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The drug tamoxifen shows evidence of genotoxicity, and induces liver tumours in rats. Covalent DNA adducts have been detected in the liver of rats treated with tamoxifen, and these arise through metabolism at the α-position to give an ester which reacts with DNA. (E)-1-(4-iodophenyl)-2-phenyl-1-[4-(2-pyrrolidinoethoxy)phenyl]-b ut-1-ene (idoxifene) is an analogue of tamoxifen in which formation of DNA adducts is greatly reduced; we could not detect any adducts in the DNA of cultured rat hepatocytes treated with 10 μM idoxifene, after analysis by the 32P-post-labelling method. The metabolite (Z)-4-(4-iodophenyl)-4-[4-(2-pyrrolidinoethoxy)phenyl]-3-phenyl-3 -buten-2-ol (α-hydroxyidoxifene) gave adducts in rat hepatocytes, but far fewer than the corresponding tamoxifen metabolite. In human hepatocytes, neither idoxifene nor tamoxifen induced detectable levels of DNA adducts. We prepared the α-acetoxy ester of idoxifene as a model for the ultimate reactive metabolite formed in rat liver. It was less reactive than α-acetoxytamoxifen, as might be expected on mechanistic grounds. It reacted with DNA in the same way, to give adducts which were probably N2-alkyldeoxyguanosines, but to a lower extent. All these results indicate that idoxifene is much less genotoxic than tamoxifen, and should therefore be a safer drug. [ABSTRACT FROM PUBLISHER]

The development of ligands for biological targets is critically dependent on the identification of sites on proteins that bind molecules with high affinity. A set of compounds, called FragLites, can identify such sites, along with the interactions required to gain affinity, by X-ray crystallography. We demonstrate the utility of FragLites in mapping the binding sites of bromodomain proteins BRD4 and ATAD2 and demonstrate that FragLite mapping is comparable to a full fragment screen in identifying ligand binding sites and key interactions. We extend the FragLite set with analogous compounds derived from amino acids (termed PepLites) that mimic the interactions of peptides. The output of the FragLite maps is shown to enable the development of ligands with leadlike potency. This work establishes the use of FragLite and PepLite screening at an early stage in ligand discovery allowing the rapid assessment of tractability of protein targets and informing downstream hit-finding.

NF-κB-inducing kinase (NIK) is a key enzyme in the noncanonical NF-κB pathway, of interest in the treatment of a variety of diseases including cancer. Validation of NIK as a drug target requires potent and selective inhibitors. The protein contains a cysteine residue at position 444 in the back pocket of the active site, unique within the kinome. Analysis of existing inhibitor scaffolds and early structure-activity relationships (SARs) led to the design of C444-targeting covalent inhibitors based on alkynyl heterocycle warheads. Mass spectrometry provided proof of the covalent mechanism, and the SAR was rationalized by computational modeling. Profiling of more potent analogues in tumor cell lines with constitutively activated NIK signaling induced a weak antiproliferative effect, suggesting that kinase inhibition may have limited impact on cancer cell growth. This study shows that alkynyl heterocycles are potential cysteine traps, which may be employed where common Michael acceptors, such as acrylamides, are not tolerated.

Inhibition of murine double minute 2 (MDM2)-p53 protein-protein interaction with small molecules has been shown to reactivate p53 and inhibit tumor growth. Here, we describe rational, structure-guided, design of novel isoindolinone-based MDM2 inhibitors. MDM2 X-ray crystallography, quantum mechanics ligand-based design, and metabolite identification all contributed toward the discovery of potent in vitro and in vivo inhibitors of the MDM2-p53 interaction with representative compounds inducing cytostasis in an SJSA-1 osteosarcoma xenograft model following once-daily oral administration.

Renewed interest in covalent inhibitors of enzymes implicated in disease states has afforded several agents targeted at protein kinases of relevance to cancers. We now report the design, synthesis and biological evaluation of 6-ethynylpurines that act as covalent inhibitors of Nek2 by capturing a cysteine residue (Cys22) close to the catalytic domain of this protein kinase. Examination of the crystal structure of the non-covalent inhibitor 3-((6-cyclohexylmethoxy-7 H -purin-2-yl)amino)benzamide in complex with Nek2 indicated that replacing the alkoxy with an ethynyl group places the terminus of the alkyne close to Cys22 and in a position compatible with the stereoelectronic requirements of a Michael addition. A series of 6-ethynylpurines was prepared and a structure activity relationship (SAR) established for inhibition of Nek2. 6-Ethynyl- N -phenyl-7 H -purin-2-amine [IC 50 0.15 μM (Nek2)] and 4-((6-ethynyl-7 H -purin-2-yl)amino)benzenesulfonamide (IC 50 0.14 μM) were selected for determination of the mode of inhibition of Nek2, which was shown to be time-dependent, not reversed by addition of ATP and negated by site directed mutagenesis of Cys22 to alanine. Replacement of the ethynyl group by ethyl or cyano abrogated activity. Variation of substituents on the N -phenyl moiety for 6-ethynylpurines gave further SAR data for Nek2 inhibition. The data showed little correlation of activity with the nature of the substituent, indicating that after sufficient initial competitive binding to Nek2 subsequent covalent modification of Cys22 occurs in all cases. A typical activity profile was that for 2-(3-((6-ethynyl-9 H -purin-2-yl)amino)phenyl)acetamide [IC 50 0.06 μM (Nek2); GI 50 (SKBR3) 2.2 μM] which exhibited >5-10-fold selectivity for Nek2 over other kinases; it also showed > 50% growth inhibition at 10 μM concentration against selected breast and leukaemia cell lines. X-ray crystallographic analysis confirmed that binding of the compound to the Nek2 ATP-binding site resulted in covalent modification of Cys22. Further studies confirmed that 2-(3-((6-ethynyl-9 H -purin-2-yl)amino)phenyl)acetamide has the attributes of a drug-like compound with good aqueous solubility, no inhibition of hERG at 25 μM and a good stability profile in human liver microsomes. It is concluded that 6-ethynylpurines are promising agents for cancer treatment by virtue of their selective inhibition of Nek2., (This journal is © The Royal Society of Chemistry 2020.)

Extracellular regulated kinase 5 (ERK5) signalling has been implicated in driving a number of cellular phenotypes including endothelial cell angiogenesis and tumour cell motility. Novel ERK5 inhibitors were identified using high throughput screening, with a series of pyrrole-2-carboxamides substituted at the 4-position with an aroyl group being found to exhibit IC 50 values in the micromolar range, but having no selectivity against p38α MAP kinase. Truncation of the N-substituent marginally enhanced potency (∼3-fold) against ERK5, but importantly attenuated inhibition of p38α. Systematic variation of the substituents on the aroyl group led to the selective inhibitor 4-(2-bromo-6-fluorobenzoyl)-N-(pyridin-3-yl)-1H-pyrrole-2-carboxamide (IC 50 0.82 μM for ERK5; IC 50  > 120 μM for p38α). The crystal structure (PDB 5O7I) of this compound in complex with ERK5 has been solved. This compound was orally bioavailable and inhibited bFGF-driven Matrigel plug angiogenesis and tumour xenograft growth. The selective ERK5 inhibitor described herein provides a lead for further development into a tool compound for more extensive studies seeking to examine the role of ERK5 signalling in cancer and other diseases., (Copyright © 2019 Elsevier Masson SAS. All rights reserved.)

