Moore, A.S.
Faisal, A.
Mak, G.W.
Miraki-Moud, F.
Bavetsias, V.
Valenti, M.
Box, G.
Hallsworth, A.
de Haven Brandon, A.
Xavier, C.P.
Stronge, R.
Pearson, A.D.
Blagg, J.
Raynaud, F.I.
Chopra, R.
Eccles, S.A.
Taussig, D.C.
Linardopoulos, S.
(2020). Quizartinib-resistant FLT3-ITD acute myeloid leukemia cells are sensitive to the FLT3-Aurora kinase inhibitor CCT241736. Blood adv,
Vol.4
(7),
pp. 1478-1491.
show abstract
full text
Internal tandem duplication of FLT3 (FLT3-ITD) is one of the most common somatic mutations in acute myeloid leukemia (AML); it causes constitutive activation of FLT3 kinase and is associated with high relapse rates and poor survival. Small-molecule inhibition of FLT3 represents an attractive therapeutic strategy for this subtype of AML, although resistance from secondary FLT3 tyrosine kinase domain (FLT3-TKD) mutations is an emerging clinical problem. CCT241736 is an orally bioavailable, selective, and potent dual inhibitor of FLT3 and Aurora kinases. FLT3-ITD+ cells with secondary FLT3-TKD mutations have high in vitro relative resistance to the FLT3 inhibitors quizartinib and sorafenib, but not to CCT241736. The mechanism of action of CCT241736 results in significant in vivo efficacy, with inhibition of tumor growth observed in efficacy studies in FLT3-ITD and FLT3-ITD-TKD human tumor xenograft models. The efficacy of CCT241736 was also confirmed in primary samples from AML patients, including those with quizartinib-resistant disease, which induces apoptosis through inhibition of both FLT3 and Aurora kinases. The unique combination of CCT241736 properties based on robust potency, dual selectivity, and significant in vivo activity indicate that CCT241736 is a bona fide clinical drug candidate for FLT3-ITD and TKD AML patients with resistance to current drugs..
Wood, F.L.
Shepherd, S.
Hayes, A.
Liu, M.
Grira, K.
Mok, Y.
Atrash, B.
Faisal, A.
Bavetsias, V.
Linardopoulos, S.
Blagg, J.
Raynaud, F.I.
(2019). Metabolism of the dual FLT-3/Aurora kinase inhibitor CCT241736 in preclinical and human in vitro models: Implication for the choice of toxicology species. Eur j pharm sci,
Vol.139,
p. 104899.
show abstract
full text
CCT241736 is a dual fms-like tyrosine kinase 3 (FLT3)/Aurora kinase inhibitor in development for the treatment of acute myeloid leukaemia. The successful development of any new drug relies on adequate safety testing including preclinical toxicology studies. Selection of an appropriate preclinical species requires a thorough understanding of the compound's metabolic clearance and pathways, as well as other pharmacokinetic and pharmacodynamic considerations. In addition, elucidation of the metabolising enzymes in human facilitates improved clinical prediction based on population pharmacokinetics and can inform drug-drug interaction studies. Intrinsic clearance (CLint) determination and metabolite profiling of CCT241736 in human and four preclinical species (dog, minipig, rat and mouse) was undertaken in cryopreserved hepatocytes and liver microsomes. Recombinant human cytochrome P450 bactosomes (rCYP) were utilised to provide reaction phenotyping data and support prediction of metabolic pathways. CCT241736 exhibited low CLint in both hepatocytes and liver microsomes of human, dog, minipig and rat, but considerably higher CLint in mouse. CYP3A4 and CYP3A5 were identified as the major enzymes responsible for biotransformation of CCT241736 in human, exclusively forming five out of seven metabolites. Minipig showed greatest similarity to human with regard to both overall metabolic profile and abundance of specific metabolites relative to parent compound, and is therefore proposed as the most appropriate toxicological species. The greatest disparity was observed between human and dog. Based on metabolic profile, either mouse or rat is a suitable rodent species for toxicology studies..
Le Bihan, Y.-.
Lanigan, R.M.
Atrash, B.
McLaughlin, M.G.
Velupillai, S.
Malcolm, A.G.
England, K.S.
Ruda, G.F.
Mok, N.Y.
Tumber, A.
Tomlin, K.
Saville, H.
Shehu, E.
McAndrew, C.
Carmichael, L.
Bennett, J.M.
Jeganathan, F.
Eve, P.
Donovan, A.
Hayes, A.
Wood, F.
Raynaud, F.I.
Fedorov, O.
Brennan, P.E.
Burke, R.
van Montfort, R.L.
Rossanese, O.W.
Blagg, J.
Bavetsias, V.
(2019). C8-substituted pyrido[3,4-d]pyrimidin-4(3H)-ones: Studies towards the identification of potent, cell penetrant Jumonji C domain containing histone lysine demethylase 4 subfamily (KDM4) inhibitors, compound profiling in cell-based target engagement assays. Eur j med chem,
Vol.177,
pp. 316-337.
show abstract
full text
Residues in the histone substrate binding sites that differ between the KDM4 and KDM5 subfamilies were identified. Subsequently, a C8-substituted pyrido[3,4-d]pyrimidin-4(3H)-one series was designed to rationally exploit these residue differences between the histone substrate binding sites in order to improve affinity for the KDM4-subfamily over KDM5-subfamily enzymes. In particular, residues E169 and V313 (KDM4A numbering) were targeted. Additionally, conformational restriction of the flexible pyridopyrimidinone C8-substituent was investigated. These approaches yielded potent and cell-penetrant dual KDM4/5-subfamily inhibitors including 19a (KDM4A and KDM5B Ki = 0.004 and 0.007 μM, respectively). Compound cellular profiling in two orthogonal target engagement assays revealed a significant reduction from biochemical to cell-based activity across multiple analogues; this decrease was shown to be consistent with 2OG competition, and suggests that sub-nanomolar biochemical potency will be required with C8-substituted pyrido[3,4-d]pyrimidin-4(3H)-one compounds to achieve sub-micromolar target inhibition in cells..
Vazquez-Rodriguez, S.
Wright, M.
Rogers, C.M.
Cribbs, A.P.
Velupillai, S.
Philpott, M.
Lee, H.
Dunford, J.E.
Huber, K.V.
Robers, M.B.
Vasta, J.D.
Thezenas, M.-.
Bonham, S.
Kessler, B.
Bennett, J.
Fedorov, O.
Raynaud, F.
Donovan, A.
Blagg, J.
Bavetsias, V.
Oppermann, U.
Bountra, C.
Kawamura, A.
Brennan, P.E.
(2019). Design, Synthesis and Characterization of Covalent KDM5 Inhibitors. Angew chem int ed engl,
Vol.58
(2),
pp. 515-519.
show abstract
full text
Histone lysine demethylases (KDMs) are involved in the dynamic regulation of gene expression and they play a critical role in several biological processes. Achieving selectivity over the different KDMs has been a major challenge for KDM inhibitor development. Here we report potent and selective KDM5 covalent inhibitors designed to target cysteine residues only present in the KDM5 sub-family. The covalent binding to the targeted proteins was confirmed by MS and time-dependent inhibition. Additional competition assays show that compounds were non 2-OG competitive. Target engagement and ChIP-seq analysis showed that the compounds inhibited the KDM5 members in cells at nano- to micromolar levels and induce a global increase of the H3K4me3 mark at transcriptional start sites..
Fearon, D.
Westwood, I.M.
van Montfort, R.L.
Bayliss, R.
Jones, K.
Bavetsias, V.
(2018). Synthesis and profiling of a 3-aminopyridin-2-one-based kinase targeted fragment library: Identification of 3-amino-5-(pyridin-4-yl)pyridin-2(1H)-one scaffold for monopolar spindle 1 (MPS1) and Aurora kinases inhibition. Bioorg med chem,
Vol.26
(11),
pp. 3021-3029.
show abstract
full text
Screening a 3-aminopyridin-2-one based fragment library against a 26-kinase panel representative of the human kinome identified 3-amino-5-(1-methyl-1H-pyrazol-4-yl)pyridin-2(1H)-one (2) and 3-amino-5-(pyridin-4-yl)pyridin-2(1H)-one (3) as ligand efficient inhibitors of the mitotic kinase Monopolar Spindle 1 (MPS1) and the Aurora kinase family. These kinases are well recognised as attractive targets for therapeutic intervention for treating cancer. Elucidation of the binding mode of these fragments and their analogues has been carried out by X-ray crystallography. Structural studies have identified key interactions with a conserved lysine residue and have highlighted potential regions of MPS1 which could be targeted to improve activity and selectivity..
Hayes, A.
Mok, N.Y.
Liu, M.
Thai, C.
Henley, A.T.
Atrash, B.
Lanigan, R.M.
Sejberg, J.
Le Bihan, Y.-.
Bavetsias, V.
Blagg, J.
Raynaud, F.I.
