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A series of biaryl pyrazole and imidazole Liver X Receptor (LXR) partial agonists has been synthesized displaying LXRβ selectivity. The LXRβ selective partial agonist 18 was identified with potent induction of ATP binding transporters ABCA1 and ABCG1 in human whole blood (EC50 = 1.2 μM, 55% efficacy). In mice 18 displayed peripheral induction of ABCA1 at 3 and 10 mpk doses with no significant elevation of plasma or hepatic triglycerides at these doses, showing an improved profile compared to a full pan-agonist.

Variously substituted indolin-2-ones were synthesized and evaluated for activity against KDR, Flt-1, FGFR-1 and PDGFR. Extension at the 5-position of the oxindole ring with ethyl piperidine (compound 7i) proved to be the most beneficial for attaining both biochemical and cellular potencies. Further optimization of 7i to balance biochemical and cellular potencies with favorable ADME/ PK properties led to the identification of 8h, a compound with a clean CYP profile, acceptable pharmacokinetic and toxicity profiles, and robust efficacy in multiple xenograft tumor models.

A series of substituted benzofuropyrimidinones with pan-PIM activities and excellent selectivity against a panel of diverse kinases is described. Initial exploration identified aryl benzofuropyrimidinones that were potent, but had cell permeability limitation. Using X-ray crystal structures of the bound PIM-1 complexes with 3, 5m, and 6d, we were able to guide the SAR and identify the alkyl benzofuropyrimidinone (6l) with good PIM potencies, permeability, and oral exposure.

CDC7 is a serine/threonine kinase that has been shown to be required for the initiation and maintenance of DNA replication. Up-regulation of CDC7 is detected in multiple tumor cell lines, with inhibition of CDC7 resulting in cell cycle arrest. In this paper, we disclose the discovery of a potent and selective CDC7 inhibitor, XL413 (14), which was advanced into Phase 1 clinical trials. Starting from advanced lead 3, described in a preceding communication, we optimized the CDC7 potency and selectivity to demonstrate in vitro CDC7 dependent cell cycle arrest and in vivo tumor growth inhibition in a Colo-205 xenograft model.

Unlike all other organisms studied to date, Bacillus subtilis expresses two different thymidylate synthases: bsTS-A and bsTS-B. bsTS-A displays enhanced enzymatic and structural thermal stability uncharacteristic of most TSs. Despite the high level of TS conservation across most species, bsTS-A shares low sequence identity (

Background: Enzymes have evolved to recognise their target substrates with exquisite selectivity and specificity. Whether fragments of the substrate – perhaps never available to the evolving enzyme – are bound in the same manner as the parent substrate addresses the fundamental basis of specificity. An understanding of the relative contributions of individual portions of ligand molecules to the enzyme-binding interaction may offer considerable insight into the principles of substrate recognition. Results: We report 12 crystal structures of Escherichia coli thymidylate synthase in complexes with available fragments of the substrate (dUMP), both with and without the presence of a cofactor analogue. The structures display considerable fidelity of binding mode and interactions. These complexes reveal several interesting features: the cofactor analogue enhances the localisation of substrate and substrate fragments near the reactive thiol; the ribose moiety reduces local disorder through additional specific enzyme–ligand interactions; the pyrimidine has multiple roles, ranging from stereospecificity to mechanistic competence; and the glycosidic linkage has an important role in the formation of a covalent attachment between substrate and enzyme. Conclusions: The requirements of ligand–protein binding can be understood in terms of the binding of separate fragments of the ligand. Fragments which are sybsystems of the natural substrate for the enzyme confer specific contributions to the binding affinity, orientation or electrostatics of the enzymatic mechanism. This ligand-binding analysis provides a complementary method to the more prevalent approaches utilising site-directed mutagenesis. In addition, these observations suggest a modular approach for rational drug design utilising chemical fragments.

