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.     

N-4-羟基苯基维甲酰胺与呋咱氮氧化物偶联物的合成及抗肿瘤活性研究

为了寻找治疗乳腺癌的药物, 将N-4-羟基苯基维甲酰胺(4-HPR)以乙酸为连接基团通过酯键或酰胺键与NO供体呋咱氮氧化物缀合, 合成了NO供体型维甲酸类化合物, 共11个新的目标化合物, 其结构经IR, MS, ¹H NMR和元素分析表征, 总收率为8.8%～12.9%. 对目标物进行体外抗乳腺癌活性测试, 结果表明, 所有目标物均具有不同程度的抗肿瘤活性, 其中8g抗肿瘤活性和对照药阿霉素相当.     

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.     

Estimation of High-Performance Liquid Chromatographic Retention Indices of Glucuronide Metabolites

Abstract

The retention indices of several glucuronide metabolites and their parent compounds were measured using a reversed-phase HPLC system. It was found that the typical glucuronide metabolite had a retention index 244 ± 31 units lower than the parent compound.     

Tissue distribution and metabolism of the putative cancer chemopreventive agent 3′,4′,5′‐trimethoxyflavonol (TMFol) in mice

3′,4′,5′‐Trimethoxyflavonol (TMFol) is a synthetic flavonol with preclinical cancer chemopreventive properties. The hypothesis was tested that, in mice, p.o. administration of TMFol results in measureable levels of the parent in target tissues. A single oral dose (240 mg/kg) was administered to mice (n = 4 per time point) with time points ranging from 5 to 1440 min. TMFol and its metabolites were identified and quantitated in all tissues by high‐performance liquid chromatography (HPLC). Plasma levels of TMFol were at the limit of quantification or below, although metabolites were identified. Peak levels of TMFol in the gastrointestinal tract and the prostate averaged 1671 ± 265 µg/g (5.3 µmol/g) and 6.0 ± 1.6 µg/g (18.4 nmol/g), and occurred 20 and 360 min post‐dose, respectively. The area under the tissue concentration–time curve (AUC) for TMFol was greater than those of the metabolites, indicating that TMFol is relatively metabolically stable. Micromolar TMFol levels are easily achieved in the prostate and gastrointestinal tract, suggesting that TMFol might exert chemopreventive efficacy at these tissue sites. Further investigations are warranted to elucidate the potential chemopreventive potency of TMFol. Copyright © 2012 John Wiley & Sons, Ltd.     

Two new epimers of C15-acetogenin, 4-epi-isolaurallene and 4-epi-itomanallene A as diastereomeric model

Abstract

Two new C₁₅-acetogenins, 4-epi-isolaurallene (1) and 4-epi-itomanallene A (2) were isolated from a population of marine red alga Laurencia nangii Masuda from Carrington Reef. The structures of these compounds were determined intensively by NMR and HRESIMS data. Their configurations were elucidated by detailed comparison of chemical shifts, germinal protons splitting and NOE correlations with known and synthesized analogues. In addition, antibacterial activities of these compounds were evaluated. These compounds would serve as diastereomeric models for future reference. Since the isolaurallene, neolaurallene, 9-acetoxy-1,10,12-tribromo-4,7:6,13-bisepoxypentadeca-1,2-diene, itomanallene A and laurendecumallene A were isolated, compounds 1 and 2 were the sixth example of C₁₅-acetogenin with dioxabicyclo[7.3.0]dodecene skeleton.     

Improved synthesis of the C-glucuronide/glycoside of 4-hydroxybenzylretinone (4-HBR)

Improvements in the synthesis of carbon-linked glucuronide/glucoside conjugates of cancer chemopreventive retinoids have been achieved starting with 2,3,4,6-tetra-O-benzyl-D-glucopyranose. The revised approach demonstrates better yields, eliminates the use of an expensive, carcinogenic protecting group reagent, and avoids much painstaking chromatography. The new approach should allow synthesis of larger quantities of the agents for detailed animal and mechanistic studies.     

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.

