Separation Of Diastereoisomers Research Articles

Diastereoisomer separation of silybin A and silybin B by semi‐preparative liquid chromatography using Box–Behnken design

AbstractThe effects of the flow rate, the injection volume, and the mobile phase ratio on the retention time and the resolution of separation of silybin were examined using Box–Behnken design on a symmetry prep C18 column. The optimum values of the operating parameters were 8 ml/min for the flow rate, 3 ml for the injection volume, and 40:60 for the mobile phase ratio. Under the optimal conditions, the retention time of silybin B was 59.552 min and the resolution of silybin A and silybin B was 1.0696, where the purities of the obtained silybin A and silybin B were both over 99% by ultra‐performance liquid chromatography. The preparative‐liquid chromatography simulation was applied with COMSOL. The optimization results of the response surface are consistent with the experiment and the simulation results. The structures of the compounds were identified by ultra‐performance liquid chromatography‐tandem mass spectrometry and ultra‐performance liquid chromatography‐ time of flight mass spectrometry.

Asymmetric Ruthenium Catalysis Enables Fluorophores with Point Chirality Displaying CPL Properties.

A novel enantiopure π-allylruthenium(IV) precatalyst allowed the enantioselective and stereospecific allylations of indoles and gave access to indolin-3-ones, containing vicinal stereogenic centers. Facile separation of diastereoisomers exhibiting opposite circularly polarized luminescence (CPL) activities in diverse solvents, including water, demonstrated the potential of these sustainable transformations and of the newly prepared molecules.

Characterizing the Diastereoisomeric Distribution of Phosphorothioate Oligonucleotides by Metal Ion Complexation Chromatography, In-Series Reversed Phase-Strong Anion Exchange Chromatography, and 31P NMR.

Replacement of a non-bridging oxygen atom of the phosphate diester linkage of an oligonucleotide by sulfur conveys pharmacokinetic benefits, such as increased nuclease resistance and enhanced protein binding. Substitution renders the internucleotide linkages chiral, and so phosphorothioate diester (PS) oligonucleotides comprise complex mixtures of diastereoisomers. Currently, chromatographic separation of individual diastereoisomers is limited to oligonucleotides that contain no more than about four or five PS linkages. The development of therapeutic PS oligonucleotides, which often contain >15 PS linkages, would be greatly aided by methods useful for assessing batch-to-batch stereo-reproducibility. To this effect, the relative sensitivities of metal ion complexation chromatography (MICC), in-series reversed phase-strong anion exchange chromatography (RP-SAX), and 31P NMR toward changes in the diastereoisomeric distributions of therapeutic PS oligonucleotides were compared. Model oligonucleotides synthesized under conditions known to impact PS stereochemistry were used to evaluate the method performance, and all three methods showed excellent sensitivity toward changes in the diastereoisomeric composition. Interactions via the solvent-accessible areas and a combination of hydrophobic and electrostatic forces may be responsible for the selectivity demonstrated by MICC and in-series RP-SAX, respectively.

The first synthesis of natural alkaloid capparine A

Abstract Substances containing a spirooxindole framework display important biological activities. Natural alkaloid capparine A [(S)-(−)-1] has an anti-inflammatory effect. In the present study, attention has been paid to the first total synthesis of natural capparine A [(S)-(−)-1]. Racemic capparine A [(±)-1] was synthesized by bromospirocyclization of 6-methoxy-1-Boc-brassinin with water, followed by oxidation of obtained spirobrassinol derivatives and removal of the Boc group. Synthesized racemic capparine A [(±)-1] was enantioresolved by derivatization with (1R,2S,5R)-menthyl chloroformate, chromatographic separation of diastereoisomers and the cleavage of the chiral auxiliary using sodium methoxide. Screening of anti-proliferative activity against human cancer cells revealed no anti-proliferative activity of the capparine A [(S)-(−)-1].

Ring-closing metathesis of prochiral oxaenediynes to racemic 4-alkenyl-2-alkynyl-3,6-dihydro-2H-pyrans.

