A nonneglectable stereochemical factor in drug development: Atropisomerism.

Chirality is one of the key factors affecting the medicinal efficacy of compounds. In addition to central chirality, sterically hindered chiral axes commonly appear in drugs and the resulting chirality is known as atropisomerism. With developments in medicinal chemistry, atropisomerism has attracted increasing attention. This review discusses the classification, biological activity, pharmacokinetics, toxicity and side effects of atropisomers, and can serve as a reference in the research and development of potential chiral drugs.

Chiral probe for mass spectrometric identification and quantitation of levodropropizine and its enantiomer, dextrodropropizine.

Acyl chlorides react rapidly with hydroxyl and amino groups in the absence of catalysts and therefore hold great promise for chiral mass spectrometry. Herein, a tandem mass spectrometry method based on derivatization with (-)-camphanic acid chloride as a simple chiral probe was developed for the highly sensitive detection and quantitation of levodropropizine and its enantiomer, namely, dextrodropropizine. The diastereomeric derivatization products were quantified based on the relative abundances of their fragment ions produced upon collision-induced dissociation in positive-ion mode. The reactive site was elucidated using nuclear magnetic resonance spectroscopy and two-dimensional total correlation spectroscopy, and the reaction mechanism was proved by stoichiometry studies. The degree of isomer recognition was investigated at different collision energies and determined as Rchiral  ≈ 1.5. Thus, this study gives the way to the mass spectrometric identification and quantitation of levodropropizine and its enantiomer, and the developed method represents a new and practical technique for rapid and sensitive determination and quality control of enantiomers of chiral drugs.

Chiral drug fluorometry based on a calix[6]arene/molecularly imprinted polymer double recognition element grafted on nano-C-dots/Ir/Au.

A luminescent double recognition nanoprobe is described as a new strategy for the selective determination of chiral molecules. C-dots/Ir/Au fluorescent nanoparticles, synthesised under hydrothermal conditions, are used as a high-performance probe in combination with a molecularly imprinted polymer (MIP) and calix[6]arene as a double recognition element. Thiolated calix[6]arene is grafted on C-dots/Ir/Au as the first recognition element, which then forms a host-guest complex with the target molecule levodopa (L-DOPA). Subsequently, an MIP is prepared on the C-dots/Ir/Au (MIP/C-dots/Ir/Au) by chemical polymerisation. After the removal of L-DOPA, double recognition imprinting cavities are formed. The fluorescence intensity at 478 nm of the nanoprobe is effectively quenched by adsorption of L-DOPA on MIP/C-dots/Ir/Au, which provides a method for L-DOPA determination. Owing to the double recognition strategy, this method has excellent selectivity which can effectively avoid interference from enantiomer D-DOPA, and a imprinting factor of 7.1 is obtained for L-DOPA. This accurate and reliable method, with a wide linear range (5 × 10-10 to 1.2 × 10-7 mol L-1) and a low limit of detection (1.45 × 10-10 mol L-1), was successfully applied to the determination of L-DOPA in real samples, giving standard recoveries of 89.7-110.0%. Thus, the proposed sensing method provides a viable approach for the determination of a single enantiomer. Graphical abstract Schematic presentation of the MIP/C-dots/Ir/Au for L-DOPA detection. A fluorescence double chiral recognition nanoprobe is prepared of C-dots/Ir/Au nanoparticles as signal probe, and a molecularly imprinted polymer (MIP) and calix[6]arene as a double recognition element. Owing to the double recognition strategy, this method has strong specificity and can effectively avoid interference from enantiomers and racemates.

Graphene oxide-assisted non-immobilized SELEX of chiral drug ephedrine aptamers and the analytical binding mechanism.

Here, we describe a study of screen characterization of aptamers targeting the chiral drug ephedrine using the non-immobilized graphene oxide (GO) SELEX. The improved method of long and short chains was here used to prepare the ssDNA library. The Resonance Rayleigh Scattering (RRS) method was first used to monitor the screening process. Through high-throughput sequencing, the genetic sequence data of 90,487 aptamers were obtained. Through the analysis of the parameters of free energy value and secondary structure prediction model of high repeatability sequence, the 10 candidate sequences were identified. Finally, a best-fit aptamer named EP08 was identified by combining the dissociation experiment. The binding affinity and binding mechanism of the aptamer and target were analyzed using an isothermal titration colorimetry (ITC) experiment and circular dichromatic (CD) experiment. The binding affinity (Kd) of the EP08 aptamer to ephedrine is approximately 2.86 ±â€¯0.24 µM. This novel DNA aptamer will help in the future development of a new method for the identification and detection of chiral drug ephedrine.

