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The chiral nature of living systems has strong implications for biologically active compounds interacting with them. Consequently, the stereoisomers of drugs can differ in both pharmacodynamic and pharmacokinetic actions. Regulatory authorities encourage the pharmaceutical industry to develop single isomers of chiral drugs. Thus, methods are required which are suitable for stereospecifically evaluating the enantiomers of a drug in quality control of enantiomerically pure drugs, e.g. the determination of the enantiomeric excess (ee). Beside the major methods, chiral HPLC and NMR spectroscopy, cyclodextrin-modified capillary electrophoresis is enjoying increasing popularity in both academic and industrial laboratories. In order to obtain a baseline separation of mixture of a minor and a major isomer which is appropriate for determining the ee, the following parameters have to be optimised: the nature and concentration of the cyclodextrin derivative, the composition of the background electrolyte, the pH value, the organic modifier, and the kind and conditioning of the capillary. In addition, the sensitivity of detection has to be taken into consideration, because a limit of detection of 0.1 percent is necessary to fulfil the requirements of the ICH guidelines. Strategies of method development will be discussed using currently reported examples.

Even though it is known that the enantiomers of a drug can exhibit different pharmacokinetic and dynamic properties, fencamfamine has been used in clinical practice as a mixture of four isomers, arising from the two centres of chirality in the molecule. The purpose of this investigation was to establish a capillary electrophoretic method for the discrimination of the isomers of fencamfamine and to determine its isomeric composition. The chiral selectors studied were uncharged α-cyclodextrin (α-CD), β-CD, heptakis (2,3-di- O -acetyl)β-cyclodextrin, γ-CD and octakis (2,3-di- O -acetyl)γ-cyclodextrin, and the negatively charged sulfobutylether-β-CD. In order to optimize the separation conditions, the selector concentration, pH values and buffer strength were varied. With Diac-β-CD and γ-CD, a very high resolution of the four isomers was found. Interestingly, the migration order of the diastereomers was different for these two selectors. In each case NMR spectroscopic studies revealed multiple isomer–cyclodextrin complexes to be responsible for the good discrimination of the isomers. In contrast to the classical inclusion complexes mostly described for CDs, fencamfamine was found to interact mainly with the outside region of the CD cavity.

NMR spectroxcopy has been used to compare the interaction of ephedrine and N-methylephedrine with β-cyclodextrin, heptakis(2,3-di-O-acetyl)β-cyclodextrin, heptakis(6-O-acetyl)β-cyclodextrin. The stoichiometry of the complexes formed between all three cyclodextrins and N-methylephedrine was found to be 1:1 by UV spectroscopy by means of the Job technique. NMR spectra of the single enantiomers of ephedrine and N-methylephedrine in the presence of all three cyclodextrins gave information about the parts of the ligands which interact differently with the host molecules and may be responsible for the chiral discrimination. To quantify the complex stabilities, binding constants were calculated from the changes in the chemical shifts of the ligand signals upon complexation. Analyses of the coupling constants of both species showed that no significant conformational change occurs upon complexation. ROESY spectra of these optical isomers with all three cyclodextrins provided detailed information about the geometry of the complexes. Different intermolecular cross-peaks between the individual isomers of ephedrine and N-Methylephedrine were found for native β-cyclodextrin and its 2,3-diacetylated derivative but not for 6-acetyl cyclodextrin. Analyses of the intramolecular cross-signals of the ligands confirmed that no significant conformational change occurs upon complexation. Chirality 9:211–219, 1997. © 1997 Wiley-Liss, Inc.

The effect of acetylation of β-cyclodextrin (CD) on the chiral discrimination of a series of phenethylamines has been investigated using capillary electrophoresis and nuclear magnetic resonance (NMR) spectroscopy. Therefore, pure, fully derivatized CDs were synthesized. Capillary electrophoresis measurements revealed that the 2,3-diacetylated CD was a better discriminator than the 6-acetylated and the native CD. NMR investigations of the complexes suggest that the phenyl ring of the phenethylamines is inserted in the cavity and the side chain interacts with the hydroxyl rim of the macrocycle. The structures of the complexes indicated by the NMR results were supported by molecular models of the derivatised CDs.

