Chemical synthesis of two novel diaryl ether dimers of estradiol-17beta

We recently detected the formation of estradiol-17beta (estradiol) dimers, linked together through a diaryl ether bond between the C-3 phenolic oxygen of one estradiol molecule and the 2- or 4-position aromatic carbon of another estradiol, following incubations of [3H]estradiol with human liver microsomes or cytochrome p450 enzymes in the presence of NADPH. Using estradiol as the starting material, we designed a four-step method for the chemical synthesis of these two estrogen dimers with the Ullmann condensation reaction as a key step. Step 1: Synthesis of 2- or 4-bromoestradiol from estradiol. Step 2: Protection of the C-3 phenolic hydroxyl group of the 2- or 4-bromoestradiol. Step 3: The Ullmann condensation reaction between the phenol-protected bromoestradiol and the estradiol potassium salt under our modified reaction conditions (with a 41% product yield). Step 4: Removal of the C-3 benzyl group by catalytic hydrogenation. The chromatographic and various spectrometric properties of the two synthesized compounds were identical to those metabolically formed by human cytochrome p450 3A4.     

Regioselective reaction of thiols with catechol estrogens and estrogen-O-quinones

Incubations of [3H]estradiol and [3H]2-hydroxyestradiol (2-OHE2) with rat liver microsomes and mushroom tyrosinase were carried out in the presence of glutathione and 2-mercaptoethanol. A ratio of about 3.5:1 for the C-4 and C-1 thioether conjugates of 2-OHE2 was observed. Chemical reaction of estradiol-2, 3-O-quinone with various thiols showed that alkyl and phenyl thiols gave about a 1:1 ratio of C-4 to C-1 thioethers. However, reaction of the O-quinone with 4-nitrothiophenol gave a C-4/C-1 ratio of 0.25 while 4-bromothiophenol gave a C-4/C-1 ratio of 4.0. These studies suggest that the regioselectivity of the reaction of thiols with estrogen catechols and O-quinones may be dependent on the nature of the thiol compounds and less on steric hindrance.     

Metabolism of 2,2'-dihydroxybiphenyl by Pseudomonas sp. strain HBP1: production and consumption of 2,2',3-trihydroxybiphenyl.

Cells of Pseudomonas sp. strain HBP1 grown on 2-hydroxy- or 2,2'-dihydroxybiphenyl contain NADH-dependent monooxygenase activity that hydroxylates 2,2'-dihydroxybiphenyl. The product of this reaction was identified as 2,2',3-trihydroxybiphenyl by 1H nuclear magnetic resonance and mass spectrometry. Furthermore, the monooxygenase activity also hydroxylates 2,2',3-trihydroxybiphenyl at the C-3' position, yielding 2,2',3,3'-tetrahydroxybiphenyl as a product. An estradiol ring cleavage dioxygenase activity that acts on both 2,2',3-tri- and 2,2',3,3'-tetrahydroxybiphenyl was partially purified. Both substrates yielded yellow meta-cleavage compounds that were identified as 2-hydroxy-6-(2-hydroxyphenyl)-6-oxo-2,4-hexadienoic acid and 2-hydroxy-6-(2,3-dihydroxyphenyl)-6-oxo-2,4-hexadienoic acid, respectively, by gas chromatography-mass spectrometry analysis of their respective trimethylsilyl derivatives. The meta-cleavage products were not stable in aqueous incubation mixtures but gave rise to their cyclization products, 3-(chroman-4-on-2-yl)pyruvate and 3-(8-hydroxychroman-4-on-2-yl)pyruvate, respectively. In contrast to the meta-cleavage compounds, which were turned over to salicylic acid and 2,3-dihydroxybenzoic acid, the cyclization products are not substrates to the meta-cleavage product hydrolase activity. NADH-dependent salicylate monooxygenase activity catalyzed the conversions of salicylic acid and 2,3-dihydroxybenzoic acid to catechol and pyrogallol, respectively. The partially purified estradiol ring cleavage dioxygenase activity that acted on the hydroxybiphenyls also produced 2-hydroxymuconic semialdehyde and 2-hydroxymuconic acid from catechol and pyrogallol, respectively.     