(2017). Pyrido[3,4-d]pyrimidin-4(3H)-one metabolism mediated by aldehyde oxidase is blocked by C2-substitution. Xenobiotica,
Vol.47
(9),
pp. 771-777.
show abstract
full text
1. We have previously described C8-substituted pyrido[3,4-d]pyrimidin-4(3H)-one derivatives as cell permeable inhibitors of the KDM4 and KDM5 subfamilies of JmjC histone lysine demethylases. 2. Although exemplar compound 1 exhibited moderate clearance in mouse liver microsomes, it was highly cleared in vivo due to metabolism by aldehyde oxidase (AO). Similar human and mouse AO-mediated metabolism was observed with the pyrido[3,4-d]pyrimidin-4(3H)-one scaffold and other C8-substituted derivatives. 3. We identified the C2-position as the oxidation site by LC-MS and 1H-NMR and showed that C2-substituted derivatives are no longer AO substrates. 4. In addition to the experimental data, these observations are supported by molecular modelling studies in the human AO protein crystal structure..
Hatch, S.B.
Yapp, C.
Montenegro, R.C.
Savitsky, P.
Gamble, V.
Tumber, A.
Ruda, G.F.
Bavetsias, V.
Fedorov, O.
Atrash, B.
Raynaud, F.
Lanigan, R.
Carmichael, L.
Tomlin, K.
Burke, R.
Westaway, S.M.
Brown, J.A.
Prinjha, R.K.
Martinez, E.D.
Oppermann, U.
Schofield, C.J.
Bountra, C.
Kawamura, A.
Blagg, J.
Brennan, P.E.
Rossanese, O.
Müller, S.
(2017). Assessing histone demethylase inhibitors in cells: lessons learned. Epigenetics chromatin,
Vol.10,
p. 9.
show abstract
full text
BACKGROUND: Histone lysine demethylases (KDMs) are of interest as drug targets due to their regulatory roles in chromatin organization and their tight associations with diseases including cancer and mental disorders. The first KDM inhibitors for KDM1 have entered clinical trials, and efforts are ongoing to develop potent, selective and cell-active 'probe' molecules for this target class. Robust cellular assays to assess the specific engagement of KDM inhibitors in cells as well as their cellular selectivity are a prerequisite for the development of high-quality inhibitors. Here we describe the use of a high-content cellular immunofluorescence assay as a method for demonstrating target engagement in cells. RESULTS: A panel of assays for the Jumonji C subfamily of KDMs was developed to encompass all major branches of the JmjC phylogenetic tree. These assays compare compound activity against wild-type KDM proteins to a catalytically inactive version of the KDM, in which residues involved in the active-site iron coordination are mutated to inactivate the enzyme activity. These mutants are critical for assessing the specific effect of KDM inhibitors and for revealing indirect effects on histone methylation status. The reported assays make use of ectopically expressed demethylases, and we demonstrate their use to profile several recently identified classes of KDM inhibitors and their structurally matched inactive controls. The generated data correlate well with assay results assessing endogenous KDM inhibition and confirm the selectivity observed in biochemical assays with isolated enzymes. We find that both cellular permeability and competition with 2-oxoglutarate affect the translation of biochemical activity to cellular inhibition. CONCLUSIONS: High-content-based immunofluorescence assays have been established for eight KDM members of the 2-oxoglutarate-dependent oxygenases covering all major branches of the JmjC-KDM phylogenetic tree. The usage of both full-length, wild-type and catalytically inactive mutant ectopically expressed protein, as well as structure-matched inactive control compounds, allowed for detection of nonspecific effects causing changes in histone methylation as a result of compound toxicity. The developed assays offer a histone lysine demethylase family-wide tool for assessing KDM inhibitors for cell activity and on-target efficacy. In addition, the presented data may inform further studies to assess the cell-based activity of histone lysine methylation inhibitors..
Bavetsias, V.
Lanigan, R.M.
Ruda, G.F.
Atrash, B.
McLaughlin, M.G.
Tumber, A.
Mok, N.Y.
Le Bihan, Y.-.
Dempster, S.
Boxall, K.J.
Jeganathan, F.
Hatch, S.B.
Savitsky, P.
Velupillai, S.
Krojer, T.
England, K.S.
Sejberg, J.
Thai, C.
Donovan, A.
Pal, A.
Scozzafava, G.
Bennett, J.M.
Kawamura, A.
Johansson, C.
Szykowska, A.
Gileadi, C.
Burgess-Brown, N.A.
von Delft, F.
Oppermann, U.
Walters, Z.
Shipley, J.
Raynaud, F.I.
Westaway, S.M.
Prinjha, R.K.
Fedorov, O.
Burke, R.
Schofield, C.J.
Westwood, I.M.
Bountra, C.
Müller, S.
van Montfort, R.L.
Brennan, P.E.
Blagg, J.
(2016). 8-Substituted Pyrido[3,4-d]pyrimidin-4(3H)-one Derivatives As Potent, Cell Permeable, KDM4 (JMJD2) and KDM5 (JARID1) Histone Lysine Demethylase Inhibitors. J med chem,
Vol.59
(4),
pp. 1388-1409.
show abstract
full text
We report the discovery of N-substituted 4-(pyridin-2-yl)thiazole-2-amine derivatives and their subsequent optimization, guided by structure-based design, to give 8-(1H-pyrazol-3-yl)pyrido[3,4-d]pyrimidin-4(3H)-ones, a series of potent JmjC histone N-methyl lysine demethylase (KDM) inhibitors which bind to Fe(II) in the active site. Substitution from C4 of the pyrazole moiety allows access to the histone peptide substrate binding site; incorporation of a conformationally constrained 4-phenylpiperidine linker gives derivatives such as 54j and 54k which demonstrate equipotent activity versus the KDM4 (JMJD2) and KDM5 (JARID1) subfamily demethylases, selectivity over representative exemplars of the KDM2, KDM3, and KDM6 subfamilies, cellular permeability in the Caco-2 assay, and, for 54k, inhibition of H3K9Me3 and H3K4Me3 demethylation in a cell-based assay..
Bavetsias, V.
Pérez-Fuertes, Y.
McIntyre, P.J.
Atrash, B.
Kosmopoulou, M.
O'Fee, L.
Burke, R.
Sun, C.
Faisal, A.
Bush, K.
Avery, S.
Henley, A.
Raynaud, F.I.
Linardopoulos, S.
Bayliss, R.
Blagg, J.
(2015). 7-(Pyrazol-4-yl)-3H-imidazo[4,5-b]pyridine-based derivatives for kinase inhibition: Co-crystallisation studies with Aurora-A reveal distinct differences in the orientation of the pyrazole N1-substituent. Bioorg med chem lett,
Vol.25
(19),
pp. 4203-4209.
show abstract
full text
Introduction of a 1-benzyl-1H-pyrazol-4-yl moiety at C7 of the imidazo[4,5-b]pyridine scaffold provided 7a which inhibited a range of kinases including Aurora-A. Modification of the benzyl group in 7a, and subsequent co-crystallisation of the resulting analogues with Aurora-A indicated distinct differences in binding mode dependent upon the pyrazole N-substituent. Compounds 7a and 14d interact with the P-loop whereas 14a and 14b engage with Thr217 in the post-hinge region. These crystallographic insights provide options for the design of compounds interacting with the DFG motif or with Thr217..
Bavetsias, V.
Linardopoulos, S.
(2015). Aurora Kinase Inhibitors: Current Status and Outlook. Front oncol,
Vol.5,
p. 278.
show abstract
The Aurora kinase family comprises of cell cycle-regulated serine/threonine kinases important for mitosis. Their activity and protein expression are cell cycle regulated, peaking during mitosis to orchestrate important mitotic processes including centrosome maturation, chromosome alignment, chromosome segregation, and cytokinesis. In humans, the Aurora kinase family consists of three members; Aurora-A, Aurora-B, and Aurora-C, which each share a conserved C-terminal catalytic domain but differ in their sub-cellular localization, substrate specificity, and function during mitosis. In addition, Aurora-A and Aurora-B have been found to be overexpressed in a wide variety of human tumors. These observations led to a number of programs among academic and pharmaceutical organizations to discovering small molecule Aurora kinase inhibitors as anti-cancer drugs. This review will summarize the known Aurora kinase inhibitors currently in the clinic, and discuss the current and future directions..
Macdonald, J.
Oldfield, V.
Bavetsias, V.
Blagg, J.
(2013). Regioselective C2-arylation of imidazo[4,5-b]pyridines. Org biomol chem,
Vol.11
(14),
pp. 2335-2347.
show abstract
We show that N3-MEM-protected imidazo[4,5-b]pyridines undergo efficient C2-functionalisation via direct C-H arylation. Twenty-two substituted imidazo[4,5-b]pyridines are prepared and iterative, selective elaboration of functionalised imidazo[4,5-b]pyridines gives 2,7- and 2,6-disubstituted derivatives in good yields from common intermediates. Mechanistic observations are consistent with a concerted-metallation-deprotonation mechanism facilitated by coordination of copper(I)iodide to the imidazo[4,5-b]pyridine..
Bavetsias, V.
Faisal, A.
Crumpler, S.
Brown, N.
Kosmopoulou, M.
