Leveraging a “Catch–Release” Logic Gate Process for the Synthesis and Nonchromatographic Purification of Thioether- or Amine-Bridged Macrocyclic Peptides

Macrocyclic peptides containing N-alkylated amino acids have emerged as a promising therapeutic modality, capable of modulating protein–protein interactions and an intracellular delivery of hydrophilic payloads. While multichannel automated solid-phase peptide synthesis (SPPS) is a practical approach for peptide synthesis, the requirement for slow and inefficient chromatographic purification of the product peptides is a significant limitation to exploring these novel compounds. Herein, we invent a “catch–release” strategy for the nonchromatographic purification of macrocyclic peptides. A traceless catch process is enabled by the invention of a dual-functionalized N-terminal acetate analogue, which serves as a handle for capture onto a purification resin and as a leaving group for macrocyclization. Displacement by a C-terminal nucleophilic side chain thus releases the desired macrocycle from the purification resin. By design, this catch/release process is a logic test for the presence of the key components required for cyclization, thus removing impurities which lack the required functionality, such as common classes of peptide impurities, including hydrolysis fragments and truncated sequences. The method was shown to be highly effective with three libraries of macrocyclic peptides, containing macrocycles of 5–20 amino acids, with either thioether- or amine-based macrocyclic linkages; in this latter class, the reported method represents an enabling technology. In all cases, the catch–release protocol afforded significant enrichment of the target peptides purity, in many cases completely obviating the need for chromatography. Importantly, we have adapted this process for automation on a standard multichannel peptide synthesizer, achieving an efficient and completely integrated synthesis and purification platform for the preparation of these important molecules

Discovery and Lead Optimization of a Novel Series of CC Chemokine Receptor 1 (CCR1)-Selective Piperidine Antagonists via Parallel Synthesis

A series of novel, potent CCR1 inhibitors was developed from a moderately active hit using an iterative parallel synthesis approach. The initial hit (composed of three subunits: an amine, a central amino acid, and an N-terminal cap) became the basis for a series of parallel chemical libraries designed to generate SAR data. Libraries were synthesized that explored each of the three subunits; the CCR1 binding data obtained revealed the following: (1) changes to the amine are not well tolerated; (2) small alkylamino acids are preferred in the center of the molecule; (3) substitutions at the N-terminus are generally well tolerated. These data were used to drive the optimization of the series, ultimately providing a lead with a CCR1 binding IC₅₀ of 28 nM (<b>48</b>). This lead demonstrates high selectivity for CCR1 over other CCR-family members, high microsomal stability, and good pharmacokinetics in mice

The Discovery of Macrocyclic XIAP Antagonists from a DNA-Programmed Chemistry Library, and Their Optimization To Give Lead Compounds with in Vivo Antitumor Activity

Affinity selection screening of macrocycle libraries derived from DNA-programmed chemistry identified XIAP BIR2 and BIR3 domain inhibitors that displace bound pro-apoptotic caspases. X-ray cocrystal structures of key compounds with XIAP BIR2 suggested potency-enhancing structural modifications. Optimization of dimeric macrocycles with similar affinity for both domains were potent pro-apoptotic agents in cancer cell lines and efficacious in shrinking tumors in a mouse xenograft model

Challenges with the Synthesis of a Macrocyclic Thioether Peptide: From Milligram to Multigram Using Solid Phase Peptide Synthesis (SPPS)

We describe an optimization and scale-up of the 45-membered macrocyclic thioether peptide BMS-986189 utilizing solid-phase peptide synthesis (SPPS). Improvements to linear peptide isolation, macrocyclization, and peptide purification were demonstrated to increase the throughput and purification of material on scale and enabled the synthesis and purification of >60 g of target peptide. Taken together, not only these improvements resulted in a 28-fold yield increase from the original SPPS approach, but also the generality of this newly developed SPPS purification sequence has found application in the synthesis and purification of other macrocyclic thioether peptides

Discovery of a Potent and Orally Bioavailable Dual Antagonist of CC Chemokine Receptors 2 and 5

We describe the hybridization of our previously reported acyclic and cyclic CC chemokine receptor 2 (CCR2) antagonists to lead to a new series of dual antagonists of CCR2 and CCR5. Installation of a γ-lactam as the spacer group and a quinazoline as a benzamide mimetic improved oral bioavailability markedly. These efforts led to the identification of <b>13d</b>, a potent and orally bioavailable dual antagonist suitable for use in both murine and monkey models of inflammation

Dimeric Macrocyclic Antagonists of Inhibitor of Apoptosis Proteins for the Treatment of Cancer

