Enantiomeric Separation of Monosubstituted Tryptophan Derivatives and Metabolites by HPLC with a Cinchona Alkaloid-Based Zwitterionic Chiral Stationary Phase and Its Application to the Evaluation of the Optical Purity of Synthesized 6-Chloro-l-Tryptophan.

6-Chlorotryptophan possesses unique bioactivity and can be used as a precursor for several bioactive compounds in medicinal chemistry. It was enantioselectively synthesized by condensing 6-chloroindole with racemic N-acetylserine, followed by enzymatic hydrolysis with l-aminoacylase (EC 3.5.1.14). The optical purity was examined by conducting high-performance liquid chromatography with a Cinchona alkaloid-based zwitterionic chiral stationary phase (CSP) [CHIRALPAK(®) ZWIX(+)], which bears a chiral trans-2-aminocyclohexanesulfonic acid moiety tagged at C-9 of the Cinchona alka-loid. The zwitterionic CSP enabled efficient enantiomeric separations of monosubstituted tryptophan derivatives 1-methyltryptophan, 5-methyltryptophan, 6-methyltryptophan, 5-methoxytryptophan, and 6-chlorotryptophan with a methanol/H2O (98/2) mobile phase containing formic acid (FA) and diethylamine (DEA) additives. The mobile phase contains 25-75 mM FA and 20-50 mM DEA, enabling good separation of the enantiomers of each tryptophan derivative (α > 1.25). Thus, the optical purity of the synthesized 6-chloro-l-tryptophan was easily determined (greater than 99.0%) using HPLC with the zwitterionic CSP.

Spectroscopic investigation for the interaction of mycophenolate mofetil with ferrous ions.

The interaction of mycophenolate mofetil (MMF) with ferrous ions (Fe(2+)) in the solid state, in water, and in polar organic solvents was investigated using (1)H-NMR, (13)C-NMR, IR, and UV-visible (Vis) spectroscopies. A red-purple colored substance was formed after grinding solid MMF and FeSO4·7H2O in a mortar. The IR spectrum of taken as a KBr tablet of the colored substance showed a new absorption band at 1651 cm(-1). Although the color disappeared when the sample was dissolved in water, it persisted in organic solvents such as MeOH or dimethyl sulfoxide (DMSO). The UV-Vis spectrum of a 0.25 mM MeOH solution of MMF showed a new absorption maximum at 507 nm in the presence of Fe(2+) ions, while an aqueous solution of the same mixture showed no significant change from the MMF solution. All the signals in the (13)C-NMR spectrum in DMSO-d6 solution were unambiguously assigned. Upon the addition of 0.5 eq. of Fe(2+) ions, all the carbon signals except those of the 2-morpholinoethyl group almost disappeared, which clearly indicated that the Fe(2+) ions were located far away from the 2-morpholinoethyl groups in the MMF molecules. On the basis of these results, we have concluded that the MMF-Fe(2+) complex is actually formed in the solid state as well as in polar organic solvents such as MeOH or DMSO.

Enantioselective desymmetrization of FTY720.

A method for enantioselective desymmetrization of N-Ac and N-Boc-FTY720 by nonenzymatic asymmetric acylation was developed. Effective enantioselective monobenzoylation using benzoyl chloride in the presence of the tetraphenylbisoxazoline (L2)-CuCl2 complex gave the desired products 3a and 3b in 52-62% yield with 64% ee.

Synthesis of VIP-lipopeptide using a new linker to modify liposomes: towards the development of a drug delivery system for active targeting.

A new component for the solid phase peptide synthesis of lipopeptide, 2-[(4R,5R)-5-({[(9H-fluoren-9-yl)methoxy]carbonylaminomethyl}-2,2-dimethyl-1,3-dioxolan-4-yl)methoxy]acetic acid (2), was designed and synthesized from (-)-2,3-O-isopropylidene-D-threitol (3) in 4 steps. The key step was the selective alkylation of 3 with benzyl bromoacetate in the presence of Cs2CO3. Vasoactive intestinal peptide (VIP)-lipopeptide (1) incorporating this linker was synthesized by solid phase peptide synthesis.

Chemoselective benzylation of unprotected anthranilic acids with benzhydryl alcohols by water-soluble Au(III)/TPPMS in water.

A novel and efficient method for the benzylation of unprotected anthranilic acids with benzhydryl alcohols using water-soluble Au(III)/TPPMS in water is developed. Water plays an important role in our catalytic system. This new protocol could be used for not only N-benzylation, but also chemoselective C-benzylation by the Friedel-Crafts reaction.