Joshi, A.
Atrash, B.
Pérez-Fuertes, Y.
Schmitt, J.A.
Boxall, K.J.
Burke, R.
Sun, C.
Avery, S.
Bush, K.
Henley, A.
Raynaud, F.I.
Workman, P.
Bayliss, R.
Linardopoulos, S.
Blagg, J.
(2013). Aurora isoform selectivity: design and synthesis of imidazo[4,5-b]pyridine derivatives as highly selective inhibitors of Aurora-A kinase in cells. J med chem,
Vol.56
(22),
pp. 9122-9135.
show abstract
Aurora-A differs from Aurora-B/C at three positions in the ATP-binding pocket (L215, T217, and R220). Exploiting these differences, crystal structures of ligand-Aurora protein interactions formed the basis of a design principle for imidazo[4,5-b]pyridine-derived Aurora-A-selective inhibitors. Guided by a computational modeling approach, appropriate C7-imidazo[4,5-b]pyridine derivatization led to the discovery of highly selective inhibitors, such as compound 28c, of Aurora-A over Aurora-B. In HCT116 human colon carcinoma cells, 28c and 40f inhibited the Aurora-A L215R and R220K mutants with IC50 values similar to those seen for the Aurora-A wild type. However, the Aurora-A T217E mutant was significantly less sensitive to inhibition by 28c and 40f compared to the Aurora-A wild type, suggesting that the T217 residue plays a critical role in governing the observed isoform selectivity for Aurora-A inhibition. These compounds are useful small-molecule chemical tools to further explore the function of Aurora-A in cells..
Naud, S.
Westwood, I.M.
Faisal, A.
Sheldrake, P.
Bavetsias, V.
Atrash, B.
Cheung, K.M.
Liu, M.
Hayes, A.
Schmitt, J.
Wood, A.
Choi, V.
Boxall, K.
Mak, G.
Gurden, M.
Valenti, M.
de Haven Brandon, A.
Henley, A.
Baker, R.
McAndrew, C.
Matijssen, B.
Burke, R.
Hoelder, S.
Eccles, S.A.
Raynaud, F.I.
Linardopoulos, S.
Van Montfort, R.L.
Blagg, J.
(2013). Structure-based design of orally bioavailable 1H-pyrrolo[3,2-c]pyridine inhibitors of the mitotic kinase monopolar spindle 1 (MPS1). J med chem,
Vol.56,
pp. 10045-10065.
Moore, A.S.
Faisal, A.
Gonzalez de Castro, D.
Bavetsias, V.
Sun, C.
Atrash, B.
Valenti, M.
de Haven Brandon, A.
Avery, S.
Mair, D.
Mirabella, F.
Swansbury, J.
Pearson, A.D.
Workman, P.
Blagg, J.
Raynaud, F.I.
Eccles, S.A.
Linardopoulos, S.
(2012). Selective FLT3 inhibition of FLT3-ITD+ acute myeloid leukaemia resulting in secondary D835Y mutation: a model for emerging clinical resistance patterns. Leukemia,
Vol.26
(7),
pp. 1462-1470.
show abstract
Acquired resistance to selective FLT3 inhibitors is an emerging clinical problem in the treatment of FLT3-ITD(+) acute myeloid leukaemia (AML). The paucity of valid pre-clinical models has restricted investigations to determine the mechanism of acquired therapeutic resistance, thereby limiting the development of effective treatments. We generated selective FLT3 inhibitor-resistant cells by treating the FLT3-ITD(+) human AML cell line MOLM-13 in vitro with the FLT3-selective inhibitor MLN518, and validated the resistant phenotype in vivo and in vitro. The resistant cells, MOLM-13-RES, harboured a new D835Y tyrosine kinase domain (TKD) mutation on the FLT3-ITD(+) allele. Acquired TKD mutations, including D835Y, have recently been identified in FLT3-ITD(+) patients relapsing after treatment with the novel FLT3 inhibitor, AC220. Consistent with this clinical pattern of resistance, MOLM-13-RES cells displayed high relative resistance to AC220 and Sorafenib. Furthermore, treatment of MOLM-13-RES cells with AC220 lead to loss of the FLT3 wild-type allele and the duplication of the FLT3-ITD-D835Y allele. Our FLT3-Aurora kinase inhibitor, CCT137690, successfully inhibited growth of FLT3-ITD-D835Y cells in vitro and in vivo, suggesting that dual FLT3-Aurora inhibition may overcome selective FLT3 inhibitor resistance, in part due to inhibition of Aurora kinase, and may benefit patients with FLT3-mutated AML..
Faisal, A.
Naud, S.
Schmitt, J.
Westwood, I.
Hayes, A.
Gurden, M.
Bavetsias, V.
Berry, T.
Mak, G.
Innocenti, P.
Cheung, J.
Sheldrake, P.
Atrash, B.
Sun, C.
Matijssen, B.
Burke, R.
Baker, R.
McAndrew, C.
Rowlands, M.
Workman, P.
Eccles, S.A.
Hoelder, S.
Raynaud, F.I.
vanMontfort, R.
Biagg, J.
Linardopoulos, S.
(2012). Characterisation of CCT251455, a novel, selective and highly potent Mps1 kinase inhibitor. Cancer research,
Vol.72.
Bavetsias, V.
Crumpler, S.
Sun, C.
Avery, S.
Atrash, B.
Faisal, A.
Moore, A.S.
Kosmopoulou, M.
Brown, N.
Sheldrake, P.W.
Bush, K.
Henley, A.
Box, G.
Valenti, M.
de Haven Brandon, A.
Raynaud, F.I.
Workman, P.
Eccles, S.A.
Bayliss, R.
Linardopoulos, S.
Blagg, J.
(2012). Optimization of imidazo[4,5-b]pyridine-based kinase inhibitors: identification of a dual FLT3/Aurora kinase inhibitor as an orally bioavailable preclinical development candidate for the treatment of acute myeloid leukemia. J med chem,
Vol.55
(20),
pp. 8721-8734.
show abstract
Optimization of the imidazo[4,5-b]pyridine-based series of Aurora kinase inhibitors led to the identification of 6-chloro-7-(4-(4-chlorobenzyl)piperazin-1-yl)-2-(1,3-dimethyl-1H-pyrazol-4-yl)-3H-imidazo[4,5-b]pyridine (27e), a potent inhibitor of Aurora kinases (Aurora-A K(d) = 7.5 nM, Aurora-B K(d) = 48 nM), FLT3 kinase (K(d) = 6.2 nM), and FLT3 mutants including FLT3-ITD (K(d) = 38 nM) and FLT3(D835Y) (K(d) = 14 nM). FLT3-ITD causes constitutive FLT3 kinase activation and is detected in 20-35% of adults and 15% of children with acute myeloid leukemia (AML), conferring a poor prognosis in both age groups. In an in vivo setting, 27e strongly inhibited the growth of a FLT3-ITD-positive AML human tumor xenograft (MV4-11) following oral administration, with in vivo biomarker modulation and plasma free drug exposures consistent with dual FLT3 and Aurora kinase inhibition. Compound 27e, an orally bioavailable dual FLT3 and Aurora kinase inhibitor, was selected as a preclinical development candidate for the treatment of human malignancies, in particular AML, in adults and children..
Faisal, A.
Vaughan, L.
Bavetsias, V.
Sun, C.
Atrash, B.
Avery, S.
Jamin, Y.
Robinson, S.P.
Workman, P.
Blagg, J.
Raynaud, F.I.
Eccles, S.A.
Chesler, L.
Linardopoulos, S.
(2011). The aurora kinase inhibitor CCT137690 downregulates MYCN and sensitizes MYCN-amplified neuroblastoma in vivo. Mol cancer ther,
Vol.10
(11),
pp. 2115-2123.
show abstract
full text
Aurora kinases regulate key stages of mitosis including centrosome maturation, spindle assembly, chromosome segregation, and cytokinesis. Aurora A and B kinase overexpression has also been associated with various human cancers, and as such, they have been extensively studied as novel antimitotic drug targets. Here, we characterize the Aurora kinase inhibitor CCT137690, a highly selective, orally bioavailable imidazo[4,5-b]pyridine derivative that inhibits Aurora A and B kinases with low nanomolar IC(50) values in both biochemical and cellular assays and exhibits antiproliferative activity against a wide range of human solid tumor cell lines. CCT137690 efficiently inhibits histone H3 and transforming acidic coiled-coil 3 phosphorylation (Aurora B and Aurora A substrates, respectively) in HCT116 and HeLa cells. Continuous exposure of tumor cells to the inhibitor causes multipolar spindle formation, chromosome misalignment, polyploidy, and apoptosis. This is accompanied by p53/p21/BAX induction, thymidine kinase 1 downregulation, and PARP cleavage. Furthermore, CCT137690 treatment of MYCN-amplified neuroblastoma cell lines inhibits cell proliferation and decreases MYCN protein expression. Importantly, in a transgenic mouse model of neuroblastoma that overexpresses MYCN protein and is predisposed to spontaneous neuroblastoma formation, this compound significantly inhibits tumor growth. The potent preclinical activity of CCT137690 suggests that this inhibitor may benefit patients with MYCN-amplified neuroblastoma..