Potent and selective EGFR inhibitors based on 5-aryl-7H-pyrrolopyrimidin-4-amines.

The epidermal growth factor receptor represents an important target in cancer therapy, and low molecular weight inhibitors based on quinazolines have reached the marked. Herein we report on a new scaffold, 5-aryl-7H-pyrrolo[2,3-d]pyrimidin-4-amines, and show that when employing (S)-phenylglycinol as C-4 substituent, potent inhibitors can be made. The two most active inhibitors have suitable druglike properties, were equipotent with Erlotinib in Ba/F3 cell studies, and showed lower cross reactivity than Erlotinib in a panel of 50 kinases.

Enantioselective synthesis of (R)-Cinacalcet via cobalt-catalysed asymmetric Negishi cross-coupling.

A novel enantioselective synthesis of (R)-cinacalcet with 99% enantiomeric excesses (ee) has been achieved. The main strategies of the approach include a gram-scale cobalt-catalysed asymmetric cross-coupling of racemic ester with arylzinc reagent, Hoffman-type rearrangement of acidamide, the amidation of chiral amine, and improving the ee of chiral amide from 87% to 99% via recrystallization.

In vitro analysis of itraconazole cis-diastereoisomers inhibition of nine cytochrome P450 enzymes: stereoselective inhibition of CYP3A.

1. Itraconazole (ITZ), an antifungal azole derivate is a chiral drug that consists of four cis-diastereoisomers ((+)-2R,4S,2'R-ITZ-A; (+)-2R,4S,2'S-ITZ-B; (-)-2S,4R,2'S-ITZ-C and (-)-2S,4R,2'R-ITZ-D) which may differ in their pharmacokinetics and pharmacodynamics. 2. As ITZ is known as a CYP3A4 inhibitor causing severe drug-drug interaction, the inhibitory potencies of its individual optical isomers towards nine drug-metabolising cytochrome P450 (including CYP3A, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP2E1), were investigated. 3. All ITZ diastereoisomers dose-dependently inhibited CYP3A activity in both used assays, midazolam and testosterone hydroxylation. The Ki values were assessed: for testosterone ITZ-A/0.085 µM; ITZ-B/0.91 µM, ITZ-C/0.20 µM and ITZ-D/0.022 µM; for midazolam ITZ-A/0.44 µM; ITZ-B/0.48 µM, ITZ-C/1.56 µM and ITZ-D/3.48 µM. The enzyme activity of CYP2C19 was moderately inhibited (IC50 30-53 µM), but in this case without large differences between the individual optical isomers. 4. The significant differences between diastereoisomers were presented. Antifungal potency of ITZ stereoisomers also differs so the potential enantiopure preparations of ITZ was not of interest.

Enantiomeric trans ß-aryl-δ-iodo-γ-lactones derived from 2,5-dimethylbenzaldehyde induce apoptosis in canine lymphoma cell lines by downregulation of anti-apoptotic Bcl-2 family members Bcl-xL and Bcl-2.

For many years, studies focused on developing new natural or synthetic compounds with antineoplastic activity have attracted the attention of researchers. An interesting group of such compounds seem to be those with both lactone moiety and an aromatic ring which, in addition to antimicrobial or antiviral activity, also exhibit antitumor properties. The study shows antitumor activity of two enantiomeric trans isomers of 5-(1-iodoethyl)-4-(2',5'-dimethylphenyl)dihydrofuran-2-one. Our aim was to determine their antitumor activity manifested as an ability to induce apoptosis in selected canine cancer cell lines as well as to evaluate differences in their strength depending on the configuration of their stereogenic centers. The enantiomers (+)-(4R,5S,6R)-1 and (-)-(4S,5R,6S)-2 were found to induce classical caspase-dependent apoptosis through downregulation of the expression of anti-apoptotic proteins Bcl-xL and Bcl-2. Although the mechanism of apoptosis induction was the same for both enantiomers, they differed in their strength, as stronger antineoplastic activity in vitro was exhibited by isomer (+)-(4R,5S,6R)-1.

Enantioselective Monoclonal Antibodies for Detecting Ketamine to Crack Down on Illicit Use.

