ScinoPharm Taiwan

D-amino acid-containing peptides (DAACPs) occur in biological and artificial environments. Since the importance of DAACPs has been recognized, various mass spectrometry-based analytical approaches have been developed. However, the capability of higher-energy collisional dissociation (HCD) fragmentation to characterize DAACP sites has not been evaluated. In this study, we compared the normalized spectra intensity under different conditions of HCD and used liraglutide along with its DAACPs as examples. Our results indicated that the difference in the intensity of y ions between DAACPs and all-L liraglutide could not only distinguish them but also localize the sites of D-amino acids in the DAACPs. Our data demonstrate the potential of using HCD for the site characterization of DAACPs, which may have great impact in biological studies and peptide drug development.

The full characterization of non-biological complex drugs (NBCDs) is not possible but analytical approaches are of urgent needs to evaluate the similarity between different lots and compare with their follow-up versions. Here, we propose a hypothesis testing-based approach to assess the similarity/difference between random amino acid copolymer drugs using liquid chromatography mass spectrometry (LC-MS) analysis. Two glatiramer acetate (GA) drugs, commercially available Copaxone and in-house synthesized SPT, and a negative control were digested by Lys-C and followed by HILIC-MS analysis. After retention time alignment and feature identification, 1627 features matched to m/z values in an elemental composition database were considered as derived from active drug ingredients. A hypothesis testing approach, sum of squared deviations test, was developed to process high-dimensional data derived from LC-MS spectra. The feasibility of this approach was first demonstrated by testing 5 versus 5 lots of Copaxone and Copaxone versus SPT, which suggested a significant similarity by obtaining the estimated 95th percentile of the distribution of the estimator (ρ (95%)) at 0.0056 (p-value=0.0026) and 0.0026 (p-value<0.0001), respectively. In contrast, the ρ (95%) was 0.036 (p-value=1.00) while comparing Copaxone and the negative control, implying a lack of similarity. We further synthesized 9 stable isotope-labeled peptides to validate the proposed amino acid sequences in the database, demonstrating the correctness in sequence identification. The quantitation variations in our analytical procedures were determined to be 6.8%-7.7%. This approach was found to have a great potential for evaluating the similarity between generic NBCDs and listed reference drugs as well as to monitor the lot-to-lot variation.

Herein we describe the design and synthesis of a novel family of bifunctional, chiral bicyclo[2.2.1]heptadiene ligands bearing aryl and amido groups, and demonstrate their usefulness in the Rh(I)-catalyzed catalytic enantioselective addition reaction of arylboronic acids to N-diphenylphosphinyl- (N-DPP-) protected aldimines. Unlike the analogous Rh(I)-catalysts comprising diene ligands substituted with aryl and carboxylic ester groups, or with aryl groups only, the addition reaction proceeded with high stereoselectivity. The protocol tolerated a range of N-DPP-aldimines and arylboronic acids offering the desired optically active N-DPP-protected amines in 31->99% yields and 91->99% ee. The synthetic utility of the method was demonstrated by the conversion of N-DPP-protected amine 3ae into the antifungal agent, bifonazole (13).

A method providing expeditious access to chiral allylic amines via a Rh(I)/bicyclo[2.2.1]heptadiene-catalyzed enantioselective alkenylation of N-tosyl aldimines with potassium alkenyltrifluoroborates under microwave irradiation is described. The rate of the asymmetric 1,2-addition reaction, conducted in the presence of 1 mol % of the catalyst, was significantly enhanced as compared to when the standard heating method was applied while still providing the corresponding products without decrease in enantioselectivity.

The preparation of chiral 4-aryl-2-pyrrolidones by conjugate addition catalyzed by a rhodium(I) complex, comprising the chiral bicyclo[2.2.1] diene ligand 1e, is reported. The reaction of various arylboronic acids and α,β-unsaturated γ-lactams 2 bearing various N-substituents proceeds in a highly stereoselective fashion (93-99.5% ee) even in the presence of only 0.5mol% of the Rh catalyst affording the corresponding addition products in 31-99% yield.

Chiral rhodium catalysts comprising 2,5-diaryl- substituted bicyclo[2.2.1]diene ligands L1-L10 were utilized in the enantioselective 1,4-addition reaction of arylboronic acids to N-substituted maleimides. In the presence of 2.5 mol % of Rh(I) /L2, enantioenriched conjugate addition adducts were isolated in 72-99 % yields with 86-98 % ee. This protocol offers a convenient method to access a variety of 3-arylsuccinimides in a highly enantioselective manner. Maleimides with readily cleavable N-protecting groups were tolerated enabling the synthesis of useful synthetic intermediates. Pyrrolidine 4, a biologically active compound, and pyrrolidine 5, an ent-precursor to an HSD-1 inhibitor, were synthesized to demonstrate the utility of this method. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

The asymmetric conjugate addition of arylboronic acids to substituted and unsubstituted β-pyrazol-1-yl (E)-tert-butyl acrylates 4 catalyzed by 5 mol % of the Rh(I)/diene 2a catalyst provided the corresponding addition products in 44-98% yield and 91->99.5% ee. The method was applied to the formal synthesis of (3S)-3-aryl-3-(pyrazol-1-yl)propanoic acid 1b with agonistic activity toward the human GPR40 G-protein coupled receptor.

