David Austen Perry

Green chemistry tools to influence a medicinal chemistry and research chemistry based organisation

Influencing and improving the environmental performance of a large multi-national pharmaceutical company can be achieved with the help of electronic education tools, backed up by site champions and strong site teams. This paper describes the development of two of those education tools.

Isozyme-nonselective N-Substituted Bipiperidylcarboxamide Acetyl-CoA Carboxylase Inhibitors Reduce Tissue Malonyl-CoA Concentrations, Inhibit Fatty Acid Synthesis, and Increase Fatty Acid Oxidation in Cultured Cells and in Experimental Animals

Inhibition of acetyl-CoA carboxylase (ACC), with its resultant inhibition of fatty acid synthesis and stimulation of fatty acid oxidation, has the potential to favorably affect the multitude of cardiovascular risk factors associated with the metabolic syndrome. To achieve maximal effectiveness, an ACC inhibitor should inhibit both the lipogenic tissue isozyme (ACC1) and the oxidative tissue isozyme (ACC2). Herein, we describe the biochemical and acute physiological properties of CP-610431, an isozyme-nonselective ACC inhibitor identified through high throughput inhibition screening, and CP-640186, an analog with improved metabolic stability. CP-610431 inhibited ACC1 and ACC2 with IC50s of ∼50 nm. Inhibition was reversible, uncompetitive with respect to ATP, and non-competitive with respect to bicarbonate, acetyl-CoA, and citrate, indicating interaction with the enzymatic carboxyl transfer reaction. CP-610431 also inhibited fatty acid synthesis, triglyceride (TG) synthesis, TG secretion, and apolipoprotein B secretion in HepG2 cells (ACC1) with EC50s of 1.6, 1.8, 3.0, and 5.7 μm, without affecting either cholesterol synthesis or apolipoprotein CIII secretion. CP-640186, also inhibited both isozymes with IC50sof ∼55 nm but was 2–3 times more potent than CP-610431 in inhibiting HepG2 cell fatty acid and TG synthesis. CP-640186 also stimulated fatty acid oxidation in C2C12 cells (ACC2) and in rat epitrochlearis muscle strips with EC50s of 57 nm and 1.3 μm. In rats, CP-640186 lowered hepatic, soleus muscle, quadriceps muscle, and cardiac muscle malonyl-CoA with ED50s of 55, 6, 15, and 8 mg/kg. Consequently, CP-640186 inhibited fatty acid synthesis in rats, CD1 mice, and ob/ob mice with ED50s of 13, 11, and 4 mg/kg, and stimulated rat whole body fatty acid oxidation with an ED50 of ∼30 mg/kg. Taken together, These observations indicate that isozyme-nonselective ACC inhibition has the potential to favorably affect risk factors associated with the metabolic syndrome.

Torcetrapib Induces Aldosterone and Cortisol Production by an Intracellular Calcium-Mediated Mechanism Independently of Cholesteryl Ester Transfer Protein Inhibition

ILLUMINATE (Investigation of Lipid Level Management to Understand its Impact in Atherosclerotic Events), the phase 3 morbidity and mortality trial of torcetrapib, a cholesteryl ester transfer protein (CETP) inhibitor, identified previously undescribed changes in plasma levels of potassium, sodium, bicarbonate, and aldosterone. A key question after this trial is whether the failure of torcetrapib was a result of CETP inhibition or of some other pharmacology of the molecule. The direct effects of torcetrapib and related molecules on adrenal steroid production were assessed in cell culture using the H295R as well as the newly developed HAC15 human adrenal carcinoma cell lines. Torcetrapib induced the synthesis of both aldosterone and cortisol in these two in vitro cell systems. Analysis of steroidogenic gene expression indicated that torcetrapib significantly induced the expression of CYP11B2 and CYP11B1, two enzymes in the last step of aldosterone and cortisol biosynthesis pathway, respectively. Transcription profiling indicated that torcetrapib and angiotensin II share overlapping pathways in regulating adrenal steroid biosynthesis. Hormone-induced steroid production is mainly mediated by two messengers, calcium and cAMP. An increase of intracellular calcium was observed after torcetrapib treatment, whereas cAMP was unchanged. Consistent with intracellular calcium being the key mediator of torcetrapibs effect in adrenal cells, calcium channel blockers completely blocked torcetrapib-induced corticoid release and calcium increase. A series of compounds structurally related to torcetrapib as well as structurally distinct compounds were profiled. The results indicate that the pressor and adrenal effects observed with torcetrapib and related molecules are independent of CETP inhibition.

