Large-scale purification of a deprotected macrocyclic peptide by supercritical fluid chromatography (SFC) integrated with liquid chromatography in discovery chemistry.

A macrocyclic peptide A was successfully purified in large quantities (∼30 g) in >95 % purity by an integrated two-step orthogonal purification process combining supercritical fluid chromatography (SFC) with medium-pressure reverse-phase liquid chromatography (MP-RPLC). MP-RPLC was used to fractionate the crude peptide A, remove unwanted trifluoroacetic acid (TFA) originating from the peptide A cleavage off the resin, and convert the peptide A into ammonium acetate salt form, prior to the final purification by SFC. A co-solvent of methanol/acetonitrile containing ammonium acetate and water in CO was developed on a Waters BEH 2-Ethylpyridine column.

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 ()-1-((2',6-Bis(difluoromethyl)-[2,4'-bipyridin]-5-yl)oxy)-2,4-dimethylpentan-2-amine (BMS-986176/LX-9211): A Highly Selective, CNS Penetrable, and Orally Active Adaptor Protein-2 Associated Kinase 1 Inhibitor in Clinical Trials for the Treatment of Neuropathic Pain.

Recent mouse knockout studies identified adapter protein-2 associated kinase 1 (AAK1) as a viable target for treating neuropathic pain. Potent small-molecule inhibitors of AAK1 have been identified and show efficacy in various rodent pain models. ()-1-((2',6-Bis(difluoromethyl)-[2,4'-bipyridin]-5-yl)oxy)-2,4-dimethylpentan-2-amine (BMS-986176/LX-9211) () was identified as a highly selective, CNS penetrant, potent AAK1 inhibitor from a novel class of bi(hetero)aryl ethers.

Long-Acting Tumor-Activated Prodrug of a TGFβR Inhibitor.

Inhibition of TGFβ signaling in concert with a checkpoint blockade has been shown to provide improved and durable antitumor immune response in mouse models. However, on-target adverse cardiovascular effects have limited the clinical use of TGFβ receptor (TGFβR) inhibitors in cancer therapy. To restrict the activity of TGFβR inhibitors to tumor tissues and thereby widen the therapeutic index, a series of tumor-activated prodrugs of a selective small molecule TGFβR1 inhibitor were prepared by appending to a serine protease substrate and a half-life extension fatty acid carbon chain.

A Stereocontrolled Synthesis of a Phosphorothioate Cyclic Dinucleotide-Based STING Agonist.

We describe a stereodefined synthesis of the newly identified non-natural phosphorothioate cyclic dinucleotide (CDN) STING agonist, BMT-390025. The new route avoids the low-yielding racemic approach using P(III)-based reagents, and the stereospecific assembly of the phosphorothioate linkages are forged via the recently invented P(V)-based platform of the so-called PSI (Ψ) reagent system. This P(V) approach allows for the complete control of chirality of the P-based linkages and enabled conclusive evidence of the absolute configuration.

Azatricyclic Inverse Agonists of RORγt That Demonstrate Efficacy in Models of Rheumatoid Arthritis and Psoriasis.

Structure-activity relationship studies directed toward the replacement of the fused phenyl ring of the lead hexahydrobenzoindole RORγt inverse agonist series represented by with heterocyclic moieties led to the identification of three novel aza analogs -. The hexahydropyrrolo[3,2-]quinoline series (X = N, Y = Z=CH) showed potency and metabolic stability comparable to series but with improved membrane permeability and serum free fraction. This structural modification was applied to the hexahydrocyclopentanaphthalene series , culminating in the discovery of as a potent and selective RORγt inverse agonist with an excellent profile, good pharmacokinetic properties, and biologic-like efficacy in preclinical models of rheumatoid arthritis and psoriasis.

Tricyclic-Carbocyclic RORγt Inverse Agonists-Discovery of BMS-986313.

SAR efforts directed at identifying RORγt inverse agonists structurally different from our clinical compound (BMS-986251) led to tricyclic-carbocyclic analogues represented by - and culminated in the identification of (BMS-986313), with structural differences distinct from . The X-ray co-crystal structure of with the ligand binding domain of RORγt revealed several key interactions, which are different from . The in vitro and in vivo PK profiles of are described.

Novel Tricyclic Pyroglutamide Derivatives as Potent RORγt Inverse Agonists Identified using a Virtual Screening Approach.