Ketamine (KT) is a chiral anesthetic agent, (R)- and (S)-enantiomers of which differ in their pharmacological properties. KT has become one of the most commonly used illicit drugs in the world, thus, rapid and feasible on-site testing is required to crack down on the illicit use. Although immunochemical approach with specific antibodies is promising for this purpose, in practice anti-KT antibodies are difficult to obtain. We here disclose generation of monoclonal antibodies against KT. Mice were immunized with either (a) commercially-available or (b) in-house-prepared KT-albumin conjugates. Splenocytes from these mouse groups (a and b) were separately fused with P3/NS1/1-Ag4-1 myeloma cells. After standard screening and cloning, we established 5 hybridoma clones: 2 were derived from group-a mice [generating Ab-KT(a)#2 and #37] and 3 were from group-b mice [generating Ab-KT(b)#9, #13, and #45]. These antibodies exhibited practical performance in competitive enzyme-linked immunosorbent assay systems. When (±)-KT·hydrochloride (HCl) was used as the competitor, dose-response curves showed midpoint values of 30 and 70 ng/assay (a-series antibodies) and 2.0-3.0 ng/assay (b-series antibodies). Remarkably, the a-series antibodies were specific for (S)-KT·HCl, while the b-series antibodies were specific for (R)-KT·HCl. Ab-KT(a)#2 (Ka, 7.5×107 M-1) and Ab-KT(b)#45 (Ka, 7.7×108 M-1) exhibited the highest enantioselectivity for each group, and cross-reactivity with the (R)- and (S)-antipodes was 1.3 and 1.7%, respectively. The hybridomas established here are also valuable as a source of genetic information for the anti-KT antibodies, which is required for progressing to next-generation technologies using genetically engineered antibodies.

Analysis of stereoisomers of chiral drug by mass spectrometry.

Chiral molecules are of great importance in the life science since individual enantiomers may differ in biological activity, mechanism, and toxicity, making it necessary to explore efficient chiral analysis methods. Chromatography approaches are often used to differentiate enantiomers while mass spectrometry (MS) was thought to be blind in chiral analysis. With the development of MS technique, it began to play a more and more crucial part in chiral observation. In this review, we will give a detailed introduction of the analysis methods related to MS for chiral drugs, including its mechanism, applications, and future development.

Chiral discrimination of ofloxacin enantiomers using DNA double helix regulated by metal ions.

DNA-based chiral selectors are constructed to discriminate ofloxacin enantiomers through metal-ion anchoring on a special DNA double helix that contains successive GC pairs. The effects of metal ions involving Mg(2+), Ni(2+), Cu(2+), Ag(+), and Pt(2+) were studied on the regulation of DNA chiral discrimination towards ofloxacin enantiomers. It is shown that DNA-Cu(II) complexes exhibit the highest enantioselectivities at the [Cu(2+)]/base ratio of 0.1. The enantiomeric excess can reach 59% in R-enantiomer after being adsorbed by the RET-Cu(II) complex. Stereoselective recognition of ofloxacin enantiomers on the double helix is tunable via external stimulus, providing a programmable desorption process to regenerate DNA. This DNA-based chiral selector exhibits excellent reusability without apparent loss of enantioselectivity after three cycles of adsorption and desorption.

Chiral-induced covalent organic framework as novel chiral stationary phase for chiral separation using open-tubular capillary electrochromatography.

As a novel class of chiral stationary phase (CPS) material, chiral covalent organic frameworks (CCOFs) have already shown great promise in open-tubular capillary electrochromatography (OT-CEC) for chiral separation. The synthesis methods of CCOFs used in OT-CEC mainly include bottom-up, post modification and chiral induction. The CCOFs synthesized by bottom-up and post modification strategies already have lots of applications in capillary electrochromatography, however, the chiral-induced synthesized via an asymmetric catalytic strategy has not yet been reported for using as the chiral stationary phase (CPS) in OT-CEC or even in chromatographic separation. Herein, the chiral-induced COF (Λ)-TpPa-1 was synthesized by asymmetric catalytic synthesis and coated on the inner surface of a capillary by an in-situ growth strategy as the CPS for chiral drug separation. The baseline separation of six enantiomers was achieved within 14 min, with a high-resolution (Rs) range from 1.85 to 6.75. Moreover, the resolution and migration time of the capillary keep stable within 160 runs, showing its superior stability and repeatability. This research provides a new idea for the development and application of novel CPS materials in the field of capillary electrochromatography separation, also shows the new application of chiral induced COFs. Furthermore, the chiral-induced CCOFs can be easily applied to other chromatographic separation fields, exhibiting its extensive application value in chiral analysis separation.