     

Simultaneous determination of the novel thiosemicarbazone anti‐cancer agent,Bp4eT,and its main phase I metabolites in plasma: Application to a pilot pharmacokinetic study in rats

Novel thiosemicarbazone metal chelators are extensively studied anti‐cancer agents with marked and selective activity against a wide variety of cancer cells, as well as human tumor xenografts in mice. This study describes the first validated LC‐MS/MS method for the simultaneous quantification of 2‐benzoylpyridine 4‐ethyl‐3‐thiosemicarbazone (Bp4eT) and its main metabolites (E/Z isomers of the semicarbazone structure, M1‐E and M1‐Z, and the amidrazone metabolite, M2) in plasma. Separation was achieved using a C₁₈ column with ammonium formate/acetonitrile mixture as the mobile phase. Plasma samples were treated using solid‐phase extraction on 96‐well plates. This method was validated over the concentration range of 0.18–2.80 μM for Bp4eT, 0.02–0.37 μM for both M1‐E and M1‐Z, and 0.10–1.60 μM for M2. This methodology was applied to the analysis of samples from in vivo experiments, allowing for the concentration–time profile to be simultaneously assessed for the parent drug and its metabolites. The current study addresses the lack of knowledge regarding the quantitative analysis of thiosemicarbazone anti‐cancer drugs and their metabolites in plasma and provides the first pharmacokinetic data on a lead compound of this class. Copyright © 2013 John Wiley & Sons, Ltd.     

An HPLC‐UV method for the simultaneous quantification of curcumin and its metabolites in plasma and lung tissue: Potential for preclinical applications

Curcumin, derived from turmeric, has been extensively investigated for its broad spectrum of biological activities. Previously reported HPLC‐UV methods have focussed on analysis of the parent compound. Here, a sensitive HPLC‐UV method was developed and partially validated, then used for the simultaneous determination of curcumin and its glucuronide and sulfate metabolites in plasma and lung tissue from mice. The assay was applied to an in vivo pharmacokinetic study comparing formulated curcumin (Meriva™) with standard curcumin. Plasma levels of glucuronide and sulfate metabolites were 5‐ and 2‐fold higher after Meriva™ administration compared with standard curcumin. In lung tissue, free curcumin was 4‐fold higher following Meriva™ administration vs standard curcumin. This assay represents a rapid, cheap method for simultaneous detection of curcumin and its major metabolites that has applicability in pre‐clinical settings.     

The detection of androstenedione abuse in sport: a mass spectrometry strategy to identify the 4‐hydroxyandrostenedione metabolite

Studies have shown that the administration of androstenedione (ADIONE) significantly increases the urinary ratio of testosterone glucuronide to epitestosterone glucuronide (T/E) – measured by gas chromatography/mass spectrometry (GC/MS) – in subjects with a normal (≈1) or naturally high (>1) initial values. However, the urinary T/E ratio has been shown not to increase in subjects with naturally low (<1) initial values. Such cases then rely on the detection of C₆‐hydroxylated metabolites shown to be indicative of ADIONE administration. While these markers may be measured in the routine GC/MS steroid profile, their relatively low urinary excretion limits the use of gas chromatography/combustion/isotope ratio mass spectrometry (GC/C/IRMS) to specifically confirm ADIONE administration based on depleted ¹³C content. A mass spectrometry strategy was used in this study to identify metabolites of ADIONE with the potential to provide compound‐specific detection. C₄‐hydroxylation was subsequently shown to be a major metabolic pathway following ADIONE administration, thereby resulting in urinary excretion of 4‐hydroxyandrostenedione (4OH‐ADIONE). Complementary analysis of 4OH‐ADIONE by GC/MS and GC/C/IRMS was used to confirm ADIONE administration. Copyright © 2008 Commonwealth of Australia. Published by John Wiley & Sons, Ltd.     

ENANTIOPURE TRIOXADECALIN DERIVED LIQUID CRYSTALS: INFLUENCE OF PHENYL SUBSTITUTION ON THE MESOGENIC PROPERTIES

Nickel(0)-catalyzed reaction of pseudo-glucal 1 with Grignard reagents derived from bromobenzene and 1-bromo-4-phenylbenzene gives the corresponding β-C-aryl glycosides 2. Desilylation and hydrogenation of 2 leads to saturated β-C-aryl glycosides 4, which can be used as chiral intermediates in the synthesis of chiral liquid crystals. The combination with p-alkoxy-substituted benzaldehyde leads to compounds 5–6. Alternatively, reaction with p-alkoxy-substituted phenylboronic acids gives the bora analogues 7–9. The mesogenic properties of these compounds are strongly influenced by the presence of an additional phenyl ring in the molecule.     

