Design, Synthesis, and In Vitro and In Vivo Evaluation of Cereblon Binding Bruton's Tyrosine Kinase (BTK) Degrader CD79b Targeted Antibody-Drug Conjugates.

Antibody-drug conjugates (ADCs) are an established modality that allow for targeted delivery of a potent molecule, or payload, to a desired site of action. ADCs, wherein the payload is a targeted protein degrader, are an emerging area in the field. Herein we describe our efforts of delivering a Bruton's tyrosine kinase (BTK) bifunctional degrader 1 via a CD79b mAb (monoclonal antibody) where the degrader is linked at the ligase binding portion of the payload via a cleavable linker to the mAb. The resulting CD79b ADCs, 3 and 4, exhibit in vitro degradation and cytotoxicity comparable with that of 1, and ADC 3 can achieve more sustained in vivo degradation than intravenously administered 1 with markedly reduced systemic exposure of the payload.

Structure-activity relationship studies on divalent naphthalene diimide G quadruplex ligands with anticancer and antiparasitic activity.

Naphthalene diimide (NDI) is a central scaffold that has been commonly used in the design of G-quadruplex (G4) ligands. Previous work revealed notable anticancer activity of a disubstituted N-methylpiperazine propyl NDI G4 ligand. Here, we explored structure-activity relationship studies around ligand bis-N,N-2,7-(3-(4-methylpiperazin-1-yl)propyl)-1,4,5,8-naphthalenetetracarboxylic diimide, maintaining the central NDI core whilst modifying the spacer and the nature of the cationic groups. We prepared new disubstituted NDI derivatives of the original compound and examined their in vitro antiproliferative and antiparasitic activity. Several N-methylpiperazine propyl NDIs showed sub-micromolar activity against Trypanosoma brucei and Leishmania major parasites with up to 30 fold selectivity versus MRC-5 cells. The best compound was a dimorpholino NDI with an IC50 of 0.17 µM against T.brucei and 40 fold selectivity versus MRC-5 cells. However, no clear correlation between G4 binding of the new NDI derivatives and antiproliferative or antiparasitic activity was observed, indicating that other mechanisms of action may be responsible for the observed biological activity.

Imide Condensation as a Strategy for the Synthesis of Core-Diversified G-Quadruplex Ligands with Anticancer and Antiparasitic Activity*.

A facile imide coupling strategy for the one-step preparation of G-quadruplex ligands with varied core chemistries is described. The G-quadruplex stabilization of a library of nine compounds was examined using FRET melting experiments, and CD, UV-Vis, fluorescence and NMR titrations, identifying several compounds that were capable of stabilizing G-quadruplex DNA with interesting selectivity profiles. The best G4 ligand was identified as compound 3, which was based on a perylene scaffold and exhibited 40-fold selectivity for a telomeric G-quadruplex over duplex DNA. Surprisingly, a tetra-substituted flexible core, compound 11, also exhibited selective stabilization of G4 DNA over duplex DNA. The anticancer and antiparasitic activity of the library was also examined, with the lead compound 3 exhibiting nanomolar inhibition of Trypanosoma brucei with 78-fold selectivity over MRC5 cells. The cellular localization of this compound was also studied via fluorescence microscopy. We found that uptake was time dependant, with localization outside the nucleus and kinetoplast that could be due to strong fluorescence quenching in the presence of small amounts of DNA.

Modular synthesis and modification of novel bifunctional dendrons.

Herein, we report the design and synthesis of two novel bifunctional dendrons bearing multiple amine termini at the periphery and an azide at the focal point. Copper-catalyzed alkyne-azide cycloaddition enabled modular dendritic scaffold assembly resulting in a first generation dendron carrying six amines and a second generation dendron carrying eighteen amines. Peripheral amines were labeled with multiple copies of a metal isotope, whereas the azide functionality at the focal point was employed in conjugation to a single anti-human CD4 antibody. We demonstrated that the highly monomeric first generation dendron-antibody conjugate selectively detected CD4+ T cells in the PMBC culture.

Divalent Naphthalene Diimide Ligands Display High Selectivity for the Human Telomeric G-quadruplex in K+ Buffer.

