Process Development of a Macrocyclic Peptide Inhibitor of PD-L1.

This article outlines the process development leading to the manufacture of 800 g of BMS-986189, a macrocyclic peptide active pharmaceutical ingredient. Multiple N-methylated unnatural amino acids posed challenges to manufacturing due to the lability of the peptide to cleavage during global side chain deprotection and precipitation steps. These issues were exacerbated upon scale-up, resulting in severe yield loss and necessitating careful impurity identification, understanding the root cause of impurity formation, and process optimization to deliver a scalable synthesis. A systematic study of macrocyclization with its dependence on concentration and pH is presented. In addition, a side chain protected peptide synthesis is discussed where the macrocyclic protected peptide is extremely labile to hydrolysis. A computational study explains the root cause of the increased lability of macrocyclic peptide over linear peptide to hydrolysis. A process solution involving the use of labile protecting groups is discussed. Overall, the article highlights the advancements achieved to enable scalable synthesis of an unusually labile macrocyclic peptide by solid-phase peptide synthesis. The sustainability metric indicates the final preparative chromatography drives a significant fraction of a high process mass intensity (PMI).

A survey-based triage tool to identify patients potentially eligible for referral to an advanced heart failure centre.

AIMS: Accurate prevalence data for ambulatory advanced heart failure (HF) in European countries remains limited. This study was designed to identify the population of patients potentially eligible for referral for assessment for advanced surgical HF therapies to a National advanced HF and cardiac transplant centre. METHODS AND RESULTS: A survey comprising 13 potential clinical markers of advanced HF was developed, modified from the 'I NEED HELP' tool from the 2018 position statement of the Heart Failure Association of the European Society of Cardiology, and distributed to all HF clinic services (secondary and tertiary units) nationwide. Each HF clinic unit was asked to complete the survey on consecutive patients over a 3 month period fulfilling the following three criteria: (i) age <65 years; (ii) ejection fraction <40% and (iii) HF of >3 months duration. As a comparison, the number of actual referrals to the advanced HF clinic were also audited over a 9 month period. In all, 21 of 26 HF clinic units participated in the survey. Across the period of inclusion, 4950 all-comer HF patients were seen across all sites. Of these, 375 (7.5%) fulfilled the inclusion criteria and were surveyed (74.4% male, median age 57 years [IQR: 11 years]). In total, 246 (66%) of the surveyed patients had ≥1 potential markers for advanced HF, representing just under 5% of the total all-comer HF population seen across the same time period. Of these, 67 patients (27%) had ≥2, 48 (20%) had 3 and 40 (16%) had ≥4 potential markers. The most frequently noted markers were ≥1 HF hospitalization or unscheduled clinic review (56%), intolerance to renin-angiotensin-aldosterone system inhibitors due to hypotension or renal dysfunction (29%) and intolerance to beta-blockers due to hypotension (27%). Almost one-quarter of patients reported NYHA Class III or IV symptoms. During the advanced HF clinic audit, the number of patients actually referred to the advanced HF clinic during the same time period was <5% of this potentially eligible cohort. CONCLUSIONS: In this index prospective National survey, approximately 5% of an all-comer routine HF clinic population and two-thirds of a pre-selected HF with reduced EF under 65 years cohort were found to have at least one clinical or biochemical marker suggesting advanced or impending advanced HF. Almost one-quarter of patients in this chronic outpatient 'snapshot' population have NYHA III-IV symptoms. This simple one-page triage survey-modified from the 'I NEED HELP' tool-is useful to identify a population potentially eligible for referral to an advanced HF centre for assessment for advanced surgical therapies, thereby aiding resource and service planning.

Microscale purification in support of high-throughput medicinal chemistry.

In recent years, successful assay miniaturization has enabled the exploration of synthesis scale reduction in pharmaceutical discovery. Miniaturization of pharmaceutical synthesis and purification allows a reduction in material consumption and shortens timelines, which ultimately reduces the cost per experiment without compromising data quality. Isolating and purifying the compounds of interest is a key step in the library synthesis process. In this manuscript we describe a high-throughput purification workflow in support of microscale (1-5 µmol or 0.5-2 mg) library synthesis. The optimized microscale purification system can routinely purify 384-well reaction plates with an analysis time of 4 min per sample. Instrument optimization, critical parameters such as column loading, delay time calibration, ultrafast pre- and post-purification analysis and library purification examples are provided.

Interlaboratory study of a supercritical fluid chromatography method for the determination of pharmaceutical impurities: Evaluation of multi-systems reproducibility.

