Synthesis and Gelling Abilities of Polyfunctional Cyclohexane-1,2-dicarboxylic Acid Bisamides: Influence of the Hydroxyl Groups.

New enantiomerically pure C16-alkyl diamides derived from trihydroxy cyclohexane-1,2-dicarboxylic acid have been synthesized from (-)-shikimic acid. The hydroxyl groups in these compounds are free or, alternatively, they present full or partial protection. Their gelling abilities towards several solvents have been tested and rationalized by means of the combined use of Hansen solubility parameters, scanning electron microscopy (SEM), and circular dichroism (CD), as well as computational calculations. All the results allowed us to account for the capability of each type of organogelator to interact with different solvents and for the main mode of aggregation. Thus, compounds with fully protected hydroxyl groups are good organogelators for methanol and ethanol. In contrast, a related compound bearing three free hydroxyl groups is insoluble in water and polar solvents including alcohols but it is able to gelate some low-polarity solvents. This last behavior can be justified by strong hydrogen bonding between molecules of organogelator, which competes advantageously with polar solvent interactions. As an intermediate case, an organogelator with two free hydroxyl groups presents an ambivalent ability to gelate both apolar and polar solvents by means of two aggregation patterns. These involve hydrogen bonding interactions of the unprotected hydroxyl groups in apolar solvents and intermolecular interactions between amide groups in polar ones.

Systematic modifications of amino acid-based organogelators for the investigation of structure-property correlations in drug delivery system.

Thermogels, used as multi-functional drug-loading materials, have properties that mainly rely on their gelator structure. Although a large variety of organogel systems are used as drug delivery carriers, relatively few have been investigated in terms of their structure-property correlations based on amino acid derivative gelators. Here, a series of amino acid based gelators were synthesized to explore the role of the gelator structure on functional properties, with the aim of establishing a connection between the molecular parameters and gel properties. By varying the three substitutions of the gelator backbone, it was found that a comprehensive interaction, consisting of hydrophobic forces, H-bonding interactions, conformational flexibility and steric repulsion, play a crucial role in determining the gelation properties. Hansen solubility parameters were employed to explore the solvent effect on the network forming and gel properties. From an analysis of the morphologies obtained from polarized optical microscope (POM), atomic force microscopic images (AFM) and scanning electron microscopy (SEM), the gelator structure was found to have an impact on the self-assembly. According to the X-ray diffraction (XRD), the possible conformations adopted by the gelators were revealed through molecular modelling. The ability to form intermolecular H-bonding is vital in molecular packing and, thus, gelation. A structure-property relationship was developed and proposed to provide a theoretical basis for controllable drug delivery implants.

pH Dependent but not P-gp Dependent Bidirectional Transport Study of S-propranolol: The Importance of Passive Diffusion.

PURPOSE: Recent controversial publications, citing studies purporting to show that P-gp mediates the transport of propranolol, proposed that passive biological membrane transport is negligible. Based on the BDDCS, the extensively metabolized-highly permeable-highly soluble BDDCS class 1 drug, propranolol, shows a high passive permeability at concentrations unrestricted by solubility that can overwhelm any potential transporter effects. Here we reinvestigate the effects of passive diffusion and carrier-mediated transport on S-propranolol. METHODS: Bidirectional permeability and inhibition of efflux transport studies were carried out in MDCK, MDCK-MDR1 and Caco-2 cell lines at different concentrations. Transcellular permeability studies were conducted at different apical pHs in the rat jejunum Ussing chamber model and PAMPA system. RESULTS: S-propranolol exhibited efflux ratios lower than 1 in MDCK, MDCK-MDR1 and Caco-2 cells. No significant differences of Papp, B->A in the presence and absence of the efflux inhibitor GG918 were observed. However, an efflux ratio of 3.63 was found at apical pH 6.5 with significant decrease in Papp, A->B and increase in Papp, B->A compared to apical pH 7.4 in Caco-2 cell lines. The pH dependent permeability was confirmed in the Ussing chamber model. S-propranolol flux was unchanged during inhibition by verapamil and rifampin. Furthermore, pH dependent permeability was also observed in the PAMPA system. CONCLUSIONS: S-propranolol does not exhibit active transport as proposed previously. The "false" positive efflux ratio can be explained by the pH partition theory. As expected, passive diffusion, but not active transport, plays the primary role in the permeability of the BDDCS class 1 drug propranolol.