The stereoselective arylation of hydroxy protected 1,6-anhydro-beta-D-glucose with arylalanes to provide beta-C-arylglucosides is reported. Modification of triarylalanes, Ar3Al, with strong Brønsted acids (HX) or AlCl3 produced more reactive arylating agents, Ar2AlX, while the incorporation of alkyl dummy ligands into the arylating agents was also viable. Me3Al and i-Bu2AlH were found useful in the in situ blocking of the C3-hydroxyl group of 2,4-di-O-TBDPS protected 1,6-anhydroglucose. The utility of the method was demonstrated by the synthesis of the SGLT2 inhibitor, canagliflozin.

The structures of C-terminally and N-terminally monoPEGylated human parathyroid hormone fragment hPTH(1-34) as well as their unmodified counterparts, poly(ethylene glycol) (PEG) and hPTH(1-34), have been studied by small-angle neutron scattering (SANS). The scattering results show that free hPTH(1-34) in 100 mM phosphate buffer (pH 7.4) aggregate into clusters. After conjugated with PEG, the PEG-peptide conjugates self-assemble into a supramolecular core-shell structure with a cylindrical shape. The PEG chains form a shell around the hPTH(1-34) core in order to shield hPTH(1-34) from the solvent. The detailed structural information of the self-assembled structures is extracted from SANS using a model of cylindrical core with shell of Gaussian chains attached to the core surface. Based on the data, due to the charge-dipole interactions between the conjugated PEG chain and the peptide, the conjugated PEG chain forms a more collapsed conformation compared to free PEG. Moreover, the size of the self-assembled structures formed by the C-terminally monoPEGylated hPTH(1-34) is about three times larger than that of the N-terminally monoPEGylated hPTH(1-34). The different aggregation numbers of the self-assembled structures, triggered by different PEGylation sites, are reported. These size discrepancy due to different PEGylation sites could potentially affect the pharmacokinetics of the hPTH(1-34) drug.

Enantiomerically enriched tosyl-protected diarylmethylamines were rapidly prepared by the asymmetric addition of arylboronic acids to N-tosyl aldimines under mild conditions in the presence of a catalyst prepared in situ from Rh(I) and a chiral diene ligand. This methodology offers access to diarylmethylamines in good yields with excellent chiral purity at room temperature using MeOH as a solvent and NEt3 as a base. Its synthetic utility was demonstrated by the preparation of (S)-1-phenyl-1,2,3,4-tetrahydroisoquinoline (14), an antagonist of the N-methyl-D-aspartate (NMDA) receptor.

For the first time, simple N-tosyl aryl aldimines, prepared from the condensation of tosyl amide and aromatic aldehydes, can be used as substrates in the rhodium catalyzed 1,2-addition reaction using alkenylboron nucleophiles. In the presence of 1.5 mol % of [RhCl(1e)]2, enantioselective addition of various potassium alkenyltrifluoroborates to aryl aldimines furnished the corresponding chiral allylic amines in 73-96% yield and 72->99.5% ee. Notably, this method efficiently provides the di-, tri-, and tetrasubstituted allylic N-tosyl amines with high asymmetric induction.

An efficient enantioselective addition of an array of arylboronic acids to various β-nitrostyrenes catalyzed by a novel and reactive rhodium-diene catalyst (S/C up to 1000) was developed, providing β,β-diarylnitroethanes in good to high yields (62-99%) with excellent enantioselectivities (85-97% ee). The method was extended to 2-heteroarylnitroolefins and 2-alkylnitroolefins similarly providing the desired products with high enantioselectivities and yields. The usefulness of this method was demonstrated in the formal synthesis of the enantiomer of the dopamine receptor agonist and antagonist, SKF 38393.

A practical and scalable process for the manufacture of cladribine (1) is described. Vorbrüggen glycosylation of doubly silylated 2-chloroadenine 2 with protected 1-O-acetyl-2-deoxy-α,β-d-ribofuranose 3 under reversible conditions in the presence of 20 mol % triflic acid in a solvent that selectively precipitated the desired β-anomer β-4a whilst leaving the unwanted α-anomer α-4a in solution to isomerise allowed good overall stereoselectivity with exclusive regioselectivity. An aging step allowed anomerisation of α-4a to β-4a, thereby improving the isolable yield of the β-anomer. Direct filtration of the product mixture without a catalyst quench or aqueous workup furnished the crude β-anomer β-4a in good yield (up to 68%) and purity (>95% by HPLC) with no regioisomers detected and only 1–3% (by HPLC) of the undesired α-anomer. Deprotection of the crude, unpurified intermediate β-4a followed by recrystallisation provided drug-grade cladribine (1). The process includes three isolation steps and was demonstrated on kilogram scales using cGMP providing 99.8–99.9% pure cladribine in up to an overall 43% yield based on 2-chloroadenine (5). In contrast to previous methods, column chromatography and/or bulky directing groups were not required in the glycosylation step, a high pressure vessel was not needed in the deprotection step, and only one dedicated recrystallisation step was necessary.

