IntroductionPyrimido[5,4-d]pyrimidines are a relatively little studied heterocyclic system, which are of interest in the context of drug development because of their structural similarity to purines. We are especially interested in inhibitors of cyclin-dependent kinases (CDKs) and have developed purine derivatives (e.g. 2-amino-6-cyclohexylmethoxypurine1) that inhibit the cyclin B1/CDK1 and cyclin A3/CDK2 complexes at concentrations in the low micromolar range.1These purines are analogues of the ‘benchmark’ inhibitors olomoucine2aand roscovitine2b,2,3although they bind to CDK2 in a different orientation.1The objective of the present study was to obtain pyrimido[5,4-d]pyrimidine analogues of the purine CDK inhibitors. Thesynthetic access to such molecules requires that the pyrimido[5,4-d]pyrimidine scaffold be manipulated in a rational way. To this end we have investigated how to control the substitution chemistry of 2,4,6,8-tetrachloropyrimido[5,4-d]pyrimidine (3, TCPP).4This compound is readily prepared from 1,5-dihydropyrimido[5,4-d]pyrimidine-2,4,6,8-(3H,7H)-tetraone4.4Besides enabling the synthesis of individual purine mimetics, a better knowledge of the chemistry of 2,4,6,8-tetrachloropyrimido[5,4-d]pyrimidines would provide an opportunity for parallel or combinatorial syntheses of a diverse array of novel compounds.TCPP is the classical precursor of 2,4,6,8-tetrasubstituted pyrimido[5,4-d]pyrimidines, including the useful coronary vasodilator dipyridamole (5, Persantin).5The substitution pattern in dipyridamole can be described asababwhereacorresponds to a substituent at the 4- and 8-positions of the pyrimido[5,4-d]pyrimidine scaffold, whereasbcorresponds to a substituent at the 2- and 6-positions (Fig. 1). The formation of pyrimido[5,4-d]pyrimidines with anababpattern of substitution is the typical outcome of two stepwise substitution reactions of TCPP, employing an excess of nucleophile in each step.6,7This is because the rates of substitution greatly favour the C-4 and C-8 positions over C-2 and C-6. For example, treatment of TCPP with an excess of benzylamine at ambient temperatureled to the formation of 4,8-bis(benzylamino)-2,6-dichloropyrimido[5,4-d]pyrimidine6a. Further reaction of this compound with an excess of ethanolamine (2-aminoethanol) at 120 °C gave 4,8-bis(benzylamino)-2,6-bis(2-hydroxyethylamino)pyrimido[5,4-d]pyrimidine6b(Scheme 1). The observed regioselectivity relates to the ability to delocalise the negative charge arising from attack of a nucleophile at C-2/C-6 or C-4/C-8. Attack at C-4/C-8 gives a species in which negative charge on nitrogen (e.g.N-3) can be delocalised into an adjacent C&z.dbd;N bond, whereas attack at C-2/C-6 does not permit this stabilising interaction.Conversion of 2,4,6,8-tetrachloropyrimido[5,4-d]pyrimidine3(TCPP) into6a–6d.Reagents and conditions: (i) 4 equiv. benzylamine, THF, rt, 20 min; (ii) ethanolamine, 120 °C 12 h; (iii) 64 equiv. ethanolamine, THF, 75 °C, 6 h; (iv) 3.9 equiv. KOH, H2, 30–40 psi, 10% Pd/C, THF, 20 h.Substitution patterns for pyrimido[5,4-d]pyrimidines.The reactions of 4,8-bis(benzylamino)-2,6-dichloropyrimido[5,4-d]pyrimidine with nucleophiles can be carried out in a stepwise fashion to afford 4,8-bis(benzylamino)-2,6-disubstituted pyrimido[5,4-d]pyrimidines, with different substituents at C-2 and C-6,i.e.anabacsubstitution pattern (cf.Fig. 1). Furthermore, we have found that the reaction of TCPP with benzylamine can be controlled to give the mono-substituted product, 4-benzylamino-2,6,8-trichloropyrimido[5,4-d]pyrimidine, in acceptable yield. This compound served as a valuable starting material for the preparation of 2,4,6,8-tetrasubstituted pyrimido[5,4-d]pyrimidines with anabcdpattern of substitution,viaa sequence of controlled nucleophilic substitutions of the remaining three chloro functions. The structures of theabacandabcdtetrasubstituted pyrimido[5,4-d]pyrimidines describedherein were validated by spectroscopic characterisation of all compounds, and for theabcdtype by an X-ray analysis of a representative end-product. Theabcdpattern of substitution was of particular interest in the context of analogues of purine CDK inhibitors and so selected compounds were evaluated as inhibitors of the cyclin B/CDK1 complex.