Concise asymmetric synthesis of configurationally stable 4-trifluoromethyl thalidomide.

BACKGROUND: Thalidomide is one of the promising multidimensional drugs that possess high activity against serious diseases such as rheumatoid arthritis, Crohn's disease, leprosy, AIDS and various cancers. However, its medicinal applications are plagued by its configurational instability, as it easily undergoes racemization under the physiological conditions (t(1/2) = 8 h at pH 7.1, 37°C in water). Consequently, the design and synthesis of configurationally stable analogs of thalidomide continue to be an important area of research in bioorganic and medicinal chemistry. DISCUSSION: 4-trifuoromethyl thalidomide-3c was identified as an important synthetic target, which was expected to be a configurationally stable analog of thalidomide. Synthetic challenges in preparation of compound 3c were truly multipronged, considering the unique steric, electronic as well as electrostatic characteristics of trifluoromethyl group, significantly affecting properties of parent amino acids. After numerous experiments and unsuccessful attempts, both (3S,4R) and (3R, 4S) enantiomers of 4-trifluoromethyl-substituted thalidomide were effectively synthesized in six steps starting from enantio- and diastereomerically pure 3-(trifluoromethyl)pyroglutamates, prepared by highly diastereoselective Michael addition reactions between achiral glycine equivalents and chiral 3-(trifluoromethyl)acrylate. CONCLUSION: We have developed a reliable asymmetric approach for preparation of hitherto unknown 4-trifluoromethyl-substituted thalidomide in (3S,4R) and (3R,4S) enantiomerically pure forms. These thalidomide derivatives were shown to be configurationally stable and therefore may serve as useful lead compounds for the development of a new generation of thalidomide-based pharmaceuticals.

A new method for determination of both thalidomide enantiomers using HPLC systems.

Although thalidomide was withdrawn due to teratogenicity and neuropathy, there is now growing clinical interest in this compound because of its immunomodulatory and anti-angiogenic properties. In 1998, thalidomide was approved by the U.S. Food and Drug Administration for the treatment of erythema nodosum leprosum (ENL), an inflammatory complication of Hansen's disease, through a restricted-use program. Thalidomide was approved for the treatment of relapsed or refractory multiple myeloma (MM) as an orphan drug in Japan. Direct deproteinization method was shown to be useful for quantitation of enantioselective thalidomide blood level. Stabilized blood was deproteinized with methanol and 2 M trichloroacetic acid. The supernatant was injected onto reverse-phase column (CHIRALPAK AD-RH). The mobile phase consisted of 10% acetonitrile, 70% methanol and 20% 0.025 m citrate buffer (pH 3.0), and the flow rate was 0.5 ml/min. Wavelength of detection was 220 nm. (-)-(S)-thalidomide and (+)-(R)-thalidomide were separated at 13.5 min and 17.6 min, respectively. The accuracy of this method was almost the same as that of the measurement technique with extraction and concentration. In clinical practice, MM patients usually take many kinds of drugs at the same time. Actually, this patient takes a lot of drugs with thalidomide. However, we found no interference of these drugs and thalidomide on the chromatogram. This simple and reliable HPLC determination method for both enantiomers of thalidomide is thought to be very useful for thalidomide studies.

Recent advances in analytical determination of thalidomide and its metabolites.

