In vitro metabolism of a novel JNK inhibitor tanzisertib: interspecies differences in oxido-reduction and characterization of enzymes involved in metabolism.

1. In vitro metabolism of Tanzisertib [(1S,4R)-4-(9-((S)tetrahydrofuran-3-yl)-8-(2,4,6-trifluorophenylamino)-9H-purin-2-ylamino) cyclohexanol], a potent, selective c-Jun amino-terminal kinase (JNK) inhibitor, was investigated in mouse, rat, rabbit, dog, monkey and human hepatocytes over 4 h. The extent of metabolism of [(14)C]tanzisertib was variable, with <10% metabolized in dog and human, <20% metabolized in rabbit and monkey and >75% metabolized in rat and mouse. Primary metabolic pathways in human and dog hepatocytes, were direct glucuronidation and oxidation of cyclohexanol to a keto metabolite, which was subsequently reduced to parent or cis-isomer, followed by glucuronidation. Rat and mouse produced oxidative metabolites and cis-isomer, including direct glucuronides and sulfates of tanzisertib and cis-isomer. 2. Enzymology of oxido-reductive pathways revealed that human aldo-keto reductases AKR1C1, 1C2, 1C3 and 1C4 were responsible for oxido-reduction of tanzisertib, CC-418424 and keto tanzisertib. Characterizations of enzyme kinetics revealed that AKR1C4 had a high affinity for reduction of keto tanzisertib to tanzisertib compared to other isoforms. These results demonstrate unique stereoselectivity of the reductive properties documented by human AKR1C enzymes for the same substrate. 3. Characterization of UGT isoenzymes in glucuronidation of tanzisertib and CC-418424 revealed that, tanzisertib glucuronide was catalyzed by: UGT1A1, 1A4, 1A10 and 2B4, while CC-418424 glucuronidation was catalyzed by UGT2B4 and 2B7.

Isosteric analogs of lenalidomide and pomalidomide: synthesis and biological activity.

A series of analogs of the immunomodulary drugs lenalidomide (1) and pomalidomide (2), in which the amino group is replaced with various isosteres, was prepared and assayed for immunomodulatory activity and activity against cancer cell lines. The 4-methyl and 4-chloro analogs 4 and 15, respectively, displayed potent inhibition of tumor necrosis factor-α (TNF-α) in LPS-stimulated hPBMC, potent stimulation of IL-2 in a human T cell co-stimulation assay, and anti-proliferative activity against the Namalwa lymphoma cell line. Both of these analogs displayed oral bioavailability in rat.

Discovery and optimization of thieno[2,3-d]pyrimidines as B-Raf inhibitors.

The serine/threonine specific protein kinase B-Raf is part of the MAPK pathway and is an interesting oncology target. We have identified thieno[2,3-d]pyrimidines as a core scaffold of small molecule B-Raf inhibitors. The SAR of analogs in this series will be described.

Aminopurine based JNK inhibitors for the prevention of ischemia reperfusion injury.

In this Letter we describe the optimization of an aminopurine lead (1) with modest potency and poor overall kinase selectivity which led to the identification of a series of potent, selective JNK inhibitors. Improvement in kinase selectivity was enabled by introduction of an aliphatic side chain at the C-2 position. CC-359 (2) was selected as a potential clinical candidate for diseases manifested by ischemia reperfusion injury.

Discovery of CC-930, an orally active anti-fibrotic JNK inhibitor.

In this Letter we describe the discovery of potent, selective, and orally active aminopurine JNK inhibitors. Improving the physico-chemical properties as well as increasing the potency and selectivity of a subseries with rat plasma exposure, led to the identification of four structurally diverse inhibitors. Differentiation based on PK profiles in multiple species as well as activity in a chronic efficacy model led to the identification of 1 (CC-930) as a development candidate, which is currently in Phase II clinical trial for IPF.

Discovery of the c-Jun N-Terminal Kinase Inhibitor CC-90001.

