Final Active Pharmaceutical Ingredient Research Articles

A Convergent Synthesis of HPK1 Inhibitor GNE-6893 via Palladium-Catalyzed Functionalization of a Tetrasubstituted Isoquinoline

A scalable convergent synthesis of GNE-6893 (1) involving Pd-catalyzed Suzuki–Miyaura cross-coupling and C–N coupling as key steps is reported. The production of the final active pharmaceutical ingredient (API) was achieved in 33% overall yield and 98 A% HPLC purity by a one-pot process of global deprotection, pH adjustment, crystallization, and isolation. Green chemistry practices were implemented in the process development of GNE-6893 (1) by utilizing a biocatalytic resolution to produce (3R,4S)-4-methyltetrahydrofuran-3-ol and replacing toxic triphosgene with a safer alternative, disuccinimidyl carbonate (DSC), to construct the carbamate penultimate intermediate to API.

Asymmetric organocatalysis in drug discovery and development for active pharmaceutical ingredients

ABSTRACT Introduction Over the last 20 years, it has become clear that organocatalysis is the third pillar of catalysis. The low reactivity in the early days of organocatalysis has been overcome with the invention of more efficient catalysts, and by harnessing enabling technologies like continuous-flow chemistry and photo-redox catalysis. Areas covered The main focus of this review is on the development over the last 10–15 years of key APIs using asymmetric organocatalysis. Due to significant engineering advances, and also due to the need for continuous manufacturing, flow and photo-redox approaches are becoming more widespread. Expert opinion Over the last 20 years, organocatalysis has been used on various occasions for accessing chiral drugs. The great advantage of using these catalysts is that the final active pharmaceutical ingredient (API) is metal-free. Also due to their inherent stability in air and water, they are very amenable to recovery via attachment to appropriate solid supports and also application in continuous flow systems. In recent years, more efficient organocatalysts have been developed, which includes the photoredox types, with much potential for chiral API synthesis.

Determination of Terpenoid Profile in Dry Cannabis Flowers and Extracts Obtained from Different Cannabis Varietes

Aim: The aim of this study was to determine the terpenoid profile in dried cannabis flowers obtained from different varieties of cannabis plant and in cannabis extracts in order to investigate quantity of terpenes lost during extraction and purification process.
 Methods: GC/MS method for determination of terpenes was verified. The concentration of terpenes was determined in dry flowers as raw material and in decarboxylated and distillated cannabis extracts, using the same GC/MS analytical method. The extraction was performed using 96% ethanol as a solvent.
 Results: The obtained results indicate that dry cannabis flowers from different cannabis plant can be distinguished only by their terpenoid profile. The use of standardized cannabis-based extracts can be confirmed by determination of terpenoid profile. The purification process of the cannabis extracts removes terpenes. The percentage of major terpen beta-Myrcene decreased from 68% in dry flower to 15% in decarboxylated and, 1.9% in distillated cannabis oil after purification. The percentage of second major terpene alpha-Pinene decreased from 15% in dry flower to 5% in decarboxylated and, 0.7% in distillated cannabis oil after purification.
 Conclusion: Terpenes act synergistically with cannabinoids. Following the monograph for quality testing of cannabis extracts in the German Pharmacopoeia, the purification process is necessary to achieve a final concentration of cannabinoids (Tetrahydrocannabinol) of more than 95% in the final active pharmaceutical ingredient. The purification process removes terpenes that have proven synergistically pharmacological effects with cannabinoids.

Continuous Flow Synthesis of Anticancer Drugs.

Continuous flow chemistry is by now an established and valued synthesis technology regularly exploited in academic and industrial laboratories to bring about the improved preparation of a variety of molecular structures. Benefits such as better heat and mass transfer, improved process control and safety, a small equipment footprint, as well as the ability to integrate in-line analysis and purification tools into telescoped sequences are often cited when comparing flow to analogous batch processes. In this short review, the latest developments regarding the exploitation of continuous flow protocols towards the synthesis of anticancer drugs are evaluated. Our efforts focus predominately on the period of 2016–2021 and highlight key case studies where either the final active pharmaceutical ingredient (API) or its building blocks were produced continuously. It is hoped that this manuscript will serve as a useful synopsis showcasing the impact of continuous flow chemistry towards the generation of important anticancer drugs.