Thalidomide, a racemate, is coming into clinical use as immuno-modulating and anti-inflammatory drug. Thalidomide was approved by the FDA in July 1998 for the treatment of erythema nodusum leprosum associated with leprosy. Recently, thalidomide is proving to be a promising drug in the treatment of a number of cancers and inflammatory diseases, such as multiple myeloma, inflammatory bowel disease (Crohn's disease), HIV and cancer associated cachexia. These effects may chiefly be exerted by S-thalidomide, but the enantiomers are inter-converted in vivo. Thalidomide is given orally, although parenteral administration would be desirable in some clinical situations. Thalidomide has been determined in formulations and, principally in biological fluids by a variety of methods such as high-performance liquid chromatography with ultraviolet detection and liquid chromatography coupled with tandem mass spectrometry. The overview includes the most relevant analytical methodologies used in its determination.

The docking of chiral epoxides on the Whelk-O1 stationary phase: a molecular dynamics study.

The docking of analytes on the Whelk-O1 chiral stationary phase is explored for two chiral epoxides in a hexane solvent. Density functional theory calculations are employed to develop flexible models for R/S-styrene oxide (phenyl oxirane) and (R,R/S,S)-stilbene oxide (2,3-diphenyl oxirane). Molecular dynamics simulations of the racemates in the presence of the Whelk-O1 chiral stationary phase reveal the distribution of the enantiomers at the interface. The importance of hydrogen bonding and ring-ring interactions is explored along with an examination of the major docking arrangements. The interactions between the Whelk-O1 molecules and the chiral epoxide enantiomers are quite distinct and consistent with the experimental elution orders [S.E. Schaus, B.D. Brandes, J.F. Larrow, M. Tokunage, K.B. Hansen, A.E. Gould, M.E. Furrow, E.N. Jacobsen, J. Am. Chem. Soc. 124 (2001) 1307] and separation factors [W.H. Pirkle, C.J. Welch, Tetrahedron: Asymm. 5 (1994) 777]. The impact of a polar solvent modifier is examined for R/S-styrene oxide where selectivity in 80:20 n-hexane:2-propanol is assessed.

Thalidomide analogs from diamines: Synthesis and evaluation as inhibitors of TNF-alpha production.

Fourteen thalidomide analogs bearing two phthalimido units were prepared in high yields (83-94%) by condensation of different diamines with phthalic or 3-nitrophthalic anhydride. An in vitro investigation of the compounds as inhibitors of the TNF-alpha production was performed. The inhibition was higher for compounds bearing amino and nitro groups and was modulated by increasing the size of the spacers between the phthalimide groups.

Chiral inversion of drugs: coincidence or principle?

2-arylpropionic acid derivatives are probably the most frequently cited drugs exhibiting the phenomenon that is best known as chiral inversion. One enantiomer of drug is converted into its antipode either in the presence of a solvent or more often in inner environment of an organism. Mechanistic studies of the metabolic chiral inversion were carried out for several drugs from NSAIDs, and a model of this inversion was suggested and subsequently confirmed. The chiral inversion of NSAIDs has been intensively studied in the context of the pharmacological and toxicological consequences. However, the group of NSAIDs is not the sole group of drugs in which the inversion phenomenon can be observed. There exist several other drugs that also display chiral inversion of one or even both of their enantiomers. These drugs belong to different pharmacotherapeutic groups as monoamine oxidase inhibitors, antiepileptic drugs, drugs used in the treatment of hyperlipoproteinemia or drugs that are effective in the treatment of leprosy. Moreover, some chiral or prochiral drugs are metabolized to give chiral metabolites that undergo chiral inversion too, which can have direct impact on pharmacological properties or toxicity of the drug. As the process of chiral inversion is affected by several factors, so the intensity of chiral inversion of individual substances and at different conditions can differ considerably. Interspecies differences and types of tissue are reported to be the main factors that were recognized to play the key role in the process of chiral inversion. Some of more recent studies have revealed that several other factors, such as the route of administration or interaction with other xenobiotics, can influence the enantiomeric conversion, too. Chiral inversion does not seem to be a phenomenon connected with only several drugs from some unique group of 2-arylpropionic acid derivatives: it is also observed in drugs with rather different chemical structures and is much more frequent than it can be realized.

Clinical pharmacokinetics of thalidomide.