Cyclic α-melanocyte-stimulating hormone (α-MSH) analogues produced by disulphide bridging (e.g. [Cys4,Cys10]α-MSH) are known to be almost equipotent to the native hormone in amphibian skin bioassays and as a consequence have been proposed as a paradigm for the active conformation of native MSH at the pigment cell MC1 receptor. However this proposal has been somewhat speculative as there is no published data comparing biological activity of cyclic MSH analogues with data on receptor binding. This study addresses this problem by comparing tyrosinase stimulatory activity with their receptor binding affinity in B16 murine melanoma cells expressing the native MC1 melanocortin receptor. Cyclic [Cys4,Cys10] α-MSH showed almost the same affinity for the MC1 receptor as α-MSH, but the linear analogue [Cys4,Cys10]α-MSH bound less strongly. Both had biological activities similar to that of the natural ligand. Introduction of D-Phe into the ring in position 7 increased both affinity and activity of the cyclic compound. The study suggests that the intrinsic efficacy of cyclic [Cys4,Cys10]α-MSH analogues is similar to native α-MSH. Our studies support the proposal that the cyclic structure serves as a good model for the active conformation of linear α-MSH.

A β-cyclodextrin bonded stationary phase was employed for the enantioresolution of propranolol and several analogues in conjunction with various polar organic mobile phases. The effects of structural alterations in the non-polar regions of the analytes were found to exert profound changes upon chiral resolution and capacity values, indicating that features which cannot hydrogen-bond with the cyclodextrin molecule still play an important role in this chiral recognition process. This was linked to a repulsive steric effect facilitating the necessary conditions for chiral resolution. The degree of ionization of the analytes and the type and concentration of organic modifier used were also seen to influence the analytes1 enantio-selectivity and capacity values. © 1996 Wiley-Liss, Inc.

Microvascular endothelial cells were isolated from the brains of C57 mice and cultured in selective growth media. The isolation and culture techniques employed in this study minimised the contamination by nonendothelial cells such as astrocytes, pericytes, and smooth muscle cells. Microvascular endothelial cells examined using phase contrast light microscopy grew as small colonies of spindle-shaped cells which merged together to form typical contact-inhibited monolayers. The endothelial origin of these cells was determined using several established characterisation techniques. Preliminary receptor binding studies at 4 degrees using [125I-Tyr2, Nle4, D-Phe7]alpha-melanocyte-stimulating hormone ([125I-Tyr2, Nle4, D-Phe7]alpha-MSH) suggested the possibility that melanocortin receptors were present on the surface of brain microvascular endothelial cells. Subsequent binding isotherms confirmed that a small population of high-affinity melanocortin receptors was expressed. The existence of a specific binding site for alpha-MSH was confirmed by photoaffinity labeling with the 4-(1-azi-2,2,2,-trifluoroethyl)benzoic acid (ATB) derivative, [125I-Tyr2, Nle4, D-Phe7, (ATB)-Lys11] alpha-MSH. SDS-PAGE analysis identified the presence of a specific band with a molecular mass of approximately 45 kDa, which was consistent with previous data on melanoma melanocortin receptors, and represented a ligand-receptor complex. This study suggests that a receptor for alpha-MSH is expressed on the extracellular surface of murine brain microvascular endothelial cells; however, the physiological role of this receptor is as yet unknown.

The enantio-resolution of propranolol and four derivatives was examined by free solution capillary electrophoresis (FSCE) using various cyclodextrin molecules. Of the three modified cyclodextrins investigated, hydroxyethyl-β-cyclodextrin provided the largest chiral resolution values for all five analytes. This may be linked to its extended hydrogen bonding chains on the cyclodextrin rim. Methyl-β-cyclodextrin and heptakis (2,3-di-O-acetyl) β-cyclodextrin gave lower maximum analyte resolutions, probably due to differences in their macrocyclic structure and hydrogen bonding ability. The presence of a bulky, non-polar alkyl grouo on the analytes was found to enhance chiral recognition. Methanol was found to have a varied effect on chiral resolutions, dependent on the type of cyclodextrin and structure of the analyte.