Background: Thymidylate synthase (TS) is critical to DNA synthesis as it catalyzes the rate limiting step in the only biosynthetic pathway for deoxythymidine monophosphate (dTMP) production. TS is therefore an important target for anti-proliferative and anti-cancer drug design. The TS enzymatic mechanism involves the reductive methylation of the substrate, deoxyuridine monophosphate (dUMP), by transfer of a methylene group from the co-factor, methylenetetrahydrofolate (CH 2 H 4 folate), resulting in the production of deoxythymidine monophosphate (dTMP) and dihydrofolate (H 2 folate). Previous drug design efforts based on co-factor analogues have produced good inhibitors of TS, but poor bioavailability and toxicity have limited their usefulness. BW1843U89, a folate analogue, is a recently developed compound which is an exceptionally strong inhibitor (K i =0.09 nM), has good bioavailability and in clinical trials thus far has not demonstrated significant toxicity. Results We report the crystal structure of E. coli TS in ternary complex with dUMP and BW1843U89 at 2.0 a resolution. Although the benzoquinazoline ring system of the inhibitor binds to TS in much the same manner as previously determined for H 2 folate and CB3717, the larger size of the ligand is accommodated by the enzyme through a local distortion of the active site, that is not strictly conserved in both monomers in the asymmetric unit. Several conserved waters that had been previously implicated in mechanistic roles have been displaced. Conclusion BW1843U89 forms a ternary complex with dUMP and competes with CH 2 H 4 folate at the active site. Inhibition of TS by BW1843U89 shows four unique aspects in its mechanism of action. BW1843U89 prevents the Michael addition of dUMP to Cys146, in contrast to the mechanisms implicated from crystallography of other quinazoline based inhibitors; displaces a catalytic water from the active site; reorders a peptide loop (Leu72–Trp83) in the active site; and is unique amongst the antifolates in inactivating TS at a stoichiometric ratio of one molecule per TS dimer. Thus, it exploits the principles of negative cooperativity that are increasingly being recognized in the catalytic mechanism of the enzyme per se . The structure suggests that this ‘half-the-sites' effect is catalytic and not related to ligand binding. Therefore BW1843U89 is both a competitive inhibitor (at the binding site) and a non-competitive inhibitor at the other site.

A water-mediated hydrogen bond network coordinated by glutamate 60(58) appears to play an important role in the thymidylate synthase (TS) reaction mechanism. We have addressed the role of glutamate 60(58) in the TS reaction by cocrystalizing the Escherichia coli TS mutant E60(58)Q with dUMP and the cofactor analog CB3717 and have determined the X-ray crystal structure to 2.5 A resolution with a final R factor of 15.2% (Rfree = 24.0%). Using difference Fourier analysis, we analyzed directly the changes that occur between wild-type and mutant structures. The structure of the mutant enzyme suggests that E60(58) is not required to properly position the ligands in the active site and that the coordinated hydrogen bond network has been disrupted in the mutant, providing an atomic resolution explanation for the impairment of the TS reaction by the E60(58)Q mutant and confirming the proposal that E60(58) coordinates this conserved hydrogen bond network. The structure also provides insight into the role of specific waters in the active site which have been suggested to be important in the TS reaction. Finally, the structure shows a unique conformation for the cofactor analog, CB3717, which has implications for structure-based drug design and sheds light on the controversy surrounding the previously observed enzymatic nonidentity between the chemically identical monomers of the TS dimer.

A series of compounds was investigated in order to determine the factors that govem the cycloaromatization reaction exhibited by dynemicin A and related molecules. It was determined that the stereochemistry at ClOa dictated the thermal reactivity of compounds equipped with an exo-epoxide. Structures containing an a-substituent were unreactive while those possessing a P-substituent underwent cyclization at 80 "C. Molecular mechanics and dynamics calculations indicate that this reactivity difference is due to a steric interaction that develops as the a-substituted compounds undergo a conformational change that appears to be necessary for aromatization to occur. Additionally, a C 10a a-substituted endo-epoxide was synthesized and shown to undergo cycloaromatization under mild acidic conditions via an epoxide opening.

We report the discovery of a series of 4-aryl-2-aminoalkylpyrimidine derivatives as potent and selective JAK2 inhibitors. High throughput screening of our in-house compound library led to the identification of hit 1, from which optimization resulted in the discovery of highly potent and selective JAK2 inhibitors. Advanced lead 10d demonstrated a significant dose-dependent pharmacodynamic and antitumor effect in a mouse xenograft model. Based upon the desirable profile of 10d (XL019) it was advanced into clinical trials.

The phosphoinositide 3-kinases (PI3Ks) have been linked to an extraordinarily diversified group of cellular functions making these enzymes compelling targets for the treatment of disease. A large body of evidence has linked PI3Kγ to the modulation of autoimmune and inflammatory processes making it an intriguing target for drug discovery. Our high-throughput screening (HTS) campaign revealed two hits that were nominated for further optimization studies. The in vitro activity of the first HTS hit, designated as the sulfonylpiperazine scaffold, was optimized utilizing structure-based design. However, nonoptimal pharmacokinetic properties precluded this series from further studies. An overlay of the X-ray structures of the sulfonylpiperazine scaffold and the second HTS hit within their complexes with PI3Kγ revealed a high degree of overlap. This feature was utilized to design a series of hybrid analogues including advanced leads such as 31 with desirable potency, selectivity, and oral bioavailability.

Five new cembradiene diterpenoids (1–5) and two previously known compounds (7 and 8) have been isolated from an undescribed Caribbean gorgonian of the genus Eunicea. The structures of these compounds were assigned on the basis of chemical and spectral studies, and the relative stereochemistry of cembradiene 3 was defined by X-ray crystallographic methods.

