Novel non-benzimidazole chk2 kinase inhibitors

In a recent paper, [Arienti, K. L.; Brunmark, A.; Axe, F. U.; McClure, K. M.; Lee, A.; Blevitt, J.; Neff, D. K.; Huang, L.; Crawford, S.; Chennagiri, R. P.; Karlsson, L.; Brietenbucher, J. G. J. Med. Chem.2005, 48, 1873], we described the discovery of a class of benzimidazole chk2 kinase inhibitors, exemplified by compound 1, which had radio-protective effects in human T-cells subjected to ionizing radiation. Here, a series of non-benzimidazole analogs intended to define the scope of the SAR about this new series of inhibitor, and allow for refinement of the binding model of these compounds to the chk2 kinase is described.     

Enaminones 10. Molecular modeling aspects of the 5-methylcyclohexenone derivatives

This article expands upon our original submission to the Eddington, N. D.; Cox, D. S.; Khurana, M.; Salama, N. N.; Stables, J. P.; Harrison, S. J.; Negussie, A.; Taylor, R. S.; Tran, U. Q.; Moore, J. A.; Barrow, J. C.; Scott, K. R. Eur. J. Med. Chem. 2003, 38, 49 on a series of twenty (20) compounds, all 5-methyl-3-[(substituted)-phenylamino]-cyclohex-2-enone derivatives. This article provides the reasons why the compounds are active/inactive. By use of computational methods, the reasons for activity/inactivity are explained.     

A new synthesis of 9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)guanine (AraF-G)

Interesting and very promising antisense properties of 2'-deoxy-2'-fluoroarabinonucleic acids ((a) Wilds, C.J.; Damha, M.J. 2'-Deoxy-2'-fluoroarabinonucleosides and oligonucleotides (2'F-ANA): synthesis and physicochemical studies. Nucl. Acids Res. 2000, 28, 3625-3635; (b) Viazovkina, E.; Mangos, M.; Elzagheid, M.I.; Damha, M.J. Current Protocols in Nucleic Acid Chemistry 2002, 4.15.1-4.15.21) (2'F-ANA) has encouraged our research group to optimize the synthetic procedures for 2'-deoxy-2'-fluoro-beta-D-arabinonucleosides (araF-N). The synthesis of araF-U, araF-T, araF-A and araF-C is straightforward, (Tann, C.H.; Brodfuehrer, P.R.; Brundidge, S.P.; Sapino, C., Jr. Howell H.G. Fluorocarbohydrates in synthesis. An efficient synthesis of 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-5-iodouracil (beta-FIAU) and 1-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)thymine (beta-FMAU). J. Org. Chem. 1985, 50, 3644-3647; Howell, H.G.; Brodfuehrer, P.R.; Brundidge, S.P.; Benigni, D.A.; Sapino, C., Jr. Antiviral nucleosides. A stereospecific, total synthesis of 2'-fluoro-2'-deoxy-beta-D-arabinofuranosyl nucleosides. J. Org. Chem. 1988, 53, 85-88; Maruyama, T.; Takamatsu, S.; Kozai, S.; Satoh, Y.; Izana, K. Synthesis of 9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)adenine bearing a selectively removable protecting group. Chem. Pharm. Bull. 1999, 47, 966-970) however, the synthesis of the guanine analogue is more complicated and affords poor to moderate yields of araF-G (4) ((a) Elzagheid, M.I.; Viazovkina, E.; Masad, M.J. Synthesis of protected 2'-deoxy-2'-fluoro-beta-D-arabinonucleosides. Synthesis of 2'-fluoroarabino nucleoside phosphoramidites and their use in the synthesis of 2'F-ANA. Current Protocols in Nucleic Acid Chemistry 2002, 1.7.1-1.7.19; (b) Tennila, T.; Azhayeva, E.; Vepsalainen, J.; Laatikainen, R.; Azhayev, A.; Mikhailopulo, I. Oligonucleotides containing 9-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-adenine and -guanine: synthesis, hybridization and antisense properties. Nucleosides, Nucleotides and Nucl. Acids 2000, 19, 1861-1884). Here we describe an efficient synthesis of araF-G (4) that involves coupling of 2-deoxy-2-fluoro-3,5-di-O-benzoyl-alpha-D-arabinofuranosyl bromide (1) with 2-chlorohypoxanthine (2) to afford 2-chloro-beta-araF-I (3) in 52% yield. Nucleoside (3) was transformed into araF-G (4) by treatment with methanolic ammonia (150 degrees C, 6 h) in 67% yield.     