Thalidomide is a racemic glutamic acid derivative approved in the US for erythema nodosum leprosum, a complication of leprosy. In addition, its use in various inflammatory and oncologic conditions is being investigated. Thalidomide interconverts between the (R)- and (S)-enantiomers in plasma, with protein binding of 55% and 65%, respectively. More than 90% of the absorbed drug is excreted in the urine and faeces within 48 hours. Thalidomide is minimally metabolised by the liver, but is spontaneously hydrolysed into numerous renally excreted products. After a single oral dose of thalidomide 200 mg (as the US-approved capsule formulation) in healthy volunteers, absorption is slow and extensive, resulting in a peak concentration (C(max)) of 1-2 mg/L at 3-4 hours after administration, absorption lag time of 30 minutes, total exposure (AUC( infinity )) of 18 mg. h/L, apparent elimination half-life of 6 hours and apparent systemic clearance of 10 L/h. Thalidomide pharmacokinetics are best described by a one-compartment model with first-order absorption and elimination. Because of the low solubility of the drug in the gastrointestinal tract, thalidomide exhibits absorption rate-limited pharmacokinetics (the 'flip-flop' phenomenon), with its elimination rate being faster than its absorption rate. The apparent elimination half-life of 6 hours therefore represents absorption, not elimination. The 'true' apparent volume of distribution was estimated to be 16L by use of the faster elimination-rate half-life. Multiple doses of thalidomide 200 mg/day over 21 days cause no change in the pharmacokinetics, with a steady-state C(max) (C(ss)(max)) of 1.2 mg/L. Simulation of 400 and 800 mg/day also shows no accumulation, with C(ss)(max) of 3.5 and 6.0 mg/L, respectively. Multiple-dose studies in cancer patients show pharmacokinetics comparable with those in healthy populations at similar dosages. Thalidomide exhibits a dose-proportional increase in AUC at doses from 50 to 400 mg. Because of the low solubility of thalidomide, C(max) is less than proportional to dose, and t(max) is prolonged with increasing dose. Age, sex and smoking have no effect on the pharmacokinetics of thalidomide, and the effect of food is minimal. Thalidomide does not alter the pharmacokinetics of oral contraceptives, and is also unlikely to interact with warfarin and grapefruit juice. Since thalidomide is mainly hydrolysed and passively excreted, its pharmacokinetics are not expected to change in patients with impaired liver or kidney function.

Studies on the volatile compounds of roasted spotted shrimp.

The aroma of spotted shrimp (Sergia lucence Hansen) was analyzed upon roasting to determine the components that constitute the characteristic roasted shrimp flavor. Our analyses resulted in the identification of ca. 200 volatiles, including high-impact sulfur and nitrogen compounds. In addition, we synthesized all possible stereoisomers of the pyrrolidine derivatives 1 and 4, and of the imine derivatives 16 and 18-20, which are very characteristic for the aroma. The odor evaluation of these chemicals revealed distinct differences, each possessing different aroma characteristics.

LC-1H NMR used for determination of the elution order of S-naproxen glucuronide isomers in two isocratic reversed-phase LC-systems.