Enhancing the synthetic utility of aldolase antibody 38C2

Three-phase partitioning (TPP) treated aldolase antibody 38C2 was evaluated for aldol reaction between p-nitrobenzaldehyde and acetone to give 4-(4'-nitrophenyl)-4-hydroxy-2-butanone. While TPP-treated 38C2 transformed 65% of p-nitrobenzaldehyde, the untreated 38C2 gave only 24% transformation in 18 h at 25 degrees C. However, since TPP-treated 38C2 also gave an additional (unidentified) product, its synthetic utility was limited. Crosslinked aggregate of 38C2, however, gave the biocatalyst which gave a single product and could be reused at 40 degrees C five times without loss of activity.     

Synthesis of estrogen methyl ethers by extractive alkylation.

A rapid method for the quantitative preparation of a number of estrogen methyl ethers is described. Estrogen in aqueous base is extracted as an ion pair with the tetrahexylammonium oin into methylene chloride where irreversible alkylation (extractive alkylation) by methyl iodide occurs. Gas chromatography (GC) - mass spectrometry (MS) provided the basis for identification of the methylated products. Estrone (1) and estradiol were easily 3-0-methylated whereas estriol gave a dimethylated product. Further experiments suggested that dimethylation of 2-hydroxyestrone in reasonable yield was possible.     

Estrogen conjugation and antibody binding interactions in surface plasmon resonance biosensing

Thioether-linked 3-mercaptopropionic acid derivatives of 17beta-estradiol and estrone were formed at the A-ring 4-position of the steroids by substitution of their 4-bromo analogues. The carboxylic acid terminal was used to link to an oligoethylene glycol (OEG) chain of 15-atoms in length. The OEG derivative of 17beta-estradiol was then in situ immobilized on a carboxymethylated dextran-coated gold sensor surface used to detect refractive index changes upon protein binding to the surface by surface plasmon propagation in a BIAcore surface plasmon resonance (SPR) instrument. Two other estradiol-OEG derivatives with Mannich reaction linkage at the 2-position and hemisuccinate linkage at the 3-position were also immobilized on the sensor surfaces for comparison. Binding performance between these immobilized different positional conjugates and monoclonal anti-estradiol antibody, raised from a 6-position conjugate, clearly demonstrated that both 2- and 4-conjugates, not conjugated through existing functional groups, gave strong antibody bindings, whereas the 3-conjugate through an existing functional group (3-OH) gave very little binding (2% compared to the 2-conjugate). Both 2- and 4-position conjugates were then applied in a highly sensitive estradiol SPR immunoassay with secondary antibody mediated signal enhancement that gave up to a 9.5-fold signal enhancement of primary antibody binding, and a detection limit of 25 pg/mL was achieved for a rapid and convenient flow-through immunoassay of estradiol.     

Method for the synthesis of uric acid derivatives

A general procedure to obtain tetra-substituted uric acid by stepwise N-alkylation is described. 2,6-Dichloropurine (1) was condensed with 1-propanol by Mitsunobu reaction to give 9-propyl congener (2). Treatment of 2 with ammonia gave adenine derivative (4a), which was converted to the 8-oxoadenine (5b) in 3 steps. Methylation of 5b proceeded site-specifically to give 6-amino-2-chloro-7,8-dihydro-7-methyl-9-propylpurin-8-one (6) as a sole product. Compound 6 was successively treated with NaNO2 and iodomethane to give 2-chloro-1,6,7,8-tetrahydro-1,7-dimethyl-9-propylpurin-6,8-dione (9) accompanied by the O6-methyl product (8) in 75% and 6.9%, respectively. After nucleophilic substitution of 9 with NaOAc, the product (11) was reacted with iodomethane to give the uric acid (12) and the 2-methoxy product (13) in 46% and 15.5%, respectively. However, the reaction of 11 with the benzylating agents gave only O-benzyl products (14a,b).     

