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 CO2 was developed on a Waters BEH 2-Ethylpyridine column. The developed SFC method was readily scaled up onto a 5 cm diameter column to process multi-gram quantities of the MP-RPLC fraction to reach > 95 % purity with a throughput/productivity of 0.96 g/h. The incorporation of SFC with MP-RPLC has been demonstrated to have a broader application in other large-scale polypeptide purifications.

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 Novel TLR7 Agonists as Systemic Agent for Combination With aPD1 for Use in Immuno-oncology.

We have designed and developed novel and selective TLR7 agonists that exhibited potent receptor activity in a cell-based reporter assay. In vitro, these agonists significantly induced secretion of cytokines IL-6, IL-1ß, IL-10, TNFa, IFNa, and IP-10 in human and mouse whole blood. Pharmacokinetic and pharmacodynamic studies in mice showed a significant secretion of IFNα and TNFα cytokines. When combined with aPD1 in a CT-26 tumor model, the lead compound showed strong synergistic antitumor activity with complete tumor regression in 8/10 mice dosed using the intravenous route. Structure-activity relationship studies enabled by structure-based designs of TLR7 agonists are disclosed.

Identification and Optimization of Small Molecule Pyrazolopyrimidine TLR7 Agonists for Applications in Immuno-oncology.

Small molecule toll-like receptor (TLR) 7 agonists have gathered considerable interest as promising therapeutic agents for applications in cancer immunotherapy. Herein, we describe the development and optimization of a series of novel TLR7 agonists through systematic structure-activity relationship studies focusing on modification of the phenylpiperidine side chain. Additional refinement of ADME properties culminated in the discovery of compound 14, which displayed nanomolar reporter assay activity and favorable drug-like properties. Compound 14 demonstrated excellent in vivo pharmacokinetic/pharmacodynamic profiles and synergistic antitumor activity when administered in combination with aPD1 antibody, suggesting opportunities of employing 14 in immuno-oncology therapies with immune checkpoint blockade agents.

Isolation and characterization of pseudo degradation products of acalabrutinib using ESI-HRMS/MS and NMR: Formation of possible geometrical isomers.

A forced degradation study of acalabrutinib (ACB), used to treat relapsed mantle cell lymphoma, was performed to identify and characterize all possible major degradation products formed under different stress conditions. The degradation products (DP) were separated using reverse phase UHPLC system on Kinetex EVO C18 column. Major DPs formed were isolated using semi-preparative HPLC and characterized by LC-ESI-HRMS/MS and NMR. ACB degraded to form seven major degradants (DP-I to DP-VII). DP-I and DP-V were formed under alkaline stress condition, whereas DP-II, DP-III, DP-VI and DP-VII were formed under both acidic and basic conditions. Further, DP-IV was formed when ACB drug was exposed to hydrogen peroxide stress condition. ACB was found to be stable when subjected to aqueous (neutral pH), thermal and UV radiation of 254 nm, as it has not shown any significant degradation under these conditions. Interestingly, two pairs of pseudo geometrical isomeric DPs (DP-II and DP-III, DP-VI and DP-VII) were observed. The plausible degradation pathway of ACB and fragmentation patterns of both ACB and major DPs were discussed.

Tuberculosis in free-ranging and captive wild animals: Pathological and molecular diagnosis with histomorphological differentiation of granulomatous lesions.

Tuberculosis (TB) is a serious zoonotic threat, impacting the human-livestock-wildlife interface globally. Here, we evaluated the status and histomorphological differentiation of TB lesions in 89 morbid cases of wild animals (bovids, cervids, carnivores, non-human primates, and pachyderms) in India. Histomorphological and molecular studies were done using Ziehl-Neelsen (ZN) staining, immunohistochemistry, and polymerase chain reaction (PCR), whereas cultural isolation was performed on selected samples. A total of 32 (35.95%) cases were confirmed as TB, comprising of 12 carnivores, 09 bovids, 06 cervids, 04 non-human primates, and a pachyderm. The TB lesions in the lungs, liver, and lymph nodes varied from the large-sized caseous nodules filled with dry cheesy material in bovids and cervids to variable-sized cavitations containing liquefied caseum in carnivores' lungs. The lungs, livers, and spleens of non-human primates exhibited small to medium-sized nodules. Histologically, lesions were divided into four categories (Types I, II, III, and IV) based on the extent of necrosis, the presence of mineralization, giant cells, and fibrous encapsulation. Extensive caseous necrosis with calcification, abundant giant cells, and thick fibroblastic encapsulation were consistent findings in the lungs, livers, and lymph nodes of bovids and cervids, whereas airway impaction with cellular exudate containing a teeming number of acid-fast bacilli and, at times, alveolar rupture leading to cavity formation was present in the lungs of carnivores. Absence of calcification and fibrous encapsulation was recorded in lungs of non-human primates. Immunohistochemical labelling with anti-early secretory antigenic target-6 (ESAT-6) and culture filtrate protein-10 (CFP-10) antibodies showed mild, moderate, and intense positive reactions in type II and III, type I, and type IV granulomatous lesions, respectively. Molecular detection by PCR revealed Mycobacterium tuberculosis (12 carnivores, 02 non-human primates and 01 pachyderm), M. bovis (02 cervids and 01 bovid) and M. orygis (02 cervids and 01 bovid). Cultural isolation confirmed M. tuberculosis in 03 carnivores and M. orygis in 02 cervids and 01 bovid. Our findings imply that TB is quite prevalent in the wildlife of India and there are considerable differences in the histomorphological lesions induced by distinct Mycobacterium species in different wild animals. The circulation of TB organisms in wild animals warrants a strict surveillance programme to identify the carrier status of these animals so that effective TB control strategies can be formulated to prevent spillover and spillback incidences at the human-livestock-wildlife interface.