The prochiral 4-(allyloxy)hepta-1,6-diynes, optionally modified in the positions 1 and 7 with an alkyl or ester group, undergo a chemoselective ring-closing enyne metathesis yielding racemic 4-alkenyl-2-alkynyl-3,6-dihydro-2H-pyrans. Among the catalysts tested, Grubbs 1st generation precatalyst in the presence of ethene (Mori conditions) gave superior results compared to the more stable Grubbs or Hoveyda–Grubbs 2nd generation precatalysts. This is probably caused by a suppression of the subsequent side-reactions of the enyne metathesis product with ethene. On the other hand, the 2nd generation precatalysts gave better yields in the absence of ethene. The metathesis products, containing both a triple bond and a conjugated system, can be successfully orthogonally modified. For example, the metathesis product of 5-(allyloxy)nona-2,7-diyne reacted chemo- and stereoselectively in a Diels–Alder reaction with N-phenylmaleimide affording the tricyclic products as a mixture of two separable diastereoisomers, the configuration of which was estimated by DFT computations. The reported enediyne metathesis paves the way to the enantioselective enyne metathesis yielding chiral building blocks for compounds with potential biological activity, e.g., norsalvinorin or cacospongionolide B.

Inositol Adenophostin: Convergent Synthesis of a Potent Agonist of d-myo-Inositol 1,4,5-Trisphosphate Receptors.

d-myo-Inositol 1,4,5-trisphosphate receptors (IP3Rs) are Ca2+ channels activated by the intracellular messenger inositol 1,4,5-trisphosphate (IP3, 1). The glyconucleotide adenophostin A (AdA, 2) is a potent agonist of IP3Rs. A recent synthesis of d-chiro-inositol adenophostin (InsAdA, 5) employed suitably protected chiral building blocks and replaced the d-glucose core by d-chiro-inositol. An alternative approach to fully chiral material is now reported using intrinsic sugar chirality to avoid early isomer resolution, involving the coupling of a protected and activated racemic myo-inositol derivative to a d-ribose derivative. Diastereoisomer separation was achieved after trans-isopropylidene group removal and the absolute ribose–inositol conjugate stereochemistry assigned with reference to the earlier synthesis. Optimization of stannylene-mediated regiospecific benzylation was explored using the model 1,2-O-isopropylidene-3,6-di-O-benzyl-myo-inositol and conditions successfully transferred to one conjugate diastereoisomer with 3:1 selectivity. However, only roughly 1:1 regiospecificity was achieved on the required diastereoisomer. The conjugate regioisomers of benzyl derivatives 39 and 40 were successfully separated and 39 was transformed subsequently to InsAdA after amination, pan-phosphorylation, and deprotection. InsAdA from this synthetic route bound with greater affinity than AdA to IP3R1 and was more potent in releasing Ca2+ from intracellular stores through IP3Rs. It is the most potent full agonist of IP3R1 known and .equipotent with material from the fully chiral synthetic route.

Chiral auxiliary-mediated synthesis of planar chiral [2.2]metacyclophanes

The synthesis of planar chiral [2.2]metacyclophanes has been readily accomplished in a single synthetic step via the directed ortho metalation of a pro-chiral substituted metacyclophane. The use of (−)-menthyl chloroformate as a chiral auxiliary allows the introduction of the useful carbonyl functional group into the aryl ring, giving access to carboxy-substituted diastereomeric mixture of planar chiral [2.2]metacyclophanes. The separation of diastereoisomers has been easily accomplished by semipreparative HPLC, allowing the structural analysis of a single diastereoisomer by X-ray crystallography and NMR spectroscopy. The structural features of the planar chiral metacyclophanes and their high inversion barriers, determined at 473 K, encourage future investigations as chiral catalysts and ligands. This synthetic route complements our previously reported enantioselective synthesis avoiding the restrictive use of (−)-sparteine as the chiral inducer.