A series of dimeric macrocyclic compounds were prepared and evaluated as antagonists for inhibitor of apoptosis proteins. The most potent analogue <b>11</b>, which binds to XIAP and c-IAP proteins with high affinity and induces caspase-3 activation and ultimately cell apoptosis, inhibits growth of human melanoma and colorectal cell lines at low nanomolar concentrations. Furthermore, compound <b>11</b> demonstrated significant antitumor activity in the A875 human melanoma xenograft model at doses as low as 2 mg/kg on a q3d schedule

Potent and Selective Agonists of Sphingosine 1‑Phosphate 1 (S1P₁): Discovery and SAR of a Novel Isoxazole Based Series

Sphingosine 1-phosphate (S1P) is the endogenous ligand for the sphingosine 1-phosphate receptors (S1P_1–5) and evokes a variety of cellular responses through their stimulation. The interaction of S1P with the S1P receptors plays a fundamental physiological role in a number of processes including vascular development and stabilization, lymphocyte migration, and proliferation. Agonism of S1P₁, in particular, has been shown to play a significant role in lymphocyte trafficking from the thymus and secondary lymphoid organs, resulting in immunosuppression. This article will detail the discovery and SAR of a potent and selective series of isoxazole based full agonists of S1P₁. Isoxazole <b>6d</b> demonstrated impressive efficacy when administered orally in a rat model of arthritis and in a mouse experimental autoimmune encephalomyelitis (EAE) model of multiple sclerosis

Discovery and Structure–Activity Relationship (SAR) of a Series of Ethanolamine-Based Direct-Acting Agonists of Sphingosine-1-phosphate (S1P₁)

Sphingosine-1-phosphate (S1P) is a bioactive sphingolipid metabolite that regulates a multitude of physiological processes such as lymphocyte trafficking, cardiac function, vascular development, and inflammation. Because of the ability of S1P₁ receptor agonists to suppress lymphocyte egress, they have great potential as therapeutic agents in a variety of autoimmune diseases. In this article, the discovery of selective, direct acting S1P₁ agonists utilizing an ethanolamine scaffold containing a terminal carboxylic acid is described. Potent S1P₁ agonists such as compounds <b>18a</b> and <b>19a</b> which have greater than 1000-fold selectivity over S1P₃ are described. These compounds efficiently reduce blood lymphocyte counts in rats through 24 h after single doses of 1 and 0.3 mpk, respectively. Pharmacodynamic properties of both compounds are discussed. Compound <b>19a</b> was further studied in two preclinical models of disease, exhibiting good efficacy in both the rat adjuvant arthritis model (AA) and the mouse experimental autoimmune encephalomyelitis model (EAE)

Small Molecule Reversible Inhibitors of Bruton’s Tyrosine Kinase (BTK): Structure–Activity Relationships Leading to the Identification of 7‑(2-Hydroxypropan-2-yl)-4-[2-methyl-3-(4-oxo-3,4-dihydroquinazolin-3-yl)phenyl]‑9<i>H</i>‑carbazole-1-carboxamide (BMS-935177)

Bruton’s tyrosine kinase (BTK) belongs to the TEC family of nonreceptor tyrosine kinases and plays a critical role in multiple cell types responsible for numerous autoimmune diseases. This article will detail the structure–activity relationships (SARs) leading to a novel second generation series of potent and selective reversible carbazole inhibitors of BTK. With an excellent pharmacokinetic profile as well as demonstrated in vivo activity and an acceptable safety profile, 7-(2-hydroxypropan-2-yl)-4-[2-methyl-3-(4-oxo-3,4-dihydro­quinazolin-3-yl)­phenyl]-9<i>H</i>-carbazole-1-carboxamide <b>6</b> (BMS-935177) was selected to advance into clinical development

Identification of Tricyclic Agonists of Sphingosine-1-phosphate Receptor 1 (S1P₁) Employing Ligand-Based Drug Design

Fingolimod (<b>1</b>) is the first approved oral therapy for the treatment of relapsing remitting multiple sclerosis. While the phosphorylated metabolite of fingolimod was found to be a nonselective S1P receptor agonist, agonism specifically of S1P₁ is responsible for the peripheral blood lymphopenia believed to be key to its efficacy. Identification of modulators that maintain activity on S1P₁ while sparing activity on other S1P receptors could offer equivalent efficacy with reduced liabilities. We disclose in this paper a ligand-based drug design approach that led to the discovery of a series of potent tricyclic agonists of S1P₁ with selectivity over S1P₃ and were efficacious in a pharmacodynamic model of suppression of circulating lymphocytes. Compound <b>10</b> had the desired pharmacokinetic (PK) and pharmacodynamic (PD) profile and demonstrated maximal efficacy when administered orally in a rat adjuvant arthritis model