Synthesis and biological evaluation of new N 2 - substituted 2 , 4 - diamino - 6 - cyclohexylmethoxy - 5 - nitrosopyrimidines and related 5 - cyano - NNO - azoxy derivatives as CDK 2 Inhibitors

The potent and selective Cyclin-Dependent Kinase 2 (CDK2) inhibitor NU6027 (6-cyclohexylmethoxy-5nitroso-2,4-diaminopyrimidine) was used as the lead for the synthesis of a series of analogues in order to provide further insights into the structure activity relationships for 2,4-diaminopyrimidine CDK2 inhibitors. Aliphatic amino substituents were introduced at position 2. The use of linear or less sterically hindered amines gave rise to compounds endowed with slightly improved activity with respect to the lead; on the other hand, the compounds were the less active when a bulkier amino substituent was used. Substitution of the 5-nitroso group with the 5-cyano-NNO-azoxy moiety afforded a new class of inhibitors whose activity against CDK2 was similar to that of the nitroso series. The most active nitroso compound was 8b (IC50 = 0.16 μM) while in the 5-cyano-NNO-azoxy series the most active compound was 9b (IC50 = 0.30 μM). Taken as a whole these new analogues of NU6027 enhance understanding of the structure activity relationships for 2,4-diaminopyrimidine CDK2 inhibitors. Cyclin-Dependent Kinases (CDKs) are serine/threonine kinases which display an abnormal activity in many kinds of tumours. [1] This family of kinases is represented by eleven members (CDK1-CDK11) and related cyclins. [2] Today, there is a great interest in small molecules able to behave as inhibitors of these enzymes as potential anticancer drugs. In the past decade, many such compounds belonging to different chemical classes have been developed. [3] Among these inhibitors, an interesting type is represented by 2,4-diamino-6-cyclohexylmethoxy-5-nitrosopyrimidine (1) (NU6027) (Chart 1), a competitive inhibitor of CDK1 and CDK2 isoforms with respect to ATP (CDK2 IC50 = 2.2 μM). [3-5] Owing to the intramolecular hydrogen bond between the adjacent 5-nitroso and 4-amino groups, this compound assumes a pseudo-purine geometry, which is reminiscent of the structure of 6-(cyclohexylmethoxy)-9H-purine (2) (NU2058) (Chart 1), an early relatively potent CDK1 and CDK2 inhibitor (CDK2 Ki = 12 μM). [6] Compound 1 can interact with the ATP-binding site of the enzymes by a triplet of hydrogen bonds (for CDK2: 2-NH2 to Leu-83 (CO), N 3 to Leu-83 (NH), 4NH2 to Glu-81 (CO)). [4] These interactions exactly reproduce those of 2. An extended series of analogues of 1 modified at the 2-,5and 6position(s) were synthesized in order to shed light on the structure-activity relationships (SAR) in this lead compound. [3, 4] In a recent paper we described a new pyrimidine scaffold, the 2,4-diamino-5-(cyano-NNOazoxy)-6-(cyclohexylmethoxy)pyrimidine (3) (Chart 1), endowed with potent inhibitory CDK2 activity. [7] This substance can be formally obtained by substitution of the nitroso group of 1 with the , which is present in the antibiotic “calvatic acid” (4-(Z)-cyano-NNO-azoxybenzoic acid) isolated for the first time from cultural broth of Calvatia lilacina. [8] . This unusual functional group has been used to design several bioactive compounds, such as antimicrobial and antitumor agents, enzyme inhibitors, and calcium channel blockers. [9-13] The cyano-NNO-azoxy moiety displays an electron withdrawing property very similar to that of the nitroso group (p NO = 0.91; p ONNCN = 0.89), whereas it is endowed with different lipophilicity (NO = -1.20; ONNCN = -0.26) and steric properties. [14,15] Chart 1. Reference compounds. A molecular modelling study has suggested a role for a conserved water molecule in stabilizing the bioactive pose of 3 in the interaction with the ATP site of the enzyme. Preliminary SAR showed that the substitution of the cyano group of 3 with other electron withdrawing moieties induced a significant decrease in activity, whilst the introduction at the 2NH2 group of a p-methylaminosulfonyl-substituted phenyl ring gave rise to a product whose potency fell in the nM range.