Analyses of 1 H and 13 C NM R spectra of 1 5 gyrase inhibitors were carried out using mainly the hydrogen-fluorine and the carbon-fluorine coupling constants. 19 F NMR chemical shifts were obtained for pefloxacin and its metabolites

The influence of single amino acid replacements by alanine on the binding affinity and biological activity of α-MSH in B16 murine melanoma cells has been studied systematically. α-MSH analogues were synthesized by solid-phase peptide synthesis and their binding affinities to the melanocortin receptor expressed by B16 mouse melanoma cells were determined using a radioreceptor assay. Biological activity of the analogues was determined by measuring tyrosinase stimulation. Relative activity and affinity data were generally in agreement with earlier results using terminal deletion fragments of α-MSH, but the alanine scan revealed important new insights into the role of individual residues. The three terminal amino acids at either end were not necessary for binding or activity, with amino acids 4–9 forming a core sequence required for receptor binding and triggering of the biological response. It was observed that replacement of the glutamic acid residue in position 5 was possible without loss of affinity or activity, whereas replacement of Met 4 resulted in a 100-fold loss of binding affinity and biological activity. Each residue within the conserved melanocortin sequence His-Phe-Arg-Trp was shown to be essential with Phe 7 , Arg 8 , and Trp 9 being the most sensitive to replacement by alanine. Generally, there was a rank correlation between binding affinity and tyrosinase stimulation within the group of analogues studied. Tyrosinase activity was less affected by alanine substitution than binding affinity, which suggests that full receptor binding is not required for maximum biological response.

The 2-methylbutanoate tetraacetate and 2-methylbutanoate pentaacetate derivatives of synadenol, a new lathyrane diterpenoid, have been isolated from Synadenium compactum N.E. Br. var. compactum(Euphorbiaceae). The structure was elucidated using single crystal X-ray crystallography in conjunction with one and two-dimensional high field NMR experiments and mass spectrometry.

An unusual cyclisation of Z -1,2,4-tris(benzamido)butene with trifluoroacetic anhydride gives Z -4-[1,3-bis(benzamido)prop-1-enyl]-2-phenyl-5-trifluoromethyloxazole ( 3 ). The structure and stereochemistry were confirmed by 1 H, 13 C and 19 F NMR, together with 1 H 1 H COSY, 1 H 1 H NOESY and 1 H → 19 F and 19 F → 1 H heteronuclear NOE experiments. Trifluoroacetylation at the less sterically hindered N -acyl-enamine position, with subsequent cyclisation and 1,4-elimination is proposed.

Trifluoroacetylation at the 5-position of 3,4-dihydro-2H-pyran and the 3-position of 4,5-dihydrofuran, followed by treatment with hydrazine, gave 3-(3-trifluoromethyl-1H-pyrazol-4-yl)propanol and 2-(3-trifluoromethyl-1H-pyrazol-4-yl)ethanol, respectively. In the latter case, an intermediate dimer was isolated. Isomeric 2-(3-trifluoromethyl-1H-pyrazol-5-yl)ethanol was formed by reaction of hydrazine with 6-benzyloxy-1,1,1-trifluorohex-3-yn-2-one and deprotection. Reaction of 3-benzyloxypropylamine with 2,5-bis(trifluoromethyl)-1,3,4-oxadiazole, followed by deprotection, afforded 3-[3,5-bis(trifluoromethyl)-4H-1,2,4-triazol-4-yl]propanol. A series of 2-trifluoromethyl-1H-benzimidazoles and 2-trifluoromethyl-3H-imidazopyridines were prepared by condensation of the appropriate ortho-arenediamine with trifluoroacetic acid. Analysis of the 19F NMR spectra of the trifluoromethylazoles and of 3-trifluoromethylpyridine in aqueous solution at different pHs enabled determination of pKa values. All the compounds evaluated had one or more pKa between 1 and 13, except the triazole. Several compounds were identified as having potential use in measuring pH in biological media by 19F NMR spectroscopy.

The alpha-melanocyte-stimulating hormone (alpha-MSH) receptor of B16 mouse melanoma cells was characterized by photoaffinity labelling using radiolabelled photoactive derivatives of alpha-MSH. A doublet band of 43-46 kDa representing a ligand-receptor complex was identified. A novel adaptation of the streptovadin/biotin-based affinity system was used to isolate the alpha-MSH receptor. A probe was synthesized which contained biotin connected to a photolabelled alpha-MSH analogue via a cleavable disulphide linker and which displayed high affinity for the alpha-MSH receptor. Streptavidin-coated magnetic beads were used as a solid support instead of an affinity column. Covalently linked probe-receptor complexes solubilized in Triton X-100 were equilibrated with the beads, and after magnetic separation and washing, specifically bound complexes were treated with dithiothreitol to cleave the disulphide bridge in the biotin-peptide spacer arm and so release the receptor-ligand complex. The identity of the isolated protein was established by SDS/PAGE analysis. Methods to achieve purification to homogeneity and to allow quantitative isolation of the receptor are discussed.

