past cell cycle
 checkpoints and transitions between successive phases of the cell cycle,
 with the CDK's acting as catalytic sub-units of the complexes.
 There are in fact a number of different cyclin proteins which, like the
 different CDK's, form a somewhat loosely related family of CDK-activating
 proteins; different CDK/cyclin complexes function at different stages of
 the cell cycle with sequential increase and decrease in cyclin expression
 during the cell cycle and cyclin degradation during M phase usually being
 an important factor in determining orderly cell cycle progression. Thus,
 progression through G1 to S phase in mammalian cells is believed to be
 regulated primarily by cyclin dependent kinases CDK2, CDK3 and CDK4 (and
 possibly also CDK6 in some cells) in association with at least cyclins D
 and E, the complexes of CDK2 and CDK4 (and possibly CDK6) with D type
 cyclins in particular playing an important role in controlling progression
 through the G1 restriction point whilst the CDK2/cyclin E complexes are
 essential for bringing about the transition from G1 into S phase. Once S
 phase is entered it is believed that further progression and entry into G2
 then requires activated complexes of CDK2 with another cyclin which is
 designated cyclin A, i.e. complexes CDK2/cyclin A. Finally, for the
 transition from G2 phase to M phase and initiation of mitosis, activated
 complexes of the cyclin dependent kinase designated CDK1 (also known as
 Cdc2) with a cyclin designated cyclin B (and also complexes of CDK1 with
 cyclin A) are required.
 In general, control of the cell cycle and activity of CDK's involves a
 series of stimulatory and inhibitory phosphorylation and dephosphorylation
 reactions, and in exercising their regulatory functions the CDK/cyclin
 complexes when activated use ATP as a substrate to phosphorylate a variety
 of other substrate cell proteins, usually on serine and threonine groups
 thereof. Control of the cell cycle may also involve inhibitors of
 CDK/cyclin complexes which block the catalytic function of these enzymes
 so as to lead to arrest of the cell cycle. Certain natural inhibitors,
 such as for example the inhibitory proteins known as p16 and p21, can
 block cell cycle progression by binding selectively to CDK/cyclin
 complexes to inactivate the latter.
 Control by inhibitors of CDK function may therefore provide a further
 mechanism for controlling cell cycle progression, and this has led to
 proposals for using CDK inhibitors as antiproliferative therapeutic
 agents, in antitumour therapy for example, for targeting abnormally
 proliferating cells and bringing about an arrest in cell cycle
 progression. This has seemed to be especially appropriate since it is
 known that severe disorders or irregularities in cell cycle progression
 frequently occur in human tumour cells, often accompanied by
 over-expression of CDK's and other proteins associated therewith. Also,
 compared to established cytologic antitumour drugs, the use of inhibitors
 of cell proliferation acting through CDK's would have the advantage of
 avoiding a direct interaction with DNA, thereby giving a reduced risk of
 secondary tumour development.
 The potential therapeutic applications and other possible uses have
 accordingly led to a search for further chemical inhibitors of CDK's,
 especially selective inhibitors that may be suitable for pharmaceutical
 use. Inhibitory activity and selectivity of selected CDK/cyclin complexes
 is generally assayed by measuring the kinase activity in phosphorylating
 the protein histone H1 (one of the major protein constituents of chromatin
 which generally provides a good CDK substrate) in the presence of the
 suspected inhibitor under test. A number of compounds having potentially
 useful CDK inhibitory properties that have been identified in this way are
 described in a review article, of which the content is incorporated herein
 by reference entitled "Chemical inhibitors of cyclin-dependent kinases" by
 Laurent Meijer published in Cell Biology (Vol. 6), October 1996. Among the
 compounds referred to in the above-mentioned article is a potent CDK1 and
 CDK2 inhibiting adenine derivative
 2-(2-hydroxyethylamino)-6-benzylamino-9-methyl-purine, named "olomoucine",
 and also a close analogue incorporating modifications at each of positions
 2, 6 and 9, namely,
 6-(benzylamino)-2(R)-[{1-(hydroxy-methyl)propyl}amino]-9-isopropylpurine.