Identifying ligand binding sites on proteins is a critical step in target-based drug discovery. Current approaches to this require resource-intensive screening of large libraries of lead-like or fragment molecules. Here, we describe an efficient and effective experimental approach to mapping interaction sites using a set of halogenated compounds expressing paired hydrogen-bonding motifs, termed FragLites. The FragLites identify productive drug-like interactions, which are identified sensitively and unambiguously by X-ray crystallography, exploiting the anomalous scattering of the halogen substituent. This mapping of protein interaction surfaces provides an assessment of druggability and can identify efficient start points for the de novo design of hit molecules incorporating the interacting motifs. The approach is illustrated by mapping cyclin-dependent kinase 2, which successfully identifies orthosteric and allosteric sites. The hits were rapidly elaborated to develop efficient lead-like molecules. Hence, the approach provides a new method of identifying ligand sites, assessing tractability and discovering new leads.

Inhibition of CDKs is an attractive approach to cancer therapy due to their vital role in cell growth and transcription. Pan-CDK inhibitors have shown some clinical benefit, and trials are ongoing. Selective CDK4 and CDK6 inhibitors have been licensed for the treatment of hormone responsive, RB-positive breast cancer in combination with antihormonal agents. Selective inhibitors of CDKs 5, 7, 8, 9 and 12 have been identified across a range of chemotypes.

ATAD2 is an ATPase that is overexpressed in a variety of cancers and associated with a poor patient prognosis. This protein has been suggested to function as a cofactor for a range of transcription factors, including the proto-oncogene MYC and the androgen receptor. ATAD2 comprises an ATPase domain, implicated in chromatin remodelling, and a bromodomain which allows it to interact with acetylated histone tails. Dissection of the functional roles of these two domains would benefit from the availability of selective, cell-permeable pharmacological probes. An in silico evaluation of the 3D structures of various bromodomains suggested that developing small molecule ligands for the bromodomain of ATAD2 is likely to be challenging, although recent reports have shown that ATAD2 bromodomain ligands can be identified. We report a structure-guided fragment-based approach to identify lead compounds for ATAD2 bromodomain inhibitor development. Our findings indicate that the ATAD2 bromodomain can accommodate fragment hits (M r < 200) that yield productive structure-activity relationships, and structure-guided design enabled the introduction of selectivity over BRD4.

Background: The emergence of novel psychoactive substances (NPS), particularly synthetic cannabinoid receptor agonists (SCRA), has involved hundreds of potentially harmful chemicals in a highly dynamic international market challenging users', clinicians', and regulators' understanding of what circulating substances are causing harm. We describe a toxicovigilance system for NPS that predicted the UK emergence and identified the clinical toxicity caused by novel indole and indazole carboxylate SCRA., Methods: To assist early accurate identification, we synthesized 5 examples of commercially unavailable indole and indazole carboxylate SCRA (FUB-NPB-22, 5F-NPB-22, 5F-SDB-005, FUB-PB-22, NM-2201). We analyzed plasma and urine samples from 160 patients presenting to emergency departments with severe toxicity after suspected NPS use during 2015 to 2016 for these and other NPS using data-independent LC-MS/MS., Results: We successfully synthesized 5 carboxylate SCRAs using established synthetic and analytical chemistry methodologies. We identified at least 1 SCRA in samples from 49 patients, including an indole or indazole carboxylate SCRA in 17 (35%), specifically 5F-PB-22 (14%), FUB PB-22 (6%), BB-22 (2%), 5F NPB-22 (20%), FUB NPB-22 (2%), and 5F-SDB-005 (4%). In these 17 patients, there was analytical evidence of other substances in 16. Clinical features included agitation and aggression (82%), reduced consciousness (76%), acidosis (47%), hallucinations and paranoid features (41%), tachycardia (35%), hypertension (29%), raised creatine kinase (24%), and seizures (12%)., Conclusions: This toxicovigilance system predicted the emergence of misuse of indole and indazole carboxylate SCRA, documented associated clinical harms, and notified relevant agencies. Toxicity appears consistent with other SCRA, including mental state disturbances and reduced consciousness., (© 2017 American Association for Clinical Chemistry.)

Nek2 (NIMA-related kinase 2) is a cell cycle-dependent serine/threonine protein kinase that regulates centrosome separation at the onset of mitosis. Overexpression of Nek2 is common in human cancers and suppression can restrict tumor cell growth and promote apoptosis. Nek2 inhibition with small molecules, therefore, offers the prospect of a new therapy for cancer. To achieve this goal, a better understanding of the requirements for selective-inhibition of Nek2 is required. 6-Alkoxypurines were identified as ATP-competitive inhibitors of Nek2 and CDK2. Comparison with CDK2-inhibitor structures indicated that judicious modification of the 6-alkoxy and 2-arylamino substituents could achieve discrimination between Nek2 and CDK2. In this study, a library of 6-cyclohexylmethoxy-2-arylaminopurines bearing carboxamide, sulfonamide and urea substituents on the 2-arylamino ring was synthesized. Few of these compounds were selective for Nek2 over CDK2, with the best result being obtained for 3-((6-(cyclohexylmethoxy)-9H-purin-2-yl)amino)-N,N-dimethylbenzamide (CDK2 IC50 = 7.0 μM; Nek2 IC50 = 0.62 μM) with >10-fold selectivity. Deletion of the 6-substituent abrogated activity against both Nek2 and CDK2. Nine compounds containing an (E)-dialkylaminovinyl substituent at C-6, all showed selectivity for Nek2, e.g. (E)-6-(2-(azepan-1-yl)vinyl)-N-phenyl-9H-purin-2-amine (CDK2 IC50 = 2.70 μM; Nek2 IC50 = 0.27 μM). Structural biology of selected compounds enabled a partial rationalization of the observed structure activity relationships and mechanism of Nek2 activation. This showed that carboxamide 11 is the first reported inhibitor of Nek2 in the DFG-in conformation.