Homo- and heteronuclear 2D NMR techniques were used to elucidate the structure of a novel pentaester of 19-hydroxyingol from Euphorbia marginata seeds. The diterpene skeleton was identified by 1H 1D and 2D COSY spectra. Heteronuclear 2D 13C1H shift correlation spectroscopy was used to assign the 13C signals. The position of the esterified hydroxymethyl group at C-10 was identified by NOE difference spectroscopy. The nature of the five ester groups and their corresponding positions on the diterpene skeleton were identified by a combination of long-range 2D 13C1H correlation experiments and 13C spectra with 1H selective decoupling.

The views expressed in this chapter are those of the authors and do not necessarily represent the views or the opinions of Medicines Control Agency, other regulatory authorities or any of their advisory committees.

Ampicillin trihydrate, sodium salt, in aqueous solution has a pH of about 8. No complete degradation pathway has been proposed to explain the degradation of ampicillin under alkaline conditions and the information available explains the formation of only certain products. The present work was carried out with the aim of providing this information. The formation of degradation products of ampicillin trihydrate, sodium salt, produced in aqueous solutions (pH 12 and 37°C) have been studied as an accelerated form of the possible degradation that may occur in aqueous solutions at pH 8. Some of the degradation products formed under these conditions were then obtained either by synthesis or by degradation of ampicillin sodium followed by isolation using semi-preparative HPLC. These compounds were characterized by 1H NMR spectroscopy. The information obtained from the experiments by HPLC and NMR spectroscopy made it possible to propose a degradation pathway for ampicillin under the conditions described above. 5R-penicilloic acid is the first degradation product of ampicillin and subsequently undergoes epimerization at C-5 to form the 55 isomer via the imine tautomer. Mechanisms for the formation of compounds previously believed to form only under acidic conditions are proposed, i.e. ampicillin penilloic acid and 2-hydroxy-3-phenylpyrazine. The formation of ampicillin polymers was detected in dilute solutions (1% w/v) within a few hours of dissolution. The presence of ampicillin penicillenic acid and ampicillin penamaldic acid was detected by 1H NMR and their main spectroscopic features determined.

The fate of alpha-melanocyte-stimulating hormone (alpha-MSH) subsequent to binding to the melanoma melanocortin MC1 receptor is of interest with regard to its potential use in targeting cytotoxic drugs or imaging to melanoma. Tools such as iodinated, photoaffinity-labelled, biotinylated and fluorescent melanocortins are required to study the fate of the ligand during its interaction with the receptor. In this study, a series of probes for the receptor based on the potent analogue, [Nle4,Dphe7]alpha-MSH, have been developed and tested for their potential usefulness. All probes contain the core melanocortin motif His-Phe-Arg-Trp. They bind the receptor readily and appear to have similar intrinsic efficacies to the endogenous peptide, so that biological activity is regulated by their receptor binding affinity. Functional photoaffinity-labelled, biotinylated and fluorescent probes are described. The biotinylated probe binds the receptor when coupled to streptavidin, although with reduced affinity.