Small alkyl amines as ion-pair reagents for the separation of positional isomers of impurities in phosphate diester oligonucleotides

In an effort to improve separation of impurities in oligonucleotide drugs, alkyl amines of different length and carbon content were evaluated as reagents in ion pair-reversed phase (IP-RP) HPLC with mass spectrometric detection. A range of columns was tested in combination with different buffers, ion-pair modifiers and varying pH adjustments. For phosphorothioate oligonucleotides, larger amines, like tributyl and hexyl amine provided the best chromatography, as small amines tended to broaden peaks due to the separation of diastereoisomers. For phosphate diester oligonucleotides, the best separations were obtained using small alkyl amines, like propyl-, isopropyl- and diethylamine. Conditions optimized for oligonucleotide sequence and type of impurity enabled full separation of the individual components of composite impurities, such as n-1, N3-(2-cyanoethyl)thymine (CNET), deaminated and 3-(2-oxopropyl)imidazopyrimidinone (OPC) impurities. The addition of long-chain alkyl acids like hexanoic acid to the IP buffer resulted in further improvements in peak separation.

Purification of Flavonolignan Diastereoisomers from Arenaria kansuensis by Two-Dimensional Liquid Chromatography Combined with Solid-Phase Extraction

Herbal plants are significant for the reason that they have a great potential in discovering drug precursors. However, how to purify compounds with higher purity from them is a question which needs to be discussed. In present study, an offline 2D reversed-phase (RP) preparative liquid chromatography coupled with solid-phase extraction (SPE) method was successfully developed for the separation of flavonolignan diastereoisomers from Arenaria kansuensis. Based on the analysis of results, the major conclusion that we have drawn from it is a RP-SPE was selected for enriching target flavonolignan sample from A. kansuensis. After that, an ODS preparative column was used for 1D preparation, and the target sample (4.6 g) was divided into five fractions with a recovery of 83.9%. Then, a C18HCE preparative column, a polar-modified RP (polar-copolymerized) type, was used for isolating flavonolignan diastereoisomers in the 2D preparation. By establishing optimal 2D chromatography, hydrophilic interaction chromatography (HILIC) columns and normal-phase (NP) columns were tested simultaneously, and the result showed that diastereoisomers are not suitable for HILIC and NP chromatography mode. Our study resulted in a tricin and five analogous derivative flavonolignans with purity >98% were successfully purified from A. kansuensis. This is the initial report of Salcolin C, Salcolin B, Tricin 4'-O-(C-veratroylglycol) ether and 5'-methoxyhydnocarpin D from A. kansuensis. In addition, it tended to be the first time that Tricin 4'-O-(C-veratroylglycol) ether is isolated from natural resource. This method has great potential for efficiently isolating flavonolignan diastereoisomers from A. kansuensis, and it shows a great prospect for the separation of flavonolignans from complex samples.

Heterometalation of 1,1'-Bis( ortho-carborane).

