Uterine stimulant action of some 16-phenoxy analogues of (+)-11-deoxyprostaglandin E1

Some ($\text{+}$)-11-deoxy-16-phenoxyprostaglandin E₁ analogues have been evaluated as uterine stimulants in the anaesthetised pregnant rat. Gastrointestinal side effects, assessed by the antagonism of morphine-induced consitpation in the mouse, were relatively low with some of these compounds, six of which had a much more favourable relative selectivity than 16,16-dimethyl1-PGE₂ methyl ester.     

Vaginally administered 16, 16-dimethyl-prostaglandin E2 as an agent for pre-operative cervical dilatation

Twenty-one women in the 10th–12th week of pregnancy were treated prior to vacuum aspiration with vaginal suppositories containing 16, 16-dimethyl-PGE₂ (free acid). An average total dose of 3.4 mg led to abortion or adequate cervical dilatation in all patients. Based upon previous experience with the compound, no prophylactic anti-emetic or anti-diarrhetic medication was given. Gastrointestinal side effects were minimal. Excessive bleeding was not observed. In two cases, slight temperature elevation was noted prior to abortion. The low incidence of side effects in combination with the effectiveness of the compound help to make this method an attractive therapeutic adjunct to vacuum aspiration beyond the 10th week of gestation. Under the experimental conditions of this study, the results suggest that vaginally administered 16, 16-dimethyl-PGE₂ can be a safe and effective method for cervical dilatation before vacuum aspiration.     

Vaginally administered 16, 16-dimethyl-prostaglandin E2 as an agent for pre-operative cervical dilatation

Twenty-one women in the 10th–12th week of pregnancy were treated prior to vacuum aspiration with vaginal suppositories containing 16, 16-dimethyl-PGE₂ (free acid). An average total dose of 3.4 mg led to abortion or adequate cervical dilatation in all patients. Based upon previous experience with the compound, no prophylactic anti-emetic or anti-diarrhetic medication was given. Gastrointestinal side effects were minimal. Excessive bleeding was not observed. In two cases, slight temperature elevation was noted prior to abortion. The low incidence of side effects in combination with the effectiveness of the compound help to make this method an attractive therapeutic adjunct to vacuum aspiration beyond the 10th week of gestation. Under the experimental conditions of this study, the results suggest that vaginally administered 16, 16-dimethyl-PGE₂ can be a safe and effective method for cervical dilatation before vacuum aspiration.     

Dose dependent inhibition of B-16 melanoma growth in vivo by a synthetic analogue of PGE2

Daily intratumor administration of 16, 16-dimethyl-PGE₂-methyl ester in two different dosages inhibited tumor growth in C57B1/6J mice bearing subcutaneous B-16 melanomas. The larger dose (20γg/day/mouse) produced a 68% decrease in tumor volume, a 69% decrease in tumor weight and a 60% decrease in the number of cells in mitotic phase. The smaller dose (10μg/day/mouse) was one fifth less effective than the 20μg dose but produced similar changes. Histological examination of tumors revealed no significant differences either in the inflammatory cell population or the amount of necrosis in the control and di-M-PGE₂-treated tumors.     

Plasma concentrations of 9-deoxo-16,16-dimethyl-9-methylene-PGE2 in Rhesus monkeys after administration by various routes

A method is described for the estimation of 9-deoxo-16,16-dimethyl-9-methylene-PGE₂ by double antibody radioimmunoassay. Plasma samples obtained from animals treated with 9-methylene-16,16-dimethyl-PGE₂,1-adamantanamine salt were extracted with diethyl ether to recover the prostaglandin. The validation of sample preparation and assay procedure are presented. Rhesus females were treated by several routes of administration and the samples assayed for drug content. Maximum blood levels were probably reached 30 minutes following subcutaneous injection and within 30 seconds of an intravenous injection. Results of the acute intravenous injection indicate an initial half-life of approximately one minute in peripheral circulation. Continuous intravenous infusion at 3 increasing doses of this compound resulted in a stepwise increase in plasma drug concentrations. Vaginal administration of 9-methylene-16,16-dimethyl-PGE₂,1-adamantanamine salt in suppositories produced a dose dependent increase in plasma drug concentration. Higher plasma drug concentrations were produced when the prostaglandin was delivered in H-15 base suppositories than in E-76 base suppositories.     

Method for quantitative analysis of 16, 16-dimethyl-prostaglandin E2 from plasma using deuterated carrier and gas chromatography-mass spectrometry.

The preparation of 3,3,4,4-²H₄16,16-dimethyl-prostaglandin E₂ (PGE₂), 16,16-dimethyl-5,6-didehydro-PGE₂, and 5,6-³H₂16,16-dimethyl-PGE₂ is described. These compounds have been used for development of a gas-liquid chromatography-mass spectrometry method for quantitative determination of corresponding nondeuterated prostaglandin. The method is based on addition of a known amount of carrier to the sample and after purification and derivation the ratio between the protium and deuterium form is measured in the mass spectrometer. With this technique 40 pg of 16,16-dimethyl-PGE₂ can be determined with a precision of ±2.6%.     

PGE restores the immune response in chemotherapy-treated,tumor-bearing mice

Two hundred seventy-one B-16 melanoma-bearing mice were randomized and treated for 4 days with either control diluent, 10 μg of 16, 16-dimethyl-PGE₂-methyl-ester (di-M-PGE₂), chemotherapy, or chemotherapy plus di-M-PGE₂. The chemotherapeutic regimens included adriamycin (7.5 mg/kg), 5-fluorouracil (250 mg/kg), nitrogen mustard (5 mg/kg), and vincristine (0.5 mg/kg). The number of plaque-forming cells and hemagglutinin titers in response to sheep erythrocytes were used as measures of humoral immunity while cellular immunity was assessed by evaluation of delayed hypersensitivity. As we previously reported, the presence of subcutaneous B-16 tumors induced substantial immunosuppression and this suppression was reversed by treatment with di-M-PGE₂. Treatment with all four chemotherapeutic agents induced profound immunosuppression. Similarly, the addition of di-M-PGE₂ to the chemotherapy protocols resulted in significant augmentation of cellular and humoral immunity.     

Chemical instability of 15-keto-13,14-dihydro-PGE2: The reason for low assay reliability

The spontaneous degradation of 15-keto-13,14-dihydro-PGE₂ was studied under various conditions. In aqueous media, dehydration rapidly occurred, particularly at high or very low pH. The acid catalyzed dehydration product was identified as 15-keto-13,14-dihydro-PGA₂. This compound was also formed at alkaline pH, however, at higher pH a bicyclic compound, 11-deoxy-13,14-dihydro-15-keto-11,16-cyclo-PGE₂, was formed in addition (cf. Fig. 1). The amounts of the latter compound increased with time and with increasing pH.In samples containing albumin, 15-keto-dihydro-PGE₂ was degraded even more rapidly than in buffer of the same pH. In addition to the formation of the dehydration products, a considerable part of the metabolite was bound to albumin to yield water soluble adducts. A similar binding occurred to a low molecular weight fraction of plasma. The compound that was bound was 15-keto-dihydro-PGA₂, whereas both 15-keto-dihydro-PGE₂ and the bicyclic product were relatively inert in this respect.Due to its chemical stability, the bicyclic degradation product, 11-deoxy-13,14-dihydro-15-keto-11,16-cyclo-PGE₂, is suggested as a suitable target for measurements instead of the labile parent compound, 15-keto-dihydro-PGE₂, or the reactive dehydration product, 15-keto-dihydro-PGA₂.     

Synthesis and glycosidation of 1-deoxy-1-[(2,2-diacylvinyl)amino]-d-fructoses

The reactions of 1-amino-1-deoxy-d-fructose acetate (1) with methyl 3-methoxy-2-methoxycarbonylacrylate and 5-methoxymethylene-2,2-dimethyl-1,3-dioxane-4,6-dione in the presence of a base afforded 1-deoxy-1-[(2,2-dimethoxycarbonylvinyl)amino]- (2 and 1-deoxy-1-[(2,2-dimethyl-4,6-dioxo-1,3-dioxan-5-ylidenemethyl)amino]-d-fructose (3), respectively, in high yields. 1-Deoxy-1-[(4,4-dimethyl-2,6-dioxocyclohexylidenemethyl)amino]-d-fructose (4) was obtained (85%) by a transamination reaction between 1 and 5,5-dimethyl-2-phenylaminomethylene-1,3-cyclohexanedione in the presence of Et₃N. The isomeric composition of equilibrium solutions of 1–4 was established by ¹³C-n.m.r. spectroscopy. For all the compounds, the β-pyranose form was the main component in D₂O; the α-furanose, the β-furanose, and, for 1, the α-pyranose forms, were also present. The major constituents of 2 in (CD₃)₂SO solution were the β- and the α-furanose forms. Acetylation of 2 afforded the tetra-acetates of the α- and β-furanose forms, the 3,4,6-triacetates of the α- and β-furanose forms, the 3,4,5-triacetate of the β-pyranose form, and 2,3,4,5,6-penta-O-acetyl-1-deoxy-1-[(2,2-dimethoxycarbonylvinyl)amino]-d-arabino-hex-1-enitol. Glycosidation of 2 with MeOHHCl afforded a mixture of methyl 1-deoxy-1-[(2,2-dimethoxycarbonylvinyl)amino]-α- (11α) and -β-d-fructofuranoside (11β), and methyl 1-deoxy-1-[(2,2-dimethoxycarbonylvinyl)-amino]-β-d-fructopyranoside (13). Compounds 11α and 13 were isolated as their tri-acetates (12 and 14, respectively). Deacetylation and removal of the N-protecting group of 12 gave methyl 1-amino-1-deoxy-α-d-fructofuranoside (∼54% from 2).     

Synthesis and antifertility activity of ω-chain phenyl- and 16-phenoxy-analogues of (±)-11-deoxyprostaglandin F1α

Some ω-chain phenyl- and 16-phenoxy- analogues of (±)-11-deoxyprostaglandin F_1α have been synthesized and evaluated for anti-fertility activity in the hamster. 11-Deoxy-16-phenoxy-17,18,19,20-tetranor-PGF_1α was the most active member of the series with an ED₅₀ equal to that of PGF_2α. 11-Deoxy-17-phenyl-18,19,20-trinor-PGF_1α, which was one third as active as PGF_2α, was more potent than the corresponding 16- and 18-phenyl compounds. Aryl ring substitution was found to lower activity, except that with the 16-phenyl compound, p-bromo and m-trifluoromethyl substitution increased the potency.The antifertility activity of the phenoxy compounds, which were poor substrates for 15-hydroxyprostaglandin dehydrogenase, was shown to correlate well with the binding affinity for the bovine corpus luteum PGF_2α receptor. Some quantitative structure-activity data supporting this finding are presented.     

1-Methoxy-, 1-deoxy-11-hydroxy- and 11-Hydroxy-1-methoxy-Δ-tetrahydrocannabinols: new selective ligands for the CB2 receptor

Three series of new cannabinoids were prepared and their affinities for the CB₁ and CB₂ cannabinoid recptors were determined. These are the 1-methoxy-3-(1′,1′-dimethylalkyl)-, 1-deoxy-11-hydroxy-3-(1′,1′-dimethylalkyl)- and 11-hydroxy-1-methoxy-3-(1′,1′-dimethylalkyl)-Δ⁸-tetrahydrocannabinols, which contain alkyl chains from dimethylethyl to dimethylheptyl appended to C-3 of the cannabinoid. All of these compounds have greater affinity for the CB₂ receptor than for the CB₁ receptor, however only 1-methoxy-3-(1′,1′-dimethylhexyl)-Δ⁸-THC (JWH-229, 6e) has effectively no affinity for the CB₁ receptor (K_i=3134±110 nM) and high affinity for CB₂ (K_i=18±2 nM).     

The differential effects of PGE on the immune response in normal and immunosuppressed mice

The effect of 16,16-dimethyl-PGE₂-methyl ester (di-M-PGE₂) on humoral and cellular immunoresponsiveness has been compared in normal mice and in mice immunosuppressed by splenectomy and thymectomy plus antithymocyte serum (ATS). Splenectomy resulted in immunosuppression manifested by augmentation of B-16 melanoma growth; this stimulatory effect was reversed by di-M-PGE₂. In animals immunosuppressed by thymectomy plus ATS, di-M-PGE₂ augmented the humoral and cellular immune responses; this was manifested by slowing of the growth of B-16 melanoma and by stimulating the number of plaque-forming cells, hemagglutinin titers, and delayed-hypersensitivity reactions to sheep erythrocytes. In contrast, in normal (nonthymectomized) mice, di-M-PGE₂ was mildly immunosuppressive. Finally, adriamycin-immunosuppressed normal mice and this suppression were reversed by the addition of di-M-PGE₂ to the treatment regimen.     

Effects of estradiol and progesterone on uterine prostaglandin levels in the pregnant rat

Prostaglandin D₂ was found to be a potent inhibitor of B-16 melanoma cell replication $\text{in vitro}$. The inhibition was dose-dependent between 3×10^?9M and 3×10^?6M (IC_50～ 0.3 μM after 6 days). On a molar basis, PGD₂ was a better inhibitor than PGA₂ or 16,16-dimethyl-PGE₂-methyl ester (di-M-PGE₂) and in higher concentrations (10^?6?10^?7M), comparable to retinoic acid. In higher concentrations, PGD₂ inhibited DNA, RNA and protein synthesis. The B-16 melanoma cell line which we used synthesized arachidonic acid metabolites which comigrated with PGA₂, PGD₂, PGE₂ and PGF_2α on a thin layer chromatography system.     

Synthesis and anti-HCV activity of β-d-2′-deoxy-2′-α-chloro-2′-β-fluoro and β-d-2′-deoxy-2′-α-bromo-2′-β-fluoro nucleosides and their phosphoramidate prodrugs

We report herein the synthesis and evaluation of a series of β-d-2′-deoxy-2′-α-chloro-2′-β-fluoro and β-d-2′-deoxy-2′-α-bromo-2′-β-fluoro nucleosides along with their corresponding phosphoramidate prodrugs. Key intermediates, lactols 11 and 12, were obtained by a diastereoselective fluorination of protected 2-deoxy-2-chloro/bromo-ribonolactones 7 and 8. All synthesized nucleosides and prodrugs were evaluated with a hepatitis C virus (HCV) subgenomic replicon system.     

Retinoic acid increases hypoxia-inducible factor-1α through intracrine prostaglandin E2 signaling in human renal proximal tubular cells HK-2

We have previously shown in HK-2 cells that ATRA (all-trans-retinoic acid) up-regulates HIF-1α (hypoxia-inducible factor-1α) in normoxia, which results in increased production of renal protector VEGF-A (vascular endothelial growth factor-A). Here we investigated the role of COXs (cyclooxygenases) in these effects and we found that, i) ATRA increased the expression of COX-1 and COX-2 mRNA and protein and the intracellular levels (but not the extracellular ones) of PGE₂. Furthermore, inhibitors of COX isoenzymes blocked ATRA-induced increase in intracellular PGE₂, HIF-1α up-regulation and increased VEGF-A production. Immunofluorescence analysis found intracellular staining for EP1-4 receptors (PGE₂ receptors). These results indicated that COX activity is critical for ATRA-induced HIF-1α up-regulation and suggested that intracellular PGE₂ could mediate the effects of ATRA; ii) Treatment with PGE₂ analog 16,16-dimethyl-PGE₂ resulted in up-regulation of HIF-1α and antagonists of EP1-4 receptors inhibited 16,16-dimethyl-PGE₂- and ATRA-induced HIF-1α up-regulation. These results confirmed that PGE₂ mediates the effects of ATRA on HIF-1α expression; iii) Prostaglandin uptake transporter inhibitor bromocresol green blocked the increase in HIF-1α expression induced by PGE₂ or by PGE₂-increasing cytokine interleukin-1β, but not by ATRA. Therefore only intracellular PGE₂ is able to increase HIF-1α expression. In conclusion, intracellular PGE₂ increases HIF-1α expression and mediates ATRA-induced HIF-1α up-regulation.     

Synthesis and evaluation of the anti-hepatitis B virus activity of 4′-Azido-thymidine analogs and 4′-Azido-2′-deoxy-5-methylcytidine analogs: structural insights for the development of a novel anti-HBV agent

Abstract

Hepatitis B virus (HBV) infection is a major worldwide health problem that requires the development of improved antiviral therapies. Here, a series of 4′-Azido-thymidine/4′-Azido-2′-deoxy-5-methylcytidine derivatives (6, 10–15) were synthesized, and their anti-HBV activities evaluated. Compounds 10–15 were synthesized via an S_NAr reaction of 18, in which the 4-position of the thymine moiety was activated as the 2,4,6-triisopropylbenzenesulfonate. Compounds 11–15 showed no antiviral activity. However, 4′-Azido thymidine (6) and 4′-Azido-2′-deoxy-5-methylcytidine (10) displayed significant anti-HBV activity (EC₅₀ = 0.63 and 5.99?µM, respectively) with no detectable cytotoxicity against MT-2 cells up to 100?µM.     

The linkage of 4-O-methyl-L-galactose in the sulphated polysaccharide of Aeodes ulvoidea

Methyl 2-acetamido-5,6-di-O-benzyl-2-deoxy-β-d-glucofuranoside (11) was obtained in six steps from the known methyl 3-O-allyl-2-benzamido-2-deoxy-5,6-O-isopropylidene-β-d-glucofuranoside. Mild acid hydrolysis, followed by benzylation gave the 5,6-dibenzyl ether. The benzamido group was exchanged for an acetamido group by strong alkaline hydrolysis, followed by N-acetylation, and the allyl group was isomerized into a 1-propenyl group that was hydrolyzed with mercuric chloride. Treatment of 11 with l-α-chloropropionic acid and with diazomethabe gave methyl 2-acetamido-5,6-di-O-benzyl-2-deoxy-3-O-[d-1-(methoxycarbonyl)ethyl]-β-d-glucofuranoside which formed on mercaptolysis the internal ester 16, further converted into 2-acetamido-4-O-acetyl-5,6-di-O-benzyl-2-deoxy-3-O-[d-1-(methoxycarbonyl)ethyl]-d-glucose diethyl dithioacetal (18) by alkaline treatment followed by esterification with diazomethane and acetylation. Attempts to remove the O-acetyl group of the corresponding dimethyl acetal 20 with sodium methoxide in mild conditions were not successful.     

Gas chromatographic — Mass spectrometric quantitation of 16,16-dimethyl-trans-Δ2-PGE1

Di-deuterated and di-tritiated 16,16-dimethyl-trans-Δ²-PGE₁ has been synthesized and used for development of a GC-MS method for quantitation of corresponding unlabelled drug in patient plasma. Although these carrier/internal standard molecules only contain 2 deuterium atoms the lower limit of detection at each injection is as low as about 40 pg.The maximum plasma levels of this drug following administration of vaginal suppositories used in clinical studies (1 mg 16,16-dimethyl-trans-Δ²-PGE₁ methyl ester in 0.8 g Witepsol S-52) were 100–350 pg/ml i.e. in the same order of magnitude as earlier seenf ro 16,16-dimethyl-PGE₂.     

A radioimmunoassay for serum 11-deoxy-17-ketosteroids

A simplified method for evaluating serum 11-deoxy-17-ketosteroids (11-deoxy-17-KS) equivalent to dehydroepiandrosterone sulfate (DHEAS) has been developed without solvolysis and chromatography. 5μl of serum or plasma was added to 1 ml of ethanol, mixed, and centrifuged. 10 or 20 μ1 of the supernatant was evaporated to dryness and incubated with anti-11-deoxy-17-KS antiserum obtained by immunizing a rabbit with DHEA-3·O·CO-BSA which was prepared from DHEA-3·O·COC1 and containing DHEAS-7α³H, pepsin-treated human immune serum globulin and bovine serum albumin. Ammonium sulfate was used to separate free from bound DHEAS-7α³H. The accuracy, precision and sensitivity were satisfactory. The blank values could not be differentiated from zero. As the antiserum reacted not only on DHEAS but also on androsterone sulfate and etiocholanolone sulfate, serum 11-deoxy-17KS obtained by the radioimmunoassay expressed nearly the sum of 100% of DHEAS, 45% of androsterone sulfate and 35% of etiocholanolone sulfate in the serum. A good correlation was found between serum 11-deoxy-17-KS and DHEAS obtained by the radioimmunoassay described in a preveous paper (1). The present radioimmunoassay is the simplest method for the evaluation of the concentrations of C₁₉ steroids in the serum.     

Role of hydroxyl group on the mesomorphism of alkyl glycosides: synthesis and thermal behavior of alkyl 6-deoxy-β-d-glucopyranosides

A homologous series of alkyl 6-deoxy-β-d-glucopyranoside amphiphiles was prepared, in an effort to identify the role of hydroxyl group in the mesomorphic behavior of alkyl glycosides. Synthesis was performed by a chlorination of the sugar moiety in alkyl β-d-glucopyranosides with methylsulfonyl chloride in DMF, followed by a metal mediated dehalogenation to secure alkyl 6-deoxy-β-d-glucopyranosides, wherein the alkyl chain length varied from C₉ to C₁₆. The mesomorphic behavior of these 6-deoxy alkyl glycosides was assessed using polarizing optical microscopy, differential scanning calorimetry and X-ray diffraction method. Whereas the lower homologues exhibited a monotropic SmA phase till sub-ambient temperatures, the higher homologues formed a plastic phase. A partial interdigitized bilayer structure of SmA phase is inferred from experimental d-spacing and computationally derived lengths of the molecules. The results were compared with those of normal alkyl glucopyranosides, retained with hydroxyl groups at C-2–C-6 carbons, and alkyl 2-deoxy-glucopyranosides, devoid of a hydroxyl group at C-2 and the comparison showed important differences in the mesomorphic behavior.