Purines and related heterocycles substituted at C-2 with 4'-sulfamoylanilino and at C-6 with a variety of groups have been synthesized with the aim of achieving selectivity of binding to CDK2 over CDK1. 6-Substituents that favor competitive inhibition at the ATP binding site of CDK2 were identified and typically exhibited 10-80-fold greater inhibition of CDK2 compared to CDK1. Most impressive was 4-((6-([1,1'-biphenyl]-3-yl)-9H-purin-2-yl)amino) benzenesulfonamide (73) that exhibited high potency toward CDK2 (IC 50 0.044 μM) but was ∼2000-fold less active toward CDK1 (IC 50 86 μM). This compound is therefore a useful tool for studies of cell cycle regulation. Crystal structures of inhibitor-kinase complexes showed that the inhibitor stabilizes a glycine-rich loop conformation that shapes the ATP ribose binding pocket and that is preferred in CDK2 but has not been observed in CDK1. This aspect of the active site may be exploited for the design of inhibitors that distinguish between CDK1 and CDK2.

Background: The phosphatidylinositol-3-kinase/mammalian target of rapamycin (PI3K/mTOR) pathway is commonly deregulated in human cancer, hence many PI3K and mTOR inhibitors have been developed and have now reached clinical trials. Similarly, CDKs have been investigated as cancer drug targets., Methods: We have synthesised and characterised a series of 6-aminopyrimidines identified from a kinase screen that inhibit PI3K and/or mTOR and/or CDK2. Kinase inhibition, tumour cell growth, cell cycle distribution, cytotoxicity and signalling experiments were undertaken in HCT116 and HT29 colorectal cancer cell lines, and in vivo HT29 efficacy studies., Results: 2,6-Diaminopyrimidines with an O(4)-cyclohexylmethyl substituent and a C-5-nitroso or cyano group (1,2,5) induced cell cycle phase alterations and were growth inhibitory (GI50<20 μM). Compound 1, but not 2 or 5, potently inhibits CDK2 (IC50=0.1 nM) as well as PI3K, and was cytotoxic at growth inhibitory concentrations. Consistent with kinase inhibition data, compound 1 reduced phospho-Rb and phospho-rS6 at GI50 concentrations. Combination of NU6102 (CDK2 inhibitor) and pictilisib (GDC-0941; pan-PI3K inhibitor) resulted in synergistic growth inhibition, and enhanced cytotoxicity in HT29 cells in vitro and HT29 tumour growth inhibition in vivo., Conclusions: These studies identified a novel series of mixed CDK2/PI3K inhibitors and demonstrate that dual targeting of CDK2 and PI3K can result in enhanced antitumour activity.

The extracellular-related kinase 5 (ERK5) is a promising target for cancer therapy. A high-throughput screen was developed for ERK5, based on the IMAP FP progressive binding system, and used to identify hits from a library of 57 617 compounds. Four distinct chemical series were evident within the screening hits. Resynthesis and reassay of the hits demonstrated that one series did not return active compounds, whereas three series returned active hits. Structure-activity studies demonstrated that the 4-benzoylpyrrole-2-carboxamide pharmacophore had excellent potential for further development. The minimum kinase binding pharmacophore was identified, and key examples demonstrated good selectivity for ERK5 over p38α kinase.

Non-genotoxic reactivation of the p53 pathway by MDM2-p53 binding antagonists is an attractive treatment strategy for wild-type TP53 cancers. To determine how resistance to MDM2/p53 binding antagonists might develop, SJSA-1 and NGP cells were exposed to growth inhibitory concentrations of chemically distinct MDM2 inhibitors, Nutlin-3 and MI-63, and clonal resistant cell lines generated. The p53 mediated responses of parental and resistant cell lines were compared. In contrast to the parental cell lines, p53 activation by Nutlin-3, MI-63 or ionizing radiation was not observed in either the SJSA-1 or the NGP derived cell lines. An identical TP53 mutation was subsequently identified in both of the SJSA-1 resistant lines, whilst one out of three identified mutations was common to both NGP derived lines. Mutation specific PCR revealed these mutations were present in parental SJSA-1 and NGP cell populations at a low frequency. Despite cross-resistance to a broad panel of MDM2/p53 binding antagonists, these MDM2-amplified and TP53 mutant cell lines remained sensitive to ionizing radiation (IR). These results indicate that MDM2/p53 binding antagonists will select for p53 mutations present in tumours at a low frequency at diagnosis, leading to resistance, but such tumours may nevertheless remain responsive to alternative therapies, including IR., Competing Interests: The authors declare no potential conflicts of interest.

Treatment of (ortho-trimethysilyl)aryl phenylsulfonates with a soluble fluoride source initiates a Truce-Smiles rearrangement leading to the formation of functionalized bi-aryls. This new carbon-carbon bond-forming reaction proceeds without recourse to transition metal catalysis, under mild reaction conditions and with good functional group compatibility.

Two libraries of substituted benzimidazoles were designed using a 'scaffold-hopping' approach based on reported MDM2-p53 inhibitors. Substituents were chosen following library enumeration and docking into an MDM2 X-ray structure. Benzimidazole libraries were prepared using an efficient solution-phase approach and screened for inhibition of the MDM2-p53 and MDMX-p53 protein-protein interactions. Key examples showed inhibitory activity against both targets., (© 2014 John Wiley & Sons A/S.)

Potent and selective small-molecule inhibitors of the p53-MDM2 interaction intended for the treatment of p53 wild-type tumors have been designed and optimized in a number of chemical series. This review details recent disclosures of compounds in advanced optimization and features key series that have given rise to clinical trial candidates. The structure-activity relationships for inhibitor classes are discussed with reference to x-ray structures, and common structural features are identified.

Small-molecule drug discovery requires reliable synthetic methods for attaching amino compounds to heterocyclic scaffolds. Trifluoroacetic acid-2,2,2-trifluoroethanol (TFA-TFE) is as an effective combination for achieving SN Ar reactions between anilines and heterocycles (e.g., purines and pyrimidines) substituted with a leaving group (fluoro-, chloro-, bromo- or alkylsulfonyl). This method provides a variety of compounds containing a "kinase-privileged fragment" associated with potent inhibition of kinases. TFE is an advantageous solvent because of its low nucleophilicity, ease of removal and ability to solubilise polar substrates. Furthermore, TFE may assist the breakdown of the Meisenheimer-Jackson intermediate by solvating the leaving group. TFA is a necessary and effective acidic catalyst, which activates the heterocycle by N-protonation without deactivating the aniline by conversion into an anilinium species. The TFA-TFE methodology is compatible with a variety of functional groups and complements organometallic alternatives, which are often disadvantageous because of the expense of reagents, the frequent need to explore diverse sets of reaction conditions, and problems with product purification. In contrast, product isolation from TFA-TFE reactions is straightforward: evaporation of the reaction mixture, basification and chromatography affords analytically pure material. A total of 45 examples are described with seven discrete heterocyclic scaffolds and 2-, 3- and 4-substituted anilines giving product yields that are normally in the range 50-90 %. Reactions can be performed with either conventional heating or microwave irradiation, with the latter often giving improved yields., (Copyright © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Evaluation of the effects of purine C-8 substitution within a series of CDK1/2-selective O(6)-cyclohexylmethylguanine derivatives revealed that potency decreases initially with increasing size of the alkyl substituent. Structural analysis showed that C-8 substitution is poorly tolerated, and to avoid unacceptable steric interactions, these compounds adopt novel binding modes. Thus, 2-amino-6-cyclohexylmethoxy-8-isopropyl-9H-purine adopts a "reverse" binding mode where the purine backbone has flipped 180°. This provided a novel lead chemotype from which we have designed more potent CDK2 inhibitors using, in the first instance, quantum mechanical energy calculations. Introduction of an ortho-tolyl or ortho-chlorophenyl group at the purine C-8 position restored the potency of these "reverse" binding mode inhibitors to that of the parent 2-amino-6-cyclohexylmethoxy-9H-purine. By contrast, the corresponding 8-(2-methyl-3-sulfamoylphenyl)-purine derivative exhibited submicromolar CDK2-inhibitory activity by virtue of engineered additional interactions with Asp86 and Lys89 in the reversed binding mode, as confirmed by X-ray crystallography.