Optimization and Development of a Scalable Palladium-Catalyzed C–H Activation Process for the Geometry-Selective Preparation of Kilograms of YLF466D, a Potent AMP-Activated Protein Kinase Activator

Adenosine 5′-monophosphate-activated protein kinase (AMPK) activator YLF466D is a promising preclinical drug candidate to treat metabolic diseases and myocardial ischemia-reperfusion injury (MIRI). Herein, we report our efforts on optimization and development of a practical and scalable process for the preparation of YLF466D in kilogram scale. The process features a palladium-catalyzed C–H activation under mild reaction conditions, geometry selectivity, effective impurities purging, and low levels of residual solvents in the final active pharmaceutical ingredient (API). We applied this process successfully to prepare more than 17 kg (3.0–3.6 kg per batch) of YLF466D to support its preclinical study.

Quantitation and speciation of residual protein within active pharmaceutical ingredients using image analysis with SDS-PAGE

Recent advances in biocatalysis and directed enzyme evolution has led to a variety of enzymatically-driven, elegant processes for active pharmaceutical ingredient (API) production. For biocatalytic processes, quantitation of any residual protein within a given API is of great importance to ensure process robustness and quality, pure pharmaceutical products. Typical analytical methods for analyzing residual enzymes within an API, such as enzyme-linked immunosorbent assays (ELISA), colorimetric assays, and liquid chromatographic techniques, are limited for determining only the concentration of known proteins and require harsh solvents with high API levels for analysis. For the first time, total residual protein content in a small molecule API was quantitated using image analysis applied to SDS-PAGE. Herein, a proposed methodology for residual protein detection, quantitation, and size-based speciation is presented, in which an orthogonal technique is offered to traditional analysis methods, such as ELISA. Results indicate that our application of the analytical methodology is able to reliably quantitate both protein standards and the total residual protein present within a final API, with good agreement as compared to traditional ELISA results. Further, speciation of the residual protein within the API provides key information concerning the individual residual proteins present, including their molecular weight, which can lead to improved process development efforts for residual protein rejection and control. This analytical methodology thus offers an alternative tool for easily identifying, quantitating, and speciating residual protein content in the presence of small molecule APIs, with potential for wide applicability across industry for biocatalytic or directed enzyme evolution efforts within process development.

Sub/supercritical Fluid Chromatography Purification and Desalting of a Cyclic Dinucleotide STING Agonist

An efficient and “endotoxin-free” purification of a cyclic dinucleotide (CDN) STING agonist was achieved to produce multigram quantities of pure BMT-390025, an active pharmaceutical ingredient (API), for toxicological studies. A two-step sub/supercritical fluid chromatography (SFC) procedure was developed for the achiral purification and desalting of the polar ionic CDN. A robust SFC process employing methanol-acetonitrile-water with ammonium acetate as co-solvent in CO2 on BEH 2-ethylpyridine was established and scaled up as the first step to achieve a successful purification. The desalting/salt-switching (i.e. removing acetate and acetamide) was conducted using methanol-water with ammonium hydroxide as co-solvent on the same column in the second step to convert the final API to the ammonium salt. Water with additive was essential to eliminating salt precipitation and improving the peak shape and resolution. Due to the extreme hydrophilicity of BMT-390025, 65% of co-solvent was needed to adequately elute the target in both steps. More than 40 g of crude API was purified and desalted producing >20 g of pure BMT-390025 as the ammonium salt which was obtained with a chemical purity of >98.5% and met the endotoxin requirement of <0.1 EU/mg. In addition, >80 g of its penultimate prior to the deprotection of the silyl group was purified at a high throughput of 6.3 g/h (0.42 g/day/g SP).

