Flow Asymmetric Propargylation: Development of Continuous Processes for the Preparation of a Chiral ß-Amino Alcohol.

The development of a flow chemistry process for asymmetric propargylation using allene gas as a reagent is reported. The connected continuous process of allene dissolution, lithiation, Li-Zn transmetallation, and asymmetric propargylation provides homopropargyl ß-amino alcohol 1 with high regio- and diastereoselectivity in high yield. This flow process enables practical use of an unstable allenyllithium intermediate. The process uses the commercially available and recyclable (1S,2R)-N-pyrrolidinyl norephedrine as a ligand to promote the highly diastereoselective (32:1) propargylation. Judicious selection of mixers based on the chemistry requirement and real-time monitoring of the process using process analytical technology (PAT) enabled stable and scalable flow chemistry runs.

Discovery and Optimization of Imidazopyridine-Based Inhibitors of Diacylglycerol Acyltransferase 2 (DGAT2).

The medicinal chemistry and preclinical biology of imidazopyridine-based inhibitors of diacylglycerol acyltransferase 2 (DGAT2) is described. A screening hit 1 with low lipophilic efficiency (LipE) was optimized through two key structural modifications: (1) identification of the pyrrolidine amide group for a significant LipE improvement, and (2) insertion of a sp(3)-hybridized carbon center in the core of the molecule for simultaneous improvement of N-glucuronidation metabolic liability and off-target pharmacology. The preclinical candidate 9 (PF-06424439) demonstrated excellent ADMET properties and decreased circulating and hepatic lipids when orally administered to dyslipidemic rodent models.

A streamlined strategy for aglycone assembly and glycosylation.

Multipurpose sugars: Carbohydrate-derived silane reagents are utilized as the reductant for nickel-catalyzed aldehyde-alkyne reductive coupling reactions and as the glycosyl donor for subsequent intramolecular glycosylation. The approach enables the assembly of the carbon-carbon framework and stereochemical features of an aglycone while simultaneously establishing the site of glycosylation.

Chemoselective hydrosilylation of hydroxyketones.

A chemoselective method for the hydrosilylation of ketones has been developed, using the combination of triphenylsilane and a catalyst prepared from Ni(COD)2 and the simple N-heterocyclic carbene IMes. The most notable feature of this method is that free hydroxyls are largely unaffected, thus providing a simple one-step procedure for the conversion of hydroxyketones to mono-protected diols, wherein the protecting group is exclusively installed on the ketone-derived hydroxyl. The process is typically high yielding with both simple ketones and more complex hydroxyketone substrates.

Synthesis of 7-oxo-dihydrospiro[indazole-5,4'-piperidine] acetyl-CoA carboxylase inhibitors.

Synthesis of oxo-dihydrospiroindazole-based acetyl-CoA carboxylase (ACC) inhibitors is reported. The dihydrospiroindazoles were assembled in a regioselective manner in six steps from substituted hydrazines and protected 4-formylpiperidine. Enhanced regioselectivity in the condensation between a keto enamine and substituted hydrazines was observed when using toluene as the solvent, leading to selective formation of 1-substituted spiroindazoles. The 2-substituted spiroindazoles were formed selectively from alkyl hydrazones by ring closure with Vilsmeier reagent. The key step in the elaboration to the final products is the conversion of an intermediate olefin to the desired ketone through elimination of HBr from an O-methyl bromohydrin. This methodology enabled the synthesis of each desired regioisomer on 50-75 g scale with minimal purification. Acylation of the resultant spirocyclic amines provided potent ACC inhibitors.

Maximizing lipophilic efficiency: the use of Free-Wilson analysis in the design of inhibitors of acetyl-CoA carboxylase.

This paper describes the design and synthesis of a novel series of dual inhibitors of acetyl-CoA carboxylase 1 and 2 (ACC1 and ACC2). Key findings include the discovery of an initial lead that was modestly potent and subsequent medicinal chemistry optimization with a focus on lipophilic efficiency (LipE) to balance overall druglike properties. Free-Wilson methodology provided a clear breakdown of the contributions of specific structural elements to the overall LipE, a rationale for prioritization of virtual compounds for synthesis, and a highly successful prediction of the LipE of the resulting analogues. Further preclinical assays, including in vivo malonyl-CoA reduction in both rat liver (ACC1) and rat muscle (ACC2), identified an advanced analogue that progressed to regulatory toxicity studies.