Recent studies have shown that irreversible inhibition of Nek2 kinase [(Never in mitosis gene a)-related kinase 2], overexpression of which is observed in several cancers, can be achieved using Michael acceptors containing an ethynyl group, which target the enzyme's cysteine 22 residue lying near the catalytic site. The model studies described herein demonstrate an analogous capture of the ethynyl moiety in a series of ethynyl-heterocycles (e.g. 6-ethynyl-N-phenyl-9H-purin-2-amine) by N-acetylcysteine methyl ester in the presence of 1,4-diazabicyclo[2.2.2]octane in either dimethyl sulfoxide or N,N-dimethylformamide. Kinetic studies showed a 50-fold range in reactivity with 7-ethynyl-N-phenyl-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-amine being the most reactive compound, whereas 4-ethynyl-N-phenyl-7H-pyrrolo[2,3-d]pyrimidin-2-amine was the least reactive. Studies of the isomeric compounds, 2-(3-((6-ethynyl-7-methyl-7H-purin-2-yl)amino)phenyl)acetamide and 2-(3-((6-ethynyl-9-methyl-9H-purin-2-yl)amino)phenyl)acetamide, revealed the N(7)-methyl isomer to be 5-fold more reactive than the 9-methyl isomer, which is ascribed to a buttressing effect in the N(7)-methyl compound. Comparison of the crystal structures of these isomers showed that the ethynyl group is significantly displaced away from the methyl group exclusively in the N(7)-methyl isomer with an sp(2) bond angle of 124°, whereas the corresponding angle in the N(9)-methyl isomer was the expected 120°. The results of this study indicate heterocyclic scaffolds that are likely to be more promising for inhibition of Nek2 and other kinases containing a reactive cysteine.

Screening identified 2-(3-((4,6-dioxo-2-thioxotetrahydropyrimidin-5(2 H )-ylidene)methyl)-2,5-dimethyl-1 H -pyrrol-1-yl)-4,5,6,7-tetrahydrobenzo[ b ]thiophene-3-carbonitrile as an MDM2-p53 inhibitor (IC 50 = 12.3 μM). MDM2-p53 and MDMX-p53 activity was seen for 5-((1-(4-chlorophenyl)-2,5-diphenyl-1 H -pyrrol-3-yl)methylene)-2-thioxodihydropyrimidine-4,6(1 H ,5 H )-dione (MDM2 IC 50 = 0.11 μM; MDMX IC 50 = 4.2 μM) and 5-((1-(4-nitrophenyl)-2,5-diphenyl-1 H -pyrrol-3-yl)methylene)pyrimidine-2,4,6(1 H ,3 H ,5 H )-trione (MDM2 IC 50 = 0.15 μM; MDMX IC 50 = 4.2 μM), and cellular activity consistent with p53 activation in MDM2 amplified cells. Further SAR studies demonstrated the requirement for the triarylpyrrole moiety for MDMX-p53 activity but not for MDM2-p53 inhibition.

Analogues of (dibenzo[b,d]thiophen-4-yl)-2-morpholino-4H-chromen-4-one (NU7441), a potent inhibitor of DNA-dependent protein kinase (DNA-PK; IC50 = 42 ± 2 nM), have been synthesized in which water-solubilizing groups [NHCO(CH₂)nNR¹R², where n = 1 or 2 and the moiety R¹R²N was derived from a library of primary and secondary amines, e.g., morpholine] were placed at the 1-position. Several of the newly synthesized compounds exhibited high potency against DNA-PK and potentiated the cytotoxicity of ionizing radiation (IR) in vitro 10-fold or more (e.g., 2-(4-ethylpiperazin-1-yl)-N-(4-(2-morpholino-4-oxo-4H-chromen-8-yl)dibenzo[b,d]thio-phen-1-yl)acetamide, 39; DNA-PK IC₅₀ = 5.0 ± 1 nM, IR dose modification ratio = 13). Furthermore, 39 was shown to potentiate not only IR in vitro but also DNA-inducing cytotoxic anticancer agents, both in vitro and in vivo. Counter-screening against other members of the phosphatidylinositol 3-kinase (PI-3K) related kinase (PIKK) family unexpectedly revealed that some of the compounds were potent mixed DNA-PK and PI-3K inhibitors.

Purines protected at N-9 by p-methoxybenzyl are methylated or ethylated in 2,2,2-trifluoroethanol at N-7 by trimethyl- or triethyl-oxonium borofluorate, respectively. Subjecting the resulting cationic species to microwave irradiation releases an N(7)-methyl- or ethyl-purine. This one-pot procedure is an efficient regiospecific method applicable to diverse substrates.

Substitution at the 7-position of the chromen-4-one pharmacophore of 8-(dibenzo[b,d]thiophen-4-yl)-2-morpholino-4H-chromen-4-one NU7441, a potent and selective DNA-dependent protein kinase (DNA-PK) inhibitor, with allyl, n-propyl or methyl enabled the resolution by chiral HPLC of atropisomers. Biological evaluation against DNA-PK of each pair of atropisomers showed a marked difference in potency, with biological activity residing exclusively in the laevorotatory enantiomer.