Dodson, C.A.
Kosmopoulou, M.
Richards, M.W.
Atrash, B.
Bavetsias, V.
Blagg, J.
Bayliss, R.
(2010). Crystal structure of an Aurora-A mutant that mimics Aurora-B bound to MLN8054: insights into selectivity and drug design. Biochemical journal,
Vol.427,
pp. 19-10.
Bouloc, N.
Large, J.M.
Kosmopoulou, M.
Sun, C.
Faisal, A.
Matteucci, M.
Reynisson, J.
Brown, N.
Atrash, B.
Blagg, J.
McDonald, E.
Linardopoulos, S.
Bayliss, R.
Bavetsias, V.
(2010). Structure-based design of imidazo[1,2-a]pyrazine derivatives as selective inhibitors of Aurora-A kinase in cells. Bioorg med chem lett,
Vol.20
(20),
pp. 5988-5993.
show abstract
Co-crystallisation of the imidazo[1,2-a]pyrazine derivative 15 (3-chloro-N-(4-morpholinophenyl)-6-(pyridin-3-yl)imidazo[1,2-a]pyrazin-8-amine) with Aurora-A provided an insight into the interactions of this class of compound with Aurora kinases. This led to the design and synthesis of potent Aurora-A inhibitors demonstrating up to 70-fold selectivity in cell-based Aurora kinase pharmacodynamic biomarker assays..
Bavetsias, V.
Large, J.M.
Sun, C.
Bouloc, N.
Kosmopoulou, M.
Matteucci, M.
Wilsher, N.E.
Martins, V.
Reynisson, J.
Atrash, B.
Faisal, A.
Urban, F.
Valenti, M.
de Haven Brandon, A.
Box, G.
Raynaud, F.I.
Workman, P.
Eccles, S.A.
Bayliss, R.
Blagg, J.
Linardopoulos, S.
McDonald, E.
(2010). Imidazo[4,5-b]pyridine derivatives as inhibitors of Aurora kinases: lead optimization studies toward the identification of an orally bioavailable preclinical development candidate. J med chem,
Vol.53
(14),
pp. 5213-5228.
show abstract
Lead optimization studies using 7 as the starting point led to a new class of imidazo[4,5-b]pyridine-based inhibitors of Aurora kinases that possessed the 1-benzylpiperazinyl motif at the 7-position, and displayed favorable in vitro properties. Cocrystallization of Aurora-A with 40c (CCT137444) provided a clear understanding into the interactions of this novel class of inhibitors with the Aurora kinases. Subsequent physicochemical property refinement by the incorporation of solubilizing groups led to the identification of 3-((4-(6-bromo-2-(4-(4-methylpiperazin-1-yl)phenyl)-3H-imidazo[4,5-b]pyridin-7-yl)piperazin-1-yl)methyl)-5-methylisoxazole (51, CCT137690) which is a potent inhibitor of Aurora kinases (Aurora-A IC(50) = 0.015 +/- 0.003 muM, Aurora-B IC(50) = 0.025 muM, Aurora-C IC(50) = 0.019 muM). Compound 51 is highly orally bioavailable, and in in vivo efficacy studies it inhibited the growth of SW620 colon carcinoma xenografts following oral administration with no observed toxicities as defined by body weight loss..
Bavetsias, V.
Blagg, J.
Bayliss, R.
(2010). Crystal structure of an Aurora-A mutant that mimics Aurora-B bound to MLN8054: insights into selectivity and drug design. Biochemical journal,
Vol.427
(Part 1),
pp. 19-28.
show abstract
full text
The production of selective protein kinase inhibitors is often frustrated by the similarity of the enzyme active sites. For this reason, it is challenging to design inhibitors that discriminate between the three Aurora kinases, which are important targets in cancer drug discovery. We have used a triple-point mutant of Aurora-A (AurA(x3)) which mimics the active site of Aurora-B to investigate the structural basis of MLN8054 selectivity. The bias toward Aurora-A inhibition by MLN8054 is fully recapitulated by AurAx3 in vitro. X-ray crystal structures of the complex suggest that the basis for the discrimination is electrostatic repulsion due to the T217E substitution, which we have confirmed using a single-point mutant. The activation loop of Aurora-A in the AurA(x3)-MLN8054 complex exhibits an unusual conformation in which Asp(274) and Phe(275) side chains point into the interior of the protein. There is to our knowledge no documented precedent for this conformation, which we have termed DFG-up. The sequence requirements of the DFG-up conformation suggest that it might be accessible to only a fraction of kinases. MLN8054 thus circumvents the problem of highly homologous active sites. Binding of MLN8054 to Aurora-A switches the character of a pocket within the active site from polar to a hydrophobic pocket, similar to what is observed in the structure of Aurora-A bound to a compound that induces DFG-out. We propose that targeting this pocket may be a productive route in the design of selective kinase inhibitors and describe the structural basis for the rational design of these compounds..
Gorsuch, S.
Bavetsias, V.
Rowlands, M.G.
Aherne, G.W.
Workman, P.
Jarman, M.
McDonald, E.
(2009). Synthesis of isothiazol-3-one derivatives as inhibitors of histone acetyltransferases (HATs). Bioorg med chem,
Vol.17
(2),
pp. 467-474.
show abstract
High-throughput screening led to the identification of isothiazolones 1 and 2 as inhibitors of histone acetyltransferase (HAT) with IC50s of 3 microM and 5 microM, respectively. Analogues of these hit compounds with variations of the N-phenyl group, and with variety of substituents at C-4, C-5 of the thiazolone ring, were prepared and assayed for inhibition of the HAT enzyme PCAF. Potency is modestly favoured when the N-aryl group is electron deficient (4-pyridyl derivative 10 has IC(50)=1.5 microM); alkyl substitution at C-4 has little effect, whilst similar substitution at C-5 causes a significant drop in potency. The ring-fused compound 38 has activity (IC(50)=6.1 microM) to encourage further exploration of this bicyclic structure. The foregoing SAR is consistent with an inhibitory mechanism involving cleavage of the S-N bond of the isothiazolone ring by a catalytically important thiol residue..
Quevedo, C.E.
Bavetsias, V.
McDonald, E.
(2009). Microwave-assisted synthesis of 2-aminonicotinic acids by reacting 2-chloronicotinic acid with amines. Tetrahedron letters,
Vol.50
(21),
pp. 2481-2483.
Gutierrez, C.D.
Bavetsias, V.
McDonald, E.
(2008). ClTi(OiPr)3-promoted reductive amination on the solid phase: combinatorial synthesis of a biaryl-based sulfonamide library. J comb chem,
Vol.10
(2),
pp. 280-284.
show abstract
A combinatorial library (9 amines x 7 sulfonyl chlorides x 13 boronic acids = 819 compounds) was produced on solid support in a four-step sequence, i.e., ClTi(O(i)Pr)3-promoted reductive amination, sulfonylation of the resin-bound amine, Suzuki cross-coupling, and acid-mediated cleavage. The library members were obtained in moderate quantity (1-8 mg) with over 70% of the sampled products greater than 90% pure according to LC-MS analysis..
Bavetsias, V.
Sun, C.
Bouloc, N.
Reynisson, J.
Workman, P.
Linardopoulos, S.
McDonald, E.
(2007). Hit generation and exploration: imidazo[4,5-b]pyridine derivatives as inhibitors of Aurora kinases. Bioorg med chem lett,
Vol.17
(23),
pp. 6567-6571.
show abstract
A hit generation and exploration approach led to the discovery of 31 (2-(4-(6-chloro-2-(4-(dimethylamino)phenyl)-3H-imidazo[4,5-b]pyridin-7-yl)piperazin-1-yl)-N-(thiazol-2-yl)acetamide), a potent, novel inhibitor of Aurora-A, Aurora-B and Aurora-C kinases with IC(50) values of 0.042, 0.198 and 0.227microM, respectively. Compound 31 inhibits cell proliferation and has good microsomal stability..
Chan, F.
Sun, C.
Perumal, M.
Nguyen, Q.-.
Bavetsias, V.
McDonald, E.
Martins, V.
Wilsher, N.E.
Raynaud, F.I.
Valenti, M.
Eccles, S.
Te Poele, R.
Workman, P.
Aboagye, E.O.
Linardopoulos, S.
