CK2 (casein kinase 2) is a very pleiotropic serine/threonine protein kinase whose abnormally high constitutive activity has often been correlated to pathological conditions with special reference to neoplasia. The two most widely used cell permeable CK2 inhibitors, TBB (4,5,6,7-tetrabromo-1H-benzotriazole) and DMAT (2-dimethylamino-4,5,6,7-tetrabromo-1H-benzimidazole), are marketed as quite specific CK2 blockers. In the present study we show, by using a panel of approx. 80 protein kinases, that DMAT and its parent compound TBI (or TBBz; 4,5,6,7-tetrabromo-1H-benzimidazole) are potent inhibitors of several other kinases, with special reference to PIM (provirus integration site for Moloney murine leukaemia virus)1, PIM2, PIM3, PKD1 (protein kinase D1), HIPK2 (homeodomain-interacting protein kinase 2) and DYRK1a (dual-specificity tyrosine-phosphorylated and -regulated kinase 1a). In contrast, TBB is significantly more selective toward CK2, although it also inhibits PIM1 and PIM3. In an attempt to improve selectivity towards CK2 a library of 68 TBB/TBI-related compounds have been tested for their ability to discriminate between CK2, PIM1, HIPK2 and DYRK1a, ending up with seven compounds whose efficacy toward CK2 is markedly higher than that toward the second most inhibited kinase. Two of these, K64 (3,4,5,6,7-pentabromo-1H-indazole) and K66 (1-carboxymethyl-2-dimethylamino-4,5,6,7-tetrabromo-benzimidazole), display an overall selectivity much higher than TBB and DMAT when tested on a panel of 80 kinases and display similar efficacy as inducers of apoptosis.

A general and stereospecific synthesis has been developed for the direct preparation of 2'-deoxy-/3-D-ribofuranosylpurine analogues including 2'-deoxyadenosine derivatives. The reaction of the sodium salt of 4-chloropyrrolo[2,3-if)pyrimidine (4) or 2,4-dichloropy rrolo [ 2,3-tf] pyrimidine (1) with l-chloro-2-deoxy-3,5-di-0-/Holuoyl-a-D-er>>r/¡ro-pentofuranose (25) provided the corresponding N-1,2'-deoxy-|8-D-ribofuranosyl blocked derivatives (5 and 2) which, on ammonolysis, gave 2'-deoxytubercidin (6) and 2-chloro-2'-deoxytubercidin (3), respectively, in good yield. This glycosylation also readily proceeds in the presence of a 2-methylthio group. Application of this glycosylation procedure to 4,6-dichloroimidazo[4,5-c]pyridine (10), 6-chloropurine ( 16), 2,6-dichloropurine ( 13), and 4-chloropyrazolo[ 3,4-t/] pyrimidine ( 19) gave 2-chloro-2'-deoxy-3-deazaadenosine ( 12), 2'-deoxyadenosine (18), 2-chloro-2'-deoxyadenosine (15), and 4-amino-l-(2-deoxy-/3-D-eryiAro-pentofuranosyl)pyrazolo-[3,4-t/] pyrimidine (21), respectively. Similarly, glycosylation and ammonolysis of 4,6-dichloro-1 /f-pyrrolo[3,2-c]pyridine ( 22) gave 4,6-dichloro-l-(2-deoxy-0-D-e/,>rAro-pentofuranosyl)pyrrolo[3,2-c]pyridine (24). This stereospecific attachment of the 2-deoxy-/3-D-ribofuranosyl moiety appears to be due to a Walden inversion at the C-l carbon of 25.

Casein kinase 2 (CK2) is a ubiquitous, essential, and highly pleiotropic protein kinase whose abnormally high constitutive activity is suspected to underlie its pathogenic potential in neoplasia and infective diseases. Thus, CK2 inhibitors designed to dissect the signaling pathways affected by this kinase, in perspective, may give rise to pharmacological tools. One of the most successful CK2 inhibitors is TBB (4,5,6,7-tetrabromobenzotriazole). Here we show that its inhibitory properties can be markedly improved by generating adducts in which N(2) is replaced by a carbon atom bound to a variety of polar functions. The most efficient inhibitor is 4,5,6,7-tetrabromo-2-(dimethylamino)benzimidazole (2c) followed by the methylsulfanyl (8), isopropylamino (2e), and amino (2a) congeners. All these compounds display K(i) values <100 nM (40 nM in the case of 2c). 2c induces apoptosis of Jurkat cells more readily than TBB (DC(50) value 2.7 vs 17 microM) and, unlike TBB, it does not display any side effect on mitochondria polarization up to 10 microM concentration. Molecular modeling of the CK2-2c complex, based on the crystal structure of the CK2-TBB complex suggests that a number of additional apolar contacts between its two methyl groups and hydrophobic residues nearby could account for its superior inhibitory properties. Consequently, 2c is even more susceptible than TBB to mutations of the unique hydrophobic residues V66 and/or I174 to alanine. We propose to adopt 2c as first choice CK2 inhibitor instead of TBB, especially for in cell studies.