Tip60 (KAT5) is a histone acetyltransferase (HAT enzyme) involved in multiple cellular processes including transcriptional regulation, DNA damage repair and cell signalling. In prostate cancer, aggressive cases over-express Tip60 which functions as an androgen receptor co-activator via direct acetylation of lysine residues within the KLKK motif of the receptor hinge region. The purpose of this study was to identify and characterise a Tip60 acetylase inhibitor. High-throughput screening revealed an isothiazole that inhibited both Tip60 and p300 HAT activity. This substance (initially identified as 4-methyl-5-bromoisothiazole) and other isothiazoles were synthesised and assayed against Tip60. Although an authentic sample of 4-methyl-5-bromoisothiazole was inactive against Tip60, in an in vitro HAT assay, 1,2-bis(isothiazol-5-yl)disulfane (NU9056) was identified as a relatively potent inhibitor (IC(50) 2 µM). Cellular activity was confirmed by analysis of acetylation of histone and non-histone proteins in a prostate cancer cell line model. NU9056 treatment inhibited cellular proliferation in a panel of prostate cancer cell lines (50% growth inhibition, 8-27 µM) and induced apoptosis via activation of caspase 3 and caspase 9 in a concentration- and time-dependent manner. Also, decreased androgen receptor, prostate specific antigen, p53 and p21 protein levels were demonstrated in response to treatment with NU9056. Furthermore, pre-treatment with NU9056 inhibited both ATM phosphorylation and Tip60 stabilization in response to ionising radiation. Based on the activity of NU9056 and the specificity of the compound towards Tip60 relative to other HAT enzymes, these chemical biology studies have identified Tip60 as a potential therapeutic target for the treatment of prostate cancer.

Structure-activity relationships for the MDM2-p53 inhibitory activity of a series of A-ring substituted 2-N-benzyl-3-(4-chlorophenyl)-3-(1-(hydroxymethyl)cyclopropyl)methoxy)isoindolinones have been investigated, giving rise to compounds with improved potency over their unsubstituted counterparts. Isoindolinone A-ring substitution with a 4-chloro group for the 4-nitrobenzyl, 4-bromobenzyl and 4-cyanobenzyl derivatives (10a-c) and substitution with a 6-tert-butyl group for the 4-nitrobenzyl derivative (10j) were found to confer additional potency. Resolution of the enantiomers of 10a showed that potent MDM2-p53 activity resided in the (-)-enantiomer ((-)-10a; IC(50)=44 ± 6 nM). The cellular activity of key compounds has been examined in cell lines with defined p53 and MDM2 status. Compounds 10a and (-)-10a increase p53 protein levels, activate p53-dependent MDM2 and p21 transcription in MDM2 amplified cells, and show improved selectivity for growth inhibition in wild type p53 cell lines over the parent compound., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Amino-substituted biphenyls were obtained by Suzuki cross-coupling of 2,6-dibromoaniline with a phenylboronic acid (substituted with Me, NO(2), OH, OMe or Cl) preferably assisted by microwave irradiation. Conversion of the amino group into a thiol preceded a base-induced intramolecular substitution, also facilitated by microwave heating, to generate the second C-S bond of the target dibenzothiophene. The 1-, 2-, 3- or 4-substituted 6-halodibenzothiophenes obtained were subjected to a palladium-mediated coupling with 2-morpholin-4-yl-8-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-4H-chromen-4-one to give the respective 6-, 7-, 8- or 9-substituted dibenzothiophen-4-ylchromenones. These compounds were evaluated as inhibitors of DNA-dependent protein kinase (DNA-PK) and compared to the parent 8-(dibenzo[b,d]thiophen-4-yl)-2-morpholin-4-yl-4H-chromen-4-one. Notably, derivatives bearing hydroxy or methoxy substituents at C-8 or C-9 retained activity, whereas substitution at C-7 lowered activity. Substitution with chloro at C-6 was not detrimental to activity, but a chloro group at C-7 or C-8 reduced potency. The data indicate permissive elaboration of hydroxyl at C-8 or C-9, enabling the possibility of improved pharmaceutical properties, whilst retaining potency against DNA-PK.

To facilitate the evaluation of CDK2 (cyclin-dependent kinase 2) as a cancer target, the in vitro and in vivo properties of NU6102 (O⁶-cyclohexylmethyl-2-(4'-sulphamoylanilino)purine) and a water soluble prodrug (NU6301) were investigated. NU6102 selectively inhibited the growth of CDK2 WT (wild type) versus KO MEFs (knockout mouse embryo fibroblasts) (GI₅₀ (concentration required to inhibit cell growth by 50%) 14 μM versus >30 μM), and was more growth-inhibitory in p53 mutant or null versus p53 WT cells (p=0.02), and in Rb (retinoblastoma protein) WT SKUT-1B versus SKUT 1 Rb deficient cells (p=0.01). In SKUT-1B cells NU6102 induced a G2 arrest, inhibition of Rb phosphorylation and cytotoxicity (LC₅₀ 2.6 μM for a 24h exposure). The prodrug NU6301 rapidly generated NU6102 in vitro in mouse plasma, and tumour NU6102 levels in vivo consistent with activity in vitro. Eight or 12 hourly dosing of 120 mg/kg NU6301 for 10 days was well tolerated in SKUT-1B tumour-bearing mice and inhibited Rb phosphorylation in tumour tissue. Two (8 hourly dosing) and 3 (12 hourly dosing) day tumour growth delay was observed (p=0.04 and p=0.007, respectively) following NU6301 administration. NU6102 and its prodrug NU6301 have pharmacological properties consistent with CDK2 inhibition, and represent useful tool molecules for the evaluation of CDK2 as a target in cancer., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The interaction between murine double minute (MDM2) and p53 is a major target in anticancer drug design. Several potent compound series, including the nutlins and spirooxindoles, have previously been established as high-affinity antagonists of MDM2. In this paper, we describe the interaction of isoindolinone inhibitors with MDM2, as characterized by nuclear magnetic resonance spectroscopy. Isoindolinone inhibitors bind specifically to the MDM2 p53 binding site and exploit all sub-pockets used by p53, nutlins and spirooxindoles. Furthermore, isoindolinones bind with low micromolar to high nanomolar affinities, with the best compound approaching the potency of nutlin-3., (© 2011 John Wiley & Sons A/S.)

Inhibition of the MDM2-p53 interaction has been shown to produce an antitumor effect, especially in MDM2 amplified tumors. The isoindolinone scaffold has proved to be versatile for the discovery of MDM2-p53 antagonists. Optimization of previously reported inhibitors, for example, NU8231 (7) and NU8165 (49), was guided by MDM2 NMR titrations, which indicated key areas of the binding interaction to be explored. Variation of the 2-N-benzyl and 3-alkoxy substituents resulted in the identification of 3-(4-chlorophenyl)-3-((1-(hydroxymethyl)cyclopropyl)methoxy)-2-(4-nitrobenzyl)isoindolin-1-one (74) as a potent MDM2-p53 inhibitor (IC(50) = 0.23 ± 0.01 μM). Resolution of the enantiomers of 74 showed that potent MDM2-p53 activity primarily resided with the (+)-R-enantiomer (74a; IC(50) = 0.17 ± 0.02 μM). The cellular activity of key compounds has been examined in cell lines with defined p53 and MDM2 status. Compound 74a activates p53, MDM2, and p21 transcription in MDM2 amplified cells and shows moderate selectivity for wild-type p53 cell lines in growth inhibition assays.