(2007). Mechanism of action of the Aurora kinase inhibitor CCT129202 and in vivo quantification of biological activity. Mol cancer ther,
Vol.6
(12 Pt 1),
pp. 3147-3157.
show abstract
The Aurora family of serine/threonine kinases is important for the regulation of centrosome maturation, chromosome segregation, and cytokinesis during mitosis. Overexpression of Aurora kinases in mammalian cells leads to genetic instability and transformation. Increased levels of Aurora kinases have also been linked to a broad range of human tumors. Here, we describe the properties of CCT129202, a representative of a structurally novel series of imidazopyridine small-molecule inhibitors of Aurora kinase activity. This compound showed high selectivity for the Aurora kinases over a panel of other kinases tested and inhibits proliferation in multiple cultured human tumor cell lines. CCT129202 causes the accumulation of human tumor cells with >or=4N DNA content, leading to apoptosis. CCT120202-treated human tumor cells showed a delay in mitosis, abrogation of nocodazole-induced mitotic arrest, and spindle defects. Growth of HCT116 xenografts in nude mice was inhibited after i.p. administration of CCT129202. We show that p21, the cyclin-dependent kinase inhibitor, is induced by CCT129202. Up-regulation of p21 by CCT129202 in HCT116 cells led to Rb hypophosphorylation and E2F inhibition, contributing to a decrease in thymidine kinase 1 transcription. This has facilitated the use of 3'-deoxy-3'[(18)F]fluorothymidine-positron emission tomography to measure noninvasively the biological activity of the Aurora kinase inhibitor CCT129202 in vivo..
Bavetsias, V.
Henderson, E.A.
McDonald, E.
(2007). Cyclopenta[g]quinazolinone-based inhibitors of thymidylate synthase targeting alpha-folate receptor overexpressing tumours: synthetic approaches to 4-{N-[(6RS)-2-hydroxymethyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl]-N-(prop-2-ynyl)amino}benzoic acid. Tetrahedron,
Vol.63
(7),
pp. 1537-1543.
show abstract
An advanced intermediate for the synthesis of cyclopenta[g]quinazolinone-based antifolates such as (6RS)-CB300945 was prepared by a convergent methodology. The oxo-functionality required for the formation of the C-6-N-10 bond in 4 was introduced in the initial steps of the synthesis, then the tricyclic ring was constructed and finally the propargyl group was introduced using the (propargyl)CO2(CO)(6)(+) complex without the need to protect the N-3-H or 2-hydroxymethyl functionalities. (c) 2006 Elsevier Ltd. All rights reserved..
Collins, I.
Caldwell, J.
Fonseca, T.
Donald, A.
Bavetsias, V.
Hunter, L.-.
Garrett, M.D.
Rowlands, M.G.
Aherne, G.W.
Davies, T.G.
Berdini, V.
Woodhead, S.J.
Davis, D.
Seavers, L.C.
Wyatt, P.G.
Workman, P.
McDonald, E.
(2006). Structure-based design of isoquinoline-5-sulfonamide inhibitors of protein kinase B. Bioorg med chem,
Vol.14
(4),
pp. 1255-1273.
show abstract
Structure-based drug design of novel isoquinoline-5-sulfonamide inhibitors of PKB as potential antitumour agents was investigated. Constrained pyrrolidine analogues that mimicked the bound conformation of linear prototypes were identified and investigated by co-crystal structure determinations with the related protein PKA. Detailed variation in the binding modes between inhibitors with similar overall conformations was observed. Potent PKB inhibitors from this series inhibited GSK3beta phosphorylation in cellular assays, consistent with inhibition of PKB kinase activity in cells..
Henderson, E.A.
Bavetsias, V.
Theti, D.S.
Wilson, S.C.
Clauss, R.
Jackman, A.L.
(2006). Targeting the alpha-folate receptor with cyclopenta[g]quinazoline-based inhibitors of thymidylate synthase. Bioorg med chem,
Vol.14
(14),
pp. 5020-5042.
show abstract
The alpha-FR has been reported to be overexpressed in many carcinomas, in particular those of the ovary and uterus. The high expression of alpha-FR in some tumours compared with normal tissues has been exploited over the last decade for folate-mediated targeting of macromolecules, anticancer drugs, imaging agents and nucleic acids to cancer cells. CB300638, a cyclopenta[g]quinazoline-based inhibitor of thymidylate synthase (TS), has been reported to have high affinity for the receptor and selectivity for alpha-FR overexpressing tumour cell lines. In this study, the structural features of the molecule, in particular modifications at the 2-position, have been investigated with respect to TS inhibition, affinity for the alpha-FR and reduced folate carrier (RFC) and activity in A431-FBP cells (transfected with human alpha-FR) compared with neo-transfected A431 cells. Compounds 1a,b, 2a,b and 3a,b were synthesised utilising multistep sequences. It was found that the 2-substituent does not affect the affinity for the alpha-FR; however, it greatly affects selectivity for A431-FBP cells, and suggests that there are factors other than TS inhibition and alpha-FR affinity that are important for the activity of these compounds. Compound 2b (2-CH2OH derivative) displayed the highest selectivity for the A431-FBP cells compared with A431 cells..
Sorrentino, R.
Libertini, S.
Pallante, P.L.
Troncone, G.
Palombini, L.
Bavetsias, V.
Spalletti-Cernia, D.
Laccetti, P.
Linardopoulos, S.
Chieffi, P.
Fusco, A.
Portella, G.
(2005). Aurora B overexpression associates with the thyroid carcinoma undifferentiated phenotype and is required for thyroid carcinoma cell proliferation. J clin endocrinol metab,
Vol.90
(2),
pp. 928-935.
show abstract
Alterations in chromosome number (aneuploidy) are common in human neoplasias. Loss of mitotic regulation is believed to induce aneuploidy in cancer cells and act as a driving force during the malignant progression. The serine/theronine protein kinases of aurora family genes play a critical role in the regulation of key cell cycle processes. Aurora B mediates chromosome segregation by ensuring orientation of sister chromatids and overexpression of Aurora B in diploid human cells NHDF (normal human diploid fibroblast) induces multinuclearity. We analyzed Aurora B expression in human thyroid carcinomas. Cell lines originating from different histotypes showed an increase in Aurora B expression. Immunohistochemical analysis of archive samples showed a high expression of Aurora B in anaplastic thyroid carcinomas; conversely, Aurora B expression was not detectable in normal thyroid tissue. Real-time PCR analysis confirmed a strong expression of Aurora B in anaplastic thyroid carcinomas. The block of Aurora B expression induced by RNA interference or by using an inhibitor of Aurora kinase activity significantly reduced the growth of thyroid anaplastic carcinoma cells..
Stimson, L.
Rowlands, M.G.
Newbatt, Y.M.
Smith, N.F.
Raynaud, F.I.
Rogers, P.
Bavetsias, V.
Gorsuch, S.
Jarman, M.
Bannister, A.
Kouzarides, T.
McDonald, E.
Workman, P.
Aherne, G.W.
(2005). Isothiazolones as inhibitors of PCAF and p300 histone acetyltransferase activity. Mol cancer ther,
Vol.4
(10),
pp. 1521-1532.
show abstract
Histone acetylation plays an important role in regulating the chromatin structure and is tightly regulated by two classes of enzyme, histone acetyltransferases (HAT) and histone deacetylases (HDAC). Deregulated HAT and HDAC activity plays a role in the development of a range of cancers. Consequently, inhibitors of these enzymes have potential as anticancer agents. Several HDAC inhibitors have been described; however, few inhibitors of HATs have been disclosed. Following a FlashPlate high-throughput screen, we identified a series of isothiazolone-based HAT inhibitors. Thirty-five N-substituted analogues inhibited both p300/cyclic AMP-responsive element binding protein-binding protein-associated factor (PCAF) and p300 (1 to >50 micromol/L, respectively) and the growth of a panel of human tumor cell lines (50% growth inhibition, 0.8 to >50 micromol/L). CCT077791 and CCT077792 decreased cellular acetylation in a time-dependent manner (2-48 hours of exposure) and a concentration-dependent manner (one to five times, 72 hours, 50% growth inhibition) in HCT116 and HT29 human colon tumor cell lines. CCT077791 reduced total acetylation of histones H3 and H4, levels of specific acetylated lysine marks, and acetylation of alpha-tubulin. Four and 24 hours of exposure to the compounds produced the same extent of growth inhibition as 72 hours of continuous exposure, suggesting that growth arrest was an early event. Chemical reactivity of these compounds, as measured by covalent protein binding and loss of HAT inhibition in the presence of DTT, indicated that reaction with thiol groups might be important in their mechanism of action. As one of the first series of small-molecule inhibitors of HAT activity, further analogue synthesis is being pursued to examine the potential scope for reducing chemical reactivity while maintaining HAT inhibition..
Gibbs, D.D.
Theti, D.S.
Wood, N.
Green, M.
Raynaud, F.
Valenti, M.
Forster, M.D.
Mitchell, F.
Bavetsias, V.
Henderson, E.
Jackman, A.L.