Mass spectrometric-based investigation of differentially protected azatryptophan derivatives using Orbitrap mass spectrometry: Differentiation of positional isomers under protonation and alkali-cationization conditions.

RATIONALE: Differentiation and structural characterization of positional isomers of differentially protected azatryptophan derivatives using electrospray ionization high-resolution tandem mass spectrometry (ESI-HRMS/MS) is important from the perspective of drug discovery research. Also, these derivatives can be used as building blocks for the synthesis of various biologically active compounds and have attracted significant attention in the field of modern drug discovery, especially peptide-based drugs, protein folding and protein-protein interactions because of their interesting spectral properties. METHODS: ESI-HRMS/MS in positive ionization mode was used to differentiate and characterize positional isomers of protected azatryptophan derivatives. RESULTS: ESI-HRMS/MS of [M + H]+ and [M + Na]+ ions of positional isomers of differentially protected azatryptophan derivatives display distinct fragmentation patterns. The MS/MS of [M + H]+ ion of isomer 1 showed an additional ion at m/z 358.0846 ([M + H-Boc-C14 H10 -HF]+ ) which was not present for 4. The fragment ion at m/z 332.0857 was observed for 1 and not for 4 which would be formed by the expulsion of butyloxycarbonyl (Boc) and fluorenylmethyloxycarbonyl (Fmoc) groups. Moreover, the ions 422.0812 and 378.0912 are found to be relatively more abundant for isomer 4 which could be probably attributed to the formation of stable ions. Similarly, other positional isomers exhibited distinct fragmentation from one another. CONCLUSIONS: The present study demonstrates that ESI-HRMS/MS can be used for differentiation and structural characterization of positional isomers of protected azatryptophan derivatives. The MS/MS of [M + H]+ and [M + Na]+ ions of these positional isomers displayed differences in their fragmentation behaviour. The impact of different substitutions at different positions (1 and 6) of protected azatryptophan derivatives (1-6) on their fragmentation behaviour was also investigated in detail. Also, the nitrogen atom at different positions in the pyrrolopyridine ring led to different fragmentation patterns.

Discovery and Preclinical Pharmacology of an Oral Bromodomain and Extra-Terminal (BET) Inhibitor Using Scaffold-Hopping and Structure-Guided Drug Design.

Inhibition of the bromodomain and extra-terminal (BET) family of adaptor proteins is an attractive strategy for targeting transcriptional regulation of key oncogenes, such as c-MYC. Starting with the screening hit 1, a combination of structure-activity relationship and protein structure-guided drug design led to the discovery of a differently oriented carbazole 9 with favorable binding to the tryptophan, proline, and phenylalanine (WPF) shelf conserved in the BET family. Identification of an additional lipophilic pocket and functional group optimization to optimize pharmacokinetic (PK) properties culminated in the discovery of 18 (BMS-986158) with excellent potency in binding and functional assays. On the basis of its favorable PK profile and robust in vivo activity in a panel of hematologic and solid tumor models, BMS-986158 was selected as a candidate for clinical evaluation.

Conformational-Analysis-Guided Discovery of 2,3-Disubstituted Pyridine IDO1 Inhibitors.