Introduction of an O-alkoxyphenyl substituent at the 8-position of the 2-morpholino-4H-chromen-4-one pharmacophore enabled regions of the ATP-binding site of DNA-dependent protein kinase (DNA-PK) to be probed further. Structure-activity relationships have been elucidated for inhibition of DNA-PK and PI3K (p110α), with N-(2-(cyclopropylmethoxy)-4-(2-morpholino-4-oxo-4H-chromen-8-yl)phenyl)-2-morpholinoacetamide 11a being identified as a potent and selective DNA-PK inhibitor (IC(50)=8 nM)., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Following the discovery of dibenzo[b,d]thiophen-4-yl)-2-morpholino-4H-chromen-4-one (NU7441) ( Leahy , J. J. J. ; Golding , B. T. ; Griffin , R. J. ; Hardcastle , I. R. ; Richardson , C. ; Rigoreau , L. ; Smith , G. C. M. Bioorg. Med. Chem. Lett. 2004 , 14 , 6083 - 6087) as a potent inhibitor (IC₅₀ = 30 nM) of DNA-dependent protein kinase (DNA-PK), we have investigated analogues in which the chromen-4-one core template has been replaced by aza-heterocyclic systems: 9-substituted 2-morpholin-4-ylpyrido[1,2-a]pyrimidin-4-ones and 8-substituted 2-morpholin-4-yl-1H-quinolin-4-ones. The 8- and 9-substituents were either dibenzothiophen-4-yl or dibenzofuran-4-yl, which were each further substituted at the 1-position with water-solubilizing groups [NHCO(CH₂)(n)NR¹R², where n = 1 or 2 and the moiety R¹R²N was derived from a library of primary and secondary amines (e.g., morpholine)]. The inhibitors were synthesized by employing a multiple-parallel approach in which the two heterocyclic components were assembled by Suzuki-Miyaura cross-coupling. Potent DNA-PK inhibitory activity was generally observed across the compound series, with structure-activity studies indicating that optimal potency resided in pyridopyrimidin-4-ones bearing a substituted dibenzothiophen-4-yl group. Several of the newly synthesized compounds (e.g., 2-morpholin-4-yl-N-[4-(2-morpholin-4-yl-4-oxo-4H-pyrido[1,2-a]pyrimidin-9-yl)dibenzothiophen-1-yl]acetamide) combined high potency against the target enzyme (DNA-PK IC₅₀ = 8 nM) with promising activity as potentiators of ionizing radiation-induced cytotoxicity in vitro.

Replacement of the core heterocycle of a defined series of chromen-4-one DNA-PK inhibitors by the isomeric chromen-2-one (coumarin) and isochromen-1-one (isocoumarin) scaffolds was investigated. Structure-activity relationships for DNA-PK inhibition were broadly consistent, albeit with a reduction of potency compared with the parent chromenone., (Copyright 2010 Elsevier Ltd. All rights reserved.)

CDK2 inhibitory structure-activity relationships have been explored for a range of 5-substituted O(4)-alkylpyrimidines. Variation of the 5-substituent in the 2,6-diaminopyrimidine series confirmed the 5-nitroso substituent as optimal, and showed that 5-formyl and 5-acetyl substituents were also tolerated at this position. A series of O(4)-alkyl-N(2)-aryl-5-substituted-6-aminopyrimidines revealed interesting structure-activity relationships. In the 5-nitroso series, the optimum O(4)-alkyl substituents were cyclohexylmethyl or sec-butyl, combined with a 2-sulfanilyl group. By contrast, in the N(2)-arylsulfonamido-5-formyl series, the cyclohexylmethyl compound showed relatively poor activity compared with the sec-butyl derivative (22j, (R)-4-(4-amino-6-sec-butoxy-5-formylpyrimidin-2-ylamino)benzenesulfonamide; CDK2 IC(50) = 0.8 nM). Similarly, in the N(2)-arylsulfonamido-5-(hydroxyiminomethyl) series the O(4)-sec-butyl substituent conferred greater potency than the cyclohexylmethyl (23c, (rac)-4-(4-amino-6-sec-butoxy-5-(hydroxyiminomethyl)pyrimidin-2-ylamino)benzenesulfonamide; CDK2 IC(50) = 7.4 nM). The 5-formyl derivatives show selectivity for CDK2 over other CDK family members, and are growth inhibitory in tumour cells (e.g. 22j, GI(50) = 0.57 microM).

The attenuated S(N)2 reactivity of the 2,2,2-trifluoroethyl group has been exploited for the synthesis of a series of 6-cyclohexylmethoxy-2-arylaminopurines in which a sulfonamide moiety was attached to the aryl ring via a methylene group. These were required as potential inhibitors of serine-threonine kinases of interest for the treatment of cancer. 3-Nitrophenylmethanesulfonyl chloride was converted into the corresponding 2,2,2-trifluoroethoxysulfonyl ester by reaction with 2,2,2-trifluoroethanol in the presence of triethylamine/4-dimethylaminopyridine. Catalytic hydrogenation of the nitro group employing 2,2,2-trifluoroethanol as solvent gave 2,2,2-trifluoroethyl 3-aminophenylmethanesulfonate, which was reacted with 6-cyclohexylmethoxy-2-fluoropurine in 2,2,2-trifluoroethanol/trifluoroacetic acid to afford 2,2,2-trifluoroethyl 3-(6-cyclohexylmethoxy-9H-purin-2-ylamino)phenylmethanesulfonate. 3-(6-Cyclohexylmethoxy-9H-purin-2-ylamino)phenylmethanesulfonamides were synthesised by microwave heating of the trifluoroethoxysulfonate with an amine and 1,8-diazabicycloundec-7-ene in tetrahydrofuran. The mechanism of this process was shown to involve an intermediate sulfene by a deuterium-labelling experiment. 3-(6-Cyclohexylmethoxy-9H-purin-2-ylamino)phenylmethanesulfonamide derivatives were assayed as inhibitors of human cyclin-dependent kinase 2. Previous structure-activity studies demonstrated that relocating the sulfonamide group of O(6)-cyclohexylmethoxy-2-(4'-sulfamoylanilino)purine from the 4- to the 3-position on the 2-arylamino ring resulted in a 40-fold reduction in potency against CDK2. In the present study, no further loss of activity was observed on introducing a methylene group between the sulfonamide and the aryl ring, 3-(6-cyclohexylmethoxy-9H-purin-2-ylamino)phenylmethanesulfonamide proving equipotent with O(6)-cyclohexylmethoxy-2-(3'-sulfamoylanilino)purine (IC(50) = 0.21 microM). N-Alkylation of the sulfonamide reduced CDK-2 inhibitory activity, while a substituted benzyl or 3-phenylpropyl group on the sulfonamide resulted in a loss of potency compared with 3-(6-cyclohexylmethoxy-9H-purin-2-ylamino)phenylmethanesulfonamide. The dimethylaminopropyl derivative, 1-[3-(6-cyclohexylmethoxy-9H-purin-2-ylamino)phenyl]-N-(3-dimethylaminopropyl)methanesulfonamide was only 2-fold less potent than 3-(6-cyclohexylmethoxy-9H-purin-2-ylamino)phenylmethanesulfonamide, suggesting an interaction between the basic dimethylamino group and the kinase. The presence of alicyclic groups on the pendant sulfonamide showed IC(50) values in the 0.5-1.5 microM range. N-(4-tert-Butylphenyl)-1-[3-(6-cyclohexylmethoxy-9H-purin-2-ylamino)phenyl]methanesulfonamide was markedly less active (IC(50) = 34 microM), suggesting a steric effect within the ATP-binding domain.