The reactive metabolite S-naproxen-beta-1-O-acyl glucuronide was purified from human urine using solid phase extraction (SPE) and preparative HPLC. The structure was confirmed by 600 MHz 1H NMR. Directly coupled 600 MHz HPLC-1H NMR was used to assign the peaks in chromatograms obtained when analysing a sample containing S-naproxen aglycone and the 1-, 2-, 3-, and 4-isomers of S-naproxen-beta-1-O-acyl glucuronide in two simple isocratic reversed phase HPLC-systems. Using mobile phase 1 (50 mM formate buffer pH 5.75/acetonitrile 75:25 v/v) the elution order was: 4-O-acyl isomers, beta-1-O-acyl glucuronide, 3-O-acyl isomers, 2-O-acyl isomers, and S-naproxen aglycone. Using mobile phase II (25 mM potassium phosphate pH 7.40/acetonitrile 80:20 v/v) the elution order was: alpha/beta-4-O-acyl isomers, S-naproxen aglycone, beta-1-O-acyl glucuronide, 3-O-acyl isomers, and alpha/beta-2-O-acyl isomers. In both systems the elution order for the 2-, 3- and 4-O-acyl isomers corresponded with previously published results for 2-, 3-, and 4-fluorobenzoic acid glucuronide isomers determined by reversed phase HPLC-1H NMR (U.G. Sidelmann, S.H. Hansen, C. Gavaghan, A.W. Nicholls, H.A.J. Carless, J.C. Lindon, I.D. Wilson, J.K. Nicholson, J. Chromatogr. B Biomed. Appl. 685 (1996) 113-122]. The alpha-1-O-acyl isomer was found to be present at approximately 3% of the initial S-naproxen-beta-1-O-acyl glucuronide concentration in the glucuronide isomer mixture after 6 h of incubation at pH 7.40 and 37 degrees C. In both HPLC systems it eluted just before the beta-1-O-acyl glucuronide well separated from other isomers. Investigators should consider the possible formation of a alpha-1-O-acyl isomer when studying glucuronide reactivity and degradation.

Novel biological response modifiers derived from thalidomide.

Thalidomide (N-alpha-phthalimidoglutarimide) was used widely as a hypnotic/sedative agent in the late 1950s and the early 1960s, but had to be withdrawn from the market because of its severe teratogenicity. In spite of this, there has been a resurgence of interest in the drug in recent years due to its potential usefulness for the treatment of various diseases, including acquired immunodeficiency syndrome (AIDS) and graft-versus-host disease (GVHD). The effectiveness of the drug in these diseases has been attributed to its specific inhibitory activity on tumor necrosis factor-alpha (TNF-alpha) production. Because TNF-alpha, a cytokine mediating host defence and immune regulation, with a wide range of activities, has deleterious pathophysiological effects in various diseases, including AIDS, tumors, rheumatoid arthritis and diabetes, its production-regulators are attractive lead compounds for novel biological response modifiers. The regulatory effect of thalidomide on TNF-alpha production has been found to be bidirectional, depending on both the cell-type and the TNF-alpha production-inducer; i.e., thalidomide possesses both enhancing and inhibiting activities on TNF-alpha production. Structural modification of thalidomide aiming at the creation of superior TNF-alpha production-regulators has afforded a number of phenyl- and benzylphthalimide analogs possessing more potent activity than thalidomide itself. The structure-activity relationships of these analogs has been investigated. The bidirectional TNF-alpha production-regulating activity is electronic state- and enantio-dependent, and both pure inhibitors and pure enhancers of TNF-alpha production has been obtained. Further structural development of the phthalimide analogs has yielded potent non-steroidal androgen antagonists.

Characteristic new members of the phthiocerol and phenolphthiocerol families from Mycobacterium ulcerans.

Diacyl phthiodiolone A and phenolphthiodiolone A lipids were isolated from two strains of Mycobacterium ulcerans. The diol units of the phthiodiolone A and phenolphthiodiolone A components were shown to have erythro stereochemistry by infrared spectroscopy and proton nuclear magnetic resonance of an acetal derivative. This stereochemistry is shared only by related diols from M. marinum, the diols from M. bovis, M. kansasii, M. leprae and M. tuberculosis having threo stereochemistry.

Conformational changes in the IgG molecule of lepromatous sera using laser Raman spectroscopy.

In the present work we report our studies on IgG separated from the serum of lepromatous patients and non-lepromatous leprosy cases using Laser Raman Spectroscopy. Striking spectral changes in LL cases have been observed in the following special regions: (a) the amide I and III, (b) the S-S and C-S stretching (c) the skeletal bending and (d) skeletal stretching regions. These changes indicate a decrease in the amount of beta-structure and a transition towards alpha-helical conformation.

The use of a potential-sensitive cyanine dye for studying ion-dependent electrogenic renal transport of organic solutes. Uptake of L-malate and D-malate by luminal-membrane vesicles.