 This latter compound is named "roscovitine" and is even more potent than
 olomoucine as a CDK inhibitor. The strong but selective CDK inhibitory
 properties of olomoucine were first described in a paper by J. Vesely et
 al entitled "Inhibition of cyclin-dependent kinases by purine analogues",
 Eur. J. Biochem. 224, 771-786 (1994), and further studies on CDK
 inhibitory properties of a range of purine compounds in the form of
 adenine derivatives, including olomoucine and roscovitine, are reported
 and discussed in a paper by L. Havlicek et al entitled "Cytokinin-Derived
 Cyclin-Dependent Kinase Inhibitors: Synthesis and cdc2 Inhibitory Activity
 of Olomoucine and Related Compounds" J. Med. Chem. (1997) 40, 408-412.
 Again, the content of these publications is to be regarded as being
 incorporated herein by reference.
 The inhibitory activity of both olomoucine and roscovitine has been shown
 to result from these compounds acting as competitive inhibitors for ATP
 binding. It may be noted that olomoucine at least is reported as having a
 total lack of inhibitory activity in relation to many common kinases other
 than CDK's. Selectivity is further manifest by the fact that both
 olomoucine and roscovitine inhibit activity of CDK1, CDK2 and CDK5, but
 neither has been found to be active against CDK4 or CDK6.
 Olomoucine in particular has been regarded as providing a lead compound for
 helping to identify and design further purine based CDK inhibitors, and
 based on structure/activity studies it was suggested in the
 above-mentioned paper of Vesely et al that N9 substitution by a
 hydrophobic residue such as methyl, 2-hydroxyethyl or isopropyl was
 important, e.g. to provide a direct hydrophobic interaction with the CDK,
 and that a side chain at C2 appeared to be essential. Similarly, in the
 paper of Havlicek et al, apart from observing that for CDK inhibitory
 activity the 1 and 7 positions, and possibly the 3 position, of the purine
 ring must remain free to permit hydrogen bonding, it was also stated that
 a polar side chain at position 2 appears to be essential and that N9
 substitution by a hydrophobic residue is also probably important for
 positive binding. Positions 2, 6 and 9 in the purine ring were identified
 as being the positions which control binding to CDK1.
 In the review article of Meijer, it is also mentioned that as a result of
 crystallization of CDK--inhibitor complexes, and in particular
 co-crystallization studies with CDK2, it has been found that inhibitors
 such as olomoucine and roscovitine localize in the ATP binding pocket
 which is located in the cleft between the small and large lobes of the CDK
 protein molecule, and that specificity was probably provided by portions
 of the inhibitor molecules interacting with the kinases outside the ATP
 binding sites.
 SUMMARY OF THE INVENTION
 The present invention has developed from an observation made in the course
 of testing various guanine derivatives for activity as inhibitors of the
 DNA repair protein O.sup.6 -methylguanine DNA-methyltransferase (MGMT)
 when it was found unexpectedly that although the compound O.sup.6
 -cyclohexylmethylguanine had very little activity as a MGMT inhibitor, it
 was nonetheless cytotoxic and showed very high inhibitory activity,
 comparable to that of olomoucine, against CDK1(cdc2)/cyclin B complexes.
 This was particularly surprising against the background discussed above in
 relation to olomoucine given that this guanine compound has no
 substituents at either the 2-NH.sub.2 position or the 9 position in the
 purine ring and that the replacement of the 6-NH by 6-O made the compound
 less like ATP with which olomoucine at least is believed to compete for
 binding sites.
 Subsequently, other guanine derivatives have been identified, more closely
 related to O.sup.6 -cyclohexylmethylguanine than to compounds such as
 olomoucine and roscovitine, which show significant CDK inhibitory
 activity, and crystallographic studies have revealed that complexes of
 CDK2 (homologous with CDK1, at least in respect of the catalytic binding
 site) with guanine derivatives such as O.sup.6 -cyclohexylmethylguanine
 and O.sup.6 -cyclohex-1-enylmethylguanine bind together in a different
 manner from complexes of CDK2 with olomoucine.