Synthesis of 2',3'-dideoxy-2'-fluoro-3'-thioarabinothymidine and its 3'-phosphoramidite derivative

An efficient method for the synthesis of 5'-O-monomethoxytrityl-2',3'-dideoxy-2'-fluoro-3'-thioarabinothymidine [(5'MMT)araF-T(3'SH), (5)] and its 3'-phosphoramidite derivative (6) suitable for automated incorporation into oligonucleotides, is demonstrated. A key step in the synthesis involves reaction of 5'-O-MMT-2,3'-O-anhydrothymidine (4) (Eleuteri, A.; Reese, C.B.; Song, Q. J. Chem. Soc. Perkin Trans. 1 1996, 2237 pp.) with sodium thioacetate to give (5'-MMT)araF-T(3'SAc) (5) (Elzagheid, M.I.; Mattila, K.; Oivanen, M.; Jones, B.C.N.M.; Cosstick, L?nnberg, H. Eur. J. Org. Chem. 2000, 1987-1991). This nucleoside was then converted to its corresponding phosphoramidite derivative, 6, as described previously ((a) Sun, S.; Yoshida, A.; Piccirilli, J.A. RNA, 1997, 3, 1352-1363; (b) Matulic-Adamic, J.; Beigelman, L. Helvetica Chemica Acta 1999, 82, 2141-2150: (c) Fettes, K.J.; O'Neil, I.; Roberts, S.M.; Cosstick, R. Nucleosides, Nucleotides and Nucl. Acids 2001, 20, 1351-1354).     

Modified low molecular weight cyclic peptides as mimetics of BDNF with improved potency,proteolytic stability and transmembrane passage in vitro

We recently reported the development of the BDNF mimetic peptide cyclo-[dPAKKR] 1 which promotes the survival of cultured sensory neurons via a trkB independent mechanism [Fletcher, J. M.; Morton, C. M.; Zwar, R. A.; Murray, S. S.; O’Leary, P. D.; Hughes, R. A. J. Biol. Chem. 2008, 283, 33375]. In the present study we prepared a series of hydrophobically-modified analogues of 1 with an eye to improving its pharmacokinetic properties. Select members of this second generation of compounds showed improved biological activity, stability in plasma, and an ability to cross model biological membranes.     

Highly potent growth hormone secretagogues

During an effort to search for more potent growth hormone secretagogues, we discovered a class of compounds of which the best compound 8 was 7-fold more active in vitro than the best compound in the series we revealed before [Tata, J. R.; Lu, Z.; Jacks, T. M.; Schleim, K. D.; Cheng, K.; Wei, L.; Chan, W.-S.; Butler, B.; Tsou, N.; Leung, K.; Chiu, S.-H. L.; Hickey, G. J.; Smith, R. G.; Patchett, A. A. Bioorg. Med. Chem. Lett.1997, 7, 2319.]. Animal studies show that compound 8 can stimulate growth hormone release at the oral dose as low as 0.06 mpk. Chemistry and biological studies are discussed.     

