A new derivative of vitamin C and its application to the synthesis of labelled ascorbic acid

1. The preparation of a mono-O-cyclohexylidene derivative of l-ascorbic acid is described. 2. The new compound is shielded by the cyclohexanone group at C-5 and C-6 of the ascorbic acid molecule, while the double bond between C-2 and C-3 is kept intact. 3. The double bond of the new derivative is more resistant to oxidation than its parent compound. 4. Ascorbic acid is easily regenerated by mild acid hydrolysis. 5. The new derivative facilitates the synthesis of (14)C-labelled vitamin C.     

Incorporation of 5-methylorcylaldehyde and methionine into the acetogenin (polyketide) gliorosein in Gliocladium roseum I.M.I. 93065

1. methyl-(14)C-labelled 1,3-dihydroxy-4,5-dimethylbenzene, 5-methylorcylaldehyde and 5-methylorsellinic acid were synthesized from orcinol and sodium [(14)C]cyanide and tested for activity as precursors of gliorosein. ring-(14)C-labelled orcylaldehyde was also prepared. 5[(14)C]-Methylorcylaldehyde was incorporated into gliorosein (36% conversion); all the radioactivity was located in the C-methyl groups. 5-Methylorsellinic acid was decarboxylated by Gliocladium roseum and the resulting phenol was secreted into the medium. 2. The formation of an enzyme-bound derivative of 5-methylorsellinic acid as the first aromatic compound in the biosynthesis of gliorosein is suggested to explain these results. 3. ring-(14)C-labelled 3,4-dihydroxy-6-methyltoluquinone was also effectively incorporated into gliorosein and related products (20% conversion). 4. Sodium [(14)C]formate and [Me-(14)C]-methionine were incorporated into gliorosein and related products (15.4 and 22.2% conversion respectively). Isolation and estimation of the radioactivity in the O-methyl and C-methyl groups in the (14)C-labelled gliorosein thus formed showed an appreciable difference in the specific activities of the two types of methyl group (14 and 15% respectively). The results in the doubly-labelled methionine experiment indicate that the C-methyl group arises in the same manner as that in ergosterol; one of the original hydrogen atoms of the methyl group is lost. This confirms that C-methylation occurs at an ethylenic group at the aliphatic level. 5. The sequence of reactions at the aromatic level leading to the formation of gliorosein is proposed as 5-methylorsellinyl-enzyme-->3-hydroxy-5-methylorsellinyl-enzyme-->3,4-dihydroxy-6-methyltoluquinol-->3,4-dimethoxy-6-methyltoluquinol-->gliorosein.     

The absolute configuration of prunioside A from Spiraea prunifolia and biological activities of related compounds

The stereochemistry of prunioside A isolated from Spiraea prunifolia was determined by chemical transformations and NMR spectral data analysis. The configurations at C-5 and C-6 were determined to be 5S and 6R by application of the modified Mosher's method, CD analysis, and 13C NMR spectroscopic data analysis of an acetonide derivative. Other compounds related to prunioside A have inhibitory effects on the synthesis of nitric oxide in LPS-stimulated macrophage-like RAW 264.7 cells.     

Syntheses of stable isotope-labeled 6 beta-hydroxycortisol, 6 beta-hydroxycortisone,and 6 beta-hydroxytestosterone