Deboronation of [8-(1'- closo-1',2'-C2B10H11)- closo-2,1,8-MC2B9H10] affords diastereoisomeric mixtures of [8-(7'- nido-7',8'-C2B9H11)- closo-2,1,8-MC2B9H10]- anions (1, M = Ru( p-cymene); 2, M = CoCp) isolated as [HNMe3]+ salts. Deprotonation of 1 and reaction with CoCl2/NaCp followed by oxidation yields [8-(1'-3'-Cp -closo-3',1',2'-CoC2B9H10)-2-( p-cymene)- closo-2,1,8-RuC2B9H10] isolated as two separable diastereoisomers, namely, 3α and 3β, the first examples of heterometalated derivatives of 1,1'-bis( ortho-carborane). Deprotonation of [7-(1'- closo-1',2'-C2B10H11)- nido-7,8-C2B9H11]-, metalation with CoCl2/NaCp* and oxidation affords the isomers [1-(1'- closo-1',2'-C2B10H11)-3-Cp*- closo-3,1,2-CoC2B9H10] (4) and [8-(1'- closo-1',2'-C2B10H11)-2-Cp*- closo-2,1,8-CoC2B9H10] (5) as well as a trace amount of the 13-vertex/12-vertex species [12-(1'- closo-1',2'-C2B10H11)-4,5-Cp*2- closo-4,5,1,12-Co2C2B9H10] (6). Reduction then reoxidation of 4 converts it to 5. Deboronation of either 4 or 5 yields a diastereoisomeric mixture of [8-(7'- nido-7',8'-C2B9H11)-2-Cp*- closo-2,1,8-CoC2B9H10]- (7), again isolated as the [HNMe3]+ salt. Deprotonation of this followed by treatment with [RuCl2( p-cymene)]2 produces [8-(1'-3'-( p-cymene)- closo-3',1',2'-RuC2B9H10)-2-Cp*- closo-2,1,8-CoC2B9H10] (8) as a mixture of two diastereoisomers in a 2:1 ratio, which could not be separated. Diastereoisomers 8 are complementary to 3α and 3β in which {CoCp} and {Ru( p-cymene)} in 3 were replaced by {Ru( p-cymene)} and {CoCp*}, respectively, in 8. Finally, thermolysis of mixture 8 in refluxing dimethoxyethane yields [8-(8'-2'-( p-cymene)- closo-2',1',8'-RuC2B9H10)-2-Cp*- closo-2,1,8-CoC2B9H10] (9), again as a 2:1 diastereoisomeric mixture that could not be separated. All new species were characterized by multinuclear NMR spectroscopy, and 3α, 3β, 4, 5, 6, and 9 were also characterized crystallographically.

A liquid chromatography-mass spectrometry study on the spirocyclization of ninhydrin with the aminothiols

Ninhydrin reacts with some aminothiols to form spiranes adducts whose optical and chromatographic properties have proved to be useful for chiral recognition. Liquid chromatography-mass spectrometry data along with spectroscopic analysis reveal that under certain conditions, in addition to the known single-spirane configuration, the spirothiazolidinic complexes can exist also as double- and mixed double-spiranes. The reaction was exploited to check the enantiomeric purity of two commercially available dosage form of D-penicillamine and to measure the aminothiol concentration in the urine sample from a subject under treatment with the drug. Separation of diastereoisomers was achieved on a C18 column in isocratic mode by using a mixture of an aqueous solution of formic acid (30 mmol/L)/acetonitrile (90:10, v/v) as a mobile phase. Diastereoisomers were detected by a fluorescence detector and mass spectrometer in short times and with a good resolution. Intra- and inter-assay reproducibility were under 4% with an average recovery of 98%. At a LOD of 0.01%, no evidence of the toxic distomer (l-enantiomer) was found in the biological sample and drugs.

Synthesis of glyceryl glycosides related to A-type prymnesin toxins

A suite of glycosylated glycerol derivatives representing various fragments of the glycosylated ichthyotoxins called prymnesins were chemically synthesised. Glycerol was used to represent a small fragment of the prymnesin backbone, and was glycosylated at the 2° position with the sugars currently reported to be present on prymnesin toxins. Neighbouring group participation was utilised to synthesise 1,2-trans-glycosides. SnCl2-promoted glycosylation with furanosyl fluorides gave 1,2-cis-furanosides with moderate stereocontrol, whilst TMSOTf promoted glycosylation with a furanosyl imidate gave a 1,2-cis-furanoside with good stereocontrol. The chemical synthesis of two larger glyceryl diglycoside fragments of prymnesin-1, glycosylated with α-ʟ-arabinopyranose and α-ᴅ-ribofuranose, is also described. As the stereochemistry of the prymnesin backbones at this region is undefined, both the 2R- and 2S- glycerol isomers were synthesised. The separated diastereoisomers were distinguished by comparing NOESY NMR with computational models.