Modern supercritical fluid chromatography (SFC) is now a well-established technique, especially in the field of pharmaceutical analysis. We recently demonstrated the transferability and the reproducibility of a SFC-UV method for pharmaceutical impurities by means of an inter-laboratory study. However, as this study involved only one brand of SFC instrumentation (Waters®), the present study extends the purpose to multi-instrumentation evaluation. Specifically, three instrument types, namely Agilent®, Shimadzu®, and Waters®, were included through 21 laboratories (n = 7 for each instrument). First, method transfer was performed to assess the separation quality and to set up the specific instrument parameters of Agilent® and Shimadzu® instruments. Second, the inter-laboratory study was performed following a protocol defined by the sending lab. Analytical results were examined regarding consistencies within- and between-laboratories criteria. Afterwards, the method reproducibility was estimated taking into account variances in replicates, between-days and between-laboratories. Reproducibility variance was larger than that observed during the first study involving only one single type of instrumentation. Indeed, we clearly observed an 'instrument type' effect. Moreover, the reproducibility variance was larger when considering all instruments than each type separately which can be attributed to the variability induced by the instrument configuration. Nevertheless, repeatability and reproducibility variances were found to be similar than those described for LC methods; i.e. reproducibility as %RSD was around 15 %. These results highlighted the robustness and the power of modern analytical SFC technologies to deliver accurate results for pharmaceutical quality control analysis.

Design and synthesis of C2-symmetric N-heterocyclic carbene precursors and metal carbenoids.

Chiral, C(2)-symmetric imidazolium and imidazolinium ions, as well as the corresponding copper- or silver-bound carbenoids, have been prepared. Structural study of these compounds by X-ray crystallography reveals a chiral pocket that surrounds the putative carbene site or the metal-carbene bond, at carbon 2, in three of the four ligands prepared. Preliminary investigation into the application of these complexes has shown one of them to be highly enantioselective in the hydrosilylation of acetophenone.

Design of a novel pyrrolidine scaffold utilized in the discovery of potent and selective human ß3 adrenergic receptor agonists.

A novel class of human ß(3)-adrenergic receptor agonists was designed in effort to improve selectivity and metabolic stability versus previous disclosed ß(3)-AR agonists. As observed, many of the ß(3)-AR agonists seem to need the acyclic ethanolamine core for agonist activity. We have synthesized derivatives that constrained this moiety by introduction of a pyrrolidine. This unique modification maintains human ß(3) functional potency with improved selectivity versus ancillary targets and also eliminates the possibility of the same oxidative metabolites formed from cleavage of the N-C bond of the ethanolamine. Compound 39 exhibited excellent functional ß(3) agonist potency across species with good pharmacokinetic properties in rat, dog, and rhesus monkeys. Early de-risking of this novel pyrrolidine core (44) via full AMES study supports further research into various new ß(3)-AR agonists containing the pyrrolidine moiety.

Fluorinated piperidine acetic acids as gamma-secretase modulators.

We report herein a novel series of difluoropiperidine acetic acids as modulators of gamma-secretase. Synthesis of 2-aryl-3,3-difluoropiperidine analogs was facilitated by a unique and selective beta-difluorination with Selectfluor. Compounds 1f and 2c were selected for in vivo assessment and demonstrated selective lowering of Abeta42 in a genetically engineered mouse model of APP processing. Moreover, in a 7-day safety study, rats treated orally with compound 1f (250mg/kg per day, AUC(0-24)=2100microMh) did not exhibit Notch-related effects.

Selective PPARgamma modulators with improved pharmacological profiles.

A series of metabolically robust N-benzyl-indole selective PPARgamma modulators with either a 3-benzoyl or 3-benzisoxazoyl moiety have been identified. In vitro, these compounds are partial agonists and exhibit reduced adipogenesis in human adipocytes. In vivo, these SPPARgammaMs result in potent glucose lowering in db/db mice and attenuate increases in heart weight and brown adipose tissue that is typically observed in rats upon treatment with PPARgamma full agonists.

Benzoyl 2-methyl indoles as selective PPARgamma modulators.

Routine screening for human PPAR ligands yielded compounds 1 and 2, both of which were sub-micromolar hPPARgamma agonists. Synthetic modifications of these leads led to a series of potent substituted 3-benzyl-2-methyl indoles, a subset of which were noted to be selective PPARgamma modulators (SPPARgammaMs). SPPARgammaM 24 displayed robust anti-diabetic activity with an improved therapeutic window in comparison to a PPARgamma full agonist in a rodent efficacy model.