Synthesis of (2S)-isopropyl-5-alkynylpyrimidin-2-ones: precursors of beta-aminoacids

The efficient palladium-catalyzed Suzuki-Miyaura cross-coupling reaction of (2S)-isopropyl-5-iodo-2,3-dihydro-4(H)-pyrimidin-4-one with, arylethynyl-, heteroarylethynyl-, and alkylethynyltrifluoroborate salts is reported. The standard protocol was evaluated and optimized in order to gain access to suitable precursors of enantiopure 2-substituted beta-amino acids. The scope and limitations of this methodology are discussed. (C) 2010 Elsevier Ltd. All rights reserved.

Nuclear Quadrupole Resonance: A Technique to Control Hydration Processes in the Pharmaceutical Industry

Pharmaceuticals can exist in many solid forms, which can have different physical and chemical properties. These solid forms include polymorphs, solvates, amorphous, and hydrates. Particularly, hydration process can be quite common since pharmaceutical solids can be in contact with water during manufacturing process and can also be exposed to water during storage. In the present work, it is proved that NQR technique is capable of detecting different hydrated forms not only in the pure raw material but also in the final product (tablets), being in this way a useful technique for quality control. This technique was also used to study the dehydration process from pentahydrate to trihydrate.

A novel labdane diterpene from Acritopappus longifolius

A novel labdane diterpene was isolated from the plant Acritopappus longifolius. The structure of this compound was established by 1D- and 2D-nuclear magnetic resonance spectroscopic techniques and mass spectrometry data. N-Methyl-4-hydroxy-trans-proline, stigmasterol-3-O-beta-D-glycoside. and the flavonoids quercetin, luteolin, kaempferol, and rutin were also isolated. (C) 2010 Elsevier Ltd. All rights reserved.

Functionalization of (2S)-Isopropyl-5-iodo-2,3-dihydro-4(H)-pyrimidin-4-ones by a Suzuki-Miyaura Cross-Coupling Reaction Using Aryltrifluoroborate Salts: Convenient Enantioselective Preparation of alpha-Substituted beta-Amino Acids

A simple protocol for the Pd(OAc)(2)-catalyzed cross-coupling reaction of 1-benzoyl-(2S)-isopropyl-5-iodo-2,3-dihydro-4(H)-pyrimidin-4-ones with potassium aryltrifluoroborates was developed. The reaction is performed at 110 degrees C with a ligand-free catalyst. In all cases, complete conversion of the 1-benzoyl-(2S)-isopropyl-5-iodo-2,3-dihydro-4(H)-pyrimidin-4-ones and aryltrifluoroborates into the C-C coupling products was observed within 30-360 min. It is noteworthy that a large variety of groups present in the potassium aryltrifluoroborates (-CF(3), -OMe, -SEt, -CN, -CHO, -Cl, -Cbz, -NCbz, -OH, -CO(2)H) could be tolerated. Hydrogenation of the endocyclic double bonds in the Suzuki-Miyaura products followed by acid hydrolysis afforded highly enantioenriched alpha-aryl-substituted beta-amino acids.

Influence of Plasma Protein Binding on Pharmacodynamics: Estimation of In Vivo Receptor Affinities of beta Blockers Using a New Mechanism-Based PK-PD Modelling Approach

The objective of this investigation was to examine in a systematic manner the influence of plasma protein binding on in vivo pharmacodynamics. Comparative pharmacokinetic-pharmacodynamic studies with four beta blockers were performed in conscious rats, using heart rate under isoprenaline-induced tachycardia as a pharmacodynamic endpoint. A recently proposed mechanism-based agonist-antagonist interaction model was used to obtain in vivo estimates of receptor affinities (K(B),(vivo)). These values were compared with in vitro affinities (K(B),(vitro)) on the basis of both total and free drug concentrations. For the total drug concentrations, the K(B),(vivo) estimates were 26, 13, 6.5 and 0.89 nM for S(-)-atenolol, S(-)-propranolol, S(-)-metoprolol and timolol. The K(B),(vivo) estimates on the basis of the free concentrations were 25, 2.0, 5.2 and 0.56 nM, respectively. The K(B),(vivo)-K(B),(vitro) correlation for total drug concentrations clearly deviated from the line of identity, especially for the most highly bound drug S(-)-propranolol (ratio K(B),(vivo)/K(B),(vitro) similar to 6.8). For the free drug, the correlation approximated the line of identity. Using this model, for beta-blockers the free plasma concentration appears to be the best predictor of in vivo pharmacodynamics. (C) 2008 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci 98:3816-3828, 2009