Tumors derived from the colonic epithelium exhibit cholesterol metabolism which is clearly different from that in fibroblasts, hepatocytes, adrenals, and ovaries. In hepatocytes and fibroblasts MEV inhibition of the rate limiting step in cholesterol synthesis HMG Co A reductase can be overcome by the uptake of LDL. Colon cancer cells however do not overcome MEV inhibition by LDL uptake but rather exhibit further growth suppression Mevinolin (Mevacor), a drug used to lower serum cholesterol levels has the advantage of accumulating in the liver to approximately 95% with the first pass. A small but variable percentage of non-sterol precursors may escape inhibition and be utilized for other pathways in the isoprenylation of certain proteins, among them members of the ras family. Mutated ras, an oncogene, is found in 40-50% of colon tumors and the expression of a functional gene product is dependent on isoprenylation for anchorage to the tumor cell membrane. d-Limonene, a relatively non-toxic monoterpene found in orange skin oil, selectively inhibits isoprenylation and also accumulates to some extent in the liver. It was hypothesized that the differences in mevalonate metabolism between hepatocytes and colon tumor cells could provide a chemotherapeutic advantage in which MEV and/or d-limonene could effectively inhibit cholesterol synthesis and post-translational modification of proteins with non-sterol cholesterol precursors in colon tumor derived hepatic metastases and thus inhibit their growth. Since each drug affects aspects of mevalonate synthesis at different points, the effects of the combination of their agents on inhibiting tumor metastases was investigated to ascertain if these could be additive. In tissue culture, MEV and d-limonene significantly inhibited the growth of CT-26, a murine transplantable colon tumor. Cholesterol synthesis assessed in these cells indicated that in lipid deficient media the following additions-25-hydroxycholesterol, and LDL significantly reduced cholesterol synthesis. Conversely, perillyl alcohol increased cholesterol synthesis 2.5 fold. In cells cultured in FBS based medium, which have an FBS control, MEV treatment reduced cholesterol synthesis to 65% of control. Perillyl alcohol increased synthesis 1.4 fold and when given in conjunction with MEV, it abolished the effects of this inhibitor. In isoprenylation studies of 14C-mevalonate incorporation into proteins, MEV impaired isoprenylation by restricting synthesis of mevalonate derived intermediates. Results of CT-26 treatment with perillyl alcohol are inconsistent with its putative role as a protein isoprenylation inhibitor. The combination of these agents indicates an additive action which requires additional investigation to elucidate their mechanism(s). Dietary MEV and d-limonene were evaluated alone and in combination for their chemotherapeutic potential in a hepatic "metastasis" model. Using splenic colonization in which CT-26 was implanted into the spleen and ultimately seeded the liver, each of these compounds were found to inhibit the growth of resultant tumors both alone and in combination by approximately 80% versus controls at 35 days post-implantation. Assessment of HMGCoA reductase in liver and tumor indicated that these agents were effective in reaching these target sites. The findings to date indicate that while d-limonene and MEV may differentially affect the same pathway, and their individual actions may appear antagonistic in vitro, their overall action individually or together, appears promising as a chemotherapeutic modality for the possible management of hepatic metastases from colon cancer.

The resolution of nine sympathomimetic phenethylamine racemates by beta-cyclodextrin and heptakis(2,3-di-O-acetyl)beta-cyclodextrin has been investigated by capillary electrophoresis and 1H NMR spectroscopy. The NMR and capillary electrophoresis results showed that beta-cyclodextrin probably formed stronger complexes with the amines than did heptakis(2,3-di-O-acetyl)beta-cyclodextrin but was a poorer chiral discrimination agent in both techniques. The addition of heptakis(2,3-di-O-acetyl)beta-cyclodextrin to the capillary electrophoresis buffer gave baseline resolution of enantiomer peaks for seven of the compounds studied while beta-cyclodextrin resolved only three of the racemates.

Membrane preparations of cells expressing the cloned rat hypothalamus melanocortin receptor, MC3, have been photoaffinity labelled using a radiolabelled photoreactive analogue of alpha-MSH, [125I-Tyr2,Nle4,D-Phe7,ATB-Lys11]alpha-MSH. SDS-PAGE followed by autoradiography showed a single band at 53-56 kDa for the native receptor or 35 kDa after deglycosylated with PNGase F, consistent with the predicted cDNA sequence. Receptor binding studies with alpha-MSH, gamma-MSH and [Nle4,D-Phe7]alpha-MSH established that alpha-MSH and gamma-MSH had similar affinities while [Nle4,D-Phe7]alpha-MSH bound 100 times more strongly. These results suggest that the receptor recognises the conserved 'core sequence' (-Met-Glu/Gly-His-Phe-Arg-Trp-) of MSH/ACTH peptides. The binding affinities of alanine-substituted analogues of alpha-MSH were determined to investigate the role of individual residues in ligand-receptor interactions. While in the terminal regions only the replacement of Tyr2 reduced the affinity of the peptide, replacement of Met4, Phe7, Arg8 and Trp9 within the peptide core led to a significant loss of affinity. Glu5 appeared unimportant for receptor recognition.