Synthesis of Carbohydrate Mimetics by Intramolecular 1,3‐Dipolar Cycloaddition of N‐(3‐Alkenyl)nitrones Derived from Unprotected d‐Aldopentoses

A concise synthesis of C‐furanosides has been achieved, starting from unprotected d‐xylose and d‐arabinose. Both sugars reacted with hydroxylamine 6 to give the respective nitrones. These underwent in‐situ intramolecular 1,3‐dipolar cycloaddition reactions to give, in each case, a pair of diastereomeric derivatives of 7‐oxa‐1‐aza‐bicyclo[2.2.1]heptane 7 and 8. The ratio of chromatographically separable diastereoisomers could be changed by carrying out the reaction in the presence of weak Lewis acid. Both d‐xylose‐derived stereoisomers 7a and 8a were converted into d‐erythro‐C‐furanosides by tosylation or mesylation followed by catalytic hydrogenolysis. On the other hand, the formation of d‐threo‐C‐furanosides from d‐arabinose derivatives 7b and 8b under similar conditions was accompanied by the formation of minor amounts of polyhydroxyquinolizidines as a result of competitive N‐alkylation. The polyhydroxyquinolizidine side‐products were useful for the assignment of the configuration of the new compounds.

Novel donepezil-like N-benzylpyridinium salt derivatives as AChE inhibitors and their corresponding dihydropyridine “bio-oxidizable” prodrugs: Synthesis, biological evaluation and structure-activity relationship

As an extension of our previous work on donepezil-based “bio-oxidizable” prodrug approach, two new classes of N-benzylpyridinium donepezil analogues in tetralone B2 and acetophenone B3 series and a new set of indanone derivatives B1 were investigated along with the corresponding dihydropyridine prodrugs A1-3. A total of fifty one N-benzylpyridinium quaternary donepezil analogues B1-3 and twenty two prodrugs A1-3 were synthesized and evaluated for their inhibitory activities against hAChE and eqBuChE. While most prodrugs A1-3 were demonstrated to be inactive against AChE (IC50 > 10 μM), a large number of the corresponding N-benzylpyridinium salt B1-3 exhibited appealing three-to-one-digit nanomolar hAChE inhibitory activities and even reaching subnanomolar activity (IC50 = 0.36 nM). In addition, in silico docking studies were conducted for several compounds to explain the more relevant in vitro results. Lastly, the influence of the two stereogenic centers in prodrugs A was also evaluated, highlighting not only marked differences in residual AChE inhibitory activity of the four separated isomers of prodrug 23h (IC50 ranging from 173 nM to 10 μM) but also significant variations of the oxidation rate between two separated diastereoisomers of prodrug 24a. This work provides useful information in the search of a preclinical candidate to conduct further development of this attractive “bio-oxidizable” prodrug strategy.

2‐(1S)‐Camphanoyloxy‐2′‐phosphanylbiphenyl Ligands – Synthesis, Structure, and Preliminary Tests in Transition‐Metal Catalysis

Diastereoisomer separation of the (1S)‐camphanic acid 2‐isopropylphenylphosphanyl‐phenyl ester 1 exemplifies the potential of (1S)‐camphanoyl chloride for enantiomer separation of hydroxyl‐functional asymmetric phosphanes. Esterification of lithium 2′‐phosphanylbiphenyl‐2‐olates, generated from the respective 2‐OH or 2‐OSiMe3 precursors 2aOH and 2b–fSi, furnished the 2‐(1S)‐camphanoyloxy‐biphenylphosphanes 3a–c as 1:1 mixtures of diastereomers with low barriers for interconversion by rotation around the C–C axis (ΔG# = 70–73 kJ mol–1 for 3a and 3c by 31P VT NMR spectroscopy). The P‐asymmetric compounds 3d–f form 1:1 mixtures of stereoisomers. There is a tendency to cocrystallization of two preferred diastereoisomers, as shown by the crystal structure analyses of 3dD and 3fD, and in solution, there is a tendency toward partial isomerization to the sterically less‐favored atropisomers. The [RhCl(cod)(3dD)] complex 4dD, however, seems stable in solution. Excess 2dLi reacted with (1S)‐camphanoyl chloride preferentially to form the (SP,Rax,1S) isomer, which was separated by crystallization as enantiopure 3dE, characterized by single‐crystal XRD. Preliminary screening tests of this ligand in Rh‐catalyzed asymmetric hydrogenations of N‐(1‐phenylvinyl)acetamide allowed high conversion and up to 59 % ee. Hydrosilylation of acetophenone proceeded with 78 % conversion and 48 % ee; Suzuki–Miyaura couplings of 1‐bromo‐2‐naphthol with PhB(OH)2, in the presence of 3b/[Pd(OAc)2], gave yields up to 98 %.