As a result of emerging biological data suggesting that within the c-Jun N-terminal kinase (JNK) family, JNK1 and not JNK2 or JNK3 may be primarily responsible for fibrosis pathology, we sought to identify JNK inhibitors with an increased JNK1 bias relative to our previous clinical compound tanzisertib (CC-930). This manuscript reports the synthesis and structure-activity relationship (SAR) studies for a novel series of JNK inhibitors demonstrating an increased JNK1 bias. SAR optimization on a series of 2,4-dialkylamino-pyrimidine-5-carboxamides resulted in the identification of compounds possessing low nanomolar JNK inhibitory potency, overall kinome selectivity, and the ability to inhibit cellular phosphorylation of the direct JNK substrate c-Jun. Optimization of physicochemical properties in this series resulted in compounds that demonstrated excellent systemic exposure following oral dosing, enabling in vivo efficacy studies and the selection of a candidate for clinical development, CC-90001, which is currently in clinical trials (Phase II) in patients with idiopathic pulmonary fibrosis (NCT03142191).

Discovery of CRBN E3 Ligase Modulator CC-92480 for the Treatment of Relapsed and Refractory Multiple Myeloma.

Many patients with multiple myeloma (MM) initially respond to treatment with modern combination regimens including immunomodulatory agents (lenalidomide and pomalidomide) and proteasome inhibitors. However, some patients lack an initial response to therapy (i.e., are refractory), and although the mean survival of MM patients has more than doubled in recent years, most patients will eventually relapse. To address this need, we explored the potential of novel cereblon E3 ligase modulators (CELMoDs) for the treatment of patients with relapsed or refractory multiple myeloma (RRMM). We found that optimization beyond potency of degradation, including degradation efficiency and kinetics, could provide efficacy in a lenalidomide-resistant setting. Guided by both phenotypic and protein degradation data, we describe a series of CELMoDs for the treatment of RRMM, culminating in the discovery of CC-92480, a novel protein degrader and the first CELMoD to enter clinical development that was specifically designed for efficient and rapid protein degradation kinetics.

A Systematic Analysis of Physicochemical and ADME Properties of All Small Molecule Kinase Inhibitors Approved by US FDA from January 2001 to October 2015.

BACKGROUND: During lead identification and optimization, the advancement criteria may be driven based on scientific principles, prior experiences, and/or by examining the path paved by approved drugs. However, accessing the discovery data on physicochemical and ADME properties of the approved kinase inhibitors is a monumental task as these are either scattered in the literature or have not been published. OBJECTIVE: Our goals were: 1) To compile the relevant data on all kinase inhibitors approved prior to 2016 for easy access by the biopharmaceutical community, 2) To provide a retrospective analysis to highlight trends and attributes which may have contributed to the "developability" of these drugs, and 3) To ignite focused debates on what constitutes "actionable", "nice-to-have", and unnecessary data. Such debates bring about more clarity on stage appropriateness of different types of information and prevent confusion due to abundance of unnecessary data, leading to more efficient and less costly drug discovery programs. METHODS: A careful and thorough analysis of different bodies of data such as published manuscripts, and available regulatory documents were employed. RESULTS: We were able to assemble a large body of data on the first thirty kinase inhibitors approved by US FDA since 2001. CONCLUSION: In conclusion, we have compiled physicochemical and ADME data on the first 30 approved kinase inhibitors and provided our retrospective analysis, which we hope is helpful in constructing advancement criteria in discovery programs. The examination of this data provides an opportunity to develop an opinion on data prioritization and stage appropriateness of assays.

Implementation of a novel ultra fast metabolic stability analysis method using exact mass TOF-MS.

AIM: Increasing numbers of compounds requiring stability data means highly optimized methods capable of rapid turnaround are desirable during early discovery. Materials and methods/results: An advanced, generic analytical workflow for metabolic stability has been developed that utilizing ballistic gradient LC (sub 1 min run times), exact mass TOF-MS (Waters Xevo-G2-XS Q-TOF) and automated data processing (Waters UNIFI software) allowed for rapid integration and interpretation of all data produced, eliminating the need for method development and manual processing. We can analyze and process 96 compounds across two species in quadruplicate in a 24-h period with no method development. CONCLUSION: An advanced bioanalytical workflow has increased our capacity threefold and reduced our instrument/processing needs threefold.

The Discovery of a Dual TTK Protein Kinase/CDC2-Like Kinase (CLK2) Inhibitor for the Treatment of Triple Negative Breast Cancer Initiated from a Phenotypic Screen.

