An integrated Qual/Quan strategy for ganglioside lipidomics using high-resolution mass spectrometry and Skyline software.

RATIONALE: Gangliosides (GS) are attractive targets in biomarker discovery because of their physiological significance in numerous human diseases including certain cancers and developmental and metabolic disorders. The robust strategy described here enables the profiling of numerous GS while obtaining quantitative data of exploratory biomarkers present in human plasma and whole blood. METHOD: The GS from human blood, human plasma, and several cell lines were extracted using a mixture of methanol and isopropanol/0.1% formic acid followed by direct analysis of the supernatant. The simultaneous Qualitative and Quantitative (Qual/Quan) approach involves micro flow (20 µL/min) high pressure liquid chromatography (HPLC)/high-resolution mass spectrometry (HRMS) and post-acquisition data processing with Skyline software for profiling numerous GS in biological matrices. The quantitative assay involves reverse-phase liquid chromatography/HRMS and calibration curves using commercially available GS. RESULTS: Protein precipitation resulted in ~60%-80% GS recovery from biological matrices. Direct injection of the extract allowed for quantification of targeted GS in human blood, plasma, and cancer cell lines. The lower limit of detection for the target analytes, GM1, GT1, GD1, spiked into 1% BSA/PBS, ranged from 1 to 10 ng/mL. Human lung cancer cell lines contained variable amounts (1-130 ng/mL) of soluble Fuc-GM1 analogs, potential biomarkers of lung cancer. CONCLUSIONS: A combination of simple extraction and micro-HPLC/HRMS allowed for quantification of GS in human serum and whole blood. Integration of HRMS with Skyline allowed for GS profiling in the same samples using post-acquisition HRMS data without the need for reanalysis. The strategy presented here is expected to play an important role in profiling exploratory GS biomarkers in discovery bioanalytical research.

Fucosyl monosialoganglioside: Quantitative analysis of specific potential biomarkers of lung cancer in biological matrices using immunocapture extraction/tandem mass spectrometry.

RATIONALE: Certain lung cancer patients express elevated Fucosyl Monosialoganglioside (Fuc-GM1) in circulation compared to control groups. Several sensitive methods involving characterization of Fuc-GM1 have been reported. However, a highly specific and sensitive method for quantifying multiple potential Fuc-GM1 biomarkers present in various biological matrices has not been reported to date. METHODS: Individual Fuc-GM1 analogs in a commercially obtained standard mixture were characterized using HPLC/UV/MS and high-resolution mass spectrometry (HRMS). Proprietary antibodies, mAb1 and mAb2, were used to selectively capture and pre-concentrate the soluble and drug-bound forms of Fuc-GM1 molecules present in human serum and whole blood, eliminating the background matrix components. Immunocapture extraction (ICE) followed by HPLC/MS/MS was used to quantify specific Fuc-GM1 analogs in biological matrices. RESULTS: The concentration of individual Fuc-GM1 analogs in the standard mixture was estimated to be 7-34%, using HPLC/UV/MS. Using the standard mixture spiked into the biological matrices (100 µL), the lower limit of quantification (LLOQ) of each analog was 0.2-0.4 ng/mL with a dynamic range of up to 200 ng/mL. The applicability of the ICE-HPLC/MS/MS method was demonstrated by detecting endogenous Fuc-GM1 analogs present in rat blood and in several lung cancer cell lines. CONCLUSIONS: This highly specific and sensitive HPLC/MS/MS method for quantifying individual potential Fuc-GM1 biomarkers in serum and whole blood can play a critical role in patient stratification strategies and during drug treatment. This method can be employed for monitoring both free (soluble) form and antibody drug-bound Fuc-GM1.

Identification of bicyclic hexafluoroisopropyl alcohol sulfonamides as retinoic acid receptor-related orphan receptor gamma (RORγ/RORc) inverse agonists. Employing structure-based drug design to improve pregnane X receptor (PXR) selectivity.