The effect of water on the large-scale supercritical fluid chromatography purification of two factor XIa active pharmaceutical ingredients

BMS-962212, a parenteral Factor XIa inhibitor, was scaled-up for toxicity studies. Two steps of supercritical fluid chromatography (SFC) were developed for the chiral resolution of the penultimate and achiral purification of final active pharmaceutical ingredient (API), BMS-962212. A robust SFC process using Chiralcel OD-H with methanol-acetonitrile as modifier in CO2 was established to achieve a stable and uninterrupted operation with reduced mobile phase viscosity and system pressure drop. More than 230 g of the racemic penultimate was chirally resolved to reach >99% chiral purity, ready for final tert-butyl ester deprotection to provide the API. There were a significant number of impurities in BMS-962212 generated from the final step that needed to be removed. In contrast to conventional SFC conditions, an SFC method exploiting water and ammonia as additives in both the mobile phase and sample solution was developed to accomplish purification and desalting (i.e. removing TFA) of the zwitterionic API in one step. Water as an additive eliminated salt precipitation and improved the resolution while ammonia contributed to the desalting, details of which will be discussed in this article. A throughput of 2 g/h was achieved, and >80 g of the crude API was purified. The same strategy was applied to another Factor XIa API (compound A) and its penultimate.

API Continuous Cooling and Antisolvent Crystallization for Kinetic Impurity Rejection in cGMP Manufacturing

Crystallization of 204 kg of final active pharmaceutical ingredient was accomplished continuously using a cascade of mixed suspension mixed product removal crystallizers in cGMP manufacturing. This article describes the journey taken to transform a set of technical to final batch crystallizations into a continuous, combined cooling and antisolvent crystallization using three stirred tank crystallizers in series. Conversion of the batch process to a continuous process was beneficial to kinetically purge a key impurity. The conversion also allowed for the direct integration of the crystallization process with upstream continuous chemistry sections. A robust control strategy was developed from early research scale all the way to cGMP manufacturing. The authors will share the tools, techniques, modeling, and equipment used and challenges overcome to ensure a safe and reliable manufacturing process. A new intermittent flow technique transferred hot solution from a continuous evaporator into the first crystallizer with no solids bearding at the end of the inlet tubing. The continuous distillation, crystallization, and slurry-off filters were a key part of a broader continuous process and new building that won an International Society for Pharmaceutical Engineering 2019 Facility of the Year Award for Innovation.

Synthesis of Vixotrigine, a Voltage- and Use-Dependent Sodium Channel Blocker. Part 2: Development of a Late-Stage Process

As vixotrigine (1) entered a later clinical phase for trigeminal neuralgia (Zakrzewska, J. M.; et al. Lancet Neurol. 2017, 16, 291−300), the development of a sustainable late-stage process was required to meet the supply needs for formulation optimization, phase 3 clinical trials, and registration stability batches (this is the expected commercial formulation). In this article, we describe how the process was streamlined from the early supply route (Giblin, G.; et al. Org. Process Res. Dev. 2020, DOI: 10.1021/acs.oprd.0c00382) and a comprehensive control strategy was established. Process improvements included improving safety and scalability for a temperature-sensitive Grignard reaction, simplifying unit operations, removal of heterogenous conditions, and route redesign to afford a high yielding, one-pot sequential alkylation and amidation. Improvement in the salt formation step, combined with wet milling, resulted in improved particle properties with enhanced flow properties of the final active pharmaceutical ingredient. The process mass intensity was improved 65% while maintaining drug substance purity at more than 99.8%. This new process has been scaled up to generate metric ton quantities of drug substance.