Triple negative breast cancer (TNBC) remains a serious unmet medical need with discouragingly high relapse rates. We report here the synthesis and structure-activity relationship (SAR) of a novel series of 2,4,5-trisubstituted-7H-pyrrolo[2,3-d]pyrimidines with potent activity against TNBC tumor cell lines. These compounds were discovered from a TNBC phenotypic screen and possess a unique dual inhibition profile targeting TTK (mitotic exit) and CLK2 (mRNA splicing). Design and optimization, driven with a TNBC tumor cell assay, identified potent and selective compounds with favorable in vitro and in vivo activity profiles and good iv PK properties. This cell-based driven SAR produced compounds with strong single agent in vivo efficacy in multiple TNBC xenograft models without significant body weight loss. These data supported the nomination of CC-671 into IND-enabling studies as a single agent TNBC therapy.

Efficiency in Drug Discovery: Liver S9 Fraction Assay As a Screen for Metabolic Stability.

BACKGROUND: A rapid and comprehensive metabolic stability screen at the top of a drug discovery flow chart serves as an effective gate in eliminating low value compounds. This imparts a significant level of efficiency and saves valuable resources. While microsomes are amenable to high throughput automation and are cost effective, their enzymatic make-up is limited to that which is contained in endoplasmic reticulum, thereby informing only on Phase I metabolism. Lack of Phase II metabolism data can become a potential liability later in the process, adversely affecting discovery projects' timelines and budget. Hepatocytes offer a full complement of metabolic enzymes and retain their cellular compartments, better representing liver metabolic function. However, hepatocyte screens are relatively expensive, labor intensive, and not easily automatable. Liver S9 fractions include Phase I and II metabolic enzymes, are relatively inexpensive, easy to use, and amenable to automation, making them a more appropriate screening system. We compare the data from the three systems and present the results. RESULTS: Liver S9 and hepatocyte stability assays binned into the same category 70-84% of the time. Microsome and hepatocyte data were in agreement 73-82% of the time. The true rate for stability versus plasma clearance was 45% for hepatocytes and 43% for S9. CONCLUSION: In our opinion, replacing liver microsome and hepatocyte assays with S9 assay for high throughput metabolic screening purposes provides the combined benefit of comprehensive and high quality data at a reasonable expense for drug discovery programs.

Boston Society's 11th Annual Applied Pharmaceutical Analysis conference.

Boston Society's 11th Annual Applied Pharmaceutical Analysis conference, Hyatt Regency Hotel, Cambridge, MA, USA, 14-16 September 2015 The Boston Society's 11th Annual Applied Pharmaceutical Analysis (APA) conference took place at the Hyatt Regency hotel in Cambridge, MA, on 14-16 September 2015. The 3-day conference affords pharmaceutical professionals, academic researchers and industry regulators the opportunity to collectively participate in meaningful and relevant discussions impacting the areas of pharmaceutical drug development. The APA conference was organized in three workshops encompassing the disciplines of regulated bioanalysis, discovery bioanalysis (encompassing new and emerging technologies) and biotransformation. The conference included a short course titled 'Bioanalytical considerations for the clinical development of antibody-drug conjugates (ADCs)', an engaging poster session, several panel and round table discussions and over 50 diverse talks from leading industry and academic scientists.

Formation of A Novel Purine Metabolite through CYP3A4 Bioactivation and Glutathione Conjugation.

BACKGROUND: The study of novel sites of metabolism is important in understanding new mechanisms of biotransformation of a particular moiety by metabolic enzymes. This information is valuable in designing metabolically-stable compounds with drug-like properties. It may also provide insights into the existence of active and reactive metabolites. METHODS: We utilized small scale incubations to generate adequate amounts of the metabolite of interest. After purification, LC-MS/MS and Proton Nuclear Magnetic Resonance (1H-NMR) were utilized to unequivocally assign the novel site of glutathione conjugation on the purine ring system. RESULTS: A proposed novel site of glutathione conjugation was investigated on a diaminopurine-containing molecule. It was demonstrated that the formation of the glutathione conjugate at the C-6 position of the purine ring system was due to the bioactivation of the compound to a di-imine intermediate by CYP3A4, followed by the nucleophilic addition of glutathione. CONCLUSION: S-glutathionylation at C-6 position of a purine was proven unequivocally. This previously unreported mechanism constitutes a novel biotransformation for purines.