Employing a virtual screening approach, we identified the pyroglutamide moiety as a nonacid replacement for the cyclohexanecarboxylic acid group which, when coupled to our previously reported conformationally locked tricyclic core, provided potent and selective RORγt inverse agonists. Structure-activity relationship optimization of the pyroglutamide moiety led to the identification of compound as a potent and selective RORγt inverse agonist, albeit with poor aqueous solubility. We took advantage of the tertiary carbinol group in to synthesize a phosphate prodrug, which provided good solubility, excellent exposures in mouse PK studies, and significant efficacy in a mouse model of psoriasis.

Driving Potency with Rotationally Stable Atropisomers: Discovery of Pyridopyrimidinedione-Carbazole Inhibitors of BTK.

Bruton's tyrosine kinase (BTK) has been shown to play a key role in the pathogenesis of autoimmunity. Therefore, the inhibition of the kinase activity of BTK with a small molecule inhibitor could offer a breakthrough in the clinical treatment of many autoimmune diseases. This Letter describes the discovery of BMS-986143 through systematic structure-activity relationship (SAR) development.

Tricyclic sulfones as potent, selective and efficacious RORγt inverse agonists - Exploring C6 and C8 SAR using late-stage functionalization.

In order to rapidly develop C6 and C8 SAR of our reported tricyclic sulfone series of RORγt inverse agonists, a late-stage bromination was employed. Although not regioselective, the bromination protocol allowed us to explore new substitution patterns/vectors that otherwise would have to be incorporated at the very beginning of the synthesis. Based on the SAR obtained from this exercise, compound 15 bearing a C8 fluorine was developed as a very potent and selective RORγt inverse agonist.

Annulation reaction enables the identification of an exocyclic amide tricyclic chemotype as retinoic acid Receptor-Related orphan receptor gamma (RORγ/RORc) inverse agonists.

RORγt is the master regulator of the IL-23/IL-17 axis, a pathway that is clinically validated for the treatment of various immunological disorders. Over the last few years, our group has reported different chemotypes that potently act as inverse agonists of RORγt. One of them, the tricyclic pyrrolidine chemotype, has demonstrated biologic-like preclinical efficacy and has led to our clinical candidate BMS-986251.

Synthesis of 1-(-Butyl) 4-Methyl (1,2,4)-2-Methylcyclohexane-1,4-dicarboxylate from Hagemann's -Butyl Ester for an Improved Synthesis of BMS-986251.

We describe an efficient synthetic route to differentially protected diester, 1-(-butyl) 4-methyl (1,2,4)-2-methylcyclohexane-1,4-dicarboxylate (+)-, via palladium-catalyzed methoxycarbonylation of an enol triflate derived from a Hagemann's ester derivative followed by a stereoselective Crabtree hydrogenation. Diester is a novel chiral synthon useful in drug discovery and was instrumental in the generation of useful SAR during a RORγt inverse agonist program. In addition, we describe a second-generation synthesis of the clinical candidate BMS-986251, using diester as a critical component.

Discovery of BMS-986251: A Clinically Viable, Potent, and Selective RORγt Inverse Agonist.

Novel tricyclic analogues were designed, synthesized, and evaluated as RORγt inverse agonists. Several of these compounds were potent in an IL-17 human whole blood assay and exhibited excellent oral bioavailability in mouse pharmacokinetic studies. This led to the identification of compound , which displayed dose-dependent inhibition of IL-17F production in a mouse IL-2/IL-23 stimulated pharmacodynamic model.

Separation of Bruton's tyrosine kinase inhibitor atropisomers by supercritical fluid chromatography.

Bruton's tyrosine kinase (BTK) plays an essential role in multiple cell types responsible for numerous autoimmune diseases, thus inhibition of BTK is anticipated to provide an effective strategy for the clinical treatment of autoimmune diseases. Preparative-scale super/subcritical fluid chromatography (SFC) separation methods for four groups of highly potent and selective BTK inhibitor atropisomers were successfully developed. Depending on the rotation barrier around the chiral axis, the compounds were prepared as a single stereochemically stable atropisomer or as an atropisomeric mixture.

Development of the Large-Scale Synthesis of Tetrahydropyran Glycine, a Precursor to the HCV NS5A Inhibitor BMS-986097.

An efficient large-scale synthesis of acid 1, a penultimate precursor to the HCV NS5A inhibitor BMS-986097, along with the final API step are described. Three routes were devised for the synthesis of 1 at the various stages of the program. The third generation route, the one that proved scalable and is the main subject of this paper, features a one-step Michael addition of t-butyl 2-((diphenylmethylene)amino)acetate (24) to (E)-benzyl 4-(1-hydroxycyclopropyl)but-2-enoate (28) followed by cyclization and chiral separation to form 27c, the core skeleton of cap piece 1.