Microstructure based simulation of the disintegration and dissolution of immediate release pharmaceutical tablets

The design of pharmaceutical tablets involves the determination of formulation parameters that define the tablet composition and internal microstructure. These parameters must be chosen so that the release of an active pharmaceutical ingredient (API) from the tablet follows a prescribed dissolution curve. In the case of immediate release formulations, the dissolution process typically consists of tablet disintegration, followed by the dissolution of the disintegration fragments. In order to find the appropriate values of formulation parameters, numerous experiments are typically required. In the present work, we propose a computational methodology for in silico design of tablet formulations with the aim of reducing the amount experimental work required during tablet design. The methodology is based on the coupling of two modelling approaches: (i) tablet fragmentation triggered by the swelling of the disintegrant is simulated by the discrete element method (DEM), and (ii) the dissolution of the resulting population of disintegration fragments is simulated using a finite volume grid-based model. The final API release curve is then obtained by the superposition of dissolution curves originating from the individual disintegration fragments. Using directly compressed tablets containing ibuprofen as the API and croscarmellose sodium as the disintegrant, the model was validated against experimental data. The fragment size distribution was evaluated by static light scattering and the dissolution profiles were obtained by a standard dissolution apparatus with UV/Vis spectroscopic detection. We demonstrate that the computational methodology is able to quantitatively predict the effect of disintegrant content and API primary particle size on the fragment size distribution and the final dissolution profiles, and is therefore useful as a tool for computer-assisted tablet formulation design.

Development of a rapid GC-FID method to simultaneously determine triethylamine, diisopropylamine, and 1,1,3,3-tetramethylguanidine residues in an active pharmaceutical ingredient

A rapid GC-FID method was developed to simultaneously determine residual levels of triethylamine (TEA), 1,1,3,3-tetramethylguanidine (TMG), and diisopropylamine (DIPA) in the synthetic route of an active pharmaceutical ingredient (API). Due to the severe absorption of amines on GC stationary phases, GC columns with various stationary phases were evaluated for optimal peak shape and reproducibility. The final conditions used the Agilent CP-Volamine column to resolve the three amines in 12 min. Various inlet liners were also screened to further improve the sensitivity of the analysis. The Restek Siltek® liner was selected to achieve the desired detectability for the method. The quantitation limits were 4, 3, and 4 μg/mL for TEA, DIPA, and TMG in the presence of API, respectively. All three amines showed good linearity (r > 0.999) and recoveries (> 90%) over the concentration range of 3 to 16 μg/mL. The testing of residual amines was initially performed at the penultimate stage of the synthesis. However, this work demonstrates that TMG can act as a proton sponge to react with salicylic acid, the counter ion of the penultimate, to form a volatile component that elutes at a different retention time. Consequently, in the final method, these three amines were monitored in the final API to circumvent the matrix interference. Key parameters of the method were qualified per method validation requirements in ICH guidelines. The method was successfully applied for batch testing during development and implemented as an in-process control procedure at manufacturing sites.

In Vitro Antioxidant, Antimicrobial and Molecular Docking Studies of Fosphenytoin and Regadenoson Impurities

Regadenoson and Fosphenytoin are USFDA approved drugs which is used for coronary vasodilator and convulsive status epileptics respectively. It is quite natural that low levels of reagents or side products are present in the final active pharmaceutical ingredient (API) or drug product as impurities. Such impurities may have unwanted toxicities, including genotoxicity and carcinogenicity. Hence, it is important to study on impurities present in both the drugs. There are 9 impurities were identified from both drugs and studied pharmacokinetic properties using Qikprop module from Schrödinger software. From the 9 compounds of both the drug’s impurities, 5 compounds obey the Lipinski rule of five and the remaining compounds are having 1 to 3 penalties. All the compounds were subjected to molecular docking study with thermo stabilised HUMAN A2A Receptor with adenosine bound protein (PDB ID: 2YDO) for regadenoson impurities and fosphenytoin impurities were docked with Human GABA-A receptor alpha1-beta2-gamma2 subtype in complex with GABA and flumazenil, conformation A protein (PDB id: 6D6U). All the compounds are showed very good interaction with docked proteins. Further selected compound subjected to in vitro Antibacterial (Gram positive, Gram negative), Antifungal and Antioxidant (DPPH and FRAP) studies.