Whereas with olomoucine it is the polar side chain on N2 of the purine ring
 that seats within the ATP ribose binding pocket of the CDK2 protein, and
 the N9 methyl substituent engages a separate hydrophobic specificity
 pocket, with N7 and 6-NH being involved in hydrogen bonding to the
 protein, in the binding mode illustrated in FIG. 2 it is the cycloalkyl
 ring of the substituent at the 6-position that seats in the ATP ribose
 binding pocket while hydrogen bond links are formed to N9, N3 and 2-NH. In
 other words, the orientation as compared with the binding of olomoucine is
 completely reversed. A similar situation obtains with the binding mode
 illustrated in FIG. 3 where the involvement of some water molecules is
 also indicated.
 It will accordingly be clear that conclusions reached in respect of
 structure/activity relationships in the adenine series of compounds
 exemplified by olomoucine and roscovitine are likely no longer to be valid
 for all purine derivatives, especially guanine derivatives.
 The compounds with which the present invention is concerned are primarily
 purine compounds which have inhibitory activity in respect of at least
 some CDK's and which bind in the manner shown in FIG. 2 (or FIG. 3) rather
 than in the manner shown in FIG. 1. Although some of these compounds are
 already known per se, they are not known in a capacity as CDK inhibitors.
 In some cases this inhibitory activity has been found to have a
 selectivity towards different CDK's which is notably different from that
 of olomoucine, and the present invention has in effect identified a new
 class of CDK inhibitors and has considerably enlarged the range of
 compounds available for use as CDK inhibitors.
 In one aspect the present invention accordingly provides pharmaceutical
 compositions for treatment of cell proliferation disorders in mammals, for
 example tumors said compositions containing as the active ingredient a
 CDK-inhibiting purine compound having the structural formula I below:
 ##STR1##
 where, in preferred embodiments,
 X is O, S or CHR.sub.X where R.sub.x is H or C.sub.1-4 alkyl;
 D is H, halo or NZ.sub.1 Z.sub.2 where Z.sub.1 and Z.sub.2 are each
 independently H or C.sub.1-4 alkyl or C.sub.1-4 hydroxyalkyl;
 A is selected from H, C.sub.1-4 alkyl, C.sub.1-4 alkoxy, hydroxy, CH.sub.2
 (CH.sub.2).sub.n OH (n=1-4, and NR.sub.a1 R.sub.a2 where R.sub.a1 and
 R.sub.a2 are each independently H or C.sub.1-4 alkyl;
 B is selected from H, C.sub.1-4 alkyl, C.sub.1-4 alkoxy, CF.sub.3, an
 optionally substituted aryl (e.g. phenyl) or an optionally substituted
 aralkyl (e.g. benzyl), and an hydroxy group that provides a C.dbd.O
 tautomer; and
 Y is or includes an optionally substituted 4- to 8-membered carbocyclic or
 heterocyclic ring.
 In some cases, however, Y may comprise an optionally substituted linear or
 branched hydrocarbon chain, especially a chain containing a double band,
 e.g. an allyl derivative as hereinafter referred to.
 So long as it is able to fit or seat in the ATP ribose binding pocket of a
 CDK protein and permit binding in the general manner depicted in FIG. 2
 rather than FIG. 1, there is a wide range of substituents likely to be
 suitable for Y. In some cases, however, it may be helpful for Y to
 comprise a ring structure that includes polar hydroxyl substituents or the
 like.
 In most embodiments Y will be a cycloalkane or cycloalkene ring, preferably
 a 5- or 6-membered ring having up to two double bonds. One or two carbon
 atoms in the ring may be replaced, however, by hetero atoms or groups,
 particularly O, S, NR' (where R' is H or C.sub.1-4 alkyl) or, in a
 cycloalkene ring, --N.dbd.. Where the ring is substituted the substituent
 or each substituent (at any position) will preferably be selected from H,
 C.sub.1-4 alkyl, OH, C.sub.1-4 alkoxy, halogen, CF.sub.3, CN, N.sub.3 and
 NR.sub.y1 R.sub.y2 where R.sub.y1 and R.sub.y2 are each independently H or
 C.sub.1-4 alkyl. Moreover, in the case where there are two substituents on
 adjacent atoms of the ring,
 ##STR2##
 these substituents P and Q may be linked to form an additional fused ring
 structure, e.g. a 4-, 5- or 6-membered carbocyclic or heterocyclic ring.