Discovery of 6-Fluoro-5-(R)-(3-(S)-(8-fluoro-1-methyl-2,4-dioxo-1,2-dihydroquinazolin-3(4H)-yl)-2-methylphenyl)-2-(S)-(2-hydroxypropan-2-yl)-2,3,4,9-tetrahydro-1H-carbazole-8-carboxamide (BMS-986142): A Reversible Inhibitor of Bruton's Tyrosine Kinase (BTK) Conformationally Constrained by Two Locked Atropisomers.

Bruton's tyrosine kinase (BTK), a nonreceptor tyrosine kinase, is a member of the Tec family of kinases. BTK plays an essential role in B cell receptor (BCR)-mediated signaling as well as Fcγ receptor signaling in monocytes and Fcε receptor signaling in mast cells and basophils, all of which have been implicated in the pathophysiology of autoimmune disease. As a result, inhibition of BTK is anticipated to provide an effective strategy for the clinical treatment of autoimmune diseases such as lupus and rheumatoid arthritis.

Additive free preparative chiral SFC separations of 2,2-dimethyl-3-aryl-propanoic acids.

A series of racemic 2,2-dimethyl-3-aryl-propanoic acids were resolved by chiral supercritical fluid chromatography (SFC) without the use of an acidic additive, trifluoroacetic acid (TFA). The use of additive-free protic methanol as co-solvent in CO was expanded to successfully resolve other series of carboxylic acid containing racemates. Large-scale SFC of racemic acid 4, 3-(1-(4-fluorophenyl)-1H-indazol-5-yl)-2,2-dimethyl-3-phenylpropanoic acid, in methanol without TFA as additive on both Chiralpak AD-H and Chiralcel OJ-H will be discussed, along with impact on throughput and solvent consumption.

Regioselective Synthesis of Substituted 4-Alkylamino and 4-Arylaminophthalazin-1(2H)-ones.

An efficient regioselective synthesis of substituted 4-alkylamino and 4-arylaminophthalazin-1(1H)-ones 5 is described. This new method features the formation of substituted phthalazin-1(1H)-ones 3 by the reaction of 2-formylbenzoic acids 1 or 3-hydroxyisobenzofuran-1(3H)-ones 2 with hydrazine to generate phthalazin-1(2H)-ones 3. Subsequent regioselective bromination of phthalazin-1(2H)-ones 3 with benzyltrimethylammonium tribromide (BTMA-Br3) followed by mixed copper-copper oxide-catalyzed amination of 4-bromophthalazin-1(2H)-ones 4 with primary amines generates aminophthalazin-1(2H)-ones in good overall yields.

From analytical methods to large scale chiral supercritical fluid chromatography using chlorinated chiral stationary phases.

While traditional non-chlorinated Cellulose- and Amylose-derivatized phases have been used successfully in supercritical fluid chromatography (SFC) to resolve a broad variety of chiral compounds, some chiral pharmaceutical compounds are not well resolved on these traditional chiral stationary phases (CSP) due to the lack of chiral selectivity. Since there are no universal CSP to resolve all chiral compounds, chlorinated CSP can be complementary to the non-chlorinated CSP. Chlorinated CSP such as 4-Chloro-3-methylphenyl-carbamatecellulose (Lux-Cellulose-4), 3-Chloro-4-methylphenyl-carbamatecellulose (Lux-Cellulose-2), 5-Chloro-2-methylphenyl-carbamateamylose (Lux-Amylose-2) and immobilized 3,5-dichlorophenyl-carbamatecellulose (Chiralpak IC) have provided a range of chiral recognition mechanisms which have allowed the authors to successfully achieve chiral SFC resolution on several structurally diverse compounds, which are not well resolved in the non-chlorinated CSP.

Analytical scale supercritical fluid fractionation and identification of single polar lipids from deoiled soybean lecithin.

The phosphatidylcholine (PC)-enriched fraction from soybean lecithin is of interest due to its critical role in both the pharmaceutical and industrial field. In this work, enhancement of the purity of the PC fraction along with other individual polar lipid fractions was achieved from crude soybean lecithin by using supercritical fluid extraction (SFE) with methanol-modified SC-CO(2). Neutral lipids were first removed from the crude sample using pure CO(2).