(2005). BGC 945, a novel tumor-selective thymidylate synthase inhibitor targeted to alpha-folate receptor-overexpressing tumors. Cancer res,
Vol.65
(24),
pp. 11721-11728.
show abstract
BGC 945 is a cyclopenta[g]quinazoline-based, thymidylate synthase inhibitor specifically transported into alpha-folate receptor (alpha-FR)-overexpressing tumors. Affinity of BGC 945 for the alpha-FR is 70% of the high-affinity ligand folic acid. In contrast to conventional antifolates, BGC 945 has low affinity for the widely expressed reduced-folate carrier (RFC). The K(i) for isolated thymidylate synthase is 1.2 nmol/L and the IC(50) for inhibition of the growth of alpha-FR-negative mouse L1210 or human A431 cells is approximately 7 micromol/L. In contrast, BGC 945 is highly potent in a range of alpha-FR-overexpressing human tumor cell lines (IC(50) approximately 1-300 nmol/L). Pharmacokinetic variables measured following i.v. injection of 100 mg/kg BGC 945 to KB tumor-bearing mice showed rapid plasma clearance (0.021 L/h) and tissue distribution. The terminal half-lives in plasma, liver, kidney, spleen, and tumor were 2, 0.6, 5, 21, and 28 hours, respectively. Tumor BGC 945 concentration at 24 hours was approximately 1 nmol/g tissue, at least 10-fold higher than that in plasma or normal tissues. Inhibition of thymidylate synthase in tissues leads to increased incorporation of 5-[(125)I]-iodo-2'-deoxyuridine ([(125)I]dUrd) into DNA. Forty-eight hours after injection of 100 mg/kg 6RS-BGC 945 ([(125)I]dUrd injected at 24 hours), tumor was the only tissue with incorporation above control level (6-fold). The RFC-mediated thymidylate synthase inhibitor plevitrexed also increased uptake of [(125)I]dUrd in tumor (10-fold) but, in contrast, also caused increased incorporation in other normal tissues such as spleen and small bowel (4.5- and 4.6-fold, respectively). These data suggest that BGC 945 selectively inhibits thymidylate synthase in alpha-FR-overexpressing tumors and should cause minimal toxicity to humans at therapeutic doses..
Gutierrez, C.D.
Bavetsias, V.
McDonald, E.
(2005). TiCl((OPr)-Pr-i)(3) and NaBH(OAc)(3): an efficient reagent combination for the reductive amination of aldehydes by electron-deficient amines. Tetrahedron letters,
Vol.46
(20),
pp. 3595-3.
McDonald, T.
Bavetsias, V.
(2005). Discovery of small molecules as anticancer agents: the role of the medicinal chemist in the discovery of new cancer therapeutics. Forum of clinical oncology,
Vol.4,
pp. 51-57.
show abstract
Discovery of small molecules as anticancer agents: the role of the medicinal chemist in the discovery of new cancer therapeutics.
Bavetsias, V.
Henderson, E.A.
McDonald, E.
(2004). 2-pivalamido-3H-pyrimidin-4-one derivatives: convenient pivalamide hydrolysis using Fe(NO3)(3) in MeOH. Tetrahedron lett,
Vol.45
(29),
pp. 5643-5644.
show abstract
A simple methodology for pivalamide (trimethylacetamide, pivaloylamino) hydrolysis has been discovered using Fe(NO3)(3) in MeOH at room temperature. The pivalamido group of 2-pivilamido-3H-pyrimidin-4-ones or fused 2-pivalamido-3H-pyrimidin-4-ones such as 2-pivilamido-3H-quinazolin-4-ones and 2-pivalamido-3H-pteridines have been hydrolysed under these conditions to afford the corresponding amine. (C) 2004 Elsevier Ltd. All rights reserved..
Theti, D.S.
Bavetsias, V.
Skelton, L.A.
Titley, J.
Gibbs, D.
Jansen, G.
Jackman, A.L.
(2003). Selective delivery of CB300638, a cyclopenta[g]quinazoline-based thymidylate synthase inhibitor into human tumor cell lines overexpressing the alpha-isoform of the folate receptor. Cancer res,
Vol.63
(13),
pp. 3612-3618.
show abstract
The alpha-isoform of the glycosylphosphatidylinositol cell membrane tethered folate receptor (alpha-FR) is overexpressed in some carcinomas (notably ovarian carcinomas) relative to normal tissues. The nonpolyglutamatable folate-based thymidylate synthase (TS) inhibitor, CB300638 (TS K(i) = 0.24 nM) displayed an IC(50) of 0.0028 microM for the inhibition of the growth of human A431-FBP cells transfected with the alpha-FR. In contrast, the IC(50) for the neotransfected A431 cells was 0.81 microM (300-fold higher). Similarly, this compound inhibited the growth of human KB cells that constitutively overexpress the alpha-FR with an IC(50) of 0.0036 microM. These data were derived from cells grown in a physiological concentration of folate (20 nM R,S-leucovorin). Incubation of KB cells with a 1 microM excess of folic acid (FA), to selectively block uptake via the alpha-FR, increased the CB300638 IC(50) to 0.39 microM. The relatively low potency of CB300638 under these conditions, or in cell lines not expressing the alpha-FR, is ascribed to its low affinity for the ubiquitously expressed folate transporter, the reduced-folate carrier (K(i) for inhibition of [(3)H]methotrexate transport >100 microM). The high potency of CB300638 in alpha-FR-overexpressing cell lines is attributable to high affinity of the alpha-FR (53% of FA) and efficient endosomal trafficking mediated by the alpha-FR. Sixteen-h exposure to CB300638 inhibited the rate of (3)H(2)O release from 5-[(3)H]dUrd (in situ TS assay) in A431, A431-FBP, and KB cells with IC(50) values of 0.1 microM, 0.005 microM, and 0.002 microM, respectively. The coaddition of 1 micro M FA increased the IC(50)s for A431-FBP and KB cells to approximately 0.1 microM consistent with alpha-FR-mediated transport of CB300638. In conclusion, alpha-FR-mediated uptake of CB300638 leads to TS and growth inhibition that is highly selective for alpha-FR overexpressing tumor cell lines. The low expression of the alpha-FR in normal tissues, particularly those sensitive to TS inhibitors, together with the low affinity of CB300638 for the reduced-folate carrier, suggests that the compound may have potential as an antitumor agent with a high therapeutic index..
Bavetsias, V.
Clauss, R.
Henderson, E.A.
(2003). Antifolate chemistry: synthesis of 4-[N-[(6RS)-2-methyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl]-N-(prop-2-ynyl)amino]benzoic acid via a (propargyl)Co2(CO)6+ complex. Org biomol chem,
Vol.1
(11),
pp. 1943-1946.
show abstract
A new route to compound 3 (4-[N-(6RS)-2-methyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta[g]quinazolin-6-yl]-N-(prop-2-ynyl)amino]benzoic acid), a crucial intermediate for the synthesis of potent inhibitors of thymidylate synthase (TS), is described. In this sequence the C6-N10 bond was constructed first, by the reductive amination of 5-acetamido-6-bromoindan-1-one 6 with tert-butyl 4-aminobenzoate, then the cyclopenta[g]quinazolinone ring was formed and the propargyl group was introduced on the N10-position using the (propargyl)Co2(CO)6+ complex as the electrophilic propargyl reagent..
Bavetsias, V.
Skelton, L.A.
Yafai, F.
Mitchell, F.
Wilson, S.C.
Allan, B.
Jackman, A.L.
(2002). The design and synthesis of water-soluble analogues of CB30865, a quinazolin-4-one-based antitumor agent. J med chem,
Vol.45
(17),
pp. 3692-3702.
show abstract
4-[N-[7-Bromo-2-methyl-4-oxo-3,4-dihydroquinazolin-6-ylmethyl]-N-(prop-2-ynyl)amino]-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..
Bavetsias, V.
Marriott, J.H.
Theti, D.S.
Melin, J.C.
Wilson, S.C.
Jackman, A.L.
(2001). Cyclopenta[g]quinazoline-based antifolates: the effect of the chirality at the 6-position on the inhibition of thymidylate synthase (TS). Bioorg med chem lett,
Vol.11
(23),
pp. 3015-3017.
show abstract
Cyclopenta[g]quinazoline-based inhibitors of thymidylate synthase (TS) possess a chiral centre at the 6-position of the molecule. The effect of this chirality on the inhibition of TS was investigated by synthesising compounds 6S-1a-c, 6R-1a-c. It was shown, in particular with the diastereoisomers 6S-1c, 6R-1c, that the inhibitory activity against TS is mainly due to the 6S diastereoisomer rather than the 6R diastereoisomer, which is virtually inactive..
Theti, D.
Melin, C.
Bavetsias, V.
Skelton, L.
Jackman, A.L.
(2000). Selective targeting of the novel cyclopenta[g]quinazoline-based thymidylate synthase (TS) inhibitors CB300638 and CB300907 to alpha-folate receptor over-expressing cells in vitro. Brit j cancer,
Vol.83,
pp. 67-67.
Bavetsias, V.
Henderson, E.A.
(2000). Synthesis of N-substituted derivatives of tert-butyl 4-aminobenzoate via a palladium-catalysed reaction. J chem res-s,
(9),
pp. 418-419.
show abstract
Amination of tert-butyl 4-bromobenzoate using PdCl2 (7% mel) and P(o-tolyl)(3) provides a convenient route to N-substituted derivatives of tert-butyl 4-aminobenzoate..
Bavetsias, V.
Marriott, J.H.
Melin, C.
Kimbell, R.
Matusiak, Z.S.
Boyle, F.T.
Jackman, A.L.