A new approach to the synthesis of 1 (DS003, BMS-599793), a small-molecule HIV entry inhibitor, is described. The initial medical chemistry route has been modified by rearranging the sequence of synthetic steps followed by replacement of the Suzuki coupling step by the Negishi conditions. Acylation of the resulting azaindole 7 under the Friedel–Crafts conditions is studied using monoesters of chlorooxalic acid in the presence of aluminum chloride. Polymorphism of 1 is also investigated to develop conditions suitable for preparation of the desired Form 1 of the target compound. The new route is further optimized and scaled up to establish a new process that is applied to the synthesis of kilogram quantites of the target active pharmaceutical ingredient.

Liquid chromatography-tandem mass spectrometry-based proteomics for peptide mapping and sequencing was used to characterize the marketed monoclonal antibody trastuzumab and compare it with two biosimilar products, mAb A containing D359E and L361M variations at the Fc site and mAb B without variants. Complete sequence coverage (100%) including disulfide linkages, glycosylations and other commonly occurring modifications (i.e., deamidation, oxidation, dehydration and K-clipping) were identified using maps generated from multi-enzyme digestions. In addition to the targeted comparison for the relative populations of targeted modification forms, a non-targeted approach was used to globally compare ion intensities in tryptic maps. The non-targeted comparison provided an extra-dimensional view to examine any possible differences related to variants or modifications. A peptide containing the two variants in mAb A, D359E and L361M, was revealed using the non-targeted comparison of the tryptic maps. In contrast, no significant differences were observed when trastuzumab was self-compared or compared with mAb B. These results were consistent with the data derived from peptide sequencing via collision induced dissociation/electron transfer dissociation. Thus, combined targeted and non-targeted approaches using powerful mass spectrometry-based proteomic tools hold great promise for the structural characterization of biosimilar products.

A process is described for the synthesis of kilogram quantities of homochiral 4-silyloxycyclopentenone (R)-1, a key intermediate useful for the synthesis of a plurality of prostaglandin analogue drugs. Cyclopentenone (R)-1 was synthesized in 14 isolated steps from furfural. Key steps in the synthesis include a Wittig reaction, Piancatelli rearrangement, and an enzymatic resolution featuring in situ recycling of the undesired enantiomer furnishing the desired homochiral alcohol in ≥99.5% ee. As a retort to the unsatisfactory coformation of about 8% at best of the trans-olefin in the Wittig reaction, a change to the order of several steps and the identification of a recrystallisable, amine salt derivative, 2, allowed the unwanted isomer to be controlled to as low as 0.2%.

In this study, the relationship of the structural stability of peptide diastereomers in elution solvents and their retention behaviors in reversed-phase chromatography (RPC) was examined to provide guidance on the solvent selection for a better separation of peptide diastereomers. We investigated the chromatographic retention behaviors of exenatide, a peptide drug for the treatment of type II diabetes mellitus and its three diastereomers using RPC and implicit molecular dynamics (MD) simulation analysis. Three diastereomers involved in the single serine residue mutation of D-form at the 11th, 32nd, and 39th residues were investigated in this study. Results show that the order of the solution structural stability of exenatide and its diastereomers is consistent with their retention order by 36 % acetonitrile/water elution. The sample loading solvent also affects the retention behaviors of exenatide peptide diastereomers in RPC column. Furthermore, a larger solution conformation energy difference of the critical pair of exenatide and its diastereomer (D-Ser39) at the elution solvent of 32 % tetrahydrofuran/water were obtained by MD simulation, and baseline separation was proved experimentally. In summary, we demonstrated that the solution structural stability-chromatographic retention relationship could be a powerful tool for elution solvent selection in peptide chromatographic purification, especially valuable for the separation of critical pair of diastereomers.

The micronization of an asthma active pharmaceutical ingredient, fluticasone propionate, using the supercritical antisolvent (SAS) process was investigated in this study. The most commonly used supercritical carbon dioxide was employed as the antisolvent. The effects of five process parameters, including operating temperature, operating pressure, solution flow rate, solution concentration, and nozzle diameter, were compared and discussed. The physical properties of micronized fluticasone propionate were examined by the particle size analyzer, the scanning electron microscope and X-ray diffractometer. The cross interaction effect of operating temperature and pressure was observed in the SAS treatment of fluticasone propionate and verified by the method of calculated mixture critical point (MCP), using the predictive Soave-Redlich-Kwong equation of state. Operation slightly above the MCP was suggested for successful micronization of fluticasone propionate.