A method is described for the preparation of two types of multi-labeled 6 beta-hydroxycortisol containing either five deuterium atoms at C-19 methyl and C-1 methylene or four 13C atoms at C-1, C-2, C-4, and C-19 in addition to the five deuterium atoms for use as analytical internal standards for gas chromatography-mass spectrometry (GC-MS). BMD derivatives of [1,1,19,19,19-2H(5)]cortisone and [1,2,4,19-13C(4),1,1,19,19,19-2H(5)]cortisone (cortisone-2H(5)-BMD and cortisone-13C(4),2H(5)-BMD) were first synthesized via indan synthon method starting from optical active 11-oxoindanylpropionic acid and labeled isopropenyl anion ([1,1,3,3,3-2H(5)]- or [1,3-13C(2),1,1,3,3,3-2H(5)]isopropenyl anion). The labeled isopropenyl anion was prepared from commercially available [1,1,1,3,3,3-2H(6)]- or [1,3-13C(2),1,1,1,3,3,3-2H(6)]acetone. Ultraviolet (UV) irradiated autoxidation at C-6 position of 3-ethyl-3,5-dienol ether derivatives of the labeled cortisone-BMDs gave 6 beta-hydroxy-[1,1,19,19,19-2H(5)]cortisone-BMD and 6 beta-hydroxy-[1,2,4,19-13C(4),1,1,19,19,19-2H(5)]cortisone-BMD, respectively, as a mixture of 6 beta- and 6 alpha-epimers in a ratio of 4:1. Separation of 6 beta- and 6 alpha-epimers by thin-layer chromatography (TLC) and subsequent hydrolysis of the BMD group at C-17 gave pure labeled 6 beta-hydroxycortisone. After protecting the keto group at C-3 of the labeled 6 beta-hydroxycortisone-BMD as semicarbazone, reduction of 11-keto group with NaBH(4) and subsequent removal of the C-3 and C-17 protecting groups gave 6beta-hydroxy-[1,1,19,19,19-2H(5)]cortisol (6 beta-hydroxycortisol-2H(5)) and 6 beta-hydroxy-[1,2,4,19-13C(4),1,1,19,19,19-2H(5)]cortisol (6 beta-hydroxycortisol-13C(4),2H(5)), respectively, as a mixture of 6 beta- and 6 alpha-epimers (6 beta:6 alpha=4.4:1). The isotopic compositions of 6 beta-hydroxycortisol-2H(5) and 6 beta-hydroxycortisol-13C(4),2H(5) were 90.9 and 92.1 at.%, respectively. Furthermore, 6 beta-hydroxy-[1 alpha,16,16,17 alpha-2H(4)]testosterone was synthesized by the UV irradiated autoxidation at C-6 position of 3-ethyl-3,5-dienol ether derivative of deuterium-labeled testosterone ([1 alpha,16,16,17 alpha-2H(4)]testosterone) obtained by using catalytic deuteration and hydrogen-deuterium exchange reactions.     

Chemical synthesis of 3β-sulfooxy-7β-hydroxy-24-nor-5-cholenoic acid: An internal standard for mass spectrometric analysis of the abnormal Δ-bile acids occurring in Niemann-Pick disease

In Niemann-Pick disease, type C1, increased amounts of 3β,7β-dihydroxy-5-cholenoic acid are reported to be present in urinary bile acids. The compound occurs as a tri-conjugate, sulfated at C-3, N-acetylglucosamidated at C-7, and N-acylamidated with taurine or glycine at C-24. For sensitive LC-MS/MS analysis of this bile acid, a suitable internal standard is needed. We report here the synthesis of a satisfactory internal standard, 3β-sulfooxy-7β-hydroxy-24-nor-5-cholenoic acid (as the disodium salt). The key reactions involved were (1) the so-called “second order” Beckmann rearrangement (one-carbon degradation at C-24) of hyodeoxycholic acid (HDCA) 3,6-diformate with sodium nitrite in a mixture of trifluoroacetic anhydride and trifluoroacetic acid, (2) simultaneous inversion at C-3 and elimination at C-6 of the ditosylate derivatives of the resulting 3α,6α-dihydroxy-24-nor-5β-cholanoic acid with potassium acetate in aqueous N,N-dimethylformamide, and (3) regioselective sulfation at C-3 of an intermediary 3β,7β-dihydroxy-24-nor-Δ⁵ derivative using sulfur trioxide-trimethylamine complex. Overall yield of the desired compound was 1.8% in 12 steps from HDCA.     