The mechanisms of uptake of dicarboxylic acids by rabbit renal luminal-membrane vesicles were studied by the use of filtration and spectrophotometric techniques as described in an accompanying paper [Kragh-Hansen, Jørgensen & Sheikh (1982) Biochem. J.208, 359-368]. Addition of l- or d-malate to dye-membrane-vesicle suspensions in the presence of Na(+) gradients (extravesicular>intravesicular) resulted in spectral curves indicative of depolarization events. The renal uptake of dicarboxylic acids was dependent on the type of Na(+)-salt anion present and could be correlated with the ability of the anions to penetrate biological membranes (i.e. Cl(-)>SO(4) (2-)>gluconate). Identical results were obtained by a filtration technique with Sartorius membrane filters. The results indicate that the dicarboxylic acids are taken up by the membrane vesicles in an electrically positive form (i.e. Na(+)/substrate coupling ratio 3:1) by an Na(+)-dependent transport system. This proposal was further supported by spectrophotometric experiments with various ionophores such as valinomycin, gramicidin and nigericin. The absorbance changes associated with simultaneous addition of l- and d-malate and spectrophotometric competition studies revealed that the two isomers are taken up by a common transport system. Spectral changes of the dye induced by addition of increasing concentrations of l- or d-malate indicated that the transport system favours the unphysiological d-form rather than the l-form of malate. Furthermore, it was observed that the affinity of both isomers for the transport system was dependent on the concentration of Na(+) in the medium.

Regulation of macromolecular synthesis in reovirus-infected L-929 cells I. Effect of L-histidinol.

The histidine analogue L-histidinol, reported by Vaughan and Hansen (1973) to establish a potent, readily reversible inhibition of eukaryotic protein synthesis in vivo, was used to investigate the regulation of macromolecular synthesis in reovirus-infected L-929 cells. The addition of L-histidinol to normal L cells led to a total inhibition of protein synthesis. The inhibition appeared to be a consequence neither of isotope dilution resulting from elevated endogenous amino acids nor of an inability of treated cells to accumulate exogenous amino acids. Addition of L-histidine to histidinol-arrested cells resulted in a complete recovery of protein synthesis. Similarly, protein synthesis in reovirus-infected L cells examined 17 h postinfection (31 C) was totally inhibited by histidinol treatment and was readily reversed by the addition of histidine. Reovirus-infected cells treated with histidinol had an essentially unaltered capacity to synthesize reovirus single-stranded RNA relative to unperturbed cultures but a diminishing ability to maintain genome RNA synthesis. Addition of L-histidine to arrested cultures led to a complete recovery of genome RNA synthesis. The L-histidinol-mediated arrest of protein synthesis was both very effective and easily reversed, suggesting the general applicability of this novel inhibitor to investigations of regulation of macromolecular synthesis in both normal and virus-infected eukaryotic cells.

Unusual effects of reducing agents on 0-diphenoloxidase of Mycobacterium leprae.

Reducing agents had no effect on the oxidation of 3,4-dihydroxyphenylalanine (DOPA) to quinone by Mycobacterium leprae; no quinone formation by o-diphenoloxidase of mammalian or plant origin was detected under similar experimental conditions. Ascorbic acid and reduced glutathione prevented further oxidation and polymerization of the quinone to melanin by M. leprae; cysteine was less effective. In the presence of reducing agents, the quinone (indole-5,6-quinone) formed from DOPA by M. leprae was not reduced back to diphenol. On the other hand, the quinone (dopachrome) produced from DOPA by mammalian or plant phenolase was rapidly decolorized by reducing agents. Oxidized glutathione and cystine had little effect on o-diphenoloxidase from all of the three sources. Cyanide, which completely inhibited mammalian and plant phenolases, had only a partial effect on the enzyme in the bacilli. Various lines of evidence suggest that the properties of o-diphenoloxidase in M. leprae are different from those of similar enzymes obtained from other sources.