(2000). Design and synthesis of Cyclopenta[g]quinazoline-based antifolates as inhibitors of thymidylate synthase and potential antitumor agents(,). J med chem,
Vol.43
(10),
pp. 1910-1926.
show abstract
Following the development of raltitrexed, the synthesis of nonpolyglutamatable inhibitors of TS that do not use the reduced folate carrier (RFC) for cellular entry should provide compounds which overcome mechanisms of resistance to folate-based inhibitors of TS that are associated with decreased/altered folylpolyglutamate synthetase (FPGS) expression and/or an impaired RFC. Examination of a computer graphics model of the humanized Escherichia coli TS enzyme with quinazoline inhibitors of TS, such as 1 bound in the active site of the enzyme, suggested that conformational restriction introduced by bridging the C9 with C7 to form a pentacycle may be beneficial for binding to TS. That led to the synthesis of a series of potent cyclopenta[g]quinazoline-based inhibitors of the enzyme in which the glutamyl residue associated with classical antifolates was replaced with a variety of glutamate-derived ligands; the most potent inhibitor being the L-Glu-gamma-D-GluT(alpha) derivative 7j. In the mouse L1210:1565 cell line (mutant RFC), the majority of these compounds had activity equal or only slightly greater compared with the parental L1210 cell line, indicating a reduced dependence on the RFC for cellular uptake in the L1210 cell line..
Marriott, J.H.
Neidle, S.
Matusiak, Z.
Bavetsias, V.
Jackman, A.L.
Melin, C.
Boyle, F.T.
(1999). Chemoenzymatic preparation of the novel antifolate thymidylate synthase inhibitor N-(4-{N-[(6S)-2-methyl-4-oxo-3,4,7,8-tetrahydro-6H-cyclopenta [g]quinazolin-6-yl]-N-(prop-2-ynyl)amino}-benzoyl)-L-glutamic acid and its glutamyl cleavage product. J chem soc perk t 1,
(11),
pp. 1495-1503.
show abstract
5-Aminoindane was converted in six steps to the cydopenta[g]quinazoline ketone 13. Condensation of 13 with diethyl 4-aminobenzoyl-L-glutamate, followed by in situ reduction, produced the secondary amine 15. N-Propargylation of 15, followed by deprotection, gave the diacid 17 as a mixture of diastereoisomers. Treatment of 17 with the bacterial enzyme carboxypeptidase G(2) resulted in removal of the L-glutamic acid residue from (6R)-17 to give a chromatographically separable mixture of the monoacid 18 and the antifolate 5 [(6S)-17], which was assayed as an inhibitor of thymidylate synthase (K-i(app) = 3 nM). Treatment of isolated diacid 5 with carboxypeptidase G(2) produced the monoacid 19 in ca. 98% enantiomeric excess. The (6S) stereochemistry of compound 19 has been established by X-ray crystal structure determination of the amide derivative 24..
Bavetsias, V.
(1999). Heterocyclic templates and nonpolyglutamatable inhibitors of thymidylate synthase as potential antitumour agents: A chemical perspective. J heterocyclic chem,
Vol.36
(4),
pp. 827-835.
Bavetsias, V.
Jackman, A.L.
(1998). Nonpolyglutamatable antifolates as inhibitors of thymidylate synthase (TS) and potential antitumour agents. Curr med chem,
Vol.5
(4),
pp. 265-288.
show abstract
Thymidylate synthase (TS), an enzyme that catalyses the conversion of dUMP to dTMP, has been the focus of interest as a target in cancer chemotherapy for more than two decades. Over the last 10 years much research has been devoted to the design and development of nonpolyglutamatable inhibitors of TS as antitumour agents, mainly to over-come resistance due to unfavourable expression of folylpolyglutamate synthetase (FPGS). Lipophilic inhibitors of the enzyme were expected not to depend on the reduced folate carrier transporter (RFC) for cellular uptake, thus avoiding resistance due to an impaired RFC. Compounds of this type can be classified in three groups: A: nonclassical lipophilic inhibitors of TS, mainly folate-based analogues lacking the glutamate side chain; B: folate-based analogues in which the glutamate side chain has been modified in such a way that polyglutamation is precluded; and C: nonpolyglutamatable glutamate-containing inhibitors of TS. Compounds of group A included 5- or 6-substituted quinazolin-4-ones, benzo[flquinazolines, imidazotetrahydroquinoline- and benz[cd]indole-based inhibitors. The second group is mainly related to a series of g-linked dipeptide derivatives of ICIl98583, or analogues of this inhibitor where the glutamate residue was replaced with a range of a-amino acids. The third group is concerned with some 7-substituted derivatives of ICI198583 and the pyrrolo[3, 2-d]pyrimidine-based inhibitor 175. A large number of structurally diverse nonpolyglutamatable inhibitors of TS were synthesised some of which were potent inhibitors of the enzyme (human or E. coli) and in vitro cell growth. Three compounds, i.e. 49 (AG 337), 83 (AG 331), 123 (ZD9331) have reached the stage of clinical evaluation..
Bavetsias, V.
(1998). A facile route to quinazolin-4(3H)-ones functionalised at the 2-position. Synthetic commun,
Vol.28
(24),
pp. 4547-4559.
show abstract
Treatment of 2-methoxyacetamidobenzonitriles or 2-chloroacetamidobenzonitrile with UHP and K2CO3 provides a convenient route to 2-methoxymethyl- or 2-chloromethylquinazolin-4(3H)-ones. In addition, demethylation of 2-methoxymethylquinazolin-4(3H)-ones with 48% HBr gives 2-hydroxymethylquinazolin-4(3H)-ones..
Bavetsias, V.
Jackman, A.L.
Marriott, J.H.
Kimbell, R.
Gibson, W.
Boyle, F.T.
Bisset, G.M.
(1997). Folate-based inhibitors of thymidylate synthase: synthesis and antitumor activity of gamma-linked sterically hindered dipeptide analogues of 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583). J med chem,
Vol.40
(10),
pp. 1495-1510.
show abstract
In an effort to synthesize inhibitors of thymidylate synthase (TS) that do not undergo polyglutamation, a series of gamma-linked sterically hindered dipeptide analogues of 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583) was prepared. A methyl, ethyl, or propargyl group was incorporated into the gamma-glutamyl amide bond of gamma-linked L,L dipeptide derivatives of ICI 198583, such as ICI 198583-gamma-L-Glu. In addition, steric bulk was introduced on either side of the gamma-glutamyl bond of ICI 198583-gamma-L-Glu or ICI 198583-gamma-L-Ala. The resulting dipeptide analogues, e.g., ICI 198583-gamma-MeGlu and ICI 198583-gamma-Aib, were apparently stable to in vivo hydrolysis but poorer inhibitors of TS and L1210 cell growth. However, introduction of 7-Me, 2'-F substitution into the quinazoline nucleus gave significant improvement in the inhibitory activity against thymidylate synthase. Compounds 28-30, the 7-Me, 2'-F derivatives of ICI 198583-gamma-MeGlu, ICI 198583-gamma-EtGlu, and ICI 198583-gamma-PgGlu, respectively, were potent inhibitors of TS (K(iapp) = 0.21-1.1 nM) and L1210 cell growth (IC50 = 0.05-0.34 microM) and were similar to that seen with the most potent gamma-linked L,D dipeptide derivatives of ICI 198583 previously synthesized. Furthermore, the low cross-resistance ratios for the L1210:R(D1694)/L1210 cell line indicated that 28-30 do not undergo polyglutamation..
Bavetsias, V.
Bisset, G.M.
Kimbell, R.
Boyle, F.T.
Jackman, A.L.
(1997). Synthesis of novel quinazoline-based antifolates with modified glutamate side chains as potential inhibitors of thymidylate synthase and antitumour agents. Tetrahedron,
Vol.53
(39),
pp. 13383-13396.
show abstract
Several novel antifolates, derivatives of 2-desamino-2-methyl-N-10-propargyl-5,8-dideazafolic acid, were synthesised as inhibitors of thymidylate synthase (TS) and antitumour agents. This was accomplished by first developing routes to the key intermediates Glu-OMe-gamma-psi-[CSNH]Glu(OEt)-OEt (8), Glu-OBut-gamma-psi[CH2NH]Glu(oBu(t))-OBut (16), Glu-OMe-gamma-psi[CN4]Gly-OMe (23) and its 2,5-disubstituted regioisomer (22), followed by DEPC coupling to 4-[N-[(3,4-dihydro-2-methyl-4-oxo-6-quinazolinyl)-methyl]-N-prop-2-ynylamino]benzoic acid (9) or 4-[N-[(3, 4-dihydro-2,7-dimethyl-4-oxo-6-quinazolinyl)methyl]-N-prop-2-ynylamino]-2- fluorobenzoic acid (24), and finally removal of the protecting groups. The resulting quinazoline-based antifolates with modified glutamate side chains, and in particular, the tetrazole derivatives 26 and 29 displayed potent TS and L1210 cell growth inhibitory activities (e.g., for 26: TS IC50 = 2.4 nM, L1210 IC50 = 1.3 mu M). (C) 1997 Elsevier Science Ltd..