[7] In this study a detailed description of the synthetic routes to a series of new N2-substituted examples of this structural class (9a-i) and of their 5-nitroso precursors (8a-i) is reported and the influence of the lateral chain at this position on the their CDK2 inhibitory activity is discussed. Scheme 1. Reagents and conditions: i) (bromomethyl)cyclohexane, 140°C W heating, 16 min; ii) mCPBA, CH2Cl2, RT, 18 h; iii) R 1 NH2, 120°C, W heating; iv) R 2 ONO, DMSO, RT; v) NH2CN, (diacetoxyiodo)benzene (IBA), CH3CN, RT, 2 h. A series of N2-substituted 2,4-diamino-6-cyclohexylmethoxy-5-nitrosopyrimidines (8a-i) was prepared using a synthetic strategy (Scheme 1) similar to that described previously by Marchetti et al. [5] Some modifications improved the reported reaction conditions. Alkylation of 6-amino-2-thioxo-2,3-dihydropyrimidin-4(1H)-one (4) was performed using (bromomethyl)cyclohexane. At variance with the previous synthetic strategy, (bromomethyl)cyclohexane was used to enable the concomitant alkylation at 2and 4positions. In addition, the use of microwave heating (W) afforded the desired product 5 in 16 min at 140°C. Oxidation of 5 with mCPBA gave the corresponding cyclohexylmethylsulfone 6. The nucleophilic displacement of cyclohexylsulfonyl group in 6 with diverse aliphatic amines was performed in an organic solvent (diglyme or THF) using microwave heating and in some cases, a Lewis acid such as Yb(OSO2CF3)3 was added to improve the yield. The N2-substituted 4-amino-6-cyclohexylmethoxypyrimidines (7a-i) were thus obtained in moderate yield. Subsequent nitrosation at the 5-position with alkyl nitrites (menthyl nitrite or amyl nitrite), gave the desired nitroso compounds 8a-i. The 5-nitroso derivative 8g was obtained after (Boc)2O protection of compound 7g, followed by nitrosation and subsequent deprotection of the amino group (Scheme 2). The final products (9a-i) were prepared by treating the nitroso derivatives (8a-i) with (diacetoxyiodo)benzene (IBA) and cyanamide (NH2CN) in dry CH3CN. [16] Scheme 2. Reagents and conditions: i) (Boc)2O, THF, RT, 2 h; ii) menthyl nitrite, DMSO, RT, 18 h; iii) TFA, CH2Cl2, RT, 2 h. In the 1 H NMR experiments reported for the 5-nitroso derivatives (8a-i), the resonance of the 4-NH2 group was split into two neat signals, which were observed at around 8 and 10 ppm, respectively. This observation was already reported in the previous work [7] and confirms the formation of an intramolecular hydrogen bond between the nitroso oxygen atom and one hydrogen of NH2 group in the 4-position. In contrast, for the cyano-NNO-azoxy derivatives (9a-i), the signal of the 4-NH2 group was reported as a broad singlet. In addition, 1 H and 13 C NMR spectra of both nitroso and cyano-NNO-azoxy derivatives presented two sets of resonances. This can be ascribed to the significant component of the C2-N bond, as a consequence of strong conjugation between the electron donating N groups (e.g. NHCH3 p = 0.84) [14] and the strong electron withdrawing groups, the cyano-NNO-azoxy and the nitroso group respectively, with consequent restricted rotation around the C2-N bond at room temperature (Figure 1). This hypothesis was confirmed by a variable temperature NMR experiment (VT-NMR) performed on compound 8a for which coalescence of the two sets of signals was observed at 125 °C. Figure 1. Electronic conjugation between the electron releasing N group and the electron withdrawing NO or cyanoNNO-azoxy groups. Table 1. Inhibitory activity of target compounds against CDK2. Compound R 1 R 2 IC50 (M) [a], [b]