From genome to drug lead: identification of a small-molecule inhibitor of the SARS virus

Virtual screening, a fast, computational approach to identify drug leads [Perola, E.; Xu, K.; Kollmeyer, T. M.; Kaufmann, S. H.; Prendergast, F. G. J. Med. Chem.2000, 43, 401; Miller, M. A. Nat. Rev. Drug Disc.2002, 1 220], is limited by a known challenge in crystallographically determining flexible regions of proteins. This approach has not been able to identify active inhibitors of the severe acute respiratory syndrome-associated coronavirus (SARS-CoV) using solely the crystal structures of a SARS-CoV cysteine proteinase with a flexible loop in the active site [Yang, H. T.; Yang, M. J.; Ding, Y.; Liu, Y. W.; Lou, Z. Y. Proc. Natl. Acad. Sci. U.S.A.2003, 100, 13190; Jenwitheesuk, E.; Samudrala, R. Bioorg. Med. Chem. Lett.2003, 13, 3989; Rajnarayanan, R. V.; Dakshanamurthy, S.; Pattabiraman, N. Biochem. Biophys. Res. Commun.2004, 321, 370; Du, Q.; Wang, S.; Wei, D.; Sirois, S.; Chou, K. Anal. Biochem.2005, 337, 262; Du, Q.; Wang, S.; Zhu, Y.; Wei, D.; Guo, H. Peptides2004, 25, 1857; Lee, V.; Wittayanarakul, K.; Remsungenen, T.; Parasuk, V.; Sompornpisut, P. Science (Asia)2003, 29, 181; Toney, J.; Navas-Martin, S.; Weiss, S.; Koeller, A. J. Med. Chem.2004, 47, 1079; Zhang, X. W.; Yap, Y. L. Bioorg. Med. Chem.2004, 12, 2517]. This article demonstrates a genome-to-drug-lead approach that uses terascale computing to model flexible regions of proteins, thus permitting the utilization of genetic information to identify drug leads expeditiously. A small-molecule inhibitor of SARS-CoV, exhibiting an effective concentration (EC50) of 23 microM in cell-based assays, was identified through virtual screening against a computer-predicted model of the cysteine proteinase. Screening against two crystal structures of the same proteinase failed to identify the 23-microM inhibitor. This study suggests that terascale computing can complement crystallography, broaden the scope of virtual screening, and accelerate the development of therapeutics to treat emerging infectious diseases such as SARS and Bird Flu.     

Stereoselective synthesis of 1,1'-linked alpha-l-lyxopyranosyl beta-d-glucopyranoside, the proposed biosynthetic precursor of the FG ring system of avilamycins

The non-reducing disaccharide beta-d-Glcp-(1<-->1)-alpha-L-Lyxp1 had been proposed to be an early intermediate during the biosynthesis of avilamycin A [Boll, R.; Hofmann, C.; Heitmann, B.; Hauser, G.; Glaser, S.; Koslowski, T.; Friedrich, T.; Bechthold, A. J. Biol. Chem.2006, 281, 14756-14763]. This work describes a comparison of two strategies for the synthesis of 1 and its 2-amino-2-deoxy analog with either the glucose or the lyxose moiety acting as the glycosyl donor. The best results in terms of stereoselectivity and yield were obtained with 2,3,4-tri-O-acetyl-alpha-L-lyxopyranosyl trichloroacetimidate 13. Reaction of 13 with 2,3,4,6-tetra-O-acetyl-D-glucopyranose gave the disaccharide as mixture of 1beta,1'alpha and 1beta,1'beta isomers in a ratio of 10:1 and a yield of 50%. Reaction of 13 and 3,4,6-tri-O-acetyl-2-azido-2-deoxy-D-glucopyranose yielded the desired 1beta,1'alpha disaccharide as a single isomer in 72% yield. Interestingly, the formation of alpha-glucosides was not observed in any case, regardless of the use of glucose as glycosyl donor or acceptor.     