[beta]-Hydroxy-F128b-Methylbutyrate (HMB) Supplementation and the Promotion of Muscle Growth and Strength

[beta]-Hydroxy [beta]-methylbutyrate (HMB), a metabolite of the essential amino acid leucine, is one of the latest dietary supplements promoted to enhance gains in strength and lean body mass associated with resistance training. Unlike anabolic hormones that induce muscle hypertrophy by increasing muscle protein synthesis, HMB is claimed to influence strength and lean body mass by acting as an anticatabolic agent, minimising protein breakdown and damage to cells that may occur with intense exercise. Research on HMB has recently tested this hypothesis, under the assumption that it may be the active compound associated with the anticatabolic effects of leucine and its metabolites. While much of the available literature is preliminary in nature and not without methodological concern, there is support for the claims made regarding HMB supplementation, at least in young, previously untrained individuals. A mechanism by which this may occur is unknown, but research undertaken to date suggests there may be a reduction in skeletal muscle damage, although this has not been assessed directly. The response of resistance trained and older individuals to HMB administration is less clear. While the results of research conducted to date appear encouraging, caution must be taken when interpreting outcomes as most manuscripts are presented in abstract form only, not having to withstand the rigors of peer review. Of the literature reviewed relating to HMB administration during resistance training, only 2 papers are full manuscripts appearing in peer reviewed journals. The remaining 8 papers are published as abstracts only, making it difficult to critically review the research. There is clearly a need for more tightly controlled, longer duration studies to verify if HMB enhances strength and muscular hypertrophy development associated with resistance training across a range of groups, including resistance trained individuals.

Solution structure of robustoxin, the lethal neurotoxin from the funnel-web spider Atrax robustus

The solution structure of robustoxin, the lethal neurotoxin from the Sydney funnel-web spider Atrax robustus, has been determined from 2D H-1 NMR data, Robustoxin is a polypeptide of 42 residues cross-linked by four disulphide bonds, the connectivities of which were determined from NMR data and trial structure calculations to be 1-15, 8-20, 14-31 and 16-42 (a 1-4/2-6/3-7/5-8 pattern), The structure consists of a small three-stranded, anti-parallel beta-sheet and a series of interlocking gamma-turns at the C-terminus. It also contains a cystine knot, thus placing it in the inhibitor cystine knot motif family of structures, which includes the omega-conotoxins and a number of plant and animal toxins and protease inhibitors. Robustoxin contains three distinct charged patches on its surface, and an extended loop that includes several aromatic and non-polar residues, Both of these structural features may play a role in its binding to the voltage-gated sodium channel. (C) 1997 Federation of European Biochemical Societies.

Validity of (-)-[H-3]-CGP 12177A as a radioligand for the 'putative beta(4)-adrenoceptor' in rat atrium