[Development of unprotected syntheses in aqueous media].

Introduction of carbon side chain at C(3)-position of indole ring was accomplished by using Pd-catalyzed allylation and vinylation. The selective vinylation at C(3)-position of 4-bromoindole was applied to the synthesis of optically active 4-bromotryptophan derivatives, which was used as a starting material for the synthesis of several optically active ergot alkaloids, which were clavicipitic acids, chanoclavine-I, costacalvine, and 1,1-dimethylallyltryptophan (DMAT). The three-step synthesis of optically active clavicipitic acids were accomplished without using a protecting group starting from 4-bromoindole and dl-serine. Some new synthetic reactions using unprotected amino acids were developed. Those were the biomimetic synthesis of tryptophan, the bromination of free aromatic amino acids, and the Pd-catalyzed N-allylation of free amino acids with allylic alcohol in aqueous media. Unique reactivity of π-allyl palladium complex or η3-(benzyl)palladium complex in aqueous media was found through Pd-catalyzed reaction of anthranilic acid, 2-aminobenzamide, and indole with allylic alcohols or benzyl alcohols, respectively.

Synthesis and biological evaluation of novel tryptoline derivatives as indoleamine 2,3-dioxygenase (IDO) inhibitors.

Indoleamine 2,3-dioxygenase (IDO) plays a significant role in several disorders such as Alzheimer's disease, age-related cataracts and tumors. A series of novel tryptoline derivatives were synthesized and evaluated for their inhibitory activity against IDO. Substituted tryptoline derivatives (11a, 11c, 11e, 12b and 12c) were demonstrated to be more potent than known inhibitor MTH-Trp. Suzuki-Miyaura cross-coupling reaction of 11a-d with phenylboronic acid proceeded in high yields. In most cases, C5 and C6 substitutions on the corresponding indole ring were well tolerated. The tryptoline derivative 11c is a promising chemical lead for the discovery of novel IDO inhibitors.

A practical regioselective synthesis of alkylthio- or arylthioindoles without the use of smelly compounds such as thiols.

A convenient method for the synthesis of 3-methylthioindoles has been established which does not use smelly compounds such as thiol derivatives. The method, which introduces an alkyl- or arylthio-group into the C(3)-position of the indole skeleton, was extended to the direct introduction of a methylthio or bromo group at the C(2)-position using 3-methylthioindoles. No dimerization occurred, and the reaction mechanism was confirmed. The products have the partial structure of potent anti-methicillin-resistant Staphylococcus aureus (anti-MRSA) bromomethylthioindoles (MC 5-8) isolated from marine algae. Furthermore, this reaction could be applied to the synthesis of 3,3-diindolyl thioether which is a core structure of Echinosulfone A.

Synthesis of a key intermediate for the preparation of FTY720 analogs.

A concise synthesis of a useful intermediate 10 for the preparation of fingolimod (FTY-720) analogs was achieved by utilizing a chemoselective Sonogashira reaction of trihalobenzene 12 with alkyne 13. The reaction proceeded with high selectivity to give alkyne 11 containing the dihalobenzene moiety in good yield. Compound 11 was converted into intermediate 10 by hydrogenation without reduction of the halogen atoms.

Development of a new distyrylbenzene-derivative amyloid-ß-aggregation and fibril formation inhibitor.

Several new amyloid-ß (Aß) aggregation inhibitors were synthesized according to our theory that a hydrophilic moiety could be attached to the Aß-recognition unit for the purpose of preventing amyloid plaque formation. A distyrylbenzene-derivative, DSB(EEX)(3), which consider the Aß recognition unit (DSB, 1,4-distyrylbenzene) and expected to bind to amyloid fibrils (ß-sheet structure), was combined with the hydrophilic aggregation disrupting element (EEX) (E, Glu; X, 2-(2-(2-aminoethoxy)ethoxy)acetic acid). This DSB(EEX)(3) compound, compared to several others synthesized similarly, was found to be the most active for reducing Aß toxicity toward IMR-32 human neuroblastoma cells. Moreover, its inhibition of Aß-aggregation or fibril formation was directly confirmed by transmission electron microscopy and atomic force microscopy. These results suggest that the Aß aggregation inhibitor DSB(EEX)(3) disrupts clumps of Aß protein and is a likely candidate for drug development to treat Alzheimer's disease.