We disclose the optimization of a high throughput screening hit to yield benzothiazine and tetrahydroquinoline sulfonamides as potent RORγt inverse agonists. However, a majority of these compounds showed potent activity against pregnane X receptor (PXR) and modest activity against liver X receptor α (LXRα). Structure-based drug design (SBDD) led to the identification of benzothiazine and tetrahydroquinoline sulfonamide analogs which completely dialed out LXRα activity and were less potent at PXR. Pharmacodynamic (PD) data for compound 35 in an IL-23 induced IL-17 mouse model is discussed along with the implications of a high Ymax in the PXR assay for long term preclinical pharmacokinetic (PK) studies.

Identification and synthesis of potent and selective pyridyl-isoxazole based agonists of sphingosine-1-phosphate 1 (S1P1).

The synthesis and structure-activity relationship (SAR) of a series of pyridyl-isoxazole based agonists of S1P1 are discussed. Compound 5b provided potent in vitro activity with selectivity, had an acceptable pharmacokinetic profile, and demonstrated efficacy in a dose dependent manner when administered orally in a rodent model of arthritis.

Strategy to improve the quantitative LC-MS analysis of molecular ions resistant to gas-phase collision induced dissociation: application to disulfide-rich cyclic peptides.

Due to observed collision induced dissociation (CID) fragmentation inefficiency, developing sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) assays for CID resistant compounds is especially challenging. As an alternative to traditional LC-MS/MS, we present here a methodology that preserves the intact analyte ion for quantification by selectively filtering ions while reducing chemical noise. Utilizing a quadrupole-Orbitrap MS, the target ion is selectively isolated while interfering matrix components undergo MS/MS fragmentation by CID, allowing noise-free detection of the analyte's surviving molecular ion. In this manner, CID affords additional selectivity during high resolution accurate mass analysis by elimination of isobaric interferences, a fundamentally different concept than the traditional approach of monitoring a target analyte's unique fragment following CID. This survivor-selected ion monitoring (survivor-SIM) approach has allowed sensitive and specific detection of disulfide-rich cyclic peptides extracted from plasma.

Inhibitors of HIV-1 attachment. Part 9: an assessment of oral prodrug approaches to improve the plasma exposure of a tetrazole-containing derivative.

7-(2H-Tetrazol-5-yl)-1H-indole 3 was found to be a potent inhibitor of HIV-1 attachment but the compound lacked oral bioavailability in rats. The cause of the low exposure was believed to be poor absorption attributed to the acidic nature of the tetrazole moiety and, in an effort to address this liability, three more lipohilic tetrazole analogs, N-acetoxymethyl 4, N-pivaloyloxymethyl 5, and N-methyl 6, were evaluated as potential oral prodrugs in rats. Prodrug 5 was ineffective in improving the plasma concentration of 3 in vivo but compound 4 provided a 15-fold enhancement of the plasma concentration of 3. Most interestingly, oral dosing of analog 6 afforded a substantial increase in the plasma concentration of the parent in rats when compared to dosing of parent. This represents a novel example of a methyl tetrazole that acts as a prodrug for a free NH tetrazole-containing compound.

Inhibitors of HIV-1 attachment. Part 7: indole-7-carboxamides as potent and orally bioavailable antiviral agents.

A series of substituted carboxamides at the indole C7 position of the previously described 4-fluoro-substituted indole HIV-1 attachment inhibitor 1 was synthesized and the SAR delineated. Heteroaryl carboxamide inhibitors that exhibited pM potency in the primary cell-based assay against a pseudotype virus expressing a JRFL envelope were identified. The simple methyl amide analog 4 displayed a promising in vitro profile, with its favorable HLM stability and membrane permeability translating into favorable pharmacokinetic properties in preclinical species.

Inhibitors of HIV-1 attachment. Part 8: the effect of C7-heteroaryl substitution on the potency, and in vitro and in vivo profiles of indole-based inhibitors.