1 We have recently suggested the existence in the heart of a 'putative beta(4)-adrenoceptor' based on the cardiostimulant effects of non-conventional partial agonists, compounds that cause cardiostimulant effects at greater concentrations than those required to block beta(1)- and Bz-adrenoceptors. We sought to obtain further evidence by establishing and validating a radioligand binding assay for this receptor with (-)-[H-3]-CGP 12177A ((-)-4-(3-tertiarybutylamino-2-hydroxypropoxy) benzimidazol-2-one) in rat atrium. We investigated (-)-[H-3]-CGP 12177A for this purpose for two reasons, because it is a nonconventional partial agonist and also because it is a hydrophilic radioligand. 2 Increasing concentrations of(-)-[H-3]-CGP 12177A, in the absence or presence of 20 mu M (-)-CGP 12177A to define non-specific binding, resulted in a biphasic saturation isotherm. Low concentrations bound to beta(1)- and beta(2)-adrenoceptors (pK(D) 9.4+/-0.1, B-max 26.9+/-3.1 fmol mg(-1) protein) and higher concentrations bound to the 'putative beta(4)-adrenoceptor' (pK(D) 7.5+/-0.1, B-max 47.7+/-4.9 fmol mg(-1) protein). In other experiments designed to exclude beta(1)- and beta(2)-adrenoceptors, (-)-[H-3]-CGP 12177A (1-200 nM) binding in the presence of 500 nM (-)-propranolol was also saturable (pK(D) 7.6+/-0.1, B-max 50.8+/-7.4 fmol mg(-1) protein). 3 The non-conventional partial agonists (-)-CGP 12177A (pK(i) 7.3+/-0.2), (+/-)-cyanopindolol (pK(i) 7.6+/-0.2), (-)-pindolol (pK(i) 6.6+/-0.1) and (+)-carazolol (pk(i), 7.2+/-0.2) and the antagonist (-)-bupranolol (pK(i) 6.6+/-0.2), all competed for (-)-[H-3]-CGP 12177A binding in the presence of 500 nM (-)-propranolol at the 'putative beta(4)-adrenoceptor', with affinities closely similar to potencies and affinities determined in organ bath studies. 4 The catecholamines competed with (-)-[H-3]-CGP 12177A at the 'putative beta(4)-adrenoceptor' in a stereoselective manner, (-)-noradrenaline (pK(iH) 6.3 +/- 0.3, pK(i), 3.5 +/- 0.1), (-)-adrenaline (pK(iH) 6.5 +/- 0.2, pK(iL) 2.9 +/- 0.1), (-)-isoprenaline (pK(iH) 6.2 +/- 0.5, pK(iL) 3.3 +/- 0.1), (+)-isoprenaline (pK(i) < 1.7), (-)-R0363 ((-)-(1-(3,4-dimethoxyphenethylamino)-3-(3,4-dihydroxyphenoxy)-2-propranol)oxalate, pK(i) 5.5 +/- 0.1). 5 The inclusion of guanosine 5-triphosphate (GTP 0.1 mM) had no effect on binding of (-)-CGP 12177A or (-)-isoprenaline to the 'putative beta(4)-adrenoceptor'. In competition binding studies, (-)-CGP 12177A competed with (-)-[H-3]-CGP 12177A for one receptor state in the absence (pK(i) 7.3 +/- 0.2) or presence of GTP (pK(i) 7.3 +/- 0.2). (-)-Isoprenaline competed with (-)-[H-3]-CGP 12177A for two states in the absence (pK(iH) 6.6 +/- 0.3, pK(iL) 3.5 +/- 0.1; % H 25 +/- 7) or presence of GTP (pK(iH) 6.2 +/- 0.5, pK(iL) 3.4 +/- 0.1; % H 37 +/- 6). In contrast, at beta(1)-adrenoceptors, GTP stabilized the low affinity state of the receptor for (-)-isoprenaline. 6 The specificity of binding to the 'putative beta(4)-adrenoceptor' was tested with compounds active at other receptors. High concentrations of the beta(4)-adrenoceptor agonists, BRL 37344 ((RR + SS)[4-[2-[[2-(3-chlorophenyl)-2-hydroxy -ethyl]amino]propyl]phenoxy]acetic acid, 6 mu M), SR 58611A (ethyl((7S)-7-[(2R)-2-(3-chlorophenyl)-2-hydroxyethylamino]-5,6,7,8-tetrahydronaphtyl-2-yloxy) acetate hydrochloride, 6 mu M), ZD 2079 ((+/-)-1-phenyl-2-(2-4-carboxymethylphenoxy)-ethylamino)ethan-1-ol, 60 mu M), CL 316243 (disodium (R,R)-5-[2-[2-(3-chlorophenyl)-2-hydroxyethyl-amino]propyl]- 1,3-benzodioxole-2,2-dicarboxylate, 60 mu M) and antagonist SR 59230A (3-(2-ethylphenoxy)-1-[(1S)-1,2,3,4-tetrahydronaphth-1-ylamino]-2S-2-propanol oxalate, 6 mu M) caused less than 22% inhibition of (-)-[H-3]-CGP 12177A binding in the presence of 500 nM (-)-propranolol. Histamine (1 mM), atropine (1 mu M), phentolamine (10 mu M), 5-HT(100 mu M) and the 5-HT4 receptor antagonist SE 207710 ((1-butyl-4-piperidinyl)-methyl 8-amino-7-iodo-1 ,4-benzodioxan-5-carboxylate, 10 nM) caused less than 26% inhibition of binding. 7 Non-conventional partial agonists, the antagonist (-)-bupranolol and catecholamines all competed for (-)-[H-3]-CGP 12177A binding in the absence of (-)-propranolol at beta(1)-adrenoceptors, with affinities (pK(i)) ranging from 1.6-3.6 log orders greater than at the 'putative beta(4)-adrenoceptor'. 8 We have established and validated a radioligand binding assay in rat atrium for the 'putative beta(4)-adrenoceptor' which is distinct from beta(1)-, beta(2)- and beta(3)-adrenoceptors. The stereoselective interaction with the catecholamines provides further support for the classification of the receptor as 'putative beta(4)-adrenoceptor'.