Bavetsias, V.
Jackman, A.L.
Kimbell, R.
Gibson, W.
Boyle, F.T.
Bisset, G.M.
(1996). Quinazoline antifolate thymidylate synthase inhibitors: gamma-linked L-D, D-D, and D-L dipeptide analogues of 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583). J med chem,
Vol.39
(1),
pp. 73-85.
show abstract
The syntheses of gamma-linked L-D, D-D, and D-L dipeptide analogues of 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583) are described. The general methodology for the synthesis of these molecules involved the preparation of the dipeptide derivatives employing solution phase peptide synthesis followed by condensation of the dipeptide free bases with the appropriate pteroic acid analogue via diethyl cyanophosphoridate (DEPC) activation. In the final step, tert-butyl esters were removed by trifluoroacetic acid (TFA) hydrolysis. Z-L-Glu-OBut-gamma-D-Ala-OBut, for example, was prepared from alpha-tert-butyl N-(benzyloxycarbonyl)-L-glutamate and tert-butyl D-alaninate via isobutyl-mixed anhydride coupling. The Z-group was removed by catalytic hydrogenolysis and the resulting dipeptide free base condensed with 2-desamino-2-methyl-N10-propargyl-5,8-dideazapteroic acid via DEPC coupling. Finally, tert-butyl esters were removed by TFA hydrolysis to give ICI 198583-gamma-D-Ala. The compounds were tested as inhibitors of thymidylate synthase and L1210 cell growth. Good enzyme and growth inhibitory activity were found with gamma-linked L-D dipeptides, the best examples being the Glu-gamma-D-Glu derivative 35 (Ki = 0.19 nM, L1210 IC50 = 0.20 +/- 0.017 microM) and the Glu-gamma-D-alpha-aminoadipate derivative 39 (Ki = 0.12 nM, L1210 IC50 = 0.13 +/- 0.063 microM). In addition, ICI 198583 L-gamma-D-linked dipeptides were resistant to enzymatic degradation in mice..
Springer, C.J.
Bavetsias, V.
Jackman, A.L.
Boyle, F.T.
Marshall, D.
Pedley, R.B.
Bisset, G.M.
(1996). Prodrugs of thymidylate synthase inhibitors: potential for antibody directed enzyme prodrug therapy (ADEPT). Anticancer drug des,
Vol.11
(8),
pp. 625-636.
show abstract
Prodrugs of quinazoline antifolate thymidylate synthase (TS) inhibitors have been designed and synthesized for use in antibody-directed enzyme prodrug therapy (ADEPT). The syntheses of the alpha-linked dipeptides of two potent thymidylate synthase inhibitors, ZD1694 [N-[5-[N-(3,4-dihydro-2-methyl-4-oxoquinazolin-6- ylmethyl)-N-methylamino]-2-thenoyl]-L-glutamic acid] and ICI198583 ¿N-[4-[N-[(2-methyl-3,4-dihydro-4-oxo-6-quinazolinyl) methyl]-N-prop-2-ynylamino]benzoyl]-L-glutamic acid¿ are described. The alpha-carboxyl of the glutamic acid has been linked through an amide bond to an L-alanine or an L-glutamic acid. The alpha-linked L-dipeptide prodrugs were designed to be activated to their corresponding thymidylate synthase inhibitors at a tumour site by prior administration of a monoclonal antibody conjugated to the enzyme carboxypeptidase A (CPA). The viability of a colorectal cell line was monitored with the potential prodrugs in the presence or absence of CPA or with the parent drugs alone. All the dipeptides had greatly decreased cytotoxicity, with a deactivation of approximately 100-fold for the ZD1694 prodrugs and approximately 20-200-fold for the ICI198583 prodrugs. Activation of the alpha-linked L-alanine dipeptides with CPA led to a cytotoxicity enhancement of approximately 10-100 fold..
Bavetsias, V.
Jackman, A.L.
Kimbell, R.
Boyle, F.T.
Bisset, G.M.
(1996). Carboxylic acid bioisosteres of gamma-linked dipeptide analogues of the folate-based thymidylate synthase (TS) inhibitor, 2-desamino-2-methyl-N-10-propargyl-5,8-dideazafolic acid (ICI 198583). Bioorg med chem lett,
Vol.6
(6),
pp. 631-636.
show abstract
Tetrazole carboxylic acid bioisosteres of gamma-linked dipeptide derivatives of 2-desamino-2-methyl-N-10-propargyl-5,8-dideazafolic acid (ICI 198583), a folate-based inhibitor of thymidylate synthase (TS), were synthesised by multistep routes starting from the appropriate pteroic acid analogue and Z-D-Ala or D-Glu. They exhibited excellent TS inhibitory activities which, however, were not accompanied by a parallel improvement in the L1210 cell growth inhibition..
Jackman, A.L.
Kimbell, R.
Brown, M.
Brunton, L.
Bisset, G.M.
Bavetsias, V.
Marsham, P.
Hughes, L.R.
Boyle, F.T.
(1995). Quinazoline-based thymidylate synthase inhibitors: relationship between structural modifications and polyglutamation. Anticancer drug des,
Vol.10
(7),
pp. 573-589.
show abstract
Quinazoline-based analogues of folic acid are a group of thymidylate synthase (TS) inhibitors that display a wide spectrum of activity for cultured tumour cells, partly due to their differential ability to form polyglutamate metabolites that are (i) more potent TS inhibitors and (ii) not readily effluxed from cells. The rate of cell membrane transport and folylpolyglutamate synthetase substrate activity influence compound polyglutamation. A series of intact-cell assays has been used to determine how specific modifications of 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolate (ICI 198583) affect compound polyglutamation. Those containing the 'classical' glutamate structure were usually, but not always, well polyglutamated intracellularly. Replacement of N10 propargyl with smaller aliphatic substituents, particularly when combined with replacement of the benzene ring with thiophene or thiazole heterocycles, was beneficial for antitumour activity through polyglutamate formation. Fluorination of the benzene, particularly if a F was adjacent to the 'bridge region' (3'F or 2',5'diF), also gave compounds with a high dependence on polyglutamation for activity. Those analogues with 2-CH2OH or NH2 substituents were poor substrates for the reduced-folate cell membrane carrier which can account for their reduced polyglutamation rate and hence growth-inhibitory activity. A large decrease or prevention of polyglutamation was achieved by the introduction of CH3, CH2CH3, Br or C1 on C7. The concomitant enhancement in TS inhibition by these modifications gave compounds active under continuous-exposure cell culture conditions. Some ICI 198583 analogues had the glutamate moiety replaced with unnatural amino acids or dipeptides. Only the L-gamma-L-glu analogue (a polyglutamate metabolite of ICI 198583) gave activity entirely attributable to polyglutamate formation..
BAVETSIAS, V.
BISSET, G.
JARMAN, M.
(1995). CONVENIENT PREPARATION OF ALPHA-TERT-BUTYL N-BLOCKED GLUTAMATES THROUGH GAMMA-ALLYL ESTER PROTECTION. Synthetic commun,
Vol.25
(7),
pp. 947-958.
show abstract
Utilisation of gamma-allyl ester protection of glutamates provides a convenient route to the synthesis of Z-Glu-OBu(t), Tr-Glu-OBu(t) and (Boc)(2)-Glu-OBu(t)..
Bisset, G.M.
Bavetsias, V.
Thornton, T.J.
Pawelczak, K.
Calvert, A.H.
Hughes, L.R.
Jackman, A.L.
(1994). The synthesis and thymidylate synthase inhibitory activity of L-gamma-L-linked dipeptide and L-gamma-amide analogues of 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583). J med chem,
Vol.37
(20),
pp. 3294-3302.
show abstract
Sixteen gamma-linked dipeptide and four L-Glu-gamma-amide analogues of 2-desamino-2-methyl-N10-propargyl-5,8-dideazafolic acid (ICI 198583) have been synthesized and evaluated as inhibitors of thymidylate synthase (TS). Z-blocked L-Glu-gamma-L-linked dipeptides and L-Glu-gamma-amides were prepared by condensing alpha-tert-butyl-N-(benzyloxycarbonyl)-L-glutamic acid with the appropriate tert-butyl-protected L-amino acid or amine. The Z group was removed by catalytic hydrogenolysis, and the resulting dipeptides or L-Glu-gamma-amides were condensed with the appropriate pteroic acid analogue trifluoroacetate salt using diethyl cyanophosphoridate as coupling reagent. Deprotection with trifluoroacetic acid in the final step gave the desired quinazoline gamma-linked dipeptides and L-Glu-gamma-amides as their trifluoroacetate salts. Nearly all the dipeptide analogues were potent inhibitors of TS, the best being ICI 198583-gamma-L-2-aminoadipate (IC50 = 2 nM). Several of these dipeptides were found to be susceptible to enzymatic hydrolysis in mice. The quinazoline monocarboxylate L-Glu-gamma-amides, lacking an alpha'-carboxyl group, are less active against TS and L1210 cell growth but are also not susceptible to enzymatic hydrolysis in mice..