 This additional ring structure may include for example up to two hetero
 atoms or groups such as O, S or NH, and it may also be substituted by one
 or more substituents, e.g. a C.sub.1-4 alkyl group or groups or a phenyl
 or substituted phenyl group. In some embodiments, Y may also be adamantyl.
 Examples of ring structures represented by Y include
 ##STR3##
 where V and W are each selected independently from
 O, S, NR' (R' is H or C.sub.1-4 alkyl)
 and CH.sub.2 (or .dbd.CH--); and
 R.sub.1 and R.sub.2 are each H or C.sub.1-4 alkyl.
 As indicated above, these ring structures can optionally bear substituents
 which may be the same or different and which may inter alia be selected
 from H, C.sub.1-4 alkyl, C.sub.1-4 alkoxy, --OH, NR.sub.y1 R.sub.y2 (where
 R.sub.y1 and R.sub.y2 are each independently H or C.sub.1-4 alkyl),
 CF.sub.3, halogen, N.sub.3, CN, optionally substituted aryl (e.g. phenyl),
 and optionally substituted aralkyl (e.g. benzyl). Also, as already
 indicated, it may be especially advantageous for the ring structure to
 have a plurality of polar substituents such as hydroxyl for example.
 Some specific examples of the structures of potentially useful CDK
 inhibitory compounds in accordance with this invention include the
 following:
 ##STR4##
 ##STR5##
 In general, the pharmaceutical compositions of this invention will contain
 an effective CDK-inhibiting non-toxic amount of the active purine
 compound, and will be formulated in accordance with any of the methods
 well known in the art of pharmacy for administration in any convenient
 manner, and may for example be presented in unit dosage form admixed with
 at least one other ingredient providing a compatible pharmaceutically
 acceptable additive, carrier, diluent or excipient.
 It will be understood that where reference is made in this specification to
 compounds of formula I such reference should be construed as extending
 also to their pharmaceutically acceptable salts and to other
 pharmaceutically acceptable bioprecursors (pro-drug forms) where relevant.
 The term "pro-drug" is used in the present specification to denote
 modified forms or derivatives of a pharmacologically active compound which
 biodegrade in vivo and become converted into said active compound after
 administration, especially oral or intravenous administration, in the
 course of therapeutic treatment of a mammal. Such pro-drugs are commonly
 chosen because of an enhanced solubility in aqueous media which helps to
 overcome formulation problems, and also in some cases to give a relatively
 slow or controlled release of the active agent.
 It should also be understood that where any of the compounds referred to
 can exist in more than one enantiomeric and/or diastereoisomeric form, all
 such forms, mixtures thereof, and their preparation and uses are within
 the scope of the invention. It should be noted, however, that
 stereochemical considerations are likely to be important and there may be
 considerable selectivity such that different enantiomers or
 diastereoisomers have significantly different inhibitory activity.
 The invention also includes of course the use of the CDK inhibiting
 compounds referred to for the manufacture of medicaments or pharmaceutical
 compositions as referred to above, and it also includes the treatment of
 abnormal cellular proliferation disorders using such medicaments or
 pharmaceutical compositions.
 Preferably, in compounds of structural formula I which are used in carrying
 out the invention, D will be an unsubstituted amino group --NH.sub.2, and
 X will be O, although in some embodiments the amino group may be mono- or
 di-substituted, with a lower alkyl group for example.
 Although it will usually be preferred that Y should comprise a saturated or
 partially saturated carbocyclic or heterocyclic ring structure, it should
 be recognised that in some cases Y may comprise an aromatic ring system
 (e.g. optionally substituted aryl or aralkyl), or even a linear or
 branched chain (preferably including a double bond as for example in allyl
 derivatives) and still provide compounds of interest as potentially
 selective CDK inhibitors that may be useful in the context of the present
 invention, especially insofar as they may be structured so as to bind with
 CDK's in substantially the same manner as depicted in FIG. 2.