Ketone hydrosilylation with sugar silanes followed by intramolecular aglycone delivery: an orthogonal glycosylation strategy.

Gettin' a little sugar-no alcohol required: A procedure for the direct glycosylation of ketones without a hydroxy intermediate enables the site-selective glycosylation of hydroxyketones at the ketone or the alcohol functionality without the use of protecting groups on the aglycone (see scheme). Site selectivity is controlled by the catalyst structure in hydrosilylation and dehydrogenative silylation reactions with sugar silanes. Bn=benzyl.

Attaching and effacing Escherichia coli downregulate DNA mismatch repair protein in vitro and are associated with colorectal adenocarcinomas in humans.

BACKGROUND: Mucosa-associated Escherichia coli are frequently found in the colonic mucosa of patients with colorectal adenocarcinoma, but rarely in healthy controls. Chronic mucosal E. coli infection has therefore been linked to colonic tumourigenesis. E. coli strains carrying eae (encoding the bacterial adhesion protein intimin) attach intimately to the intestinal mucosa and are classed as attaching and effacing E. coli (AEEC). Enteropathogenic Escherichia coli (EPEC) are the most common form of AEEC identified in man. EPEC utilise a type III secretion system to translocate effector proteins into host cells and infection induces wide-ranging effects on the host cell proteome. We hypothesised that EPEC infection could influence molecular pathways involved in colorectal tumourigenesis. METHODOLOGY/PRINCIPAL FINDINGS: When co-cultured with human colorectal cell lines, EPEC dramatically downregulated the expression of key DNA mismatch repair proteins MSH2 and MLH1 in an attachment specific manner. Cytochrome c staining and TUNEL analysis confirmed that this effect was not a consequence of apoptosis/necrosis. Ex vivo human colonic mucosa was co-cultured with EPEC and probed by immunofluorescence to locate adherent bacteria. EPEC entered 10% of colonic crypts and adhered to crypt epithelial cells, often in the proliferative compartment. Adenocarcinoma and normal colonic mucosa from colorectal cancer patients (n = 20) was probed by immunofluorescence and PCR for AEEC. Mucosa-associated E. coli were found on 10/20 (50%) adenocarcinomas and 3/20 (15%) normal mucosa samples (P<0.05). AEEC were detected on 5/20 (25%) adenocarcinomas, but not normal mucosa samples (P<0.05). SIGNIFICANCE/CONCLUSIONS: The ability of EPEC to downregulate DNA mismatch repair proteins represents a novel gene-environment interaction that could increase the susceptibility of colonic epithelial cells to mutations and therefore promote colonic tumourigenesis. The potential role of AEEC in colorectal tumourigenesis warrants further investigation.

Truncation of MBD4 predisposes to reciprocal chromosomal translocations and alters the response to therapeutic agents in colon cancer cells.

We previously identified a novel genomic instability phenotype of multiple reciprocal chromosomal translocations in a MLH1-defective, microsatellite unstable (MSI) colon cancer cell line (HCA7) and, further, showed that it was unlikely to be directly caused by the mismatch repair (MMR) defect in this cell line. To gain insight into the molecular basis to this novel translocation phenotype, we examined coding and splice-site nucleotide repeat tracts in DNA repair genes for mutations by direct sequencing together with RT-PCR expression analysis of the associated transcript. The material was a selected panel of 8 MSI cell lines including HCA7. A strong candidate identified through this approach was MBD4 as it showed a homozygous truncating mutation associated with substantial loss of the transcript in HCA7 not seen in the other lines. In previous published studies, heterozygous MBD4 mutations were observed in up to 89% of sporadic MSI microdissected colon tumor foci. Using MFISH, we show that over-expression of the truncated MBD4 (+MBD4(tru)) in DLD1, a MSH6 defective, MSI human colon carcinoma cell line predisposed these cells to acquire structural chromosomal rearrangements including multiple reciprocal translocations after irradiation, reminiscent of those seen in HCA7. We also show that over-expression of MBD4(tru) in DLD1 alters the colony survival after exposure to cisplatin or etoposide. These data suggest a wide role for MBD4 in DNA damage response and maintaining chromosomal stability.

