Predicting and tuning physicochemical properties in lead optimization: amine basicities

This review describes simple and useful concepts for predicting and tuning the pK(a) values of basic amine centers, a crucial step in the optimization of physical and ADME properties of many lead structures in drug-discovery research. The article starts with a case study of tricyclic thrombin inhibitors featuring a tertiary amine center with pK(a) values that can be tuned over a wide range, from the usual value of around 10 to below 2 by (remote) neighboring functionalities commonly encountered in medicinal chemistry. Next, the changes in pK(a) of acyclic and cyclic amines upon substitution by fluorine, oxygen, nitrogen, and sulfur functionalities, as well as carbonyl and carboxyl derivatives are systematically analyzed, leading to the derivation of simple rules for pK(a) prediction. Electronic and stereoelectronic effects in cyclic amines are discussed, and the emerging computational methods for pK(a) predictions are briefly surveyed. The rules for tuning amine basicities should not only be of interest in drug-discovery research, but also to the development of new crop-protection agents, new amine ligands for organometallic complexes, and in particular, to the growing field of amine-based organocatalysis.     

1-O-alkyl-2-(ω-oxo)acyl-sn-glycerols from shark oil and human milk fat are potential precursors of PAF mimics and GHB

This study examines the feasibility that peroxidation and lipolysis of 1-O-alkyl-2,3-diacyl-sn-glycerols (DAGE) found in shark liver oil and human milk fat constitutes a potential source of dietary precursors of platelet activating factor (PAF) mimics and of gamma-hydroxybutyrate (GHB). Purified DAGE were converted into 1-O-alkyl-2-acyl-sn-glycerols by pancreatic lipase, without isomerization, and transformed into 1-O-alkyl-2-oxoacyl-sn-glycerols by mild autooxidation. The various core aldehydes without derivatization, as well as the corresponding dinitrophenylhydrazones, were characterized by chromatographic retention time and diagnostic ions by online electrospray mass spectrometry. Core aldehydes of oxidized shark liver oil yielded 23 molecular species of 1-O-alkyl-sn-glycerols with short-chain sn-2 oxoacyl groups, ranging from 4 to 13 carbons, some unsaturated. Autooxidation of human milk fat yielded 1-O-octadecyl-2-(9-oxo)nonanoyl-sn-glycerol, as the major core aldehyde. Because diradylglycerols with short fatty chains are absorbed in the intestine and react with cytidine diphosphate-choline in the enterocytes, it is concluded that formation of such PAF mimics as 1-O-alkyl-2-(ω-oxo)acyl-sn-glycerophosphocholine from unsaturated dietary DAGE is a realistic possibility. Likewise, a C₄ core alcohol produced by aldol-keto reduction of a C₄ core aldehyde constitutes a dietary precursor of the neuromodulator and recreational drug GHB, which has not been previously pointed out.     

An efficient method for the synthesis of peptide aldehyde libraries employed in the discovery of reversible SARS coronavirus main protease (SARS-CoV Mpro) inhibitors

A method for the parallel solid-phase synthesis of peptide aldehydes has been developed. Protected amino acid aldehydes obtained by the racemization-free oxidation of amino alcohols with Dess-Martin periodinane were immobilized on threonyl resins as oxazolidines. Following Boc protection of the ring nitrogen to yield the N-protected oxazolidine linker, peptide synthesis was performed efficiently on this resin. A peptide aldehyde library was designed for targeting the SARS coronavirus main protease, SARS-CoV M(pro)(also known as 3CL(pro)), on the basis of three different reported binding modes and supported by virtual screening. A set of 25 peptide aldehydes was prepared by this method and investigated in inhibition assays against SARS-CoV M(pro). Several potent inhibitors were found with IC(50) values in the low micromolar range. An IC(50) of 7.5 muM was found for AcNSTSQ-H and AcESTLQ-H. Interestingly, the most potent inhibitors seem to bind to SARS-CoV M(pro) in a noncanonical binding mode.     

Kinetics of Thermal Dehydroxylation and Carbonation of Magnesium Hydroxide

The kinetics of simultaneous dehydroxylation and carbonation of precipitated Mg(OH)₂ were studied using isothermal and nonisothermal thermogravimetric analyses. Specimens were analyzed using X-ray diffraction, transmission electron microscopy, and through measurements of the volume of carbon dioxide evolved in a subsequent reaction with hydrochloric acid. From 275° to 475°C, the kinetics of isothermal dehydroxylation in helium were best fit to a contracting-sphere model, yielding an activation energy of 146 kJ/mol, which was greater than values reported in the literature for isothermal dehydroxylation under vacuum (53–126 kJ/mol). The carbonation kinetics were complicated by the fact that dehydroxylation occurred simultaneously. The overall kinetics also could be fit to a contracting-sphere model, yielding a net activation energy of 304 kJ/mol. The most rapid carbonation kinetics occurred near 375°C. At this temperature, Mg(OH)₂ underwent rapid dehydroxylation and subsequent phase transformation, whereas thermodynamics favored the formation of carbonate. During carbonation, MgCO₃ precipitated on the surface of disrupted Mg(OH)₂ crystals acting as a kinetic barrier to both the outward diffusion of H₂O and the inward diffusion of CO₂.     