Selective G-quadruplex ligands offer great promise for the development of anti-cancer therapies. A novel series of divalent cationic naphthalene diimide ligands that selectively bind to the hybrid form of the human telomeric G-quadruplex in K+ buffer are described herein. We demonstrate that an imidazolium-bearing mannoside-conjugate is the most selective ligand to date for this quadruplex against several other quadruplex and duplex structures. We also show that a similarly selective methylpiperazine-bearing ligand was more toxic to HeLa cancer cells than doxorubicin, whilst exhibiting three times less toxicity towards fetal lung fibroblasts WI-38.

Discovery of novel pyrrolidineoxy-substituted heteroaromatics as potent and selective PI3K delta inhibitors with improved physicochemical properties.

In the recent years, PI3Kδ has emerged as a promising target for the treatment of B- and T-cell mediated inflammatory diseases. We present a cellular assay activity analysis for our previously reported 4,6-diaryl quinazoline PI3Kδ inhibitor series that suggests an optimal logP range between 2 and 3. We discovered novel analogues in this lipophilicity space that feature a chiral pyrrolidineoxy-group as a replacement for the position-4 aromatic ring of 4,6-diaryl quinazolines. These Fsp3 enriched derivatives retain potency and selectivity towards PI3Kδ. Compared to 4,6-diaryl quinazolines, their permeability profile is improved and molecular weight as well as PSA are reduced. These modifications offer additional possibilities for derivative generation in a favorable physicochemical property space and thus increase the chances to identify a clinical candidate.

Transient targeting of phosphoinositide 3-kinase acts as a roadblock in mast cells' route to allergy.

BACKGROUND: Tissue mast cell numbers are dynamically regulated by recruitment of progenitors from the vasculature. It is unclear whether progenitors are recruited during allergic sensitization and whether recruitment promotes allergic responses. OBJECTIVE: We sought to (1) determine the effect of mast cell recruitment on acute allergic responses and (2) to define the role of phosphoinositide 3-kinase (PI3K) isoforms in sequential steps to allergic responses. METHODS: Gene-targeted mice for PI3Kγ or PI3Kδ or mice treated with isoform-specific PI3K inhibitors (a novel PI3Kγ-specific inhibitor [NVS-PI3-4] and the PI3Kδ inhibitor IC87114) were used to monitor IgE-mediated mast cell recruitment, migration, adhesion by means of intravital microscopy, degranulation, TNF-α release, and subsequent endothelial cell activation in vivo or in bone marrow-derived mast cells. RESULTS: Functional PI3Kγ, but not PI3Kδ, was crucial for mast cell accumulation in IgE-challenged skin, TNF-α release from IgE/antigen-stimulated mast cells, and mast cell/endothelial interactions and chemotaxis. PI3Kγ-deficient bone marrow-derived mast cells did not adhere to the endothelium in TNF-α-treated cremaster muscle, whereas PI3Kδ was not required. Depletion of TNF-α blocked IgE-induced mast cell recruitment, which links tissue mast cell-derived cytokine release to endothelial activation and mast cell recruitment. Interference with mast cell recruitment protected against anaphylaxis and was superior to blockage of tissue mast cell degranulation. CONCLUSIONS: Interference with mast cell recruitment to exacerbated tissues provides a novel strategy to alleviate allergic reactions and surpassed attenuation of tissue mast cell degranulation. This results in prolonged drug action and allows for reduction of drug doses required to block anaphylaxis, an important feature for drugs targeting inflammatory disease in general.

Discovery of New Binders for DCAF1, an Emerging Ligase Target in the Targeted Protein Degradation Field.

In this study, we describe the rapid identification of potent binders for the WD40 repeat domain (WDR) of DCAF1. This was achieved by two rounds of iterative focused screening of a small set of compounds selected on the basis of internal WDR domain knowledge followed by hit expansion. Subsequent structure-based design led to nanomolar potency binders with a clear exit vector enabling DCAF1-based bifunctional degrader exploration.

Discovery and Toxicological Profiling of Aminopyridines as Orally Bioavailable Selective Inhibitors of PI3-Kinase γ.

Using a novel physiologically relevant in vitro human whole blood neutrophil shape change assay, an aminopyrazine series of selective PI3Kγ inhibitors was identified and prioritized for further optimization. Severe solubility limitations associated with the series leading to low oral bioavailability and poor exposures, especially at higher doses, were overcome by moving to an aminopyridine core. Compound 33, with the optimal balance of on-target activity, selectivity, and pharmacokinetic parameters, progressed into in vivo studies and demonstrated good efficacy (10 mg/kg) in a rat model of airway inflammation. Sufficient exposures were achieved at high doses to support toxicological studies, where unexpected inflammatory cell infiltrates in cardiovascular tissue prevented further compound development.