As part of the SAR profiling of the indole-oxoacetic piperazinyl benzamide class of HIV-1 attachment inhibitors, substitution at the C7 position of the lead 4-fluoroindole 2 with various 5- and 6-membered heteroaryl moieties was explored. Highly potent (picomolar) inhibitors of pseudotyped HIV-1 in a primary, cell-based assay were identified and select examples were shown to possess nanomolar inhibitory activity against M- and T-tropic viruses in cell culture. These C7-heteroaryl-indole analogs maintained the ligand efficiency (LE) of 2 and were also lipophilic efficient as measured by LLE and LELP. Pharmacokinetic studies of this class of inhibitor in rats showed that several possessed substantially improved IV clearance and half-lives compared to 2. Oral exposure in the rat correlated with membrane permeability as measured in a Caco-2 assay where the highly permeable 1,2,4-oxadiazole analog 13 exhibited the highest exposure.

Quantification of Linrodostat and its metabolites: Overcoming bioanalytical challenges in support of a discovery Indoleamine 2,3 dioxygenase program.

BMS-986205 (Linrodostat) is a small molecule inhibitor of Indoleamine 2, 3 dioxygenase (IDO) that is currently being evaluated in clinical trials for the oral treatment of advanced cancer. Initially, there were concerns regarding possible toxicity following administration, since BMS-986205 undergoes metabolism to form 4-chloroaniline. However, it was later determined that the downstream metabolites of 4-chloroaniline might be a greater concern. To evaluate the potential toxicity of these metabolites, a sensitive LC-MS/MS analytical method was needed to quantify both the parent compound and multiple metabolites. This presented a challenge since the method required the analysis of multiple analytes while still retaining the analytical sensitivity required to support studies. By utilizing a multi-function analytical method, we were able to quantify the necessary analytes using a complex LC-MS/MS-based method including the application of both negative and positive electrospray ionization.

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.

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. This compound benefits from defined chirality derived from two rotationally stable atropisomeric axes, providing a potent and selective single atropisomer with desirable efficacy and tolerability profiles.

Identification of N-Methyl Nicotinamide and N-Methyl Pyridazine-3-Carboxamide Pseudokinase Domain Ligands as Highly Selective Allosteric Inhibitors of Tyrosine Kinase 2 (TYK2).

As a member of the Janus (JAK) family of nonreceptor tyrosine kinases, TYK2 plays an important role in mediating the signaling of pro-inflammatory cytokines including IL-12, IL-23, and type 1 interferons. The nicotinamide 4, identified by a SPA-based high-throughput screen targeting the TYK2 pseudokinase domain, potently inhibits IL-23 and IFNα signaling in cellular assays. The described work details the optimization of this poorly selective hit (4) to potent and selective molecules such as 47 and 48. The discoveries described herein were critical to the eventual identification of the clinical TYK2 JH2 inhibitor (see following report in this issue). Compound 48 provided robust inhibition in a mouse IL-12-induced IFNγ pharmacodynamic model as well as efficacy in an IL-23 and IL-12-dependent mouse colitis model. These results demonstrate the ability of TYK2 JH2 domain binders to provide a highly selective alternative to conventional TYK2 orthosteric inhibitors.

Highly Selective Inhibition of Tyrosine Kinase 2 (TYK2) for the Treatment of Autoimmune Diseases: Discovery of the Allosteric Inhibitor BMS-986165.

Small molecule JAK inhibitors have emerged as a major therapeutic advancement in treating autoimmune diseases. The discovery of isoform selective JAK inhibitors that traditionally target the catalytically active site of this kinase family has been a formidable challenge. Our strategy to achieve high selectivity for TYK2 relies on targeting the TYK2 pseudokinase (JH2) domain. Herein we report the late stage optimization efforts including a structure-guided design and water displacement strategy that led to the discovery of BMS-986165 (11) as a high affinity JH2 ligand and potent allosteric inhibitor of TYK2. In addition to unprecedented JAK isoform and kinome selectivity, 11 shows excellent pharmacokinetic properties with minimal profiling liabilities and is efficacious in several murine models of autoimmune disease. On the basis of these findings, 11 appears differentiated from all other reported JAK inhibitors and has been advanced as the first pseudokinase-directed therapeutic in clinical development as an oral treatment for autoimmune diseases.