Photon-impenetrable, electron-permeable: the carbon nanotube forest as a medium for multiphoton thermal-photoemission.

Combining the photoelectric and thermionic mechanisms to generate free electrons has been of great interest since the early days of quantum physics as exemplified by the Fowler-DuBridge theory, and recently proposed for highly efficient solar conversion. We present experimental evidence of this combined effect over the entire range spanning room-temperature photoemission to thermionic emission. Remarkably, the optical stimulus alone is responsible for both heating and photoemission at the same time. Moreover, the current depends on optical intensity quadratically, indicating two-photon photoemission, for intensities of ca. 1-50 W/cm(2), which are orders of magnitude below the intensities required for two-photon photoemission from bulk metals. This surprising behavior appears to be enabled by the internal nanostructure of the carbon nanotube forest, which captures photons effectively, yet allows electrons to escape easily.

CC-223, a Potent and Selective Inhibitor of mTOR Kinase: In Vitro and In Vivo Characterization.

mTOR is a serine/threonine kinase that regulates cell growth, metabolism, proliferation, and survival. mTOR complex-1 (mTORC1) and mTOR complex-2 (mTORC2) are critical mediators of the PI3K-AKT pathway, which is frequently mutated in many cancers, leading to hyperactivation of mTOR signaling. Although rapamycin analogues, allosteric inhibitors that target only the mTORC1 complex, have shown some clinical activity, it is hypothesized that mTOR kinase inhibitors, blocking both mTORC1 and mTORC2 signaling, will have expanded therapeutic potential. Here, we describe the preclinical characterization of CC-223. CC-223 is a potent, selective, and orally bioavailable inhibitor of mTOR kinase, demonstrating inhibition of mTORC1 (pS6RP and p4EBP1) and mTORC2 [pAKT(S473)] in cellular systems. Growth inhibitory activity was demonstrated in hematologic and solid tumor cell lines. mTOR kinase inhibition in cells, by CC-223, resulted in more complete inhibition of the mTOR pathway biomarkers and improved antiproliferative activity as compared with rapamycin. Growth inhibitory activity and apoptosis was demonstrated in a panel of hematologic cancer cell lines. Correlative analysis revealed that IRF4 expression level associates with resistance, whereas mTOR pathway activation seems to associate with sensitivity. Treatment with CC-223 afforded in vivo tumor biomarker inhibition in tumor-bearing mice, after a single oral dose. CC-223 exhibited dose-dependent tumor growth inhibition in multiple solid tumor xenografts. Significant inhibition of mTOR pathway markers pS6RP and pAKT in CC-223-treated tumors suggests that the observed antitumor activity of CC-223 was mediated through inhibition of both mTORC1 and mTORC2. CC-223 is currently in phase I clinical trials.

Discovery of mammalian target of rapamycin (mTOR) kinase inhibitor CC-223.

We report here the synthesis and structure-activity relationship (SAR) of a novel series of mammalian target of rapamycin (mTOR) kinase inhibitors. A series of 4,6- or 1,7-disubstituted-3,4-dihydropyrazino[2,3-b]pyrazine-2(1H)-ones were optimized for in vivo efficacy. These efforts resulted in the identification of compounds with excellent mTOR kinase inhibitory potency, with exquisite kinase selectivity over the related lipid kinase PI3K. The improved PK properties of this series allowed for exploration of in vivo efficacy and ultimately the selection of CC-223 for clinical development.

Optimization of a Series of Triazole Containing Mammalian Target of Rapamycin (mTOR) Kinase Inhibitors and the Discovery of CC-115.

We report here the synthesis and structure-activity relationship (SAR) of a novel series of triazole containing mammalian target of rapamycin (mTOR) kinase inhibitors. SAR studies examining the potency, selectivity, and PK parameters for a series of triazole containing 4,6- or 1,7-disubstituted-3,4-dihydropyrazino[2,3-b]pyrazine-2(1H)-ones resulted in the identification of triazole containing mTOR kinase inhibitors with improved PK properties. Potent compounds from this series were found to block both mTORC1(pS6) and mTORC2(pAktS473) signaling in PC-3 cancer cells, in vitro and in vivo. When assessed in efficacy models, analogs exhibited dose-dependent efficacy in tumor xenograft models. This work resulted in the selection of CC-115 for clinical development.