Structural elucidation of a dimeric impurity in the process development of ceftolozane using LC/HRMS and 2D-NMR

Ceftolozane is MSD’s 5th generation cephalosporin antibiotic with broad-spectrum gram-negative activity. While refining the synthetic route to the drug substance, a dimeric impurity was observed in the final active pharmaceutical ingredient (API) of ceftolozane. Through impurity enrichment and preparative isolation, the structure of the impurity was subsequently established through LC/HRMS, HRMSMS, H/D exchange and 2D-NMR studies. A kinetic study of the impurity formation was conducted to determine the best conditions to control it in the final chemical process.

Synthesis of Selective Estrogen Receptor Degrader GDC-0810 via Stereocontrolled Assembly of a Tetrasubstituted All-Carbon Olefin.

We report an efficient synthesis of GDC-0810 on the basis of a sequence involving a highly stereoselective lithium tert-butoxide-mediated enolization-tosylation (≥95:5 E: Z) and a Pd-catalyzed Suzuki-Miyaura cross-coupling as key steps. Global deprotection, pyrrolidine salt formation, and final active pharmaceutical ingredient (API) form control/isolation produced GDC-0810 free acid in a 40% overall yield with >99.0% purity as ascertained by HPLC analysis.

Nuclear magnetic resonance assessment of controlled hydrolysis of the capsular polysaccharides of Streptococcus pneumoniae serotypes 19A and 19F

ABSTRACTSeveral glycoconjugate-based vaccines are inserted nowadays in infant vaccination schedules of most countries. In an initial stage, polysaccharides are modified under controlled conditions. The assessment of polysaccharide regions available for conjugation process after the hydrolysis is crucial to predict the conjugation results for achieving the final active pharmaceutical ingredient. Two hydrolyzed capsular polysaccharides were analyzed by a heteronuclear single-quantum correlation experiment. It was adjusted for assessing 3JP-H to check the position of phosphate mono-ester residue after the hydrolysis process. All evaluated polysaccharides have been shown to remain with the anomeric position available for subsequent conjugation. The adjusted spectroscopic experiment was shown to be effective to study the hydrolysis of phosphate di-ester linked to capsular polysaccharides.

Macrocyclic glycopeptide chiral selectors bonded to core-shell particles enables enantiopurity analysis of the entire verubecestat synthetic route

Verubecestat is an inhibitor of β-site amyloid precursor protein cleaving enzyme 1 (BACE1) being evaluated in clinical trials for the treatment of Alzheimer’s disease. Synthetic route development involves diastereoselective transformations with a need for enantiomeric excess (ee) determination of each intermediate and final active pharmaceutical ingredient (API). The analytical technical package of validated methods relies on enantioselective SFC and RPLC separations using multiple 3 and 5 μm coated polysaccharide-based chiral stationary phases (CSPs) and mobile phases combinations. Evaluation of recently developed chiral columns revealed a single chiral selector (Teicoplanin) bonded to 2.7 μm core-shell particles using H3PO4 in H2O/ACN and triethylammonium acetate: methanol based eluents at different isocratic compositions allowed good enatioseparation of all verubecestat intermediates. EE determination of verubecestat is easily performed on NicoShell, another macrocyclic glycopeptide chiral selector bonded to 2.7 μm superficially porous particles. This approach enables fast and reliable enantiopurity analysis of the entire verubecestat synthetic route using only two chiral columns and mobile phases on a conventional HPLC system, simplifying technical package preparation, method validation and transfer to manufacturing facilities.