The identification of a novel alternatively spliced form of the MBD4 DNA glycosylase.

Methyl-CpG binding protein 4 (MBD4) is a mismatch-specific G:T and G:U DNA glycosylase. During an analysis of MBD4 expression in HeLa cells we noted the presence of an unexpectedly short reverse transcribed product. This cDNA lacked the region encoding the methyl-binding domain and exon 3 of MBD4 but retained the glycosylase domain. Sequence comparison indicates the existence of a previously unreported cryptic splice site in the MBD4 genomic sequence thus illuminating a mechanism whereby a glycosylase acquired a methyl-binding capacity, thus targeting potential mutagenic CpG sites. In vitro assays of this highly purified species, refolded in arginine rich conditions, confirmed that this unique, short version of MBD4 possessed uracil DNA glycosylase but not thymine DNA glycosylase activity. We conclude that the identification of a transcript encoding a short version of MBD4 indicates that MBD4 expression may be more complex than previously reported, and is worthy of further investigation.

Solvent-controlled intramolecular [2 + 2] photocycloadditions of alpha-substituted enones.

The regio- and stereoselectivity of intramolecular [2 + 2] photocycloadditions of 2'-hydroxyenones are shown to be solvent-dependent. In the presence of aprotic solvents, 2'-hydroxyenones undergo photocycloadditions in a manner consistent with the presence of an intramolecular hydrogen bond between the carbonyl group and the tether's hydroxy functionality. In protic solvents, intermolecular interactions appear to disrupt the intramolecular hydrogen bond, providing products with complementary diastereoselectivity. If the facial accessibility of the alpha-tethered olefin is limited, the cycloadditions proceed to give head-to-tail or head-to-head regioisomers, depending on the nature of the solvent employed.

Positional and functional mapping of a neuroblastoma differentiation gene on chromosome 11.

BACKGROUND: Loss of chromosome 11q defines a subset of high-stage aggressive neuroblastomas. Deletions are typically large and mapping efforts have thus far not lead to a well defined consensus region, which hampers the identification of positional candidate tumour suppressor genes. In a previous study, functional evidence for a neuroblastoma suppressor gene on chromosome 11 was obtained through microcell mediated chromosome transfer, indicated by differentiation of neuroblastoma cells with loss of distal 11q upon introduction of chromosome 11. Interestingly, some of these microcell hybrid clones were shown to harbour deletions in the transferred chromosome 11. We decided to further exploit this model system as a means to identify candidate tumour suppressor or differentiation genes located on chromosome 11. RESULTS: In a first step, we performed high-resolution array CGH DNA copy-number analysis in order to evaluate the chromosome 11 status in the hybrids. Several deletions in both parental and transferred chromosomes in the investigated microcell hybrids were observed. Subsequent correlation of these deletion events with the observed morphological changes lead to the delineation of three putative regions on chromosome 11: 11q25, 11p13-->11p15.1 and 11p15.3, that may harbour the responsible differentiation gene. CONCLUSION: Using an available model system, we were able to put forward some candidate regions that may be involved in neuroblastoma. Additional studies will be required to clarify the putative role of the genes located in these chromosomal segments in the observed differentiation phenotype specifically or in neuroblastoma pathogenesis in general.

Intramolecular [2 + 2] photocycloaddition/thermal fragmentation: formally "allowed" and "forbidden"pathways toward 5-8-5 ring systems.

The thermal fragmentation of highly functionalized, linear polycyclobutanes with a cis,syn,cis-relative stereochemistry is shown to offer a rapid entry into the dicyclopenta[a,d]cyclooctenyl (5-8-5) ring system. The thermolysis of polyfused cyclobutanes with a cis,syn,cis- or a cis,anti,cis-relationship proceeds in a formally "symmetry-allowed" manner through the intermediacy of a cis,trans-cyclooctadiene. When a bridging tether used to establish the cis,syn,cis-stereochemistry in the intramolecular [2 + 2] photocyclization is present in the thermolysis step, however, the result of a formally "symmetry-forbidden" fragmentation is observed yielding cis,cis-cyclooctadiene-containing 5-8-5 products. In general, the stereochemical observations noted in these fragmentations offer new opportunities for accessing a variety of stereochemical relationships in these 5-8-5 ring systems.