Glu375Gln and Asp225Val mutants: about the nature of the covalent linkages between heme group and apo-Protein in bovine lactoperoxidase

In analogy with studies previously reported for myeloperoxidase (Kooter, I. M.; Moguilevsky, N.; Bollen, A.; Van der Veen, L. A.; Otto, C.; Dekker, H. L.; Wever, R. J. Biol. Chem. 1999, 274, 26794), we examined for bovine lactoperoxidase the effect of mutation of Asp225 and Glu375, the residues thought to be responsible for the covalent binding of the heme group to the apoprotein. Starting from the plasmid encoding rbLPO (Watanabe, S.; Varsalona, F.; Yoo, Y.; Guillaume, J. P.; Bollen, A.; Shimazaki, K.; Moguilevsky, N. FEBS Letters 1998, 441, 476), which was engineered to carry mutations in correspondence of those residues, the mutants Asp225Val and Glu375Gln were expressed in CHO cells and their products purified and characterized. Unequivocal evidence about the existence of ester linkages as well as their relative contribution to the specific spectroscopic and catalytic properties of bLPO is here discussed.     

Structural basis for the activity of the RSK-specific inhibitor, SL0101

Inappropriate activity of p90 ribosomal S6 kinase (RSK) has been implicated in various human cancers as well as other pathologies. We previously reported the isolation, characterization, and synthesis of the natural product kaempferol 3-O-(3',4'-di-O-acetyl-alpha-l-rhamnopyranoside), termed SL0101 [Smith, J. A.; Poteet-Smith, C. E.; Xu, Y.; Errington, T. M.; Hecht, S. M.; Lannigan, D. A. Cancer Res., 2005, 65, 1027-1034: Xu, Y.-M; Smith, J. A.; Lannigan, D. A.; Hecht, S. M. Bioorg. Med. Chem., 2006, 14, 3974-3977: Maloney, D. J.; Hecht, S. M. Org. Lett., 2005, 7, 1097-1099]. SL0101 is a potent and specific inhibitor of RSK; therefore, we performed an analysis of the structural basis for the inhibitory activity of this lead compound. In in vitro kinase assays we found that acylation of the rhamnose moiety and the 4', 5, and 7-hydroxyl groups are responsible for maintaining a high affinity interaction of RSK with SL0101. It is likely that the hydroxyl groups facilitate RSK binding through their ability to form hydrogen bonds. To determine whether the SL0101 derivatives were specific for inhibition of RSK we analyzed their ability to preferentially inhibit the growth of the human breast cancer line, MCF-7, compared to the normal human breast line, MCF-10A. We have previously validated this differential growth assay as a convenient readout for analyzing the specificity of RSK inhibitors [Smith, J. A.; Maloney, D. J.; Clark, D. E.; Xu, Y.-M.; Hecht, S. M.; Lannigan, D. A. Bioorg. Med. Chem., 2006, 14, 6034-6042]. We found that acylation of the rhamnose moiety was essential for maintaining the selectivity for RSK inhibition in intact cells. Further, the efficacy of SL0101 in intact cells is limited by cellular uptake as well as possible hydrolysis of the acetyl groups on the rhamnose moiety by ubiquitous intracellular esterases. These studies should facilitate the development of a RSK inhibitor, based on the SL0101 pharmacophore, as an anti-cancer chemotherapeutic agent.     

Synthesis and evaluation of 4-(substituted styryl/alkenyl)-3,5-bis(4-hydroxyphenyl)-isoxazoles as ligands for the estrogen receptor

A series of 3,5-bis (4-hydroxyphenyl) isoxazoles bearing a styryl/alkyl vinyl group at the 4-position were prepared and evaluated as ligands for the estrogen receptor-alpha (ERα). The target compounds were prepared using the Suzuki reaction to couple an iodo-isoxazole intermediate with a series of styryl/alkenyl boronic acids, followed by O-demethylation. The products were evaluated for their estrogen receptor-α ligand binding domain (ERα-LBD) binding affinity using a competitive binding assay. The 4-(4-hydroxystyryl) derivative 4h displays binding properties similar to those of the previously described pyrazole class of ER ligands, indicating that the ERα-LBD tolerates the presence of the added vinyl group at the 4-position of the isoxazole ring.     