Determination of the stereoisomeric impurities of sitafloxacin by capillary electrophoresis with dual chiral additives

Because of the bioactivity against the human topoisomerase II, the stereoisomeric impurities of sitafloxacin should be controlled in the process of manufactory. In the present work, a capillary electrophoresis (CE) method was developed and validated for simultaneous determination of three stereoisomeric impurities of sitafloxacin. The separation with high resolution not only for the separation of enantiomers, but also for the separation of diastereoisomers was achieved by using a background electrolyte composed of dual chiral selectors, namely γ-cyclodextrin (γ-CD) and Cu2+-d-phenylalanine (D-Phe) complex. The combination of two chiral selectors is indispensable to gain a high separation selectivity due to the cooperativity effect of different chiral discrimination modes: inclusion complexation (γ-CD) and ligand exchange (Cu2+-d-Phe). The concentrations of γ-CD, Cu2+ and D-Phe were found to be critical to the separation. Because two chiral selectors were involved in the enantiomer separation system, multiple factors and their interaction should be simultaneously optimized by using the response surface methodology (RSM) with a face centred central composite design (FCCD). The obtained optimized separation conditions were as follows: 15mmol/L dipotassium hydrogenphosphate solution (pH 4.5) containing 15mmol/L D-Phe, 20mmol/L CuSO4 and 20mmol/L γ-CD, separation voltage 15kV. The method was then validated and the robustness of the method was tested. Under the optimized conditions, as low as 0.1% (m/m) stereoisomeric impurities of sitafloxacin can be determined by the method.

Phosphonoamidate prodrugs of C5-substituted pyrimidine acyclic nucleosides for antiviral therapy

Acyclic nucleoside phosphonates (ANPs) are nowadays one of the key drugs in the treatment of DNA virus and retrovirus infections. In this work, we report the synthesis and antiviral evaluation of phosphonoamidate and diamidates prodrugs of C5-pyrimidine acyclic nucleosides derivatives functionalized with but-2-enyl- chain. In the phosphonoamidate series, the most active compound 15, showed sub-micromolar activity against varicella zoster virus (VZV) (EC50 = 0.09–0.5 μM) and μM activity against human cytomegalovirus (HCMV) and herpes simplex virus (HSV). Separation of single diastereoisomers for compound 14, showed that 14b had better anti-herpesvirus activity and no cytotoxicity compared to the diastereoisomeric mixture 14. Very interestingly, phosphonodiamidate 21 showed anti-herpesvirus activity with excellent activity against wild type and thymidine kinase-deficient (TK−) VZV strains (EC50 = 0.47 and 0.2 μM, respectively) and HCMV (EC50 = 3.5–7.2 μM) without any cytotoxicity (CC50 > 100).

Phosphorothioate analogs of P1,P3-di(nucleosid-5′-yl) triphosphates: Synthesis, assignment of the absolute configuration at P-atoms and P-stereodependent recognition by Fhit hydrolase