Expression of Sonic hedgehog pathway genes is altered in colonic neoplasia.

The Hedgehog (Hh) signalling pathway is crucial for normal development and patterning of numerous human organs including the gut. Hh proteins are also expressed during gastric gland development and gastric epithelial differentiation in adults. Recently, dysregulation of these developmentally important genes has been implicated in cancer, leading to the present study of the expression of Hh signalling proteins in colon cancer. In this study, normal colon and colonic lesions (hyperplastic polyp, adenoma, and colonic adenocarcinoma) were examined by immunohistochemistry using antibodies against Hh signalling molecules: the secreted protein Sonic hedgehog (SHH), its receptor Patched (PTCH), and the PTCH-associated transmembrane protein Smoothened (SMOH). The study shows that Hh signalling pathway members are expressed in normal colonic epithelium. SHH was expressed at the top of the crypts and in a few basally located cells, while PTCH was detected in the neuroendocrine cells and SMOH at the brush border of superficial epithelium. RT-PCR analysis of laser-microdissected crypts from normal human colon confirmed that mRNAs encoding these proteins were expressed in colonic epithelium. Expression of SHH, PTCH, and SMOH was up-regulated in hyperplastic polyps, adenomas, and adenocarcinomas of the colon, and SHH expression correlated with increased expression of the proliferation marker Ki-67 in all lesions examined. To address whether the Hh signalling pathway is functional in the gut, the effect of Shh on epithelial cells in vitro was explored by treating primary murine colonocytes with either Shh peptide or neutralizing anti-Shh antibody. The proportion of cells in the S-phase was assessed by bromodeoxyuridine (BrdU) incorporation. It was found that exogenous Shh promotes cell proliferation in colonocytes, while anti-Shh inhibits proliferation, suggesting that Shh is required during proliferation of epithelial cells in vitro. It is suggested that SHH is required during epithelial proliferation in the colon and that there is a possible role for Hh signalling in epithelial colon tumour progression in vivo.

Functional Smoothened is required for expression of GLI3 in colorectal carcinoma cells.

We investigated a role for Hedgehog signalling in colon cancer by studying transcription of members of the pathway in human colorectal carcinoma cell lines. We determined the methylation status and screened the gene encoding the Hedgehog receptor-associated protein Smoothened (SMO) for putative mutations. In three cell lines lacking SMO expression the SMO promoter was fully methylated and the transcription factor GLI3 was not expressed. Two additional cell lines both having one methylated SMO allele and expressing mutant SMO did not express GLI3. Our results suggest that expression of wild-type SMO is required for expression of GLI3 by a mechanism that is independent of conventional Hedgehog signalling.

MBD1, MBD2 and CGBP genes at chromosome 18q21 are infrequently mutated in human colon and lung cancers.

The genes MBD1 and MBD2 encode methyl-CpG binding proteins that suppress transcription from methylated promoters. In contrast, CGBP encodes a protein that binds promoters containing unmethylated CpG and stimulates transcription. All three are located on human chromosome 18q21, a region of frequent loss of heterozygosity in several cancers. These genes therefore represent candidate tumour suppressor genes, whose loss of function could affect the normal regulation of gene expression, whether by lack of complete suppression of genes normally silenced (via loss of MBD1 and MBD2) or by some loss of activation of genes normally expressed (via loss of CGBP), either way contributing to the tumorigenic phenotype. We have confirmed by fluorescent in situ hybridization that MBD1 and MBD2 bracket the DCC locus giving a gene order of MBD1/CGBP-DCC 5'-DCC 3'-MBD2. Mutation analyses by single-stranded conformation polymorphism in colon and lung cancer cell lines and primary tumours revealed a small number of mutations, suggesting only a limited role of these genes in human tumorigenesis.