有机锡9-芴酮-4-甲酸酯的合成、结构及抗癌活性

合成了3个有机锡9-芴酮-4-甲酸酯:三苯基锡9-芴酮-4-甲酸酯[(C₆H₅)₃Sn(C₁₄H₇O₃)](1)、三环己基锡9-芴酮-4-甲酸酯[(C₆H₁₁)₃Sn(C₁₄H₇O₃)](2)和三(2-甲基-2-苯基丙基)锡9-芴酮-4-甲酸酯[(C₆H₅C(CH₃)₂CH₂)₃Sn(C₁₄H₇O₃)](3)。通过元素分析、红外光谱、核磁共振谱(¹H、¹³C和¹¹⁹Sn)、热重分析进行了表征;用单晶X射线衍射方法测定了化合物的晶体结构,并对其进行了量子化学计算和体外抗...     

Antimicrobial substances from the rare actinomycete Nonomuraea rhodomycinica NR4-ASC07T

Nonomuraea rhodomycinica NR4-ASC07^T is a rare actinomycete isolated from soil in Sirindhorn peat swamp forest. The crude extract of its culture broth exhibited antimicrobial and anticancer against diverse human pathogens and cancer cells. The chemical investigation of the crude extract led to the isolation of two new metabolites named nonomuric acid (1) and 3-hydroxy deoxydaunorubicinol aglycone (2), along with two known bioactive compounds [ε-rhodomycinone (3) and 7-deoxy-13-dihydrocarminomycinone (4)]. Compounds 1 and 3 showed antimalarial activity with the half maximal inhibitory concentration (IC₅₀) of 8.00 and 8.88 μg mL^?1, respectively. Compound 4 inhibited growth of Mycobacterium tuberculosis and Bacillus cereus at the minimum inhibitory concentrations of 50.0 and 12.50 μg mL^?1, respectively. Every compound exhibited cytotoxicity against cancer cells tested at IC₅₀ ≥ 6.34 μg mL^?1. These finding are the first report of bioactive metabolites produced by strain NR4-ASC07^T, suggesting that rare actinomycetes are yet promising sources for novel drug discovery.     

Four new anthraquinones from a soil actinomycete Streptomyces sp. WS-13394 and their bioactivities

Further chemical study of secondary metabolites from the soil actinomycete Streptomyces sp. WS-13394 resulted in the isolation of four new alkylated anthraquinone analogues (5–8). Their structures were elucidated on the basis of extensive spectroscopic analysis, including HR-ESI-MS, 1D and 2D NMR. The new compounds, together with analogues obtained before (1–4), were tested for their in vitro cytotoxicity against Huh-7 and SGC-7901.     

Application of the Cleavable Isocyanide in Efficient Approach to Pyroglutamic Acid Analogues with Potential Biological Activity

Two efficient procedures have been developed for the synthesis of pyroglutamic acid analogues 28, 29, and 34. According to the first method the Ugi (4C3C) reaction is followed by a post-transformation reaction, and the second method involves the Michael addition reaction. The present methodologies demonstrate the applicability of 1-(2,2-dimethoxyethyl)-2-isocyanobenzene (15) as a cleavable isocyanide in the Ugi/ post-transformation reaction and a strong nucleophile in the Michael addition reaction. The framework of pyroglutamic acid analogues has been constructed by the selective cleavage of the C-terminal amide bond and nucleophilic addition to the activated α,β-unsaturated carbonyl group.

     

Analysis of Anisodine and Identification of Twenty of Its Metabolites in Rat Urine by Liquid Chromatography–Tandem Mass Spectrometry

A simple and rapid high-performance liquid chromatographic-electrospray ionization (ESI) tandem mass spectrometric method has been developed for elucidation of the structures of the metabolites of anisodine in rat urine after administration of a single dose (20 mg). Different extraction techniques (free fraction, acid hydrolysis, and enzyme hydrolysis) were compared for investigation of the metabolism of anisodine. After extraction the pretreated samples were injected into a reversed-phase C₁₈ column with 60:40 (v/v) methanol–0.01% triethylamine solution (2 mM, adjusted to pH 3.5 with formic acid) as mobile phase. Detection was by on-line MS-MS. Identification of the metabolites and elucidation of their structure were performed by comparing changes in molecular masses (ΔM), retention-times, and spectral patterns of product ions with those of the parent drug. At least twenty metabolites (norscopine, scopine, α-hydroxytropic acid, aponoranisodine, apoanisodine, noranisodine, anisodine N-oxide, hydroxyanisodine, hydroxyanisodine N-oxide, methoxyanisodine, hydroxymethoxyanisodine, trihydroxyanisodine, dihydroxymethoxyanisodine, hydroxydimethoxyanisodine, glucuronide conjugates, and sulfate conjugates of noranisodine, hydroxyanisodine and the parent drug) and the parent drug were found in the urine after ingestion of 20 mg anisodine by healthy rats. Anisodine N-oxide, hydroxyanisodine, and the parent drug were detected in rat urine for up 120 h after ingestion of the drug.     