Pd-catalyzed benzylic C-H amidation with benzyl alcohols in water: a strategy to construct quinazolinones.

A novel method for the synthesis of 4-phenylquinazolinones via a palladium-catalyzed domino reaction of o-aminobenzamides with benzyl alcohols is developed. This protocol involves N-benzylation, benzylic C-H amidation, and dehydrogenation in water, which may play an important role in the smooth generation of the (η(3)-benzyl)palladium species by activation of the hydroxyl group of the benzyl alcohol.

Palladium-catalyzed S-benzylation of unprotected mercaptobenzoic acid with benzyl alcohols in water.

Palladium-catalyzed S-benzylation of unprotected mercaptobenzoic acids with benzyl alcohols gave only S-benzylated mercaptobenzoic acids in good yields. Water may play an important role for the smooth generation of the (η(3)-benzyl)palladium species by activation of the hydroxyl group of the benzyl alcohol.

Palladium-catalyzed benzylation of unprotected anthranilic acids with benzyl alcohols in water.

Palladium-catalyzed benzylation of unprotected anthranilic acids with benzyl alcohols in the presence of Pd(OAc)(2) (5 mol %) and sodium diphenylphosphinobenzene-3-sulfonate (TPPMS, 10 mol %) in water at 120 °C for 16 h gave only dibenzylated anthranilic acids in good yields. Water may play important roles for the smooth generation of the (η(3)-benzyl)palladium species by activation of the hydroxyl group of the benzyl alcohol.

Palladium-catalyzed mono-N-allylation of unprotected anthranilic acids with allylic alcohols in aqueous media.

Palladium-catalyzed N-allylation of anthranilic acids 1a-j with allyl alcohol 2a in the presence of Pd(OAc)(2), sodium diphenylphosphinobenzene-3-sulfonate (TPPMS) in THF-H(2)O at room temperature gave only mono-N-allylated anthranilic acids 3a-j in good yields (70-98%). The reactions of 4-bromoanthranilic acid 1i with 2-methyl-3-buten-2-ol 2b showed complete chemoselectivity in N-allylation (neutral conditions) and C-vinylation (basic conditions). In our catalytic system, the keys to success are use of an unprotected anthranilic acid as a starting material and the presence of water in the reaction medium. The carboxyl group of anthranilic acid and water may play important roles for the smooth generation of the π-allyl palladium species by activation of the hydroxyl group of the allylic alcohol.

Inhibition of D-amino acid oxidase activity by antipsychotic drugs evaluated by a fluorometric assay using D-kynurenine as substrate.

A facile fluorometric assay using D-kynurenine as a substrate was utilized for evaluating the inhibition of D-amino acid oxidase (DAAO), which is one of the products of a susceptibility gene for schizophrenia, by commercial antipsychotic drugs, namely, chlorpromazine (CPZ), carbamazepine, sulpiride, quetiapine, and imipramine. CPZ inhibited DAAO (65.8 ± 13.2 µM, n = 3) as reported previously, and other drugs also inhibited DAAO activity. Among these, quetiapine had the smallest IC(50) value (19.5 ± 2.60 µM, n = 3). The proposed assay can be useful for the evaluation or screening of DAAO-inhibitory drugs.

Palladium-catalyzed mono-N-allylation of unprotected amino acids with 1,1-dimethylallyl alcohol in water.

Palladium-catalyzed N-allylation of unprotected amino acids with 1,1-dimethylallyl alcohol were carried out. The reaction in the presence of Pd(OAc)(2) (5 mol%), sodium diphenylphosphinobenzene-3-sulfonate (TPPMS, 10 mol%), and AcONa (2 equiv) in water at 120 °C for 16 h in a sealed tube gave only mono-N-allylated amino acids in good yield.

Preparation of liposomes modified with lipopeptides using a supercritical carbon dioxide reverse-phase evaporation method.