Synthesis and transformations of new annulated pyranosides using the Pauson-Khand reaction

The synthesis and transformations of new annulated pyranosides are described. These adducts were prepared by Pauson-Khand reaction on differently functionalized prop-2-ynyl-2,3-dideoxy-alpha-D-erythro-hex-2-enopyranosides (1-8). Compound 1 with a free hydroxyl group at C-4 afforded significant amounts of the hydrogenolysis product 12 in addition to the normal adduct 13. The C-4 O-protected similar precursors (2-8) gave PK products in yields ranging from 39 to 63%. Pauson-Khand adduct 19 provided intermediate 23 after selective manipulation. The oxidation plus decarbonylation synthetic sequence applied to intermediate 23 gave a poor yield of compound 24 using Wilkinson's catalyst. The t-butyl hydroperoxide promoted decarbonylation of product 23 afforded formate 25 in a typical Baeyer-Villiger rearrangement. The Ferrier-II reaction on intermediate 45, readily available from compound 9, afforded the hydrindane-type derivative 46 in 34% yield using a Ferrier-II type reaction.     

Estrogen 1,2-epoxides or estrogen quinones/semiquinones

Metabolic activation of estradiol leading to the formation of catechol estrogens is a prerequisite for its genotoxic activity. Both estrogen-o-quinones/semiquinones and estrogen 1,2-epoxides have been proposed to be responsible for this activity. Incubations of [3H]estradiol and [3H]1 alpha,2 alpha-epoxy-4-estrene-3-one-17 beta-ol (ketotautomer of estradiol 1,2-epoxide) with rat liver microsomal and cytosol preparations were carried out in the presence of SKF 525A, ascorbic acid, glutathione and cysteine. Ascorbic acid decreased binding to proteins and aqueous-soluble fraction with both [3H] estradiol and [3H]epoxyestrenolone in incubations with microsomes but no effect with cytosol fraction. Incubations of microsomes with thiols gave water-soluble metabolites which were characterized as 1(4)-thioether derivatives of 2-hydroxyestradiol and incubations of [3H]epoxyestrenolone with cytosol and thiols gave estradiol-2-thioether. Incubations with ascorbic acid and thiols resulted in decreased formation of water-soluble metabolites in microsomal incubations but not in cytosol incubations. These studies indicate that the major pathway for irreversible binding of estrogens to macromolecules involves estrogen-o-quinones/semiquinones and not estrogen 1, 2-epoxide.     

The stereoselectivity of the Paternò-Büchi reaction between tertiary 2-furylmethanol derivatives and aromatic carbonyl compounds: on the nature of the hydroxy directing effect.

The photochemical reaction of 1-(2-furyl)-1-phenylethanol with benzaldehyde gave a mixture of regioisomeric products. The adduct obtained on the more hindered side of the molecule was obtained with complete diastereoselectivity. The same substrate with benzophenone gave only one product with a diastereoisomeric excess of 48%. The reaction of 2-(2-furyl)-3,3-dimethylbutan-2-ol with benzaldehyde and benzophenone gave the corresponding adducts on the more hindered side of the molecule with diastereoisomeric excesses of 42 and 71%, respectively. These results, and also those obtained using 2-furylphenylmethanol with benzophenone and acetone (complete diastereoselectivity and absence of diastereoselectivity, respectively), were explained assuming the attack of the excited carbonyl compound on the same side as the hydroxy group, through the formation of a hydrogen bond or of a complex. This type of attack gave the biradical intermediate in preferential conformations. The relative energies of these conformers account for the observed diastereoselectivity.     