Synthesis and X-ray structures of sulfate esters of fructose and its isopropylidene derivatives. Part 1: 2,3:4,5-di-O-isopropylidene-beta-D-fructopyranose 1-sulfate and 4,5-O-isopropylidene-beta-D-fructopyranose 1-sulfate

2,3:4,5-Di-O-isopropylidene-beta-D-fructopyranose 1-sulfate have been synthesized by treatment of 2,3:4,5-di-O-isopropylidene-beta-D-fructopyranose with pyridine-sulfur trioxide complex. Direct hydrolysis of the isopropylidene group at C-4, C-5 gave 2,3-O-isopropylidene-beta-D-fructopyranose 1-sulfate. The crystal and molecular structures of ammonium (1a) and potassium (1b) salts of diisopropylidene derivative and ammonium (2) salt of monoisopropylidene derivative were determined by X-ray crystallography. Data for 1a and 1b were collected in 120 K and in 150 K for 2. All salts crystallized in P2(1)2(1)2(1) space group. There are three independent anions in asymmetric unit in 1b. Pyranose rings in the diisopropylidene derivative salts studied adopt 2S(0) twist boat conformation, whereas in the monoisopropylidene exists in a slightly distorted chair conformation (4C(1)). A staggered conformation is preferred by the sulfate group as indicated by values of C-(ester)-S-O(terminal) torsion angles: -173.2(4) degrees in 1a, 175.1(6) degrees in anion A of 1b, 170.8(6) degrees in anion C of 1b and 177.9(2) degrees in 2. However, strong interactions such as potassium-oxygen and H-bonds may affect the geometry: in anion B of 1b the value of the torsion angle is 139.4(6) degrees.     

Synthesis of novel europium-labeled estradiol derivatives for time-resolved fluoroimmunoassays.

The O-(5-carboxypentyl)-, O-(4-aminobutyl)-, O-(6-aminohexyl)oximes of 2- and 4-formylestradiol as well as the 4-carboxyethylthioether derivative of estradiol were synthesized. These estradiol derivatives were characterized using IR-, 1H-, and 13C NMR spectroscopy. All synthesized estradiol derivatives were labeled with europium chelates. These "tracers" were purified and tested in a competitive time-resolved fluoroimmunoassay with monoclonal antibody (SSI 57-2) raised against the 6-O-(carboxymethyl)oxime-bovine serum albumin derivative of estradiol. All the tested europium-labeled estradiol-4-derivatives were bound by the antibody, whereas tracers linked via position 2 were not recognized by this antibody. It was observed that tracers conjugated via C-4 gave more sensitive standard curves than tracers conjugated via C-6. Especially, the estradiol-4-thioether derivative was found to be highly useful in time-resolved fluoroimmunoassays of estradiol while using this antibody.     

The structure of the molybdenum cofactor. Characterization of di-(carboxamidomethyl)molybdopterin from sulfite oxidase and xanthine oxidase

A di-(carboxamidomethyl) derivative of molybdopterin, the organic component of the molybdenum cofactor, has been prepared under conditions favoring retention of all of the structural features of the molecule. The specific radioactivity of [1-14C]iodoacetamide incorporated relative to the amount of phosphate indicated two alkylation sites per pterin. Energy-dispersive x-ray analysis of the derivative showed the presence of 2 sulfurs in the derivative. An exact mass corresponding to the molecular formula C14H18N7O5S2 was obtained for the MH+ ion of the alkylated, dephosphorylated compound by fast atom bombardment mass spectroscopy. 1H NMR spectra of the phosphorylated and dephosphorylated forms of alkylated molybdopterin, in conjunction with the other data, have provided strong corroboration of the validity of the proposed structure of molybdopterin (Johnson, J. L., and Rajagopalan, K. V. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 6856-6860) as a 6-alkylpterin with a 4-carbon side chain containing an enedithiol on C-1' and C-2', a secondary alcohol on C-3', and a phosphorylated primary alcohol on C-4'. As isolated, the di-(carboxamido-methyl)molybdopterin was found to be a 5,6,7,8-tetrahydropterin.     

New class of potent antinociceptive and antiplatelet 10H-phenothiazine-1-acylhydrazone derivatives

In this work, we reported the synthesis and evaluation of the analgesic, antiinflammatory, and antiplatelet properties of new phenothiazine-attached acylhydrazone derivatives (6), designed exploring the molecular hybridization approach between antipsychotic chlorpromazine (4) and other heterocyclic derivatives (3) and (5) developed at LASSBio. Target compounds were synthesized in very good yields exploiting diphenylamine (7) as starting material, through regioselective functionalization of the C-1 position of 10H-phenothiazine ring. The evaluation of platelet antiaggregating profile lead us to identify a new potent prototype of antiplatelet derivative, that is (6a) (IC(50)=2.3 microM), which acts in arachidonic acid pathway probably by inhibition of platelet COX-1 enzyme. Additionally, the change of para-substituent group of acylhydrazone framework permitted us to identify hydrophilic carboxylate derivative (6g) and hydrophobic bromo derivative (6b) as two new leads of analgesics more active than dipyrone used as standard and with selective peripheral or central mechanism of action.     

Use of [2-14C]glucose and [5-14C]glucose for evaluating the mechanism and quantitative significance of the ''liver-cell'' pentose cycle.

1. Expressions are derived for the steady-state measurement of the quantitative contribution of the liver-type pentose phosphate cycle to glucose metabolism by tissues. One method requires the metabolism of [5-14C]glucose followed by the isolation and degradation of glucose 6-phosphate. The second procedure involves the metabolism of [2-14C]glucose and the isolation and degradation of a triose phosphate derivative, usually lactate or glycerol. 2. Measurements of 14C in C-2 and C-5 of glucose 6-phosphate are required and the values of the C-2/C-5 ratios can be used to calculate the quantitative contribution of the L-type pentose cycle in all tissues. 3. The measurement of 14C in C-1, C-2 and C-3 of triose phosphate derivatives can be used to calculate the quantitative contribution of the L-type pentose cycle relative to glycolysis. 4. The effect of transaldolase and transketolase exchange reactions, reactions of gluconeogenesis and non-oxidative formation of pentose 5-phosphate, isotopic equilibration of triose phosphate pools and isotopic equilibration of fructose 6-phosphate and glucose 6-phosphate, which could interfere with a clear interpretation of the data using [2-14C]- and [5-14C]glucose are discussed.     

Non-oxidative synthesis of pentose 5-phosphate from hexose 6-phosphate and triose phosphate by the L-type pentose pathway

1. Ribose 5-phosphate was non-oxidatively synthesized from glucose 6-phosphate and triose phosphate by an enzyme extract prepared from rat liver (RLEP). Analysis of the intermediates by GLC, ion-exchange chromatography and specific enzymatic analysis, revealed the presence of the following intermediates of the L-type pentose pathway: altro-heptulose 1,7-bisphosphate, arabinose 5-phosphate and D-glycero D-ido octulose 8-phosphate. 2. With either [1-14C] or [2-14C]glucose 6-phosphate as diagnostic substrates, the distribution of 14C in ribose 5-phosphate was determined. At early time intervals (0.5-8 hr), [1-14C]glucose 6-phosphate introduced 14C into C-1, C-3 and C-5 of ribose 5-phosphate, at 17 hr 14C was confined to C-1. With [2-14C]glucose 6-phosphate as substrate, 14C was confined to C-2, C-3 and C-5 of ribose 5-phosphate during early times (0.5-8 hr), while at 17 hr 14C was located in C-2. 3. The transketolase exchange reaction, [14C]ribose 5-phosphate + altro-heptulose 7-phosphate in equilibrium ribose 5-phosphate + [14C]altro-heptulose 7-phosphate, was demonstrated for the first time using purified transketolase, its activity was measured and it is proposed to play a major role in the relocation of 14C into C-3 and C-5 or ribose 5-phosphate during the prediction labelling experiments. 4. The coupled transketolase-transaldolase reactions, 2 fructose 6-phosphate in equilibrium altro-heptulose 7-phosphate + xylulose 5-phosphate and 2 altro-heptulose 7-phosphate in equilibrium fructose 6-phosphate + D-glycero D-altro octulose 8-phosphate were demonstrated with purified enzymes, but are concluded to play a minor role in the non-oxidative synthesis of pentose 5-phosphate and octulose phosphate by (RLEP). 5. The formation of gem diol and dimers of erythrose 4-phosphate is proposed to account in part for the failure to detect monomeric erythrose 4-phosphate in the carbon balance studies. 6. The equilibrium value for the pentose pathway acting by the reverse mode in vitro was measured and contrasted with the value for the pathway acting in the forward direction. The initial specific rates of the pentose pathway reactions in vitro for the reverse and forward directions are measured. 7. The study which includes carbon balance, time course changes and 14C prediction labelling experiments reports a comprehensive investigation of the mechanism of the pentose pathway acting reversibly.     

Five biflavonoids from Calycopteris floribunda (Combretaceae)

The structures of five biflavonoids, 6"-demethoxyneocalycopterone (1), calyflorenone C (2), 6"-epi-calyflorenone B (3), 6"-epi-calyflorenone C (4) and calyflorenone D (5) from the green parts of Calycopteris floribunda were established by NMR and MS. Their NMR and chiroptical properties (CD, [alpha]20D ) were compared with those of the known C. f. biflavonoids 6-11. Compound 1 represents a calycopterone derivative, 2-5 have a calyflorenone skeleton. With regard to one chiral center (C-6"), 4 and 3 are the respective epimers of 2 and 11.     

Synthesis and biological evaluation of novel 1 beta-methylcarbapenems with isothiazoloethenyl side chains

The synthesis of novel 1 beta-methylcarbapenems 1a,b bearing isothiazoloethenyl moieties at C-5 position of pyrrolidine ring and their biological evaluation are described. Both compounds showed potent and well-balanced antibacterial activity as well as high stability to DHP-I. Especially, 5-isothiazole derivative 1a exhibited excellent DHP-I stability and advanced pharmacokinetics profiles, compared to 5-isoxazole derivative 2, imipenem, and meropenem.     

N6-(1-carboxyethyl)lysine formation by Streptococcus lactis. Purification, synthesis, and stereochemical structure

During growth in an arginine-deficient (chemically defined) medium, cells of Streptococcus lactis K1 formed significant amounts of a previously undetected ninhydrin-positive compound. This intracellular compound did not cochromatograph with any of a wide range of amino acids or amino acid analogs tested. However, by two-dimensional thin layer chromatography, the unknown compound migrated close to the recently discovered N5-(1-carboxyethyl)ornithine (Thompson, J., Curtis, M. A., and Miller, S. P. F. (1986) J. Bacteriol. 167, 522-529; Miller, S. P. F., and Thompson, J. (1987) J. Biol. Chem. 262, 16109-16115). The purified compound behaved as a neutral amino acid and eluted between valine and methionine in the amino acid analyzer. The results of 1H NMR spectroscopy suggested the presence of a lysine backbone and a coupled methyl-methine unit in the molecule, and 13C NMR showed that there were nine carbon atoms, of which two (C-1 and C-7) were carboxyl carbons. The simplest structure compatible with the physicochemical data was that of an alkylated derivative of lysine. The identity of this new amino acid, N6-(1-carboxyethyl)lysine, was confirmed by chemical synthesis. In vivo labeling experiments conducted using L[U-14C]lysine and [epsilon-15N]lysine showed that exogenous lysine served as the precursor of intracellular N6-(1-carboxyethyl)lysine and that the epsilon-amino N atom was conserved during biosynthesis of the lysine derivative. Of the two possible diastereomers (2S,8S or 2S,8R) of N6-(1-carboxyethyl)lysine, comparative 13C NMR spectroscopy established that the amino acid produced by S. lactis K1 was exclusively of the 2S,8S configuration.     