Biosynthesis of (S)-1-(1-naphthyl) ethanol by microbial ketoreductase.

(S)-1-(1-naphthyl) ethanol (SNE) is a chiral drug intermediate for the production of mevinic acid analog, a potent cholesterol agent. It acts as an HMG-CoA reductase inhibitor and is hence used in the synthesis of statins. Statins are lipid-lowering drugs used to lower cholesterol in the body. In our present study, we carried out whole-cell bioreduction of 1-Acetonaphthone to enantiopure SNE using selected microorganisms acquired by soil acclimation technique. The microorganism which exhibited higher bioreduction activity was determined using high-performance liquid chromatography (HPLC), and it was identified as Pichia kudriavzevii by ITS primer sequencing. After optimizing the parameters, Pichia sp. produced SNE with good conversion (75%), yield (67%), and excellent enantiomeric excess (100%). The microbial enzyme showed higher activity at 24-h-old supernatant. The crude and partially purified enzyme exhibited a specific activity of 51.13 U/mL and 62.72 U/mL, respectively, with a 1.22 purification fold.

Preparation and evaluation of molecularly imprinted polymer liquid chromatography column for the separation of Cathine enantiomers.

In this study molecular imprinting technology was employed to prepare a specific affinity sorbent for the resolution of Cathine, a chiral drug product. The molecularly imprinted polymer (MIP) was prepared by non-covalent molecular imprinting with either (+) or (-)-Cathine (threo-2-amino-1-hydroxy-1-phenyl propane; norpseudoephedrine) as the template. Methacrylic acid and ethylene glycol di-methacrylate were copolymerized in the presence of the template molecule. The bulk polymerization was carried out in chloroform with 2,2'-azobisisobutyronitrile as the initiator, at 5 °C and under UV radiation. The resulting MIP was ground into powders, which were slurry packed into analytical columns. After removal of template molecules, the MIP-packed columns were found to be effective for the resolution of (±)-Cathine racemates. The separation factor for the enantiomers ranged between 1.5 and 2.4 when the column was packed with MIP prepared with (+)-Cathine as the template. A separation factor ranging from 1.6 to 2.9 could be achieved from the column packed with MIP, prepared with (-)-Cathine as the template. Although the separation factor was higher with that previously obtained from reversed-phase column chromatography following derivatization with a chiral agent, elution peaks were broader due to the heterogeneity of binding sites on MIP particles and the possible non-specific interaction.

Preparation and characterization of a chiral molecularly imprinted polymer with a novel functional monomer for controlled release of S-sulpiride.

A novel molecularly imprinted polymer (MIP) with chiral recognition affinity to S-sulpiride (S-SUL) enantiomer was prepared by using newly synthesized N-acryloyl-tryptophan (ATrp) as function monomer, S-SUL as the template molecule, and ethyleneglycol dimethacrylate (EGDMA) as the cross linker. Under the optimized synthesis conditions, the MIP was synthesized by bulk polymerization according to the molar ratio of 1:4 of S-SUL to ATrp, and structurally characterized by Fourier transform infrared spectroscopy (FT-IR), scanning electron microscope (SEM) and laser particle analysis. The results illustrated that the MIP offered uniform, loose and porous structure. The adsorption performance of the MIP was evaluated by the isotherm and kinetic models, and the adsorption isotherm conformed to the Freundlich model. The maximum adsorption capacity, selectivity factor and enantioselectivity coefficient to S-SUL were respectively 226.2389 µmol/g, 2.34 and 11.66. Based on the chiral recognition specificity, the drug release experiments demonstrated that the MIP as controlled and sustained release carrier could inhibit the release rate of S-enantiomer compared to the tablet without the MIP, exhibiting the potential of the MIP synthesized in chiral drug delivery.

Preparation and evaluation of chiral open-tubular columns supported with zeolite silica nanoparticles and single/dual chiral selectors using capillary electrochromatography with amperometric detection.

In this work, novel stationary phase coatings by zeolite SiO2NPs coupled with ß-cyclodextrin (ß-CD) or ß-CD/L-phenylalanine were developed for chiral open-tubular capillary electrochromatography (OT-CEC). The OT columns were prepared taking advantage of the strong adhesion of polydopamine in one-step method. Scanning electron micrography and electroosmotic flow were used to characterize the prepared single/dual-selector OT columns. Chiral separation of four chiral analytes (catechin/epicatechin, ephedrine/pseudoephedrine, ritodrine and salbutamol) was carried out in order to evaluate the performance of the prepared columns in OT-CEC with amperometric detection system. In terms of migration time, peak area, resolution, and selectivity factor of catechin/epicatechin and salbutamol, the run-to-run, day-to-day, and column-to-column repeatability were within 8.9%. Under the optimum conditions, the developed methods were applied for the analyses of Chinese herbal medicine Catechu herbs and salbutamol aerosol samples.