 Although a number of the CDK inhibitor compounds herein disclosed are
 already known per se as previously pointed out, some of the compounds are
 believed to be novel and to constitute new chemical entities. Examples of
 such novel compounds which have been made include
 O.sup.6 -Ribofuranosylguanine
 2-amino-6-(2-tetrahydro-furanyl)-methyloxypurine
 2-amino-6-adamantyl-methyloxypurine
 O.sup.6 -Galactosylguanine
 2-amino-6-(2-naphthyl)-methyloxypurine
 2-amino-6-(2-tetrahydropyranyl)-methyloxypurine
 2-amino-6-(1-naphthyl)-methyloxypurine
 O.sup.6 -(2,2-Dimethyl-1,3-dioxolane-4-methoxy)guanine
 O.sup.6 -(1,4-Dioxaspiro[4.5]decane-2-methoxy)guanine
 Examples of compounds which are at present especially preferred for use in
 carrying out the invention, and which include the most potent CDK
 inhibitors identified, at least when assayed in vitro against CDK1 and/or
 CDK2, are the following:
 2-amino-6-(3-methyl-2-oxo)butyloxypurine ethylene acetal
 2-amino-6-cyclohexyl-methyloxypurine
 (O.sup.6 -cyclohexylmethylguanine)
 2-amino-6-cyclopentyl-methyloxypurine
 (O.sup.6 -cyclopentylmethylguanine)
 2-amino-6-cyclohex-3-enylmethyloxypurine
 2-amino-6-cyclopent-1-enylmethyloxypurine
 (O.sup.6 -Cyclopentenylmethylguanine)
 2-amino-6-(1-cyclohexenyl)-methyloxypurine
 (O.sup.6 -Cyclohexenylmethylguanine)
 2-amino-6-perillyloxymethylpurine
 Biological Activity
 Assays are available for testing the inhibitory activity of the compounds
 of interest against a range of CDK/cyclin complexes, including CDKI/cyclin
 A, CDK1/cyclin B, CDK1/cyclin F, CDK2/cyclin A, CDK2/cyclin E, CDK4/cyclin
 D, CDK5/35 and CDK6/cyclin D3, and it is of particular interest to note
 the selectivity of some of the compounds against different CDK's.
 Test results showing CDK inhibitory activity values measured for some of
 the compounds that have been prepared are shown in Table 1 at the end of
 the present description. Where the compounds exist in different
 enantiomorphic forms, the assays have generally been carried out on
 racemic mixtures. Apart from reference compounds, the compounds listed are
 accompanied by an NU reference or identification code number. Table 1
 includes the compounds which at present are the most preferred of those
 that have been prepared, although as yet not all have been fully tested.
 Four compounds, NU2036, NU2037, NU2038 and NU2051, are included in this
 Table 1 primarily to show how activity drastically diminishes if there are
 side chains at N9 or N7, or a halo substituent at C2.
 As will be seen, in a number of cases the inhibitory assays have been
 carried out and data has been obtained in respect of CDK2 and/or CDK4. as
 well as in respect of CDK1. It is of some considerable importance to note
 that some of these compounds, unlike the previously known CDK inhibitors
 olomoucine and roscovitine, exhibit very significant selectivity as
 between CDK1 and CDK2. Also, some also exhibit significant activity
 against CDK4.
 In general, the studies carried out fully support the belief that CDK
 inhibitory characteristics of compounds tested reflect an ability of these
 compounds to act as effective antitumour drugs.
 The inhibition assays have been carried out using methods based on those
 described in the paper hereinbefore referred to of J. Vesely et al and in
 the paper of L. Azzi et al (1992) Eur. J. Biochem. 203, 353-360. By way of
 example, however, a typical protocol is summarised below.
 CDK Assay Example
 Reagents.
 Buffer C (containing 60 mM b-glycerophosphate, 30 mM nitrophenyl phosphate,
 25 mM MOPS pH 7.0, 5 mM EGTA, 15 mM MgCl.sub.2, 1 MM MgCl.sub.2 and 0.1 mM
 sodium orthovanadate) is made up as follows: