Preparation of 4-Retinamidophenyl- and 4-Retinamidobenzyl-C-glycosyl and C-Glucuronosyl Analogues of the Glucuronide of 4-Hydroxyphenyl-Retinamide as Potential Stable Cancer Chemopreventive Agents

Abstract

Glucuronide metabolites of retinoic acid and its analogues have been suggested to be active cancer chemopreventive analogues of the parent molecules. However, these metabolites are susceptible to β-glucuronidase and acid-catalyzed cleavage and it is not clear whether these carbohydrate conjugates must be hydrolyzed back to the parent molecule to show activity. Thus, the multistep syntheses of stable C-glycosyl and C-glucuronosyl analogues of the known glucuronide metabolite of the breast cancer chemopreventive agent 4-hydroxyphenylretinamide (4-HPR) are outlined. The chemical and enzymatic stability of these compounds has been evaluated relative to the glucuronide of 4-HPR.     

Metabolite Identification of Myricetin in Rats Using HPLC Coupled with ESI-MS

Myricetin, a naturally occurring flavonol, shows multifarious pharmacological activities, e.g., antidiabetic, antioxidant, anti-inflammatory, antitumor, and liver protection effects. In order to obtain an understanding of the myricetin’s metabolism in vivo, a rapid and sensitive method by high-performance liquid chromatography coupled with electrospray-ionization mass spectrometry (HPLC-MSⁿ) techniques was employed to investigate the biotransformation in rats after oral administration of myricetin. Recognition and structural exposition of the metabolites were operated by comparing the changes in molecular mass (ΔM) and MSⁿ spectra with the parent drug. As a result, the parent compound and seven metabolites were found in rat plasma, urine, and feces. In addition, besides 3,5-dihydroxyphenylacetic acid (M1) and 3,4,5-trihydroxyphenylacetic acid (M2), five other compounds were first discovered in the metabolite research of myricetin. These results indicated that, besides ring-fission, there were methylate (M3, M4, M5) and glucuronide (M6, M7) biotransformations of myricetin occurring in vivo.

     

Bioanalytical challenges and strategies for accurately measuring acyl glucuronide metabolites in biological fluids

Bioanalysis of unstable compounds such as acyl glucuronide metabolites represents a great analytical challenge owing to poor analyte stability in biological matrices. The primary goal for bioanalytical assay development is to minimize the breakdown of acyl glucuronide metabolite into its parent aglycone during sample collection, transportation, storage and analysis. Samples need to be stabilized ex vivo immediately after sample collection to minimize potential breakdown and thus to ensure accurate concentration measurement of both acyl glucuronide metabolite and its parent aglycone. In this review paper, formation of acyl glucuronide metabolites, the importance of establishing acyl glucuronide exposure measurement and safety coverage, optimization of sample pretreatment to stabilize the acyl glucuronide metabolites, current analytical strategy of assaying them as well as considerations for regulatory filings are discussed. It is important to identify acyl glucuronide metabolites that are capable of undergoing hydrolysis and pH-dependent intra-molecular migration as well as covalently binding to plasma and tissue proteins which can cause toxicity in vivo in the early stages of drug development. Carefully planning analytical experiments, identifying structures of acyl glucuronides and monitoring their concentrations in early drug development can help assess the risks associated with their exposures and potentially predict their concentrations in human circulation.     

RECENT IMPROVEMENTS TOWARDS THE SYNTHESIS OF THE C-GLUCURONOSYL CANCER CHEMOPREVENTIVE (β-d-GLUCOPYRANOSYLURONATE)-4-RETINAMIDOPHENYLMETHANE

Evidence suggests that the glucuronide conjugates of retinoids may be active metabolites of the parent compounds with potential utility as drugs.¹ We have recently shown that the O-glucuronide metabolite 1 of the synthetic retinoid N-4-hydroxyphenylretinamide (2) shows reduced toxicity and a significant chemopreventive advantage relative to the parent retinoid in a carcinogen-induced rat mammary cancer model.² In efforts to determine whether these conjugates function as intact molecules or must be hydrolyzed back to 2, we designed and synthesized a series of C-glycosyl and C-glucuronosyl analogues of 1 ³. Our syntheses of these compounds are lengthy (10-12 steps) and lead to the desired products in less than 4% overall yields. Limitations on the availability of these compounds led us to evaluate them using a modified protocol for the carcinogen-induced rat mammary tumor model. While many of our analogues showed cancer chemopreventive activity in this assay, C-glucuronosyl analogue 3 has proven to be the most effective analogue of 2 we have yet evaluated in this model.⁴ Unfortunately, compound 3 was prepared in the poorest overall yield among these analogues. However, its potent activity and low toxicity make it an important candidate for expanded biological studies. Because additional studies of this important new compound will benefit from improvements in its synthesis, we briefly describe below recent dramatic improvements we have made in the preparation of 3.     

Analysis of Salicylamide and Its Metabolites in Blood and Urine by HPLC

Abstract

Sensitive liquid chromatographic assays for salicylamide and its metabolites in urine and plasma were developed to facilitate pharmacokinetic studies of the drug's metabolism. The drug and its hydroxylated metabolite, gentisamide, were extracted and concentrated prior to separation on a small-bore reverse-phase column. Conjugated metabolites were assayed separately using reverse-phase ion-pair chromatography. An accurate method of assay calibration in the absence of pure metabolite standards was developed using radioactively-labelled parent drug. In addition one of the metabolites, salicylamide sulfate, was isolated by ion-pair extraction and purified. A significant species difference in salicylamide metabolism was observed. In the dog the drug is almost exclusively (90%) metabolized to its sulfate conjugate, while in humans the glucuronide conjugates of salicylamide (50%) and gentisamide (15%) predominate over salicylamide sulfate (30%).     

Convenient Syntheses of Daunomycinone‐7‐D‐Glucuronides and Doxorubicinone‐7‐D‐Glucuronides

Abstract

The first synthesis of new doxorubicin and daunomycin analogs containing glucuronic acid moieties instead of daunosamine are described. The desired products, daunomycinone‐7‐D‐glucuronide (DM7G, 10) and doxorubicinone‐7‐D‐glucuronide (DX7G, 11) were conveniently prepared through the glycosylation at 7‐hydroxyl group of daunomycinone (4) or 14‐acetoxydoxorubicinone (6) with glucuronic acid derivative 7 by the Koenigs‐Knorr procedure followed by alkaline deacetylation using aqueous LiOH solution and amberlite cation exchange material. The anomeric configuration and conformation of all products were fully characterized by assignment of ¹H NMR chemical shifts and H‐H coupling constants based on reported literatures.     

Separation of Diethylstilbestrol and Derivatives in Biological Fluids and Tissues by High Pressure Liquid Chromatography

Abstract

A method was developed to identify radiolabeled DES and DES metabolites in biological fluids and tissues. After a rapid initial clean-up step, the biological samples were analyzed with a single-column, reversed-phase, highpressure liquid chromatography system. The parent compound and metabolites were simultaneously resolved and tentatively identified by comparing their retention times to those of known standards. Positive identification of some metabolites was achieved by field-desorption and capillary-column mass-spectrometric analysis. The method was found to be rapid and reproducible, and sample recovery was >80%.     

Elucidation of the Structures of Metabolites of Picroside II in Rat Bile by LC�CESI�CIT�CMS

Picroside II is one of the main active constituents of Picrorhiza kurroa, which has hepatoprotective, anticholestatic, antioxidant, and immune-modulating activity. To gain an understanding of the biotransformation of picroside II in vivo, liquid chromatography?Celectrospray ionization ion-trap mass spectrometry (LC?CESI?CIT?CMS) was used to investigate the metabolism of picroside II in rats after intravenous administration of a single dose. This method could simultaneously determine picroside II and its metabolites in rat bile. The bile samples were purified by use of a C₁₈ solid-phase extraction (SPE) cartridge and were separated on a Hypersil ODS2 C₁₈ analytical column. Two phase II metabolites of picroside II in rat bile were characterized, and elucidation of their structures was performed by comparing changes in molecular masses (??M), retention times, and MS² spectral patterns of metabolites with those of the parent drug. Two metabolites identified for the first time in this research were glucuronide and sulfate conjugates.     

Preparation of CI‐1027 Glucuronide: Metabolite of a Remarkable Lipid‐Modulating Agent

Abstract

The preparation of the 1β‐glucuronide of the diacid, CI‐1027, is described. The key synthetic steps are coupling of the mono‐protected acid with bromoglucuronide, deacetylation, and enzymatic hydrolysis.     

含羟基化合物及其葡萄糖醛酸苷类反相高效液相色谱法保留指数预测

采用体外孵有生物合成方法,获得葡萄糖醛酸耷类代谢物,并建立了其保留指数预测方法,在实验条件下,误差小于12％。     

Separation of Cobalt (III) Bis(ethylenediamine) Amino Acid Complexes by Reversed Phase HPLC

Abstract

Methods for the rapid analysis of amino acid cobalt (III) bis(ethylenediamine) complexes by reversed phase high performance liquid chromatography (HPLC) are described with mobile phases containing the pairing ions, p-toluenesulphonate and hexanesulphonate. Under these conditions, the amino acid cobalt (III) bis(ethylenediamine) complexes elute in order of the relative hydrophobicities of the parent amino acids which suggests that the amino acid side chain makes a significant contribution to the retention mechanism. At high sample loadings, these complexes shows a concentration dependent peak splitting effect divergent to that normally experienced with inadequate buffering capacity of the pairing ion reagent.     

The Application of Perdeuterated Polycyclic Aromatic Hydrocarbons (PAH) as Internal Standards for the Liquid Chromatographic Determination of PAH in a Petroleum Crude Oil and Other Complex Mixtures

Abstract

A sequential liquid chromatographic (LC) procedure for the determination of polycyclic aromatic hydrocarbons (PAH) in a petroleum crude oil and other complex mixtures is described. The procedure includes normal-phase LC on an aminosilane column to isolate fractions containing isomeric PAH and reversed-phase LC on a polymeric C₁₈ column to separate the individual PAH isomers. Appropriate perdeuterated PAH are added to the sample so that each isomeric fraction will contain one internal standard. The perdeuterated PAH are excellent internal standards for this sequential LC procedure. Perdeuterated PAH have normal-phase and reversed-phase LC retention characteristics similar to those of the parent PAH. In the normal-phase LC separation, the perdeuterated PAH elute in the same fraction as the parent PAH. In the reversed-phase LC separation, the perdeuterated PAH elute first and are generally resolved from the parent PAH. The optimized spectrofluorometric detection of each PAH analyte is accomplished by programming appropriate sets of excitation and emission wavelengths to correspond with the elation time of each analyte on the polymeric C₁₈ column. The analytical results obtained from this procedure for the analysis of a shale oil sample [Standard Reference Material (SRM) 1580] and a petroleum crude oil (SRM 1582) are compared to values obtained by gas chromatography - mass spectrometry.     

A brief review: HPLC methods to directly detect drug glucuronides in biological matrices (Part I)

Detection of a drug and its glucuronide metabolite(s) is of great importance in interpretive forensic and clinical toxicology. Until recently, glucuronides were determined by cleavage of the glucuronide with an enzyme (e.g., β-glucuronidase) to yield the parent compound, which was subsequently detected, or via derivatization to a more volatile or detectable analogue. Direct detection of the glucuronide conjugates overcomes the critical limitations of approaches that involve enzymatic cleavage procedures and/or derivatization. This review will focus on direct methods to determine glucuronides of various potentially abused drugs in human biological matrices. More specifically, this review will be restricted to methods that involve liquid chromatography (LC) coupled with various detectors. Papers are presented in chronological order for each compound class to emphasize the evolution of methodology and continued importance of the application.     

High Pressure Liquid Chromatographic Assay for Salicylic Acid and its Metabolites in Rat Urine

Abstract

An HPLC method for the determination of salicylic acid (SA), gentisic acid (GA), salicyluric acid (SU), and salicyl acyl glucuronide (SAG) in rat urine was developed. The method consisted of extracting SA, GA, and SU from acidified urine into 50:50 mixture of ethyl acetate and butyl chloride. Salicyl acyl glucuronide was extracted from neutral urine after conversion to salicyl hydroxamic acid with hydroxylamine. Salicyl phenolic glucuronide was estimated indirectly as the difference between total salicylate and sum of the four constituents mentioned above. Chromatographic separation was done on a C₁₈ column with U.V. detection at 310 nm using a mobile phase consisting of 5–10% acetonitrile in 3% glacial acetic acid. The extraction recovery of these compounds from spiked urine ranged from 90–108%. The detection limits were 10 μg/ml for GA, SU and SA, and 2.5 μg/ml for SHA. The method was applied to the study of salicylic acid metabolism in the rat.     

Structure-Retention Behavior of a Morpholine Hydrochloride Pharmaceutical Intermediate

Summary A vant Hoff plot of the retention factor of (2R, 3S)-2-({(1R)-1-[3,5-bis(trifluoromethyl)phenyl]ethyl}oxy)-3-(4-fluorophenyl) morpholine hydrochloride (I), on a C-18 stationary phase, was found to exhibit significant non-linearity. Similar plots for II and III, which are structurally-related to compound I, displayed a smaller degree of non-linearity and were driven by predominately enthalpic and entropic retention processes, respectively. It was hypothesized that the retention behavior of I could be a combination of interactions rising from the shared functional groups of II and III. In addition, it was found that the curvature in the plot for I was enhanced for the homologous compounds IV and V which have extended aromaticity at the 3-position of the morpholine ring. In general, it was observed that as the size of the molecule increased, more entropic retention character was exhibited. However, molecular geometry was also found to play a key role in governing the retention properties of this class of compounds.     

Bioactive glycosides from the African medicinal plant Boerhavia erecta L.

Phytochemical studies of the previously unexplored stem of Boerhavia erecta from Burkina Faso, resulted in the isolation of an unreported glycoside 4, 2,3-dihydroxypropylbenzoate-3-O-β-[4″-methoxy] glucuronide as well as seven known glycosides (1–3, 5–8). The major isolate 5 and 8 indicated a significant inhibition against HIV integrase (IC₅₀ 10 and 22 μg/mL, respectively). The extracts and isolates were also tested for anti-malarial activity, but insignificant activity was observed.     

STEREOCHEMISTRIES OF SOME REACTIONS OF CYCLIC STEREOISOMETRIC PHOSPHITES

Abstract

The reaction of the isomeric phosphites, 1 and 2, with ozone have been shown to be stereospecific and to proceed with retention of configuration about phosphorus. Similarly ozonization of mixtures of 3 and 4 were found to be stereospecific or very nearly so with retention of configuration about phosphorus. The mechanistic implications of these findings are discussed. Reactions of 1 and 2 with neopentyl and t-butyl hypochlorites proceed in a stereochemically random manner. The formation of a pentacoordinated intermediate is implicated. Reactions of a mixture of 1 and 2 with ethyl thiyl radicals provided phosphorothionates with complete retention of configuration.     

High Performance Liquid Chromatographic Analysis of Tolmetin,Indomethacin and Sulindac in Plasma

Abstract

Isocratic and gradient reversed phase high-performance liquid chromatographic (HPLC) methods for the quantitation of tolmetin, indomethacin, and sulindac and their respective metabolites in plasma were developed. Only the determination of the parent drugs was possible using the isocratic technique. Specific, simultaneous determination of each drug and its respective metabolites was achieved using the gradient technique. The effect of pH and ionic concentration of the mobile phase on retention time was studied. Statistical analysis demonstrated excellent precision and linearity over the following ranges: 1–40, 0.1–3, and 0.1–3 ug/ml plasma for tolmetin, indomethacin, and sulindac respectively. Both methods have been applied to the analysis of patient samples.     

Facile Synthesis of Several Oleanane-Type Triterpenoid Saponins

Facile synthesis of the natural oleanane-type triterpenoid saponin 1 isolated from Panax japonicus C. A. Meyer var major (Burk.) C. Y. Wu et K. M. Feng and its derivatives is reported. The glucuronide residue in saponins at a later stage via TEMPO-mediated oxidation of the primary-OH has been achieved. All structures of new compounds were established by means of ¹H NMR, ¹³C NMR, HRMS, and optical rotation.     

A Liquid Chromatographic Electrochemical Assay for S-2-(3-Aminopropylamino) Ethylphosphorothioate (WR2721) in Human Plasma

Abstract

A liquid chromatographic electrochemical method for the determination of the radioprotective drug WR2721 in human plasma has been developed. This method includes the use of a Hg/Au electrochemical detector for the direct measurement of WR2721 concentration. An analog of WR2721, S-3-{4-aminobutylamino} propylphosphorothioate (WR80855) is the internal standard. The retention times for WR2721 and WR80855 are approximately 4.5 and 9 minutes, respectively. WR1065, the free sulfhydryl metabolite of WR2721, is retained on the column under the described chromatographic conditions and therefore does not interefere with the determination of the parent drug. With modification of the mobile phase WR1065 is eluted from the column at a retention time of approximately 20 minutes. This method has good linearity, precision and accuracy, and is free from interference from endogenous plasma substances. Preliminary results showing the applicability of this method to human pharmacokinetic studies and to investigating the enzymatic hydrolysis of WR2721 are presented.