IDO1 inhibitors have shown promise as immunotherapies for the treatment of a variety of cancers, including metastatic melanoma and renal cell carcinoma. We recently reported the identification of several novel heme-displacing IDO1 inhibitors, including the clinical molecules linrodostat (BMS-986205) and BMS-986242. Both molecules contain quinolines that, while being present in successful medicines, are known to be potentially susceptible to oxidative metabolism. Efforts to swap this quinoline with an alternative aromatic system led to the discovery of 2,3-disubstituted pyridines as suitable replacements. Further optimization, which included lowering ClogP in combination with strategic fluorine incorporation, led to the discovery of compound 29, a potent, selective IDO1 inhibitor with robust pharmacodynamic activity in a mouse xenograft model.

Molecular mechanism of interspecies differences in the binding affinity of TD139 to Galectin-3.

Galectin-3 (Gal-3), a ß-galactoside-binding lectin, has been implicated in a plethora of pathological disorders including fibrosis, inflammation, cancer and metabolic diseases. TD139-a thio-digalactoside inhibitor developed by Galecto Biotech as a potential therapeutic for idiopathic pulmonary fibrosis-is the most advanced small-molecule Gal-3 inhibitor in clinical studies. It binds to human Gal-3 with high affinity but has lower affinity towards mouse and rat homologs, which is also manifested in the differential inhibition of Gal-3 function. Using biophysical methods and high-resolution X-ray co-crystal structures of TD139 and Gal-3 proteins, we demonstrate that a single amino acid change corresponding to A146 in human Gal-3 is sufficient for the observed reduction in the binding affinity of TD139 in rodents. Site-directed mutagenesis of A146V (in human Gal-3) and V160A (in mouse Gal-3) was sufficient to interchange the affinities, mainly by affecting the off rates of the inhibitor binding. In addition, molecular dynamics simulations of both wild-type and mutant structures revealed the sustained favorable noncovalent interactions between the fluorophenyl ring and the active site A146 (human Gal-3 and mouse V160A) that corroborate the finding from biophysical studies. Current findings have ramifications in the context of optimization of drug candidates against Gal-3.

Chemical Modification of Linkers Provides Stable Linker-Payloads for the Generation of Antibody-Drug Conjugates.

Stability of antibody-drug conjugates (ADCs) in mouse serum is one of the critical requirements for the evaluation of ADCs in mouse tumor models. Described herein is a strategy to address the mouse serum instability of uncialamycin linker-payloads through various chemical approaches that involve modification of different parts of the linker and payload. This effort ultimately led to the identification of a m-amide p-aminobenzyl carbamate (MA-PABC) group that resulted in linkers with dramatic improvement of mouse serum stability without affecting the desired proteolytic cleavage.

Design, Synthesis, and Structure-Activity Relationships of Novel Tetrahydroisoquinolino Benzodiazepine Dimer Antitumor Agents and Their Application in Antibody-Drug Conjugates.

A series of tetrahydroisoquinoline-based benzodiazepine dimers were synthesized and tested for in vitro cytotoxicity against a panel of cancer cell lines. Structure-activity relationship investigation of various spacers guided by molecular modeling studies helped to identify compounds with picomolar activity. Payload 17 was conjugated to anti-mesothelin and anti-fucosylated monosialotetrahexosylganglioside (FucGM1) antibodies using lysosome-cleavable valine-citrulline dipeptide linkers via heterogeneous lysine conjugation and bacterial transglutaminase-mediated site-specific conjugation. In vitro, these antibody drug conjugates (ADCs) exhibited significant cytotoxic and target-mediated selectivity on human cancer cell lines. The pharmacokinetics and efficacy of these ADCs were further evaluated in gastric and lung cancer xenograft models in mice. Consistent pharmacokinetic profiles, high target specificity, and robust antitumor activity were observed in these models after a single dose of the ADC-46 (0.02 µmol/kg).

Aryl Ether-Derived Sphingosine-1-Phosphate Receptor (S1P1) Modulators: Optimization of the PK, PD, and Safety Profiles.

Efforts aimed at increasing the in vivo potency and reducing the elimination half-life of 1 and 2 led to the identification of aryl ether and thioether-derived bicyclic S1P1 differentiated modulators 3-6. The effects of analogs 3-6 on lymphocyte reduction in the rat (desired pharmacology) along with pulmonary- and cardiovascular-related effects (undesired pharmacology) are described. Optimization of the overall properties in the aryl ether series yielded 3d, and the predicted margin of safety against the cardiovascular effects of 3d would be large enough for human studies. Importantly, compared to 1 and 2, compound 3d had a better profile in both potency (ED50 < 0.05 mg/kg) and predicted human half-life (t 1/2 ∼ 5 days).

Discovery of BMS-986260, a Potent, Selective, and Orally Bioavailable TGFßR1 Inhibitor as an Immuno-oncology Agent.