Autoimmune pathways in mice and humans are blocked by pharmacological stabilization of the TYK2 pseudokinase domain.

TYK2 is a nonreceptor tyrosine kinase involved in adaptive and innate immune responses. A deactivating coding variant has previously been shown to prevent receptor-stimulated activation of this kinase and provides high protection from several common autoimmune diseases but without immunodeficiency. An agent that recapitulates the phenotype of this deactivating coding variant may therefore represent an important advancement in the treatment of autoimmunity. BMS-986165 is a potent oral agent that similarly blocks receptor-stimulated activation of TYK2 allosterically and with high selectivity and potency afforded through optimized binding to a regulatory domain of the protein. Signaling and functional responses in human TH17, TH1, B cells, and myeloid cells integral to autoimmunity were blocked by BMS-986165, both in vitro and in vivo in a phase 1 clinical trial. BMS-986165 demonstrated robust efficacy, consistent with blockade of multiple autoimmune pathways, in murine models of lupus nephritis and inflammatory bowel disease, supporting its therapeutic potential for multiple immune-mediated diseases.

Identification of Imidazo[1,2-b]pyridazine Derivatives as Potent, Selective, and Orally Active Tyk2 JH2 Inhibitors.

In sharp contrast to a previously reported series of 6-anilino imidazopyridazine based Tyk2 JH2 ligands, 6-((2-oxo-N1-substituted-1,2-dihydropyridin-3-yl)amino)imidazo[1,2-b]pyridazine analogs were found to display dramatically improved metabolic stability. The N1-substituent on 2-oxo-1,2-dihydropyridine ring can be a variety of alkyl, aryl, and heteroaryl groups, but among them, 2-pyridyl provided much enhanced Caco-2 permeability, attributed to its ability to form intramolecular hydrogen bonds. Further structure-activity relationship studies at the C3 position led to the identification of highly potent and selective Tyk2 JH2 inhibitor 6, which proved to be highly effective in inhibiting IFNγ production in a rat pharmacodynamics model and fully efficacious in a rat adjuvant arthritis model.

Identification and Preclinical Pharmacology of ((1 R,3 S)-1-Amino-3-(( S)-6-(2-methoxyphenethyl)-5,6,7,8-tetrahydronaphthalen-2-yl)cyclopentyl)methanol (BMS-986166): A Differentiated Sphingosine-1-phosphate Receptor 1 (S1P1) Modulator Advanced into Clinical Trials.

Recently, our research group reported the identification of BMS-986104 (2) as a differentiated S1P1 receptor modulator. In comparison to fingolimod (1), a full agonist of S1P1 currently marketed for the treatment of relapse remitting multiple sclerosis (RRMS), 2 offers several potential advantages having demonstrated improved safety multiples in preclinical evaluations against undesired pulmonary and cardiovascular effects. In clinical trials, 2 was found to exhibit a pharmacokinetic half-life ( T1/2) longer than that of 1, as well as a reduced formation of the phosphate metabolite that is required for activity against S1P1. Herein, we describe our efforts to discover highly potent, partial agonists of S1P1 with a shorter T1/2 and increased in vivo phosphate metabolite formation. These efforts culminated in the discovery of BMS-986166 (14a), which was advanced to human clinical evaluation. The pharmacokinetic/pharmacodynamic (PK/PD) relationship as well as pulmonary and cardiovascular safety assessments are discussed. Furthermore, efficacy of 14a in multiple preclinical models of autoimmune diseases are presented.

Discovery of Branebrutinib (BMS-986195): A Strategy for Identifying a Highly Potent and Selective Covalent Inhibitor Providing Rapid in Vivo Inactivation of Bruton's Tyrosine Kinase (BTK).