Crystal Structures of Cholesteryl Ester Transfer Protein in Complex with Inhibitors

Background: Human cholesteryl ester transfer protein (CETP) transfers cholesteryl esters from high-density to low-density lipoprotein particles. Results: Crystallographic, mutagenesis, and biochemical studies illuminated inhibition mechanisms of CETP by torcetrapib and a structurally distinct compound, ((2R)-3-{[4-(4-chloro-3-ethylphenoxy)pyrimidin-2-yl][3-(1,1,2,2-tetrafluoroethoxy)benzyl]amino}-1,1,1-trifluoropropan-2-ol. Conclusion: These small molecules inhibit CETP through blocking its lipid tunnel. Significance: Potential polar interactions at compound binding site may be utilized in design of inhibitors with improved physical properties. Human plasma cholesteryl ester transfer protein (CETP) transports cholesteryl ester from the antiatherogenic high-density lipoproteins (HDL) to the proatherogenic low-density and very low-density lipoproteins (LDL and VLDL). Inhibition of CETP has been shown to raise human plasma HDL cholesterol (HDL-C) levels and is potentially a novel approach for the prevention of cardiovascular diseases. Here, we report the crystal structures of CETP in complex with torcetrapib, a CETP inhibitor that has been tested in phase 3 clinical trials, and compound 2, an analog from a structurally distinct inhibitor series. In both crystal structures, the inhibitors are buried deeply within the protein, shifting the bound cholesteryl ester in the N-terminal pocket of the long hydrophobic tunnel and displacing the phospholipid from that pocket. The lipids in the C-terminal pocket of the hydrophobic tunnel remain unchanged. The inhibitors are positioned near the narrowing neck of the hydrophobic tunnel of CETP and thus block the connection between the N- and C-terminal pockets. These structures illuminate the unusual inhibition mechanism of these compounds and support the tunnel mechanism for neutral lipid transfer by CETP. These highly lipophilic inhibitors bind mainly through extensive hydrophobic interactions with the protein and the shifted cholesteryl ester molecule. However, polar residues, such as Ser-230 and His-232, are also found in the inhibitor binding site. An enhanced understanding of the inhibitor binding site may provide opportunities to design novel CETP inhibitors possessing more drug-like physical properties, distinct modes of action, or alternative pharmacological profiles.

N-benzylimidazole carboxamides as potent, orally active stearoylCoA desaturase-1 inhibitors.

A potent, small molecule inhibitor with a favorable pharmacokinetic profile to allow for sustained SCD inhibition in vivo was identified. Starting from a low MW acyl guanidine (5a), identified with a RapidFire High-Throughput Mass Spectrometry (RF-MS) assay, iterative library design was used to rapidly probe the amide and tail regions of the molecule. Singleton synthesis was used to probe core changes. Biological evaluation of a SCD inhibitor (5b) included in vitro potency at SCD-1 and in vivo modulation of the plasma desaturation index (DI) in rats on a low essential fatty acid (LEFA) diet. In addition to dose-dependent decrease in DI, effects on rodent ocular tissue were noted. Therefore, in rat, these SCD inhibitors only recapitulate a portion of phenotype exhibited by the SCD-1 knockout mouse.