Ohioensins A (1), B (2), C (3), D (4), and E (6), containing the novel polycyclic skeleton of (7bβ,-12bα,14cα)-7b,12b,13,14c-tetrahydro-14H-benzo[c]naphtho[2,1,8-mna]xanthen-14-one,have been isolated from the moss Polytrichum ohioense (Polytrichaceae) following bioassay-directed fractionation. The structures and relative stereochemistry of ohioensins were established on the basis of spectral analysis (UV, IR, MS, 2D NMR, and CD), chemical correlation, and X-ray diffraction

Five new cembradiene diterpenoids (1–5) and two previously known compounds (7 and 8) have been isolated from an undescribed Caribbean gorgonian of the genus Eunicea. The structures of these compounds were assigned on the basis of chemical and spectral studies, and the relative stereochemistry of cembradiene 3 was defined by X-ray crystallographic methods.

A series of compounds was investigated in order to determine the factors that govem the cycloaromatization reaction exhibited by dynemicin A and related molecules. It was determined that the stereochemistry at ClOa dictated the thermal reactivity of compounds equipped with an exo-epoxide. Structures containing an a-substituent were unreactive while those possessing a P-substituent underwent cyclization at 80 "C. Molecular mechanics and dynamics calculations indicate that this reactivity difference is due to a steric interaction that develops as the a-substituted compounds undergo a conformational change that appears to be necessary for aromatization to occur. Additionally, a C 10a a-substituted endo-epoxide was synthesized and shown to undergo cycloaromatization under mild acidic conditions via an epoxide opening.

Emeniveol (1), a new pollen growth inhibitor, was isolated from the fungus, Emericella nivea. The structure was elucidated from the X-ray crystallographic analysis as well as homonuclear decoupling, COSY, COLOC and HMBC experiments, and chemical reaction.

The solid and solution state structures of aplasmomycin C, a boroncontaining antibiotic, were studied by single crystal X-ray diffraction and NMR techniques. This unusual antibiotic was shown to have a complex and as yet imperfectly understood series of conformations.

A convergent asymmetric synthesis of the antinoeplastic macrolide antibiotic cytovaricin has been achieved through the synthesis and coupling of the illustrated spiroketal and polyol glycoside subunits. All absolute steroechemical relationships within the target structure were ultimately controlled by the use of asymmetric aldol, alkylation, or epoxidation methodology. Union of the two subnits was accomplished by Julia-Lythgoe trans olefination, providing direct access to a suitable macrolactonization substrate

Serpins exhibit a range of physiological roles and can contribute to certain disease states dependent on their various conformations. Understanding the mechanisms of the large-scale conformational reorganizations of serpins may lead to a better understanding of their roles in various cardiovascular diseases. We have studied the serpin, plasminogen activator inhibitor 1 (PAI-1), in both the active and the latent state and found that anionic halide ions may play a role in the active-to-latent structural transition. Crystallographic analysis of a stable mutant form of active PAI-1 identified an anion-binding site between the central beta-sheet and a small surface domain. A chloride ion was modeled in this site, and its identity was confirmed by soaking crystals in a bromide-containing solution and calculating a crystallographic difference map. The anion thus located forms a 4-fold ligated linchpin that tethers the surface domain to the central beta-sheet into which the reactive center loop must insert during the active-to-latent transition. Timecourse experiments measuring active PAI-1 stability in the presence of various halide ions showed a clear trend for stabilization of the active form with F(-)Cl(-)Br(-)I(-). We propose that the "stickiness" of this pin (i.e., the electronegativity of the anion) contributes to the energetics of the active-to-latent transition in the PAI-1 serpin.

Thymidylate synthase is an attractive target for antiproliferative drug design because of its key role in the synthesis of DNA. As such, the enzyme has been widely targeted for anticancer applications. In principle, TS should also be a good target for drugs used to fight infectious disease. In practice, TS is highly conserved across species, and it has proven to be difficult to develop inhibitors that are selective for microbial TS enzymes over the human enzyme. Using the structure of TS from Lactobacillus casei in complex with the nonsubstrate analogue phenolphthalein, inhibitors were designed to take advantage of features of the bacterial enzyme that differ from those of the human enzyme. Upon synthesis and testing, these inhibitors were found to be up to 40-fold selective for the bacterial enzyme over the human enzyme. The crystal structures of two of these inhibitors in complex with TS suggested the design of further compounds. Subsequent synthesis and testing showed that these second-round compounds inhibit the bacterial enzyme at sub-micromolar concentrations, while the human enzyme was not inhibited at detectable levels (selectivities of 100-1000-fold or greater). Although these inhibitors share chemical similarities, X-ray crystal structures reveal that the analogues bind to the enzyme in substantially different orientations. Site-directed mutagenesis experiments suggest that the individual inhibitors may adopt multiple configurations in their complexes with TS.