Bruton's tyrosine kinase (BTK), a non-receptor tyrosine kinase, is a member of the Tec family of kinases and is essential for B cell receptor (BCR) mediated signaling. BTK also plays a critical role in the downstream signaling pathways for the Fcγ receptor in monocytes, the Fcε receptor in granulocytes, and the RANK receptor in osteoclasts. As a result, pharmacological inhibition of BTK is anticipated to provide an effective strategy for the clinical treatment of autoimmune diseases such as rheumatoid arthritis and lupus. This article will outline the evolution of our strategy to identify a covalent, irreversible inhibitor of BTK that has the intrinsic potency, selectivity, and pharmacokinetic properties necessary to provide a rapid rate of inactivation systemically following a very low dose. With excellent in vivo efficacy and a very desirable tolerability profile, 5a (branebrutinib, BMS-986195) has advanced into clinical studies.

The Guinea pig as a preclinical model for demonstrating the efficacy and safety of statins.

Statins, because of their excellent efficacy and manageable safety profile, represent a key component in the current armamentarium for the treatment of hypercholesterolemia. Nonetheless, myopathy remains a safety concern for this important drug class. Cerivastatin was withdrawn from the market for myotoxicity safety concerns. BMS-423526 [{(3R,5S)-7-[4-(4-fluorophenyl)-6,7-dihydro-2-(1-methylethyl)-5H-benzo[6,7]cyclohepta[1,2-b]pyridin-3-yl]-3,5-dihydroxy-heptenoic acid} sodium salt], similar to cerivastatin in potency and lipophilicity, was terminated in early clinical development due to an unacceptable myotoxicity profile. In this report, we describe the guinea pig as a model of statin-induced cholesterol lowering and myotoxicity and show that this model can distinguish statins with unacceptable myotoxicity profiles from statins with acceptable safety profiles. In our guinea pig model, both cerivastatin and BMS-423526 induced myotoxicity at doses near the ED(50) for total cholesterol (TC) lowering in plasma. In contrast, wide differences between myotoxic and TC-lowering doses were established for the currently marketed, more hydrophilic statins, pravastatin, rosuvastatin, and atorvastatin. This in vivo model compared favorably to an in vitro model, which used statin inhibition of cholesterol synthesis in rat hepatocytes and L6 myoblasts as surrogates of potential efficacy and toxicity, respectively. Our conclusion is that the guinea pig is a useful preclinical in vivo model for demonstrating whether a statin is likely to have an acceptable therapeutic safety margin.

Discovery of brivanib alaninate ((S)-((R)-1-(4-(4-fluoro-2-methyl-1H-indol-5-yloxy)-5-methylpyrrolo[2,1-f][1,2,4]triazin-6-yloxy)propan-2-yl)2-aminopropanoate), a novel prodrug of dual vascular endothelial growth factor receptor-2 and fibroblast growth factor receptor-1 kinase inhibitor (BMS-540215).

A series of amino acid ester prodrugs of the dual VEGFR-2/FGFR-1 kinase inhibitor 1 (BMS-540215) was prepared in an effort to improve the aqueous solubility and oral bioavailability of the parent compound. These prodrugs were evaluated for their ability to liberate parent drug 1 in in vitro and in vivo systems. The l-alanine prodrug 8 (also known as brivanib alaninate/BMS-582664) was selected as a development candidate and is presently in phase II clinical trials.

Discovery and preclinical studies of 5-isopropyl-6-(5-methyl-1,3,4-oxadiazol-2-yl)-N-(2-methyl-1H-pyrrolo[2,3-b]pyridin-5-yl)pyrrolo[2,1-f][1,2,4]triazin-4-amine (BMS-645737), an in vivo active potent VEGFR-2 inhibitor.

We report herein a series of substituted N-(1H-pyrrolo[2,3-b]pyridin-5-yl)pyrrolo[2,1-f][1,2,4]triazin-4-amines as inhibitors of vascular endothelial growth factor receptor-2 tyrosine kinase. Through structure-activity relationship studies, biochemical potency, pharmacokinetics, and kinase selectivity were optimized to afford BMS-645737 (13), a compound with good preclinical in vivo activity against human tumor xenograft models.