Synthesizing Chiral Drug Intermediates by Biocatalysis.

The chiral feature is a critical factor for the efficacy and safety of many therapeutic agents. At present, about 57% of marketed drugs are chiral drugs and about 99% of purified natural products are chiral compounds. There has been a tremendous potential of functional microorganisms and biocatalysts derived from them for the bioconversion of synthetic chemicals into drugs with high enantio-, chemo-, and regio-selectivities. Biocatalysis is becoming a key subassembly in the medicinal chemist's toolbox. In fact, the intermediates of many important therapeutic agents such as sitagliptin, pregabalin, ragaglitazar, paclitaxel, epothilone, abacavir, atorvastatin, rosuvastatin, and omapatrilat have been successfully synthesized via biocatalysis. In this review, various biocatalytic systems that enable to synthesize these chiral drug intermediates are updated and discussed regarding their potential application in the pharmaceutical industry. Further development and increased utilization of biocatalysis for production of drugs with emphasis on green chemistry can be expected.

[Preparation and evaluation of diureido bridged ß-cyclodextrin-bonded chiral stationary phase by high-performance liquid chromatography].

A diureido bridged ß-cyclodextrin dimer was synthesized by the reaction of 6-deoxy-6-hydroxyethylamino-ß-cyclodextrin with hexamethylene diisocyanate. The dimer was then bonded onto silica to obtain a novel diureido bridged ß-cyclodextrin-bonded stationary phase (UBCDP). The structure of diureido bridged stationary phase was characterized by infrared spectroscopy, mass spectrometry, and elemental analysis. By using racemic drugs and pesticides as probes (flavanones, triazoles, and dansylated amino acids), the chromatographic properties of UBCDP were evaluated. The separation mechanism was elucidated by comparison with a single ß-cyclodextrin chiral stationary phase (CDCSP). The composition, pH value of the mobile phase and column temperature were optimized. The results showed that the UBCDP can resolve 25 chiral compounds, especially 2'-hydroxyflavanone, hexaconazole and dansyl leucine with high resolutions (Rs, 1.52-4.35), and the larger volume hesperidin could also be separated. This trend was related to the synergistic inclusion of bridged cyclodextrins. CDCSP can separate only a small number of enantiomers with low resolutions. UBCDP exhibited better enantio-separation ability and hence had potential application in chiral drugs and chiral pesticide residues analysis.

Construction of ß-Cyclodextrin Covalent Organic Framework-Modified Chiral Stationary Phase for Chiral Separation.

Chiral covalent organic frameworks (CCOFs), built by the condensation reactions of organic building blocks with enantiomeric purity and linking subunits, have emerged as a marvelous category of porous crystalline material. In addition to stability and good porosity, CCOFs possess remarkable enantioselectivity, which would be exploited for asymmetric catalysis and chiral separation. ß-cyclodextrin (ß-CD) and its derivatives, a group of supramolecules with a hydrophobic cavity, have been widely applied to molecular specific recognitions. In this work, the ß-CD covalent organic framework (COF) was exploited to construct chiral stationary phase (CSP) for chiral drugs analysis for the first time. We fabricated ß-CD COF via the condensation reaction of heptakis(6-amino-6-deoxy)-ß-CD and terephthalaldehyde at room temperature. ß-CD COF-modified capillary columns were subsequently prepared by a photopolymerization method with shortened time and applied for separation of chiral drugs on capillary electrochromatography with good resolution and repeatability. Baseline separation for six enantiomers was achieved and the precisions (relative standard deviations) for intraday, interday, and column-to-column were <2.1%, 4.5%, and 7.3%, respectively. The results reveal that CCOFs-coated capillary columns show great prospect for chromatographic separation of chiral drugs.

Role of Chirality in Drugs: An Overview.

Stereochemistry has occupied a great role in the manufacture and development of pharmaceuticals. Chiral properties play an important role in the determination of pharmacological actions of the drug. In recent years, there is a considerable interest in chiral separation to isolate and examine both enantiomers. This article provides an overview about the stereochemistry and its role in drugs, and also, offers approved isolation methods for enantiomeric pairs.