Synthesis of 7alpha-substituted derivatives of 17beta-estradiol

Estrogen receptor (ER) pure antagonists such as ICI-182,780 (fulvestrant) are effective alternatives to tamoxifen (an ER antagonist/weak partial agonist) in the treatment of postmenopausal, receptor-positive human breast cancers. Structurally, these pure antagonists contain the basic core structure of 17beta-estradiol (E(2)) with a long side chain attached to its C-7alpha position. We explored and compared in this study various synthetic routes for preparing a number of C-7alpha-substituted derivatives of E(2), which are highly useful for the design and synthesis of high-affinity ER antagonists, ER-based imaging ligands, and other ER-based multi-functional agents. Using E(2) as the starting material and 1-iodo-6-benzyloxyhexane as a precursor for the C-7alpha side chain, a seven-step synthetic procedure afforded 3,17beta-bis(acetoxy)-7alpha-(6-hydroxyhexanyl)-estra-1,3,5(10)-triene (one of the derivatives prepared) in an overall yield of approximately 45% as compared to other known procedures that afforded substantially lower overall yield (8-27%). The synthetic steps for this representative compound include: (1) protection of the C-3 and C-17beta hydroxyls of E(2) using methoxymethyl groups; (2) hydroxylation of the C-6 position of the bismethoxymethyl ether of E(2); (3) Swern oxidation of the C-6 hydroxy to the ketone group; (4) C-7alpha alkylation of the C-6 ketone derivative of E(2); (5) deprotection of the two methoxymethyl groups; (6) reprotection of the C-3 and C-6 free hydroxyls with acetyl groups; (7) removal of the C-6 ketone and the benzyl group on the side chain by catalytic hydrogenation in acetic acid. As predicted, two of the representative C-7alpha-substituted derivatives of E(2) synthesized in the present study retained strong binding affinities (close to those of E(2) and ICI-182,780) for the human ERalpha and ERbeta subtypes as determined using the radioligand-receptor binding assays.     

Selective metalation of 6-methylpurines: synthesis of 6-fluoromethylpurines and related nucleosides

A selective metalation at the 6-CH3 over C-8 of 6-methylpurine derivative 6 was observed with softer counter cation (Na+ or K+) of the base, while the harder Li+ showed no selectivity. In the presence of N-fluorobenzenesulfonamide (NFSI), this property was utilized for the synthesis of 6-fluoromethylpurine derivatives 4 and 5 as potential toxins for suicide gene therapy.     

The biosynthesis of streptomycin. The origin of the C-formyl group of streptose

1. The biosynthesis of streptomycin in Streptomyces griseus has been studied by adding d-[3,4-(14)C(2)]glucose or d-[1,3-(14)C(2)]glucose to the growth medium and degrading the streptomycin produced. 2. The results suggest that the C-3' branch carbon atom of l-streptose arises from C-3 of d-glucose. 3. The mechanism of biosynthesis of streptose from glucose is discussed. It probably involves an intramolecular rearrangement of a 6-deoxy-4-oxyhexose derivative, and it is suggested that the nucleoside diphosphate sugar derivative hitherto recognized as an intermediate in the biosynthesis of l-rhamnose might participate in such a rearrangement.     

Design and synthesis of 2-acetamidomethyl derivatives of isofagomine as potential inhibitors of human lysosomal beta-hexosaminidases

As part of a program towards the development of specific inhibitors of human lysosomal beta-hexosaminidase for use as chemical chaperones in therapy of G(M2) gangliosidosis related diseases, the synthesis of 2-acetamidomethyl derivatives of isofagomine has been undertaken. Key event in this synthesis is the conversion of a C-2 substituted gluconolactone derivative into the corresponding lactam, followed by reduction to the corresponding amine. The 1-N-imino-2 acetamidomethyl derivative 5 proved to be a rather selective inhibitor with a K(i) of 2.4 microM for homogenate of human spleen lysosomal beta-hexosaminidase.     

Synthesis and biological activity of [Leu5]enkephalin derivatives containing D-glucose

The synthesis of some [Leu5]enkephalin derivatives is described in which D-glucose has been linked to the opioid pentapeptide through the ester bond involving the carboxyl function at the C-terminal with C-1 or C-6 of the D-glucopyranose moiety. Enkephalin derivatives were assayed for opioid activity and found to be full agonists in bioassays based on inhibition of electrically evoked contractions of the guinea pig ileum (GPI) and of the mouse vas deferens (MVD). The obtained results suggest that the opioid activity of the tested glucoconjugates depend upon the ester bond position in the molecule. Whereas 1-O conjugate 5 was somewhat more potent than [Leu5]enkephalin in the GPI assay, the 6-O conjugates, with the exception of 1-O-benzyl derivative 11, were considerably less potent. All enkephalin derivatives were delta-receptor selective; in particular, the acetylated analog 8 was three times more delta-receptor selective than [Leu5]enkephalin.