Novel imidazole-based TGFßR1 inhibitors were identified and optimized for potency, selectivity, and pharmacokinetic and physicochemical characteristics. Herein, we report the discovery, optimization, and evaluation of a potent, selective, and orally bioavailable TGFßR1 inhibitor, 10 (BMS-986260). This compound demonstrated functional activity in multiple TGFß-dependent cellular assays, excellent kinome selectivity, favorable pharmacokinetic properties, and curative in vivo efficacy in combination with anti-PD-1 antibody in murine colorectal cancer (CRC) models. Since daily dosing of TGFßR1 inhibitors is known to cause class-based cardiovascular (CV) toxicities in preclinical species, a dosing holiday schedule in the anti-PD-1 combination efficacy studies was explored. An intermittent dosing regimen of 3 days on and 4 days off allowed mitigation of CV toxicities in one month dog and rat toxicology studies and also provided similar efficacy as once daily dosing.

Design, synthesis and biological evaluation of phenol-linked uncialamycin antibody-drug conjugates.

Uncialamycin is one of the structurally simpler and newer members of enediyne family of natural products. It exhibits highly potent activity against several types of bacteria and cancer cells. Described herein is a strategy for the targeted delivery of this cytotoxic agent to tumors using an antibody-drug conjugate (ADC) approach. Central to the design of ADC were the generation of potent and chemically stable uncialamycin analogues and attachment of protease cleavable linkers to newly realized phenolic handles to prepare linker-payloads. Conjugation of the linker-payloads to tumor targeting antibody, in vitro activity and in vivo evaluation are presented.

Discovery of a Highly Selective JAK2 Inhibitor, BMS-911543, for the Treatment of Myeloproliferative Neoplasms.

JAK2 kinase inhibitors are a promising new class of agents for the treatment of myeloproliferative neoplasms and have potential for the treatment of other diseases possessing a deregulated JAK2-STAT pathway. X-ray structure and ADME guided refinement of C-4 heterocycles to address metabolic liability present in dialkylthiazole 1 led to the discovery of a clinical candidate, BMS-911543 (11), with excellent kinome selectivity, in vivo PD activity, and safety profile.

Discovery of Clinical Candidate BMS-906024: A Potent Pan-Notch Inhibitor for the Treatment of Leukemia and Solid Tumors.

Structure-activity relationships in a series of (2-oxo-1,4-benzodiazepin-3-yl)-succinamides identified highly potent inhibitors of γ-secretase mediated signaling of Notch1/2/3/4 receptors. On the basis of its robust in vivo efficacy at tolerated doses in Notch driven leukemia and solid tumor xenograft models, 12 (BMS-906024) was selected as a candidate for clinical evaluation.

BMS-871: a novel orally active pan-Notch inhibitor as an anticancer agent.

This Letter describes synthesis, SAR, and biological activity of (2-oxo-1,4-benzodiazepin-3-yl)-succinamides as inhibitors of γ-secretase mediated signaling of Notch receptors. Optimization of this series led to the identification of BMS-871 (compound 30) which displayed robust in vivo efficacy in Notch-dependent leukemia and solid tumor xenograft models.

Identification of 1-{2-[4-chloro-1'-(2,2-dimethylpropyl)-7-hydroxy-1,2-dihydrospiro[indole-3,4'-piperidine]-1-yl]phenyl}-3-{5-chloro-[1,3]thiazolo[5,4-b]pyridin-2-yl}urea, a potent, efficacious and orally bioavailable P2Y(1) antagonist as an antiplatelet agent.

Spiropiperidine indoline-substituted diaryl ureas had been identified as antagonists of the P2Y1 receptor. Enhancements in potency were realized through the introduction of a 7-hydroxyl substitution on the spiropiperidinylindoline chemotype. SAR studies were conducted to improve PK and potency, resulting in the identification of compound 3e, a potent, orally bioavailable P2Y1 antagonist with a suitable PK profile in preclinical species. Compound 3e demonstrated a robust antithrombotic effect in vivo and improved bleeding risk profile compared to the P2Y12 antagonist clopidogrel in rat efficacy/bleeding models.

A Rare Case of Nephrocolic Fistula Resulting from Radio Frequency Ablation (RFA) of Renal Cell Carcinoma.

Nephrocolic fistula is a rare, abnormal fistulous connection between the urinary system (kidney/ureters) and colon. Different benign and malignant etiologies are implicated in the formation of a nephrocolic fistula. Even though conservative treatment options have been tried recently (especially for benign etiologies), surgical resection has been the treatment of choice and should be pursued if conservative management fails. We report the first case of a nephrocolic fistula after a radiofrequency ablation of a renal cell carcinoma, which required surgical resection after conservative management failed.