Improved, Solvent Free, Atom Efficient Commercial Process for the Synthesis of Diphenhydramine Hydrochloride

Background: Literature reports various methods for the synthesis of diphenhydramine hydrochloride each of these has one or the other drawback such as low as ~39% atom efficiency or high reaction temperature, usage of corrosive bromine and formation of hazardous by product or else it requires strong scrubbing system which generates huge amounts of inorganic waste, in some cases molar equivalents of P-toluenesulfonic acid was used which was further eliminated as waste with low product output and thus not economically viable, literature also reports continuous flow process which requires PFA tube reactor with very high temperature ~175°C addition to this it also uses IPA-HCl for salt preparation which is prone for the formation of genotoxic impurity 2-chloropropane. Methods: Objective of present work is to overcome the limitations studied in literature to improve the process in terms of atom efficiency, cost effectiveness, robust, commercially viable, industrially scalable, minimum environmental hazards considering principles of green chemistry. We have developed the process in such as way that atom efficiency at halogenation stage is enhanced by ~93% compared to ~39% when diphenylmethane reacts with bromine. HCl gas generated in-situ or if needed supplied to prepare diphenhydramine hydrochloride salt instead of using IPA-HCl to eliminate chances of formation of genotoxic impurity if any. Conclusion: In order to achieve the continuous improvement process, we have developed modified process for the synthesis of diphenhydramine hydrochloride which elimination of use of corrosive bromine, overall time cycle reduced by ~40%, overall process atom efficiency enhanced by ~45%, eliminated chance of formation of genotoxic impurity by avoiding use of IPA-HCl solvent for final stage, by carrying out reaction in either water as a universal green solvent or solvent free conditions with high yield to obtain pharmacopeial quality of the final active pharmaceutical ingredient. We assert to have a better process in terms of atom efficiency, minimal wastage, operability and quality. Keywords: Diphenhydramine, solvent free reaction, atom efficiency, bromine, Active Pharmaceutical Ingredient (API).

Trace level determination of chloroacetyl chloride and degradation products by derivatization gas chromatography

A gas chromatographic procedure has been developed for the trace determination of chloroacetyl chloride (CAC) and two of its impurities: methyl chloroacetate (MCA) and chloroacetic acid (CAA). All three compounds are derivatized using piperidine in dichloroethane prior to their analysis via gas chromatography coupled with a flame ionization detection (GC-FID). Recoveries of each compound were assessed in two different pharmaceutical matrices (intermediate and final active pharmaceutical ingredient) and ranged from 75 to 125%. The limit of quantitation has been determined to be 0.10% wt/wt for CAA and 0.03% wt/wt for CAC and MCA. The linearity ranged from 0.03 to 5.00% wt/wt for CAC and MCA and from 0.10 to 5.00% wt/wt for CAA, with correlation coefficients from 0.9995 to 1.0000. Repeatability was evaluated at LOQ and at 5.00% wt/wt and was found to be between 1.4–3.0%.

Combining NMR Spectroscopy and Chemometrics to Monitor Structural Features of Crude Hep-arin

Because of the complexity and global nature of the heparin supply chain, the control of heparin quality during manufacturing steps is essential to ensure the safety of the final active pharmaceutical ingredient (API). For this reason, there is a need to develop consistent analytical methods able to assess the quality of heparin early in production (i.e., as the crude heparin before it is purified to API under cGMP conditions). Although a number of analytical techniques have been applied to characterize heparin APIs, few of them have been applied for crude heparin structure and composition analyses. Here, to address this issue, NMR spectroscopy and chemometrics were applied to characterize 88 crude heparin samples. The samples were also analyzed by strong anion exchange HPLC (SAX-HPLC) as an orthogonal check of the purity levels of the crudes analyzed by NMR. The HPLC data showed that the chemometric analysis of the NMR data differentiated the samples based on their purity. These orthogonal approaches differentiated samples according their glycosaminoglycan (GAG) composition and their mono and disaccharide composition and structure for each GAG family (e.g., heparin/heparan, dermatan sulfate, and chondroitin sulfate A). Moreover, quantitative HSQC and multivariate analysis (PCA) were used to distinguish between crude heparin of different animal and tissue sources.