Real-Time Imaging and Quantification of Peptide Uptake in Vitro and in Vivo.

Peptides constitute an important class of drugs for the treatment of multiple metabolic, oncological, and neurodegenerative diseases, and several hundred novel therapeutic peptides are currently in the preclinical and clinical stages of development. However, many leads fail to advance clinically because of poor cellular membrane and tissue permeability. Therefore, assessment of the ability of a peptide to cross cellular membranes is critical when developing novel peptide-based therapeutics. Current methods to assess peptide cellular permeability are limited by multiple factors, such as the need to introduce rather large modifications (e.g., fluorescent dyes) that require complex chemical reactions as well as an inability to provide kinetic information on the internalization of a compound or distinguish between internalized and membrane-bound compounds. In addition, many of these methods are based on end point assays and require multiple sample manipulation steps. Herein, we report a novel "Split Luciferin Peptide" (SLP) assay that enables the real-time noninvasive imaging and quantification of peptide uptake both in vitro and in vivo using a very sensitive bioluminescence readout. This method is based on a straightforward, stable chemical modification of the peptide of interest with a d-cysteine tag that preserves the overall peptidic character of the original molecule. This method can be easily adapted for screening peptide libraries and can thus become an important tool for preclinical peptide drug development.

Discovery of CDZ173 (Leniolisib), Representing a Structurally Novel Class of PI3K Delta-Selective Inhibitors.

The predominant expression of phosphoinositide 3-kinase δ (PI3Kδ) in leukocytes and its critical role in B and T cell functions led to the hypothesis that selective inhibitors of this isoform would have potential as therapeutics for the treatment of allergic and inflammatory disease. Targeting specifically PI3Kδ should avoid potential side effects associated with the ubiquitously expressed PI3Kα and ß isoforms. We disclose how morphing the heterocyclic core of previously discovered 4,6-diaryl quinazolines to a significantly less lipophilic 5,6,7,8-tetrahydropyrido[4,3-d]pyrimidine, followed by replacement of one of the phenyl groups with a pyrrolidine-3-amine, led to a compound series with an optimal on-target profile and good ADME properties. A final lipophilicity adjustment led to the discovery of CDZ173 (leniolisib), a potent PI3Kδ selective inhibitor with suitable properties and efficacy for clinical development as an anti-inflammatory therapeutic. In vitro, CDZ173 inhibits a large spectrum of immune cell functions, as demonstrated in B and T cells, neutrophils, monocytes, basophils, plasmocytoid dendritic cells, and mast cells. In vivo, CDZ173 inhibits B cell activation in rats and monkeys in a concentration- and time-dependent manner. After prophylactic or therapeutic dosing, CDZ173 potently inhibited antigen-specific antibody production and reduced disease symptoms in a rat collagen-induced arthritis model. Structurally, CDZ173 differs significantly from the first generation of PI3Kδ and PI3Kγδ-selective clinical compounds. Therefore, CDZ173 could differentiate by a more favorable safety profile. CDZ173 is currently in clinical studies in patients suffering from primary Sjögren's syndrome and in APDS/PASLI, a disease caused by gain-of-function mutations of PI3Kδ.

Discovery and Pharmacological Characterization of Novel Quinazoline-Based PI3K Delta-Selective Inhibitors.

Inhibition of the lipid kinase PI3Kδ is a promising principle to treat B and T cell driven inflammatory diseases. Using a scaffold deconstruction-reconstruction strategy, we identified 4-aryl quinazolines that were optimized into potent PI3Kδ isoform selective analogues with good pharmacokinetic properties. With compound 11, we illustrate that biochemical PI3Kδ inhibition translates into modulation of isoform-dependent immune cell function (human, rat, and mouse). After oral administration of compound 11 to rats, proximal PD markers are inhibited, and dose-dependent efficacy in a mechanistic plaque forming cell assay could be demonstrated.

Hematopoiesis and RAS-driven myeloid leukemia differentially require PI3K isoform p110α.