The influence of the terminal amino acids of alpha-MSH on its biological action in B16 murine melanoma cells has been systematically studied. Fragments of alpha-MSH lacking various sequences of terminal residues were synthesized by solid-phase peptide synthesis and their binding affinity to melanoma cells was measured using a radioreceptor assay. Biological activity was determined by measuring both tyrosinase activity and melanogenesis. The relative affinities and activities of the fragments generally followed the same pattern as found previously in other assay systems (frog and lizard bioassay and Cloudman S91 mouse melanoma), with the three amino acids at each terminal not being essential for binding and biological activity, although the C-terminal amino acids 11-13 are more important than those in the N-terminus. The differences in biological activity between the fragments can be explained by their relative binding affinities for the receptor.

An unusual cyclisation of Z -1,2,4-tris(benzamido)butene with trifluoroacetic anhydride gives Z -4-[1,3-bis(benzamido)prop-1-enyl]-2-phenyl-5-trifluoromethyloxazole ( 3 ). The structure and stereochemistry were confirmed by 1 H, 13 C and 19 F NMR, together with 1 H 1 H COSY, 1 H 1 H NOESY and 1 H → 19 F and 19 F → 1 H heteronuclear NOE experiments. Trifluoroacetylation at the less sterically hindered N -acyl-enamine position, with subsequent cyclisation and 1,4-elimination is proposed.

Trifluoroacetylation at the 5-position of 3,4-dihydro-2H-pyran and the 3-position of 4,5-dihydrofuran, followed by treatment with hydrazine, gave 3-(3-trifluoromethyl-1H-pyrazol-4-yl)propanol and 2-(3-trifluoromethyl-1H-pyrazol-4-yl)ethanol, respectively. In the latter case, an intermediate dimer was isolated. Isomeric 2-(3-trifluoromethyl-1H-pyrazol-5-yl)ethanol was formed by reaction of hydrazine with 6-benzyloxy-1,1,1-trifluorohex-3-yn-2-one and deprotection. Reaction of 3-benzyloxypropylamine with 2,5-bis(trifluoromethyl)-1,3,4-oxadiazole, followed by deprotection, afforded 3-[3,5-bis(trifluoromethyl)-4H-1,2,4-triazol-4-yl]propanol. A series of 2-trifluoromethyl-1H-benzimidazoles and 2-trifluoromethyl-3H-imidazopyridines were prepared by condensation of the appropriate ortho-arenediamine with trifluoroacetic acid. Analysis of the 19F NMR spectra of the trifluoromethylazoles and of 3-trifluoromethylpyridine in aqueous solution at different pHs enabled determination of pKa values. All the compounds evaluated had one or more pKa between 1 and 13, except the triazole. Several compounds were identified as having potential use in measuring pH in biological media by 19F NMR spectroscopy.

The 2-methylbutanoate tetraacetate and 2-methylbutanoate pentaacetate derivatives of synadenol, a new lathyrane diterpenoid, have been isolated from Synadenium compactum N.E. Br. var. compactum(Euphorbiaceae). The structure was elucidated using single crystal X-ray crystallography in conjunction with one and two-dimensional high field NMR experiments and mass spectrometry.

The (1)H NMR characteristics of the majority of penicillin and cephalosporin beta-lactam antibiotics in world-wide clinical use are presented. Some of the data are novel and include several high resolution (220, 400 MHz) spectra. The influence of solvent and ionisation state upon spectral parameters is discussed and a scheme of analysis proposed for identifying an unknown beta-lactam sample. Spectral features of common degradation products of benzylpenicillin and other penicillins are provided and the use of (1)H NMR spectroscopy in monitoring the breakdown of penicillin antibiotics described. Other aspects discussed are NMR studies of the stereochemistry, association and protein binding of beta-lactam antibiotics.

The 13C NMR features of most β-lactam antibiotics of the penicillin and cephalosporin groups in clinical use are presented and reviewed. Data on precursor and breakdown products are included in the survey, with particular reference to benzylpenicillin. Penicillin and cephalosporin spectra are discussed, and points of similarity and difference emphasized. The potential of the data for solving problems of analysis, stability, degradation and biological structure–activity relationships is summarized.