12-Membered crown ethers. Lipophilic derivatives of benzo- and naphtho-12-crown-4 and their membrane electrode properties. Crystal structures of benzo-12-crown-4 NaI and KI complexes

Lipophilic derivatives of benzo-12-crown-4 and naphtho-12-crown-4 have been synthesized. The behavior of the parent compounds and their derivatives in membrane ion-selective electrodes have been studied. Selectivity changes have been observed with the rise in lipophilicity. Crystal structures of the NaI and KI complexes of benzo-12-crown-4 (1 and2) have been determined by X-ray analysis. The alkali metal and iodide ions are in direct contact in2 but not in1. Compound1 [Na(benzo-12-crown-4)₂]·I is triclinic, witha=13.368(8),b=10.727(7),c=10.325(4) Å; =73.56(4),=77.73(4), =108.70(5)°;Z=2, space group is . Compound2 [K(benzo-12-crown-4)₂·I] is monoclinic, witha=15.807(8),b=12.043(4),c=15.601(6) Å,=117.74(3)°;Z=4, space groupC2/c. In both compounds the cations interact with all oxygen atoms of two crown ether molecules. Correlation of the crystal structures and behavior of the crown ethers in ion-selective membrane electrodes is discussed. Supplementary Data related to this article have been deposited with the British Library as Supplementary Publication No. 82185 (15 pages).     

Enzyme mediated-transesterification of verbascoside and evaluation of antifungal activity of synthesised compounds

Enzymatic acylation of verbascoside, a polyhydroxylated natural product, has been reported in this study using five different commercial lipases and taking p-nitrophenyl alkanoates as acyl donors. Out of these enzymes, the immobilised Candida antarctica lipase B was found as the enzyme of choice. Mono- and di-acylated products were formed, with mono as major product indicating high regioselective nature of such transformations. A series of acyl esters of verbascoside have been synthesised by this enzymatic transesterification methodology. The lipophilicity of the synthesised analogues was also checked. The analogues were further subjected to synergistic antifungal activity with amphotericin B (AmB) against Candida albicans. Fourfold reduction in minimum inhibitory concentration of AmB was observed with few synthesised analogues such as verbascoside 4″-octanoate (3b), verbascoside 4″-palmitate (3d) and verbascoside 4″,4′-dipalmitate (4d) at a concentration of 0.5 μg/mL.     

Contemporary Detection Of 4-Demeth-Oxydaunorubicin And Its Metabolites 13-Dihydro-4-Demethoxydaunorubicin And4-Demethoxydaunorubicinone By Reverse Phase High-Performance Liquid Chromatography

Abstract

The aim of this work was to optimize a simple analytical method for a complete pharmacological and toxicological follow up of patients treated with a new orally active daunorubicin analog, the 4-demethoxydaunorubicin. For this reason the Chromatographic properties of the unchanged drug, its reduced metabolite 13-dihydro-4-demethoxydaunorubicin and its aglicone metabolite 4-demethoxydaunorubicinone have been investigated. Extraction of these compounds from biological fluids has been carried out using ethyl acetate and buthanol. Separation has been achieved in a C₁₈ reverse phase column by iso-cratic eluition with a mobil phase consisting of acetonitrile:methanol :phosphate buffer 40:10:50, pH 4.7. Drug and metabolites can be quantitated at nanogram level by fluorescence detection.

The appearance of a further compound, identified as the 13-dihydro-4-deme-thoxydaunorubicinone, was noted when whole blood instead of plasma was utilized while developing the assay. Aldo-cheto reductases of red blood cells could be responsible for the reduction of the 4-demethoxydaunorubicinone in its 13-dihydroderivative.