Comparison of the interactions of transferrin receptor and transferrin receptor 2 with transferrin and the hereditary hemochromatosis protein HFE

The transferrin receptor (TfR) interacts with two proteins important for iron metabolism, transferrin (Tf) and HFE, the protein mutated in hereditary hemochromatosis. A second receptor for Tf, TfR2, was recently identified and found to be functional for iron uptake in transfected cells (Kawabata, H., Germain, R. S., Vuong, P. T., Nakamaki, T., Said, J. W., and Koeffler, H. P. (2000) J. Biol. Chem. 275, 16618-16625). TfR2 has a pattern of expression and regulation that is distinct from TfR, and mutations in TfR2 have been recognized as the cause of a non-HFE linked form of hemochromatosis (Camaschella, C., Roetto, A., Cali, A., De Gobbi, M., Garozzo, G., Carella, M., Majorano, N., Totaro, A., and Gasparini, P. (2000) Nat. Genet. 25, 14-15). To investigate the relationship between TfR, TfR2, Tf, and HFE, we performed a series of binding experiments using soluble forms of these proteins. We find no detectable binding between TfR2 and HFE by co-immunoprecipitation or using a surface plasmon resonance-based assay. The affinity of TfR2 for iron-loaded Tf was determined to be 27 nm, 25-fold lower than the affinity of TfR for Tf. These results imply that HFE regulates Tf-mediated iron uptake only from the classical TfR and that TfR2 does not compete for HFE binding in cells expressing both forms of TfR.     

Novel 7α-alkoxy-17α-(4'-halophenylethynyl)estradiols as potential SPECT/PET imaging agents for estrogen receptor expressing tumours: Synthesis and binding affinity evaluation

In order to develop potential radiolabelled probes for imaging estrogen receptor (ER) positive tumours, we have synthesized and characterized a series of novel 7α-alkoxy-17α-(4'-iodophenylethynyl)estra-1,3,5(10)-triene-3,17β-diols and 7α-alkoxy-17α-(4'-fluorophenylethynyl)estra-1,3,5(10)-triene-3,17β-diols. The fluoro-substituted compounds showed a higher ER binding affinity than the corresponding iodo-derivatives, where 7α-methoxy- and 17α-(4'-fluorophenylethynyl)estra-1,3,5(10)-triene-3,17β-diol showed the highest ER binding affinities (RBA=80.9% and 78.9%, respectively), among the halophenylethynyl compounds studied and should be further explored as potential PET biomarkers for imaging of ER expressing tumours.     

The saccharide-hydrazide linkage: molecular and crystal structures of the semicarbazide derivatives of D-glucose, D-galactose, and D-xylose, including a 'forbidden' conformation of the galactose derivative.

The X-ray crystal structures of the semicarbazide derivatives of D-glucose, D-galactose, and D-xylose are described. All are glycopyranosyl derivatives in the solid state. The glucose semicarbazide crystallizes as a dihydrate. Two different conformations of the galactose semicarbazide are found, one having the gg side-chain orientation unfavorable for monosaccharides having the 4C(1)-D-galacto configuration. In the two other known examples, this conformation is stabilized by an intramolecular hydrogen bond, but in the current structure, the hydrogen bonds involving the side chain are intermolecular. In these semicarbazides the N [bond] N [bond] C[double bond] O torsional angle is approximately 180 degrees, in contrast to the angles of 0.60 and 13.9 degrees in the two solid-state conformations of 2-benzoyl-1-(alpha-D-xylopyranosyl)hydrazine [Ernholt, B. V.; Thomsen, I. B.; Lohse, A.; Plesner, I. W.; Jensen, K. B.; Hazell, R. G.; Liang, X.; Jakobsen, A.; Bols, M. Chem. Eur. J. 2000, 6, 278-287]. The water molecules and the carbonyl oxygen are heavily involved in H-bonding.