Substitution at the 3-position of the dibenzothiophen-4-yl ring of 8-(dibenzo[b,d]thiophen-4-yl)-2-morpholino-4H-chromen-4-one NU7441, a potent and selective DNA-dependent protein kinase (DNA-PK) inhibitor, with propyl, allyl or methyl enabled the separation by chiral HPLC of atropisomers. This is a consequence of restricted rotation about the dibenzothiophene-chromenone bond. Biological evaluation against DNA-PK of the pairs of atropisomers showed a marked difference in potency, with only one enantiomer being biologically active.

In this study we present a method for defining the binding modes of a set of structurally related isoindolinone inhibitors of the MDM2-p53 interaction. This approach derives the location and orientation of isoindolinone binding, based on an analysis of the patterns of magnitude and direction of chemical shift perturbations for a series of inhibitors of the MDM2-p53 interaction. The MDM2-p53 complex is an attractive target for therapeutic intervention in cancer cells with intact tumor suppressor p53, as it offers the possibility of releasing p53 by blocking the MDM2-p53 binding site with a small molecule antagonist to promote apoptosis. Isoindolinones are a novel class of MDM2-antagonists of moderate affinity, which still require the development of more potent candidates for clinical applications. As the applicability of conventional structural methods to this system is limited by a number of fundamental factors, the exploitation of the information contained in chemical shift perturbations has offered a useful route to obtaining structural information to guide the development of more potent compounds. For a set of 12 structurally related isoindolinones, the data suggests 4 different orientations of binding, caused by subtle changes in the chemical structure of the inhibitors.

The synthesis and biological evaluation of libraries of 8-biarylchromen-4-ones enabled the elucidation of structure-activity relationships for inhibition of the DNA-dependent protein kinase (DNA-PK), with 8-(3-(thiophen-2-yl)phenyl)chromen-4-one and 8-(3-(thiophen-3-yl)phenyl)chromen-4-one being especially potent inhibitors.

8-Substituted 2-morpholin-4-yl-quinolin-4-ones and 9-substituted 2-morpholin-4-yl-pyrido[1,2-a]pyrimidin-4-ones with selected aryl and heteroaryl groups as the substituent have been synthesised as potential inhibitors of DNA-dependent protein kinase. A multiple-parallel approach, employing Suzuki cross-coupling methodology, was utilised in the preparation of 8-substituted 2-morpholin-4-yl-quinolin-4-ones. For this purpose 8-bromo-2-morpholin-4-yl-quinolin-4-one was required as an intermediate. This compound was obtained by adapting a literature route in which thermal cyclocondensation of (2-bromoanilino)-morpholin-4-yl-5-methylene-2,2-dimethyl[1,3]dioxane-4,6-dione afforded 8-bromo-2-morpholin-4-yl-quinolin-4-one. A multiple-parallel approach, employing Suzuki cross-coupling methodology, was also utilised to prepare 9-substituted 2-morpholin-4-yl-pyrido[1,2-a]pyrimidin-4-ones using 9-hydroxy-2-morpholin-4-yl-pyrido[1,2-a]pyrimidin-4-one O-trifluoromethanesulfonate as an intermediate. 8-Substituted 2-morpholin-4-yl-quinolin-4-ones and 9-substituted 2-morpholin-4-yl-pyrido[1,2-a]pyrimidin-4-ones were both inhibitors of DNA-dependent protein kinase. When the substituent was dibenzothiophen-4-yl, dibenzofuran-4-yl or biphen-3-yl, IC50 values in the low nanomolar range were observed. Interestingly, the pyridopyrimidinones and quinolinones were essentially equipotent with the corresponding 8-substituted 2-morpholin-4-yl-chromen-4-ones previously reported (I. R. Hardcastle, X. Cockcroft, N. J. Curtin, M. Desage El-Murr, J. J. J. Leahy, M. Stockley, B. T. Golding, L. Rigoreau, C. Richardson, G. C. M. Smith and R. J. Griffin, J. Med. Chem., 2005, 48, 7829-7846).

An efficient synthesis of 2-substituted O(4)-cyclohexylmethyl-5-nitroso-6-aminopyrimidines from 6-amino-2-mercaptopyrimidin-4-ol has been developed and used to prepare a range of derivatives for evaluation as inhibitors of cyclin-dependent kinase 2 (CDK2). The structure-activity relationships (SARs) are similar to those observed for the corresponding O(6)-cyclohexylmethoxypurine series with the 2-arylsulfonamide and 2-arylcarboxamide derivatives showing excellent potency. Two compounds, 4-(6-amino-4-cyclohexylmethoxy-5-nitrosopyrimidin-2-ylamino)-N-(2-hydroxyethyl)benzenesulfonamide (7q) and 4-(6-amino-4-cyclohexylmethoxy-5-nitrosopyrimidin-2-ylamino)-N-(2,3-dihydroxypropyl)benzenesulfonamide (7s), were the most potent with IC50 values of 0.7 +/- 0.1 and 0.8 +/- 0.0 nM against CDK2, respectively. The SARs determined in this study are discussed with reference to the crystal structure of 4-(6-amino-4-cyclohexylmethoxy-5-nitrosopyrimidin-2-ylamino)-N-(2,3-dihydroxypropyl)benzenesulfonamide (7j) bound to phosphorylated CDK2/cyclin A.

Structure-activity relationships have been investigated for inhibition of DNA-dependent protein kinase (DNA-PK) and ATM kinase by a series of pyran-2-ones, pyran-4-ones, thiopyran-4-ones, and pyridin-4-ones. A wide range of IC50 values were observed for pyranones and thiopyranones substituted at the 6-position, with the 3- and 5-positions proving intolerant to substitution. Related pyran-2-ones, pyran-4-ones, and thiopyran-4-ones showed similar IC50 values against DNA-PK, whereas the pyridin-4-one system proved, in general, ineffective at inhibiting DNA-PK. Extended libraries exploring the 6-position of 2-morpholino-pyran-4-ones and 2-morpholino-thiopyrano-4-ones identified the first highly potent and selective ATM inhibitor 2-morpholin-4-yl-6-thianthren-1-yl-pyran-4-one (151C; ATM; IC50=13 nM) and revealed constrained SARs for ATM inhibition compared with DNA-PK. One of the most potent DNA-PK inhibitors identified, 2-(4-methoxyphenyl)-6-(morpholin-4-yl)pyran-4-one (16; DNA-PK; IC50=220 nM) effectively sensitized HeLa cells to the topoisomerase II inhibitor etoposide in vitro.