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CK2 is a highly pleiotropic Ser/Thr protein kinase that is able to promote cell survival and enhance the tumour phenotype under specific circumstances. We have determined the crystal structure of three new complexes with tetrabromobenzimidazole derivatives that display K(i) values between 0.15 and 0.30 microM. A comparative analysis of these data with those of four other inhibitors of the same family revealed the presence of some highly conserved water molecules in the ATP-binding site. These waters reside near Lys68, in an area with a positive electrostatic potential that is able to attract and orient negatively charged ligands. The presence of this positive region and two unique bulky residues that are typical of CK2, Ile66 and Ile174, play a critical role in determining the ligand orientation and binding selectivity.

CK2 is a very pleiotropic protein kinase whose high constitutive activity is suspected to cooperate to neoplasia. Here, the crystal structure of the complexes between CK2 and three selective tetrabromo-benzimidazole derivatives inhibiting CK2 with Ki values between 40 and 400 nM are presented. The ligands bind to the CK2 active site in a different way with respect to the parent compound TBB. They enter more deeply into the cavity, establishing halogen bonds with the backbone of Glu114 and Val116 in the hinge region. A detailed analysis of the interactions highlights a major role of the hydrophobic effect in establishing the rank of potency within this class of inhibitors and shows that polar interactions are responsible for the different orientation of the molecules in the active site.

CK2 denotes a pleiotropic, constitutively active protein kinase whose abnormally high level in many cancer cells is held as an example of "non oncogene addiction". A wide spectrum of cell permeable, fairly specific ATP site-directed CK2 inhibitors are currently available which are proving useful to dissect its biological functions and which share the property of inducing apoptosis of cancer cells with no comparable effect on their "normal" counterparts. One of these, CX-4945, has recently entered clinical trials for the treatment of advanced solid tumors, Castelman's disease and multiple myeloma. The solution of a wide range of 3D structures of inhibitors bound to the catalytic subunits of CK2 reveals that their efficacy substantially relies on hydrophobic interactions within a cavity which is smaller than in other protein kinases. Accordingly the potency of tetra-halogenated benzimidazoles increases upon replacement of chlorine by bromine and, even more, by iodine, and decreases if two unique bulky side chains on CK2 (Val66 and Ile174) are mutated to alanines. Many CK2 inhibitors have been tested on a panel of more than 60 kinases providing Promiscuity Scores useful to evaluate their selectivity, the lowest value (9.47), denoting highest selectivity, being displayed by quinalizarin. The observation that CK2 inhibitors with medium/high promiscuity scores share the ability to inhibit a group of protein kinases as effectively as CK2 discloses the possibility of using their scaffolds for the rational development of selective inhibitors of these kinases, with special reference to PIMs, DYRKs, HIPK2, PKD and ERK8.

Ultraviolet and infrared absorption spectroscopy, in aqueous and non-aqueous media, have been employed to study the tautomerism of 9-substituted isoguanines, including the nucleoside iso­ guanosine. With the aid of a series of model compounds, it was shown that 9-substituted isogua­ nines, and isoguanosine, in aqueous medium are predominantly in the form N(1)H,2-keto-6-amino. In dioxane solution the tautomeric equilibrium is shifted in the direction of the enol form. The shift towards this form is accentuated for those analogues in which the exocyclic amino group is methylated. With the aid of N6,N6,9-trimethylisoguanine, and its 9-octyl analogue, the tautomeric constant was studied as a function of concentration, temperature, and solvent polarity, and the results applied to evaluate the tautomeric equilibria of 9-methylisoguanine and isoguanosine as a function of these variables. In general the enol form is favoured by a decrease in solvent polarity, by a decrease in concentration in dioxane, or an increase in temperature in chloroform solution. Syntheses are described for several N6-amino and methylamino derivatives of 2-methoxy-9-methylpurine, and 3-methyl-5-oxo-7,8-dihydroimidazo (2,1-i) purine, which served as an analogue of the unavailable 1,9-dimethylisoguanine.