Two new imidazole alkaloids were isolated from a Micronesian sponge, Leucetta sp. Kealiiquinone (2) is a naphtoquinone which appears to be biogenetically derived from 1, which is a dibenzyl-substituted imidazole. Pyronaamidine (1) is cytotoxic against KB cells, MIC=5 μg/mL

In thymidylate synthase (TS), the invariant residue Asp-221 provides the only side chain that hydrogen bonds to the pterin ring of the cofactor, 5,10-methylene-5,6,7,8-tetrahydrofolate. All mutants of D221 except cysteine abolish activity. We have determined the crystal structures of two ternary complexes of the Escherichia coli mutant D221N. In a complex with dUMP and the antifolate 10- propargyl-5,8-dideazafolate (CB3717), dUMP is covalently bound to the active site cysteine, as usual. CB3717, which has no imidazolidine ring, is also bound in the usual productive orientation, but is less ordered than in wild-type complexes. The side chain of Asn-221 still hydrogen bonds to N3 of the quinazoline ring of CB3717, which must be in the enol form. In contrast, the structure of D221N with 5-fluoro-dUMP and 5,10-methylene-5,6,7,8-tetrahydrofolate shows the cofactor bound in two partially occupied, nonproductive binding sites. In both binding modes, the cofactor has a closed imidazolidine ring and adopts the solution conformation of the unbound cofactor. In one of the binding sites, the pterin ring is turned around such that Asn-221 hydrogen bonds to the unprotonated N1 instead of the protonated N3 of the cofactor. This orientation blocks the conformational change required for forming covalent ternary complexes. Taken together, the two crystal structures suggest that the hydrogen bond between the side chain of Asp-221 and N3 of the cofactor is most critical during the early steps of cofactor binding, where it enforces the correct orientation of the pterin ring. Proper orientation of the cofactor appears to be a prerequisite for opening the imidazolidine ring prior to formation of the covalent steady-state intermediate in catalysis.

Erratum to ‘‘Discovery of XL413, a potent and selective CDC7 inhibitor’’ [Bioorg. Med. Chem. Lett. 22 (2012) 3727–3731] Elena S. Koltun ⇑, Amy Lew Tsuhako, David S. Brown, Naing Aay, Arlyn Arcalas, Vicky Chan, Hongwang Du, Stefan Engst, Kim Ferguson, Maurizio Franzini, Adam Galan, Charles R. Holst, Ping Huang, Brian Kane, Moon H. Kim, Jia Li, David Markby, Manisha Mohan, Kevin Noson, Arthur Plonowski, Steven J. Richards, Scott Robertson, Kenneth Shaw, Gordon Stott, Thomas J. Stout, Jenny Young, Peiwen Yu, Cristiana A. Zaharia, Wentao Zhang, Peiwen Zhou, John M. Nuss, Wei Xu, Patrick C. Kearney

The three-dimensional structure of the unusual fatty acid metabolite neohalicholactone, isolated from the marine sponge Halinchondria okadai Kadota, has been determined by X-ray crystallography.

The structures of four new drimane-type sesquiterpenes 1–4 produced in liquid cultures of the fairy ring fungus, Marasmiusoreades, have been determined by a combination of chemical and spectroscopic methods. The structure assigned to one of these sesquiterpenes (1) has been confirmed by an X-ray crystallographic study. An unprecedented feature of all four sesquiterpenes is the biosynthetic elaboration of C8–C12 and C15 of the drimane skeleton to a dioxabicyclooctane moiety. In addition, two of the sesquiterpenes possess an uncommon α orientation of the hydroxyl groups at C3. Keywords: sesquiterpenes, fungal metabolites, drimanes, fairy ring mushroom, crystal structure.

Duodenal ulcers were produced in rats by subcutaneous infusion for 48 hr of either of the following secretagogues: histamine, carbachol, pentagastrin. When any two of these secretagogues were administered together, synergism was observed in the ulcerogenic effect. Synergism, or potentiation, is defined as an effect produced by two agents given together that is greater than the sum of the effects produced by each agent when given separately. The ulcers were located opposite to the mesenteric attachment, were often multiple, and extended as far as 4 cm from the pylorus. Their severity ranged from shallow erosions to perforation with peritonitis. When two secretagogues were infused at the same time, the incidence of animals with ulcers, their severity, number, and distance from the pylorus were related to the doses given. Pentagastrin, when administered alone, was the least ulcerogenic agent, but when pentagastrin was given with either histamine or carbachol, the ulcerogenic effect was potentiated. Although the duodenum was consistently ulcerated, the stomach remained intact, except in some animals receiving high doses of secretagogues and dying or dead from peritonitis. It is concluded that: (1) the rat can develop duodenal ulcers; and (2) secretagogues, when infused for 48 hr and especially when combined, can produce such ulcers; (3) the mechanism by which these ulcers develop remains to be elucidated.