Although liposomes are considered to be one of the most promising carriers for drug delivery systems (DDS), they have drawbacks such as insufficient drug-entrapment efficiency and long-term stability. The objectives of this study are to improve the trapping efficiency by addition of lipopeptides (LPs), and using a supercritical CO(2) reverse-phase evaporation (SCRPE) process, along with incorporation of PEG-modified phospholipids to improve long-term stability. In this study, bovine serum albumin (BSA) was used as a model drug substance for entrapment by liposomes. Improvements in the entrapment efficiency and stability of liposomes were achieved by modification with LPs and use of a SCRPE preparation process. The BSA-entrapment efficiency of liposomes modified with cationic LPs with arginine residues, as a result of their ionic interactions, was six times that of liposomes prepared by the Bangham method. Use of a SCRPE method along with LP modification further enhanced entrapment and enabled spontaneous formation of unilamellar liposomes with long-term stability. Liposomes consisting of DPPC/Chol/C(16)-Arg2/DSPE-PEG2000 (60/30/5/5), with up to 70% entrapment efficiency for BSA and a stability level of 90% for over 40 h, were obtained. DSC and SAXS analyses indicated that certain amounts of LP in the DPPC induced phase-transitional and structural changes in the lamellar membrane, and these changes improved the DDS carrier properties.The SCRPE method provides organic-solvent-free liposomes, and the LPs for the liposome modification are derivatives of amino acids and fatty acids, which are sustainable and biocompatible materials. This study therefore suggests that there are opportunities for the development of novel DDS carriers with excellent performance and which address environmental concerns.

Development of an enzyme-linked immunosorbent assay system to determine the presence of antibodies specific for taxane structures.

Taxanes, which are widely used in treatment of numerous cancer types, are well-known to induce hypersensitivity reactions (HSR), especially in the case of paclitaxel. Although the cause of the HSR is commonly thought to be a non-immunological direct effect of the diluent which is used to dissolve paclitaxel, some reports suggest the possibility of the presence of an immunological reaction to the common taxane structure. The aim of this study was to establish a method to determine the presence of anti-taxane antibodies in body fluids of patients who have previously received paclitaxel, in order to estimate the risk of the occurrence of HSR to other taxane compounds, such as docetaxel. To prepare an enzyme-linked immunosorbent assay (ELISA) plate for determining taxanes, 10-deacetylbaccatin III (DAB) was first succinylated by use of dimethylaminopyridine and succinic anhydride in dried pyridine. After the succinylation reaction, three different products were obtained, and these were confirmed as 7-succinoyl DAB (7-DAB), 10-succinoyl DAB (10-DAB), and 7,10-disuccinoyl DAB (7,10-DAB) by (1)H-NMR analysis. Each of these three products was conjugated with bovine serum albumin (BSA), and adsorbed on an ELISA plate. By using a commercially available anti-taxane monoclonal antibody as a model antibody, the detection limit of the anti-taxane antibodies on the 7-DAB-BSA-, 10-DAB-BSA-, and 7,10-DAB-BSA-conjugated ELISA plate was estimated as 0.3, 1 and 10 ng/ml, respectively. The ELISA system established in this study may therefore be useful for estimating the risk of HSR to taxanes in a patient prior to the use of these drugs.

Mechanism for the direct synthesis of tryptophan from indole and serine: a useful NMR technique for the detection of a reactive intermediate in the reaction mixture.

The reaction mechanism for the biomimetic synthesis of tryptophan from indole and serine in the presence of Ac(2)O in AcOH was investigated. Although the time-course (1)H-NMR spectra of the reaction of 5-methoxyindole with N-acetylserine were measured in the presence of (CD(3)CO)(2)O in CD(3)CO(2)D, the reactive intermediate could not be detected. This reaction was conducted without 5-methoxyindole in order to elucidate the reactive intermediate, but the intermediate could not be isolated from the reaction mixture. Since the intermediate would be expected to have a very short life time, and therefore be very difficult to detect by conventional analytical methods, the structure of the intermediate was elucidated using a 2D-NMR technique, diffusion-ordered spectroscopy (DOSY). Two intermediates were detected and confirmed to be 2-methyl-4-methyleneoxazol-5(4H)-one and 2-methyl-4-hydroxymethyloxazol-5(4H)-one. The present results demonstrated that DOSY is a powerful tool for the detection of unstable intermediates.

Synthesis and evaluation of a novel liposome containing BPA-peptide conjugate for BNCT.

We aimed at securing sufficient concentrations of (10)B in boron neutron capture therapy (BNCT) by developing a new drug delivery system. We have designed and developed a novel lipid analog and succeeded in using it to develop the new boron component liposome. It consisted of three different kinds of amino acid derivatives and two fatty acids, and could react directly with the peptide synthesized first on resin by Fmoc solid-phase synthesis. In this study, lipid analog conjugated with HIV-TAT peptide (domain of human immunodeficiency virus TAT protein) and boronophenylalanine (BPA) was synthesized and successfully incorporated into liposomes.