Absence of reactive intermediates in the formation of catechol estrogens by rat liver microsomes

Release of 3H2O from regiospecifically labeled estradiol was measured during 2-hydroxylation of this estrogen by rat liver microsomes. The amount of tritium remaining in the isolated catechol estrogen was also determined. Virtually all the tritium was removed from C-2 during the reaction confirming the absence of an NIH shift. About 20% of the tritium at C-1 was also lost without any such change occurring at C-4 or C-6,7 of the steroid molecule. These findings provide evidence for the formation of an arene oxide or o-semiquinone intermediate during the conversion of estradiol to 2-hydroxyestradiol. No indication of adduct formation at either C-1 or C-4 during this biotransformation was obtained although the 2-hydroxylated product was able to react with a nucleophile such as glutathione. The different regiospecificity of tritium loss in the generation of catechol estrogens and in their subsequent reaction leads to the important conclusion that the reactive intermediates in the two processes must be different. The possible role of catechol estrogens in neoplastic transformation is discussed.     

Tricyclic furanoid dichloroacetyl orthoesters of D-mannose from 1,2-O-trichloroethylidene-beta-D-mannofuranose

1,2-O-(R)-Trichloroethylidene-beta-D-mannofuranose (1) was obtained from the reaction of D-mannose with chloral. Reaction of 1 with potassium tert-butoxide (3 Mequiv) gave the thermodynamically stable 1,2,5-O-orthodichloroacetyl-beta-D-mannofuranose as the sole product whereas 1.5 Mequiv of reagent gave the kinetically controlled 1,2,3-O-orthodichloroacetyl-beta-D-mannofuranose (10) as the main product. Orthoester 10 gave the 5,6-isopropylidene derivative, which was also obtained from the reaction of 5,6-O-isopropylidene-1,2-O-(R)-trichloroethylidene-beta-D-mannofuranose with potassium tert-butoxide (1.5 Mequiv). These novel orthoesters are expected to prove useful as protecting groups and as building blocks in the formations of new mannofuranisidic units.     

Synthesis of 5'-O-beta-D-glucopyranosyl and 5'-O-beta-D-galactopyranosyl derivatives of ribavirin

The synthesis of 5'-O-beta-D-glucopyranosyl and 5'-O-beta-D-galactopyranosyl derivatives (13 and 15, respectively) of the antiviral agent ribavirin are described. Direct glycosylation of 2',3'-O-isopropylideneribavirin with either tetra-O-acetyl-alpha-D-glucopyranosyl bromide (4) or tetra-O-acetyl-alpha-D-galactopyranosyl bromide (8) under Koenigs-Knorr conditions (i.e., silver carbonate, silver perchlorate, and Drierite in dichloromethane) followed by O-deacetylation of the reaction product gave the corresponding ortho esters. However, treatment of 2',3'-di-O-acetyl-5'-O-tritylribavirin (11) with 4 under the Bredereck modification of the Koenigs-Knorr reaction (i.e., silver perchlorate and Drierite in nitromethane) and subsequent deacetylation furnished the desired 1-(5-O-beta-D-glucopyranosyl-beta-D-ribofuranosyl)-1,2,4-triazole-3-carb oxamide (13). Similarly, reaction of 11 with 8 in the presence of AgClO4, and deprotection of the condensation product, gave 5'-O-beta-D-galactopyranosylribavirin (15). The beta-anomeric configuration of the D-glucosyl and D-galactosyl groups of 13 and 15 was assigned by 1H-n.m.r. studies.     

Effect of exogenous prostaglandin and/or estrogen on luteolysis after electrocauterization of the largest follicles at the end of the bovine estrous cycle

In cows, electrocauterization of large follicles at the end of the luteal phase, lengthened the life span of corpora lutea. Injection of 5 mg of estradiol valerate or of 1 mg of an analogue of prostaglandin F(2alpha) induced luteolysis; however, the injection of estrogen was associated with precocious estrus without either ovulation or corpus luteum growth. Injection of both estradiol valerate and prostaglandin analogue gave the same results as estradiol valerate alone. Deferred luteolysis, observed after electrocauterization of large follicles, seemed to be due to the withdrawal of estrogens and the consequent lack of prostaglandin F(2alpha) production.     

Cyclopentane-nucleobase coupling in the synthesis of carbocyclic L-nucleosides: is a SN2-reaction an alternative to the Mitsunobu-reaction?

Several carbocyclic L-nucleosides have been synthesized by coupling a cyclopentane-system with heterocycles according to a modified Mitsunobu-protocol. This reaction gave two regioisomers, the N1-alkylated product and an unwanted O(2)-product. A simple S(N)2-reaction has been investigated as an alternative for such couplings.     

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.     

Anomeric O-acylation of Kdo using alkyl and aryl isocyanates

To develop a convenient method for the preparation of an alpha-Kdo derivative carrying a functional spacer at the reducing end, we examined anomeric O-acylation using Kdo and halogenated alkyl/aryl isocyanates as nucleophile and electrophiles, respectively. Reaction of a Kdo derivative with 2-chloroethyl isocyanate in the presence of DMAP gave an alpha-spiro product (82%) and an alpha-Kdo derivative of a dimeric isocyanate adduct (10%). Similar reaction with 4-(chloromethyl)phenyl isocyanate gave only the corresponding alpha-spiro product (81%). The NMR data show that the pyranose rings of both the alkyl and aryl spiro products adopt the 5C2 conformation. Thus, we accomplished alpha-selective anomeric O-acylation by coupling the Kdo derivative with alkyl and aryl isocyanates.     

Oxidation of pyrimidine bases and their derivatives by sodium peroxodisulfate

Treatment of 1,3-dimethyluracil (1) with sodium peroxodisulfate in water at 80 degrees C under nitrogen gave 5-hydroxy-1,3-dimethyluracil (6) as a main product. Under similar conditions, oxidation of 5-fluoro-1,3-dimethyluracil (2) gave a 6,6'-dimeric compound (10) together with other products. Similar oxidation of 5-bromo-1,3-dimethyluracil (3) and 1,3,6-trimethyluracil (4) was also investigated. Furthermore, reaction of 1,3-dimethylthymine (5) and adenine in the presence of sodium peroxodisulfate gave coupling products (12) and (13).     

The in vitro and in vivo reaction at the N7-position of guanine of the ultimate carcinogen derived from benzolalpyrene.

The previously reported reaction at N2- and N7- of guanine following addition of 7 alpha,8 beta-dihydroxy-9 beta, 10 beta-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene (anti-BPDE) to an aqueous solution of DNA has been studied in more detail. The extent of reaction and the relative yields of N2- and N7-products was measured over the range of pH 4--7. The depurination following reaction at the N7-position of guanine was found to have a half-life of 3 h. Reaction of the isomeric 7 alpha,8 beta-dihydroxy-9 alpha, 10 alpha-epoxy-7,8,9,10-tetrahydrobenzo[a]-pyrene (syn-BPDE) with DNA gave the expected N2- and no N7-guanine product. When either benzo[a]pyrene or anti-BPDE was added to mouse embryo or Chinese hamster V79 cells respectively, a major N2-guanine product and a very minor adenine product were isolated from the DNA, but no N7-guanine product was detected.     

The Synthesis of Some 5-Vinyluracil-Nucleoside Analogues

Abstract

The reaction of 5-iodouridine with esters of acrylic acid in the palladium-catalysed Heck reaction was used to generate a series of esters of the acid (E) -5- (2-carboxyvinyl)uridine in poor to moderate yield. An alternative method starts from the acid by protection of the sugar moiety followed by in situ generation of the acid chloride then ester formation followed by simultaneous sugar deprotection. Treatment of the methyl ester with ammonia and methylamine gave the corresponding amides, while treatment with dimethylamine gave 5-(1-dimethylamino-2-carboxy)ethyluridine as the major product.