Proposing advancement criteria for efficient DMPK triage of new chemical entities.

With the goal of refining our discovery DMPK workflow, we conducted a retrospective analysis on internal Celgene compounds by calculating the physicochemical properties and gathering data from several assays including solubility, rat and human liver S9 stability, Caco-2 permeability, and rat intravenous (iv.) and oral pharmacokinetics. Our analysis identified plasma clearance to be most statistically relevant for prediction of oral exposure. In rat, compounds with rat S9 stability of ≥70% at 60 min and a plasma clearance of ≤43 ml/min/kg had the greatest chance of achieving oral exposures above 3 µM.h. Compounds with the dual advantage of plasma clearance ≤43 ml/min/kg and Caco-2 permeability ≥8 × 10(-6) cm/s or efflux ratio ≤8 were highly likely to achieve those oral exposures. Implementation of these criteria leads to a significant increase in efficiency, good pharmacokinetic properties, cost savings and a reduction in the use of animals.

A proposed screening paradigm for discovery of covalent inhibitor drugs.

The in vitro and in vivo preclinical ADME properties of 10 clinically late stage or marketed covalent inhibitors were evaluated in order to define advancement criteria for discovery of future drugs in this arena. Our studies revealed the following: After incubating with S9 fractions for 30 minutes, the rat and human in vitro stability for these compounds ranged from 1% to 100%. The blood stability ranged from 30% to 100%. There was a broad range of CYP inhibition with prevalence for time-dependent inhibition of at least one enzyme. The Caco-2 permeability (A→B) ranged from negligible (0.6 x 10(-6) cm/s) to highly permeable (31 x 10(-6) cm/s) and the efflux ratio also varied widely (0.2-30). Most of the compounds were highly protein bound in both rat and human with binding ≥ 90%. Rat plasma clearance for the 10 compounds ranged from slow (11 mL/min/kg) to very rapid (350 mL/min/kg). The Vss ranged from low (0.67 L/kg) to very high (115 L/kg). MRT's also ranged from short (0.5 hr) to long (7.4 hr). The oral exposures also showed a very broad range with CMax's ranging from 0.01-77 µM and exposure levels ranging from 0.03-106 µM.hr. In conclusion, the wide range in in vitro and in vivo ADME data makes these particular ADME assays non-discriminatory in the selection of promising compounds. In our opinion, non-traditional assays such as target mass modification, target confirmation by amino acid sequencing, cellular target occupancy, and target turnover rate data in combination with the pharmacokinetic profiles are the critical considerations for progression of irreversible compounds in early discovery.

Small molecule kinase inhibitors approved by the FDA from 2000 to 2011: a systematic review of preclinical ADME data.

INTRODUCTION: Fourteen drugs targeting protein kinases have been approved by the United States Food and Drug Administration (FDA) between the years 2000 and 2011. While the mechanisms of action and clinical data have been reported, the preclinical absorption, distribution, metabolism and elimination (ADME) data on these compounds are not readily available. AREAS COVERED: This review summarizes the structural, physicochemical and preclinical ADME properties of the 14 kinase inhibitors. For this purpose, the authors identified common preclinical ADME features of the majority of these inhibitors. EXPERT OPINION: The authors believe engendering a combination of the following attributes will greatly improve one's chance of discovering kinase inhibitors with a successful development path: i) Lipinski Rule of Five and Veber's rules, ii) most basic pKa values ≤ 9, iii) low to moderate clearance values for each species and iv) moderate to high Caco-2 permeability. Furthermore, they recommend primarily focusing on in vitro hepatic stability and rodent in vivo disposition properties at early screening stage and using Caco-2 permeability/efflux ratio to salvage compounds with rapid rodent plasma clearance. In vitro drug-drug interaction data should be evaluated in the context of the dosing route, dosing regimen, elimination pathways, enzyme phenotyping profile, efficacious exposure and the target disease.