The crystal structures of a number of fungicidal azolylmethanes are compared. In the benzyl compounds [1-aryl-4,4-dimethyl-2-(1,2,4-triazol-1-yl)pentan-3-ones, the corresponding pentan-3-ols, and 1-(4-chlorophenyl)-3-(2-fluorophenyl)-2-(1,2,4-triazol-1-yl)propan-1-one], the benzyl and tert-butyl groups (or 4-chlorophenyl group) are trans, whereas in the analogous phenoxy compounds [1-(4-chlorophenoxy)-3,3-dimethyl-1-(1,2,4-triazol-1-yl)butan-2-one and the corresponding butan-2-ol], the tert-butyl groups are trans to the triazole and gauche to the phenoxy group. Coupling constants, determined by nuclear magnetic resonance (n.m.r.) spectroscopy, suggest that for some compounds there is one dominant solution conformation. Intramolecular hydrogen bonding, observed by infrared spectroscopy in two of the compounds, supports the findings by n.m.r. For some compounds, the crystal and solution conformations appear to be very similar, whereas in others they are quite different. Published data on the relative activity of the enantiomers of the benzyl- and phenoxy-compounds are discussed, but differences in the relative activity of enantiomers in the two series cannot be readily rationalised. It is concluded that different enantiomers may have different modes of binding at the active site.

C15H17C1FN30, Mr= 309"8, orthorhombic, P212121, a = 19-813 (8), b = 13-699 (6), c = 5.777 (3)A, V= 1568.0A 3, Z=4, Din= 1.29 (2), Dx=1.31Mgm -3, MoKa, 2=0.71069A, g= 0.213 mm -~, F(000)=648, room temperature, final R =0.053 for 1003 reflections classed as observed. C atom 1 of the phenyl ring and C atoms 1 to 4 of the pentanone function adopt an extended 'W' con- formation with the atoms essentially coplanar. The triazolyl ring, which shows significant delocalization (N--N bond length 1.357 (9) /k; C--N 1.324- 1.337 ,/k), and the chlorofluorophenyl ring are twisted by 86 (1) and 58 (1) °, respectively, out of this plane. The exocyclic angles of the triazolyl ring are very asymmetric with C-N-C (130.6 (7) °) significantly larger than C-N-N (119.1 (6)°). The F atom is disordered being distributed (80,20% occupancy) be- tween positions 2 and 6 in the phenyl ring. The title compound was recrystallized from a racemic mixture but the absolute configuration ((2S)-enantiomer) of the molecules in the crystal under investigation was established by comparison of wR values for the determined and the inverted structure.

Publisher Summary This chapter discusses the applications of modern-high-field nuclear magnetic resonance (NMR) spectroscopy in medicinal chemistry. One of the most useful aspects of NMR compared with other types of spectroscopy is the elucidation of spatial geometry. Modern high-field instruments give spectra in which the fine structure of the signals is more clearly resolved but may still not always show first-order coupling. The analysis of coupling constants can yield stereochemical and conformational data, examples of which are given in the chapter. The difficulty of interpreting crowded one-dimensional spectra has been tackled by the development of experiments that display spectral data in two dimensions. Some of these experiments can be difficult to appreciate fully other than in theoretical terms. However, this should not preclude the medicinal chemist from making use of the spectra obtained for the purposes of structure elucidation. The most dramatic developments have been in the field of biological NMR. The introduction of very high-field NMR spectrometers has increased the resolution of complex spectra of biomolecules, such as peptides, proteins, and polysaccharides while the application of two-dimensional experiments has aided spectral assignment.

The preparation and stereochemical characterization of (±)-1, trans 2,6-trimethyl-4-phenyl-4-propionoxypiperidine hydrochloride and its (+)- and (−)-antipodes are described. The absolute configurations of the antipodes were established by X-ray analysis of the corresponding (−)-4-piperidinol hydrobromide. In antinociceptive tests on mice and rats, the (+)-2S,6S ester proved the more potent antipode by factors of at least 10 (rats) and 20 (mice), a result consistent with earlier proposals made about the probable uptake conformation of pethidine reversed esters at opioid receptors.

High-field NMR (13C and 1H) studies of phenindamine are reported which establish structures of the free base and some of its salts in the solute condition. The base exists as a mixture of two isomers which differ in double bond position (9–9a or 4a–9a) while most salts are 9–9a isomers. The clinically employed tartrate (Thephorin) is exceptional in being a 4a–9a ene. Salts of both double bond type exist in solution as mixtures of protonated epimers of variable epimeric ratio, that of the tartrate in D2O being approximately 1:1.