Identification of potent, selective, CNS-targeted inverse agonists of the ghrelin receptor

The optimization for selectivity and central receptor occupancy for a series of spirocyclic azetidine-piperidine inverse agonists of the ghrelin receptor is described. Decreased mAChR muscarinic M2 binding was achieved by use of a chiral indane in place of a substituted benzylic group. Compounds with desirable balance of human in vitro clearance and ex vivo central receptor occupancy were discovered by incorporation of heterocycles. Specifically, heteroaryl rings with nitrogen(s) vicinal to the indane linkage provided the most attractive overall properties.

Structure-pharmacokinetic relationship of in vivo rat biliary excretion.

Accurately measuring and predicting biliary excretion would be extremely valuable in evaluating the contribution of biliary excretion to the total systemic clearance, understanding potential mechanisms of hepatobiliary toxicity as well as potentials for drug–drug interactions in drug discovery. In this study, in vivo rat biliary excretion of drug‐like molecules was measured using bile duct cannulated rats. Literature biliary excretion data with similar experimental conditions were collected. A predictive quantitative structure–pharmacokinetic relationship (QSPR) model was developed using genetic algorithm guided principal component regression analysis and 2D molecular descriptors. In the derived model, hydrophobicity expressed with calculated distribution coefficients (cLogD) is the most important molecular property correlating biliary excretion. The derived model has been validated using literature data, and should be useful in estimating biliary excretion potentials of molecules in drug discovery. Copyright

Cyclic homopentapeptides. 1. Analogs of tuberactinomycins and capreomycin with activity against vancomycin-resistant enterococci and Pasteurella

Abstract A 6a-(3′,4′-dichlorophenylamino) analog of viomycin was uncovered by a high-throughput screen against the animal health pathogen Pasteurella haemolytica, and has served as a novel lead structure for our infectious disease programs. We report herein the synthesis and activity of analogs of tuberactinomycins and capreomycin that are active against Pasteurella spp., methicillin-resistant Staphylococcus aureus, and vancomycin-resistant enterococci. This paper describes the synthesis and activity of some C-6a-substituted analogs of tuberactinomycins and capreomycin, which are active against Pasteurella spp., methicillin-resistant Staphylococcus aureus, and vancomycin-resistant enterococci.

An experimental strategy towards optimising directed biosynthesis of communesin analogues by Penicillium marinum in submerged fermentation.

It was previously demonstrated that a fungus producing communesin alkaloids, subsequently identified as Penicillium marinum, could also accept 6-fluoro analogues of tryptophan or tryptamine to form mono-fluoro-communesin analogues in addition to communesins. A strategy to increase the relative yield of analogues by mutation to impair decarboxylation of tryptophan has been studied. Four mutants with much reduced activity of tryptophan decarboxylase, and other phenotypic change, were selected from 1500 colonies from spores that survived a 99% kill treatment with N-methyl N-nitro N-nitrosoguanidine. Tlc assessment of cell-associated products from standard submerged fermentations showed that one non-sporing mutant apparently produced little or no communesins, but productivity was restored when grown in a medium supplemented with glutamine. However, more sensitive mass spectrometric analysis detected both communesins A and B in mycelium grown on a rich, yeast extract-sucrose agar, showing that deletion of communesin biosynthesis was not absolute. It was concluded that mutagenesis had generally achieved its objective, but that new literature on a putative role of aurantioclavine in communesin biosynthesis presented an additional challenge to integrate the prenylation of tryptophan before its decarboxylation, which is a characteristic of ergot alkaloid biosynthesis.

C-13β-ACYLOXYMILBEMYCINS, A NEW FAMILY OF MACROLIDES

A family of novel milbemycins possessing C-13 beta-acyloxy substitution was produced by Streptomyces hygroscopicus ATCC 53718. These compounds were detected by HPLC diode array analysis and possess anthelmintic and ectoparasiticidal activity. The origin of the oxygen atom at C-13 is discussed.