Di(nucleosid-5′-yl) polyphosphates (NPnN) are involved in various biological processes, and constitute signaling molecules in the intermolecular purinergic systems. They exert tumor suppression function and are substrates for specific hydrolases (e.g., HIT proteins). Their structural analogs may serve as molecular probes and potential therapeutic agents. Three P1,P3-bis-thio-analogs of symmetrical di(nucleosid-5′-yl) triphosphates (NP3N) bearing adenosine, guanosine or ribavirin residues (6, 7 and 8, respectively), were obtained by direct condensation of corresponding base-protected nucleoside-5′-O-(2-thio-1,3,2-oxathiaphospholane) with anhydrous phosphoric acid in the presence of DBU. Deprotected products 6 and 8 were separated into individual P-diastereoisomers, whereas 7 was partially separated to yield diastereomerically enriched fractions. The absolute configuration at P-stereogenic centers in the separated diastereoisomers was assigned by RP-HPLC analysis of the products of enzymatic digestion with snake venom phosphodiesterase. The Fhit-assisted hydrolysis rates for 6 and 7 are by 2–3 orders of magnitude lower than that for the reference AP3A, and depend on the configuration of the stereogenic phosphorus atoms, while 8 occurred to be resistant to this cleavage.

Diastereoisomeric forms of 11-ethyl-6,11-dihydro-5H-dibenzo[b,e]azepine-6-carboxamide: syntheses and the molecular and supramolecular structure of the minor form (6RS,11RS)-11-ethyl-6,11-dihydro-5H-dibenzo[b,e]azepine-6-carboxamide.

Compounds containing the tricyclic dibenzo[b,e]azepine system have potential activity in the treatment of a number of diseases. Continuing with our studies on the synthesis of new small and potentially bioactive molecules, a synthetic route, involving acid-catalysed intramolecular Friedel-Crafts cyclization, to the readily separable diastereoisomers of 11-ethyl-6,11-dihydro-5H-dibenzo[b,e]azepine-6-carboxamide, a potentially useful precursor in the synthesis of analogues of some anti-allergenic, antidepressant and antihistaminic drugs currently in use, has been developed starting from 2-allylphenylamine and methyl 2-bromo-2-phenylacetate and proceeding via racemic methyl 2-[(2-allylphenyl)amino]-2-phenylacetate (A) and racemic 2-[(2-allylphenyl)amino]-2-phenylacetamide (B), to give the two diastereoisomers (I) and (II), C17H18N2O. Isomers (I) and (II), and their precursors (A) and (B), have all been fully characterized spectroscopically. Structure analysis of the minor isomer (I) shows that it has the (6RS,11RS) configuration, and that the azepine ring adopts a conformation intermediate between the boat and twist-boat forms, with the carboxamide and ethyl substituents both occupying quasi-equatorial sites. The molecules of (I) are linked by a combination of N-H...O, N-H...π(arene) and C-H...π(arene) hydrogen bonds to form complex sheets. Comparisons are made with the structures of some related compounds.

Development and validation of a novel LC–MS/MS method for simultaneous determination of abiraterone and its seven steroidal metabolites in human serum: Innovation in separation of diastereoisomers without use of a chiral column

Abiraterone acetate (AA), the prodrug of abiraterone, is FDA-approved for the treatment of castration-resistant prostate cancer. Abiraterone is metabolized in patients to a more potent analogue, D4A. However, we have recently reported that this analogue is further metabolized to additional metabolites in patients treated with AA. Here, we present a liquid chromatography-tandem mass spectrometry method developed to resolve and detect abiraterone and its seven metabolites in human serum using an AB Sciex Qtrap 5500 mass analyzer coupled with a Shimadzu Nexera UPLC station. Analytes and the internal standard (abiraterone-d4) were extracted from human serum using the liquid–liquid extraction procedure. The analytes were separated using a Zorbax Eclipse Plus C18 150×2.1mm, 3.5μm column at 40°C and an isocratic mobile phase 35% A (0.1% formic acid in water), 65% B (0.1% formic acid in methanol:acetonitrile; 60:40). Electrospray ionization in positive mode was applied with multiple reaction monitoring in a total run time of 13min. Abiraterone detection was linear in the range 2–400ng/mL and all metabolites from 0.1–20ng/mL. The method was validated following US FDA guidelines for bioanalytical method validation, and all the metabolite results were within the acceptance limits. Despite the similarity in structure and mass transition between the metabolites, the validated method separated all the metabolites, including diastereomers, to allow accurate identification and quantitation of each compound.