CB30865

IC50: 2.8 nM for W1L2; 156 nM for isolated mammalian TS

CB30865 is a highly potent cytotoxic agent. The compound inhibits isolated mammalian thymidylate synthase (TS), but this inhibition is insufficient to account for its cellular toxicity. Thymidylate synthase (TS) is a critical enzyme in the de novo synthesis of thymidylate (dTTP) and has long been recognized as a target for chemotherapeutic intervention.

In vitro: CB30865 was the most potent growth inhibitory agent (IC50 values in the range 1–100 nM for several mouse and human cell types). Against W1L2 cells, the analogues of CB300179 and CB300189 demonstrated reduced potency in the presence of exogenous thymidine (dThd), and against a W1L2:C1 TS overproducing cell line. In contrast, CB30865 retained activity in these systems. No cell cycle redistribution was observed following exposure (4–48 h) to an equitoxic concentration of CB30865 [1].

In vivo: The in vivo evaluation of CB30865 was hampered because of its low aqueous solubility (<1 μM at pH 6). This low aqueous solubility of CB30865 initiated a search for more water-soluble analogues to allow the in vivo evaluation of this class of compounds. It was envisaged that aqueous solubility could be increased by the introduction of amino functionalities (e.g., N-methylpiperazine) at the 2-position of the quinazolin-4-one ring [2].

Clinical trial: No clinical data are available

References:
[1] Bavetsias V, Skelton LA, Yafai F, Mitchell F, Wilson SC, Allan B, Jackman AL.  The design and synthesis of water-soluble analogues of CB30865, a quinazolin-4-one-based antitumor agent. J Med Chem. 2002;45(17):3692-702.
[2] Skelton LA, Ormerod MG, Titley J, Kimbell R, Brunton LA, Jackman AL.  A novel class of lipophilic quinazoline-based folic acid analogues: cytotoxic agents with a folate-independent locus. Br J Cancer. 1999;79(11-12):1692-701.

Cell cycle effects of CB30865, a lipophilic quinazoline-based analogue of the antifolate thymidylate synthase inhibitor ICI 198583 with an undefined mechanism of action.[Pubmed:9725559]

Cytometry. 1998 Sep 1;33(1):56-66.

CB30865 (p-[N-(7-bromo-3,4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl+ ++)-N-(prop-2-ynyl)amino]-N-(3-pyridylmethyl)benzamide) is a quinazoline-based pyridine-containing compound that emerged from a programme aimed at the development of thymidylate synthase (TS) inhibitors as anticancer agents. Its structure is based on the antifolate ICI 198583, but with a pyridine ring replacing the glutamate. Despite its structure, CB30865 does not act in vitro via inhibition of TS or, apparently, other known folate-dependent pathways, and extensive mechanistic studies suggest that it acts via a novel locus with respect to conventional antitumour agents. However, CB30865 is highly potent against a variety of human tumour cell lines (e.g., 50%-inhibitory concentration [IC50] values in the 1-10 nM range). Thus, the cell cycle effects of CB30865 were investigated. DNA histogram analysis of W1L2 human lymphoblastoid, L1210 murine leukaemia, and CH1 human ovarian cells (propidium iodide staining) has demonstrated that CB30865 does not cause a phase-specific arrest at concentrations that have been shown to inhibit colony formation. This is unexpected for an anticancer agent. In W1L2 cells, using bromodeoxyuridine (BrdUrd) labelling and bivariate Hoechst/ propidium iodide staining, it was revealed that 0.003-0.15 microM CB30865 (1-50 x 72 h IC50) caused cells to arrest in all phases of the cell cycle simultaneously after 20-24 h exposure. This effect was also observed in CH1 and L1210 cells, though the arrest was at slightly different times. Thus, using this technique, it has been demonstrated that CB30865 induces an unusual and delayed cell cycle arrest, which provides further evidence for a novel locus of action for this compound.

Chemical proteomics identifies Nampt as the target of CB30865, an orphan cytotoxic compound.[Pubmed:20609415]

Chem Biol. 2010 Jun 25;17(6):659-64.

Drug discovery based on cellular phenotypes is impeded by the challenge of identifying the molecular target. To alleviate this problem, we developed a chemical proteomic process to identify cellular proteins that bind to small molecules. CB30865 is a potent (subnanomolar) and selective cytotoxic compound of previously unknown mechanism of action. By combining chemical proteomics with biochemical and cellular pharmacology we have determined that CB30865 cytotoxicity is due to subnanomolar inhibition of nicotinamide phosphoribosyltransferase (Nampt), an enzyme present in the NAD biosynthetic pathway. Cancer cells develop dependence on Nampt due to increased energy requirements and the elevated activity of NAD consuming enzymes such as sirtuins and mono and poly(ADP-ribose) polymerases (PARPs). These findings suggest new chemical starting points for Nampt inhibitors and further implicate this enzyme as a target in cancer.

The design and synthesis of water-soluble analogues of CB30865, a quinazolin-4-one-based antitumor agent.[Pubmed:12166942]

J Med Chem. 2002 Aug 15;45(17):3692-702.

4-[N-[7-Bromo-2-methyl-4-oxo-3,4-dihydroquinazolin-6-ylmethyl]-N-(prop-2-ynyl)ami no]-N-(3-pyridylmethyl)benzamide (CB30865) is a quinazolin-4-one antitumor agent whose high growth-inhibitory activity (W1L2 IC(50) = 2.8 +/- 0.50 nM) is believed to have a folate-independent locus of action. In addition, CB30865 represents a class of compounds with unique biochemical characteristics such as a delayed, non-phase specific, cell-cycle arrest. The low aqueous solubility of CB30865 prompted a search for more water-soluble analogues for in vivo evaluation of this class of compounds. It was thought that aqueous solubility could be increased by the introduction of amino functionalities at the 2-position of the quinazolin-4-one ring. A variety of compounds (5a-j, 31a-c, 32, and 33) were synthesized in a linear fashion starting from 3-chloro-4-methylaniline. Most of these compounds (e.g., 5a, 5b, 5g) were significantly more water-soluble than CB30865 (636 microM for 5a at pH 6 and 992 microM for 5g at pH 6). In addition, some of them were up to 6-fold more cytotoxic than CB30865 (e.g., for 5a, W1L2 IC(50) = 0.49 +/- 0.24 nM) and retained its novel biochemical characteristics.