Endogenous Nitric-Oxide Synthase Inhibitor ADMA after Acute Brain Injury

Previous results on nitric oxide (NO) metabolism after traumatic brain injury (TBI) show variations in NO availability and controversial effects of exogenous nitric oxide synthase (NOS)-inhibitors. Furthermore, elevated levels of the endogenous NOS inhibitor asymmetric dimethylarginine (ADMA) were reported in cerebro-spinal fluid (CSF) after traumatic subarachnoid hemorrhage (SAH). Therefore, we examined whether ADMA and the enzymes involved in NO- and ADMA-metabolism are expressed in brain tissue after TBI and if time-dependent changes occur. TBI was induced by controlled cortical impact injury (CCII) and neurological performance was monitored. Expression of NOS, ADMA, dimethylarginine dimethylaminohydrolases (DDAH) and protein-arginine methyltransferase 1 (PRMT1) was determined by immunostaining in different brain regions and at various time-points after CCII. ADMA and PRMT1 expression decreased in all animals after TBI compared to the control group, while DDAH1 and DDAH2 expression increased in comparison to controls. Furthermore, perilesionally ADMA is positively correlated with neuroscore performance, while DDAH1 and DDAH2 are negatively correlated. ADMA and its metabolizing enzymes show significant temporal changes after TBI and may be new targets in TBI treatment.     

The G-Protein-Coupled Estrogen Receptor (GPER/GPR30) in Ovarian Granulosa Cell Tumors

Ovarian granulosa cell tumors (GCTs) are thought to arise from cells of the ovarian follicle and comprise a rare entity of ovarian masses. We recently identified the G-protein-coupled estrogen receptor (GPER/GPR30) to be present in granulosa cells, to be regulated by gonadotropins in epithelial ovarian cancer and to be differentially expressed throughout folliculogenesis. Thus, supposing a possible role of GPER in GCTs, this study aimed to analyze GPER in GCTs. GPER immunoreactivity in GCTs (n = 26; n (primary diagnosis) = 15, n (recurrence) = 11) was studied and correlated with the main clinicopathological variables. Positive GPER staining was identified in 53.8% (14/26) of GCTs and there was no significant relation of GPER with tumor size or lymph node status. Those cases presenting with strong GPER intensity at primary diagnosis showed a significant reduced overall survival (p = 0.002). Due to the fact that GPER is regulated by estrogens, as well as gonadotropins, GPER may also be affected by endocrine therapies applied to GCT patients. Moreover, with our data supposing GPER to be associated with GCT prognosis, GPER might be considered as a possible confounder when assessing the efficacy of hormone-based therapeutic approaches in GCTs.     

Mechanical properties of L929 cells measured by atomic force microscopy: Effects of anticytoskeletal drugs and membrane crosslinking

To shed light on the architecture of the cytoskeleton, we used the atomic force microscope (AFM) to measure the elasticity, viscoelasticity, and plasticity of L929 cells. The initial elastic response (Young's modulus ～ 4,000 Pa) of the cells to an applied force was followed by a slow compression of the cytoskeleton (τ_1/2 ≈ 10 s). When force application was terminated, the cytoskeleton underwent a sudden partial decompression and a subsequent slow, incomplete recovery. The role of the cytoskeletal elements in cell mechanics was accessed in AFM measurements carried out on cells treated with cytochalasin D, nocodazole, or col-cemid. Cytochalasin D treatment reduced both elasticity (～45%) and cytoplasmic viscosity (～65%), whereas cells treated with nocodazole or colcemid exhibited a marked increase in elasticity (～100%) and a slight increase in viscosity (～15%). The AFM force measurements also provided evidence that the cell membrane and the cytoskeleton are mechanically coupled. Tightly adherent cells were stiffer than cells that were loosely attached. Moreover, cells crosslinked with either glutaraldehyde, 3,3 ‘-dithiobis’sul-fosuccinimidylpropionate] (DTSSP), or Concanavalin A were more rigid than untreated cells. It is of interest that cells crosslinked with Concanavalin A, but not DTSSP, displayed plastic behaviors that may reflect the induction of cytoskeletal reorganization by Concanavalin A.     

Effects of introduced aspartic and glutamic acid residues on the Pi substrate specificity, pH dependence and stability of carboxypeptidase Y

Carboxypeptidase Y is a serine carboxypeptidase isolated fromSaccharomyces cerevisiae with a preference for Cterminal hydrophobicamino acid residues. In order to alter the inherent substratespecificity of CPD-Y into one for basic amino acid residuesin P'₁, we have introduced Asp and/or Glu residues at a numberof selected positions within the Si binding site. Hie effectsof these substitutions on the substrate specificity, pH dependenceand protein stability have been evaluated. The results presentedhere demonstrate that it is possible to obtain significant changesin the substrate preference by introducing charged amino acidsinto the framework provided by an enzyme with a quite differentspecificity. The introduced acidic amino acid residues providea marked pH dependence of the (k_{cat/Km)FA-A-R-OH/(kcatm})_FA-A-R-OHratio. The change in stability upon introduction of Asp/Gluresidues can be correlated to the difference in the mean buriedsurfac surface area between the substituted and the substitutingamino acid. Thus, the effects of acidic amino acid residueson the protein stability depend upon whether the introducedamino acid protrudes from the solvent accessible surface asdefined by the surrounding residues in the wild type enzymeor is submerged below.