Synthesis, structure and magnetism of the oxalato-bridged chromium(III) complex [NBu4n](4)[Cr-2(ox)(5)]center dot 2CHCl(3)

The binuclear complex [NBu4n](4)[Cr-2(ox)(5)]. 2CHCl(3) has been prepared by an ion-exchange procedure employing Dowex 50WX2 cation-exchange resin in the n-butylammonium form and potassium tris(oxalato)chromate(III). The dimeric complex was characterised by a crystal structure determination: monoclinic, space group C2/c, a = 29.241(7), b = 15.192(2), c = 22.026(5) Angstrom, beta = 94.07(1)degrees, Z = 4. The magnetic susceptibility (300-4.2 K) indicated that the chromium(III) sites were antiferromagnetically coupled (J = -3.1 cm(-1)).

Synthesis, NMR studies and conformational analysis of oxazolidine derivatives of the beta-adrenoreceptor antagonists metoprolol, atenolol and timolol

Formaldehyde-derived oxazolidine derivatives 4-7 of the beta-adrenoreceptor antagonists metoprolol 1, atenolol 2 and timolol 3 have been synthesised. Conformational analysis of 1-3 and the oxazolidine derivatives 4-7 has been performed using H-1 NMR spectroscopy and computational methods. The H-1 NMR studies show that for the aryloxypropanolamine beta-adrenoreceptor antagonists there is a predominance of the conformer in which the amine group is approximately antiperiplanar or trans to the aryloxymethylene group. Both H-1 NMR data and theoretical studies indicate that the oxazolidine derivatives 4-7 and the aryloxypropanolamine beta-adrenoreceptor antagonists 1-3 adopt similar conformations around the beta-amino alcohol moiety. Thus, oxazolidine ring formation does not dramatically alter the preferred conformation adopted by the beta-amino alcohol moiety of 1-3. Oxazolidine derivatives of aryloxypropanolamine beta-adrenoreceptor antagonists may therefore be appropriate as prodrugs, or semi-rigid analogues, when greater lipophilicity is required for drug delivery.

Mutational analysis of the cystathionine beta-synthase gene: A splicing mutation, two missense mutations and an insertion in patients with homocystinuria

RT-PCR and direct sequence analyses were used to define mutations in the cystathionine beta-synthase (CBS) gene in two unrelated male patients with vitamin B6 nonresponsive homocystinuria. Both patients were compound heterozygotes for CBS alleles containing point mutations. One patient had a maternally derived G-->A transition in the splice-donor site of intron 1, resulting in aberrant splicing of CBS mRNA. The other allele contained a missense mutation resulting in the previously reported E144K mutant CBS protein. The second patient had a maternally derived 4 bp insertion in exon 17, predicted to cause a CBS peptide of altered amino acid sequence. A 494G-->A transition was found in exon 4 of the other allele, predicting a C165Y substitution. Expression of recombinant CBS protein, containing the C165Y mutation, had no detectable catalytic activity. Each mutation was confirmed in genomic DNA. (C) 1998 Wiley-Liss, Inc.

New stereoselective routes to macrocyclic ligands

Reaction of bis(ethane-1,2-diamine)copper(II) with acetaldehyde and nitromethane in methanol leads, stereoselectively, to the new macrocyclic complex (trans-5(R),7(R),12(S),14(S))-tetramethyl-6,13-dinitro-1,4,8,11-tetraazacyclotetradecane)copper(II) perchlorate alpha-[CuL1](ClO4)(2) in good yield. Reduction of the nitro groups affords the hexaamine (L-2), which was crystallized as [H4L2](ClO4)(4) . 2H(2)O and characterized by an X-ray crystal structure study (monoclinic P2(1)/n, a = 9.763(2) Angstrom, b = 12.1988(7) Angstrom, c = 13.036(2) Angstrom, beta = 105.668(7)degrees, Z = 2) and complexed with Cu-II to produce the complex beta-[Cu(H2L2)](ClO4)(4) . 2H(2)O, which has also been characterized by X-ray crystallography (monoclinic P2(1)/n, a = 9.717(4) Angstrom, b = 12.174(2) Angstrom, c = 13.036(5) Angstrom, beta = 106.51(2)degrees, Z = 2). Reaction of alpha-[CuL1](2+) with either basic hydrogen peroxide or dilute nitrous acid leads to mild reduction of the nitro groups to afford the ketoxime L-3 as its N-based isomeric Cu-II complexes, trans-I [CuL3](ClO4)(2) and trans-II [Cu(L-3)Cl]Cl . 7H(2)O, the latter of which has been characterized structurally: triclinic, <P(1)over bar> a = 10.8441(5) Angstrom, b = 11.6632(9) Angstrom, c = 11.8723(9) Angstrom, alpha = 113.634(7)degrees, beta = 95.744(5), gamma = 94.851(5)degrees Z = 2. Variations in the configurations of the coordinated amines in [CuL1](2+), [CuL2](2+), and [CuL3](2+) have a profound effect on the spectroscopy and electrochemistry of their complexes.