The genes encoding RAS family members are frequently mutated in juvenile myelomonocytic leukemia (JMML) and acute myeloid leukemia (AML). RAS proteins are difficult to target pharmacologically; therefore, targeting the downstream PI3K and RAF/MEK/ERK pathways represents a promising approach to treat RAS-addicted tumors. The p110α isoform of PI3K (encoded by Pik3ca) is an essential effector of oncogenic KRAS in murine lung tumors, but it is unknown whether p110α contributes to leukemia. To specifically examine the role of p110α in murine hematopoiesis and in leukemia, we conditionally deleted p110α in HSCs using the Cre-loxP system. Postnatal deletion of p110α resulted in mild anemia without affecting HSC self-renewal; however, deletion of p110α in mice with KRASG12D-associated JMML markedly delayed their death. Furthermore, the p110α-selective inhibitor BYL719 inhibited growth factor-independent KRASG12D BM colony formation and sensitized cells to a low dose of the MEK inhibitor MEK162. Furthermore, combined inhibition of p110α and MEK effectively reduced proliferation of RAS-mutated AML cell lines and disease in an AML murine xenograft model. Together, our data indicate that RAS-mutated myeloid leukemias are dependent on the PI3K isoform p110α, and combined pharmacologic inhibition of p110α and MEK could be an effective therapeutic strategy for JMML and AML.

Novel lactam NK1 antagonists with anti-emetic activity.

A series of 4,4-disubstituted cyclohexylamine NK(1) antagonists containing a lactam ring is described. The compounds are brain penetrant and activity is demonstrated in a ferret emesis model.

1H NMR study of the conformation and absolute stereochemistry of two spirocyclic NK-1 antagonists.

The binding affinities at the human NK-1 receptor of two spirocyclic compounds were found to be similar despite being epimeric at a key stereocentre. This unexpected result prompted a thorough investigation of the solution conformations of the two compounds. This revealed that a conformational switch in the tetrahydrofuran ring enabled the C-3-aryl group to be equatorial in both cases, leading to a similar juxtaposition of the aryl rings.

4,4-Disubstituted cyclohexylamine NK(1) receptor antagonists I.

A series of novel 4,4-disubstituted cyclohexylamine based NK(1) antagonists is described. The effect of changes to the C(1)-C(4) relative stereochemistry on the cyclohexane ring and replacements for the flexible linker are discussed, leading to the identification of compounds with high affinity and good in vivo duration of action.

Functional properties of the high-affinity TRPV1 (VR1) vanilloid receptor antagonist (4-hydroxy-5-iodo-3-methoxyphenylacetate ester) iodo-resiniferatoxin.

We have synthesized iodinated resiniferatoxin bearing a 4-hydroxy-5-iodo-3-methoxyphenylacetate ester (I-RTX) and have characterized its activity on rat and human TRPV1 (VR1) receptors, as well as in behavioral assays of nociception. In whole cell patch-clamp recordings from transfected cells the functional activity of I-RTX was determined. Currents activated by capsaicin exhibited characteristic outward rectification and were antagonized by capsazepine and I-RTX. On rat TRPV1 the affinity of I-RTX was 800-fold higher than that of capsazepine (IC50 = 0.7 and 562 nM, respectively) and 10-fold higher on rat versus human receptors (IC50 = 0.7 and 5.4 nM, respectively). The same difference was observed when comparing the inhibition of [3H]RTX binding to rat and human TRPV1 membranes for both RTX and I-RTX. Additional pharmacological differences were revealed using protons as the stimulus. Under these conditions capsazepine only partly blocked currents through rat TRPV1 receptors (by 70 to 80% block), yet was a full antagonist on human receptors. In contrast, I-RTX completely blocked proton-induced currents in both species and that activated by noxious heat. I-RTX also blocked capsaicin-induced firing of C-fibers in a rat in vitro skin-nerve assay. Despite this activity and the high affinity of I-RTX for rat TRPV1, only capsazepine proved to be an effective antagonist of capsaicin-induced paw flinching in rats. Thus, although I-RTX has limited utility for in vivo behavioral studies it is a high-affinity TRPV1 receptor antagonist that will be useful to characterize the functional properties of cloned and native vanilloid receptor subtypes in vitro.

4,4-Disubstituted cyclohexylamine NK(1) receptor antagonists II.

A series of novel 4,4-disubstituted cyclohexylamines as NK(1) receptor antagonists is described: modifications to the amine moiety retain NK(1) receptor binding affinity whilst disrupting I(Kr) affinity.