The safety-catch principle has been applied for the development of a new method for protecting sulfonic acids. 2,2-Dimethylsuccinic acid was reduced to 2,2-dimethylbutane-1,4-diol, which was selectively silylated to give 4-(tert-butyldiphenylsilanyloxy)-2,2-dimethylbutan-1-ol. Reaction of the latter compound with 2-chloroethanesulfonyl chloride in the presence of triethylamine afforded 4-(tert-butyldiphenylsilyloxy)-2,2-dimethylbutyl ethenesulfonate directly. The ethenesulfonate underwent Michael-type addition with secondary amines to give protected derivatives of taurine (2-aminoethanesulfonic acid). Deprotection was achieved on treatment with tetrabutylammonium fluoride, whereby cleavage of the silicon-oxygen bond led to an intermediate alkoxide that immediately cyclised to 2,2-dimethyltetrahydrofuran with liberation of a sulfonate. Pure sulfonic acids were obtained from the crude product by ion exchange chromatography on a strongly basic resin, which was eluted with aqueous acetic acid. The method developed should be generally applicable to the protection of sulfonic acids and is amenable to a multiparallel format.

[Structure: see text] 2-morpholin-4-yl-4-oxo-4H-chromen-8-yl 2,2,2-trifluoromethanesulfonate is a key intermediate for the synthesis of the DNA-dependent protein kinase (DNA-PK) inhibitor 8-dibenzothiophen-4-yl-2-morpholin-4-yl-chromen-4-one (NU7441). Two improved methods for the synthesis of this triflate have been developed: (A) in 35% overall yield, through modification of the published route, and (B) in 15% overall yield, by a new route employing a Baker-Venkataraman rearrangement to enable generation of the chromenone scaffold. Both syntheses depend on the judicious use of allyl protecting groups.

From a set of weakly potent lead compounds, using in silico screening and small library synthesis, a series of 2-alkyl-3-aryl-3-alkoxyisoindolinones has been identified as inhibitors of the MDM2-p53 interaction. Two of the most potent compounds, 2-benzyl-3-(4-chlorophenyl)-3-(3-hydroxypropoxy)-2,3-dihydroisoindol-1-one (76; IC(50) = 15.9 +/- 0.8 microM) and 3-(4-chlorophenyl)-3-(4-hydroxy-3,5-dimethoxybenzyloxy)-2-propyl-2,3-dihydroisoindol-1-one (79; IC(50) = 5.3 +/- 0.9 microM), induced p53-dependent gene transcription, in a dose-dependent manner, in the MDM2 amplified, SJSA human sarcoma cell line.

beta-Piperidinoethylsulfides are oxidized by m-chloroperbenzoic acid to intermediates containing both N-oxide and sulfone functions. These undergo a Cope-type elimination to a vinylsulfone that can be captured by amines to afford beta-aminoethylsulfones. When a beta-aminoethylsulfone group is linked to the 4-position of a phenyl group attached at N-2 of O6-cyclohexylmethylguanine, the resulting derivatives are inhibitors of the cyclin-dependent kinase CDK2. One of the most potent inhibitors (IC50 = 45 nM) contained a N-3-hydroxypropyl group on the aminoethylsulfonyl substituent. The crystal structure of this inhibitor bound to CDK2/cyclin A was determined and shows an unusual network of hydrogen bonds. The synthetic methodology developed can be utilized in multiple-parallel format and has numerous potential applications in medicinal chemistry.

Structure-activity relationships for inhibition of DNA-dependent protein kinase (DNA-PK) have been defined for substituted chromen-4-ones. For the 2-amino-substituted benzo[h]chromen-4-ones, a morpholine substituent at this position was essential for activity. Small libraries of 6- and 7-alkoxy-substituted chromen-4-ones showed that a number of 7-alkoxy-substituted chromenones displayed improved activity. Focused libraries incorporating 6-, 7-, and 8-aryl and heteroaryl substituents were prepared. In these cases, 6- and 7-substitution was disfavored, whereas 8-substitution was largely tolerated. Surprisingly, two compounds, 2-N-morpholino-8-dibenzofuranyl-chromen-4-one (NU7427, 32{38}) and the 2-N-morpholino-8-dibenzothiophenyl-chromen-4-one (NU7441, 32{26}) were excellent inhibitors (IC50 vs DNA-PK = 40 and 13 nM, respectively). The ring-saturated analogue 2-N-morpholino-8-(6',7',8',9'-tetrahydrodibenzothiophene)chromen-4-one, 36, retained potent activity (IC50 vs DNA-PK = 23 nM). The dibenzothiophene 32{38} sensitized HeLa cells to ionizing radiation in vitro, with dose modification factors of 2.5 at 10% survival being observed at 0.5 microM. The cytotoxicity of the topoisomerase II inhibitor etoposide was also potentiated.

Cyclin-dependent kinases (CDK) play a crucial role in the control of the cell cycle. Aberrations in the control of cell cycle progression occur in the majority of human malignancies; hence, CDKs are promising targets for anticancer therapy. Here, we define the cellular effects of the novel CDK inhibitor NU6140, alone or in association with paclitaxel, with respect to inhibition of cell proliferation and cell cycle progression and induction of apoptosis in HeLa cervical carcinoma cells and in comparison with purvalanol A. Both CDK inhibitors induced a concentration-dependent cell cycle arrest at the G(2)-M phase and an increase in the apoptotic rate, with a concomitant down-regulation of the antiapoptotic protein survivin, a member of the inhibitors of apoptosis protein family. Notably, the addition of NU6140 to paclitaxel-treated cells resulted in markedly increased cytotoxic effect and apoptotic response in comparison with the paclitaxel-purvalanol A combination (86 +/- 11% and 37 +/- 8%, respectively). Similarly, the extent of caspase-9 and caspase-3 activation in paclitaxel-NU6140-treated cells was approximately 4-fold higher than after the paclitaxel-purvalanol A combination. Moreover, an almost complete abrogation of the expression of the active, Thr(34)-phosphorylated form of survivin was observed in cells exposed to the paclitaxel-NU6140 combination. A synergistic effect of the paclitaxel-NU6140 combination, as a consequence of survivin inhibition and increased activation of caspase-9 and caspase-3, was also observed in OAW42/e ovarian cancer line but not in the derived OAW42/Surv subline ectopically expressing survivin. Results from this study indicate that NU6140 significantly potentiates the apoptotic effect of paclitaxel, with inhibition of survivin expression/phosphorylation as the potential mechanism.