(-)-CGP 12177 causes cardiostimulation and binds to cardiac putative beta(4)-adrenoceptors in both wild-type and beta(3)-adrenoceptor knockout mice

Some blockers of beta(1)- and beta(2)-adrenoceptors cause cardiostimulant effects through an atypical beta-adrenoceptor (putative beta(4)-adrenoceptor) that resembles the beta(3)-adrenoceptor. It is likely but not proven that the putative beta(4)-adrenoceptor is genetically distinct from the beta(3)-adrenoceptor. We therefore investigated whether or not the cardiac atypical beta-adrenoceptor could mediate agonist effects in mice lacking a functional beta(3)-adrenoceptor gene (beta(3)KO). (-)-CGP 12177, a beta(1)- and beta(2)-adrenoceptor blocker that causes agonist effects through both beta(3)-adrenoceptors and cardiac putative beta(4)-adrenoceptors, caused cardiostimulant effects that were not different in atria from wild-type (WT) mice and beta(3)KO mice. The effects of (-)-CGP 12177 were resistant to blockade by (-)-propranolol (200 nM) but were blocked by (-)-bupranolol (1 mu M) with an equilibrium dissociation constant of 15 nM in WT and 17 nM in beta(3)KO. (-)-[H-3]CGP 12177 labeled a similar density of the putative beta(4)-adrenoceptor in ventricular membranes from the hearts of both WT (B-max = 52 fmol/mg protein) and beta(3)KO (B-max = 53 fmol/mg protein) mice. The affinity of (-)-[H-3]CGP 12177 for the cardiac putative beta(4)-adrenoceptor was not different between WT (K-d = 46 nM) and beta(3)KO (K-d = 40 nM). These results provide definitive evidence that the cardiac putative beta(4)-adrenoceptor is distinct from the beta(3)-adrenoceptor.

Lemon oil to beta-cyclodextrin ratio effect on the inclusion efficiency of beta-cyclodextrin and the retention of oil volatiles in the complex

Microencapsulation of lemon oil was undertaken with beta-cyclodextrin using a precipitation method at the five lemon oil to beta-cyclodextrin ratios of 3:97, 6:94, 9:91, 12:88, and 15:85 (w/w) in order to determine the effect of the ratio of lemon oil to beta-cyclodextrin on the inclusion efficiency of beta-cyclodextrin for encapsulating oil volatiles. The retention of lemon oil volatiles reached a maximum at the lemon oil to beta-cyclodextrin ratio of 6:94; however, the maximum inclusion capacity of beta-cyclodextrin and a maximum powder recovery were achieved at the ratio of 12:88, in which the beta-cyclodextrin complex contained 9.68% (w/w) lemon oil. The profile and proportion of selected flavor compounds in the beta-cyclodextrin complex and the starting lemon oil were not significantly different.

Optimising fed-batch production of recombinant proteins using the baculovirus expression vector system

Fed-batch culture can offer significant improvement in recombinant protein production compared to batch culture in the baculovirus expression vector system (BEVS), as shown by Nguyen et al. (1993) and Bedard et al. (1994) among others. However, a thorough analysis of fed-batch culture to determine its limits in improving recombinant protein production over batch culture has yet to be performed. In this work, this issue is addressed by the optimisation of single-addition fed-batch culture. This type of fed-batch culture involves the manual addition of a multi-component nutrient feed to batch culture before infection with the baculovirus. The nutrient feed consists of yeastolate ultrafiltrate, lipids, amino acids, vitamins, trace elements, and glucose, which were added to batch cultures of Spodoptera frugiperda (Sf9) cells before infection with a recombinant Autographa californica nuclear polyhedrosis virus (Ac-NPV) expressing beta-galactosidase (beta-Gal). The fed-batch production of beta-Gal was optimised using response surface methods (RSM). The optimisation was performed in two stages, starting with a screening procedure to determine the most important variables and ending with a central-composite experiment to obtain a response surface model of volumetric beta-Gal production. The predicted optimum volumetric yield of beta-Gal in fed-batch culture was 2.4-fold that of the best yields in batch culture. This result was confirmed by a statistical analysis of the best fed-batch and batch data (with average beta-Gal yields of 1.2 and 0.5 g/L, respectively) obtained from this laboratory. The response surface model generated can be used to design a more economical fed-batch operation, in which nutrient feed volumes are minimised while maintaining acceptable improvements in beta-Gal yield. (C) 1998 John Wiley & Sons, Inc.