Discovery of novel N-acylpyrazoles as potent and selective thrombin inhibitors.

Direct oral anticoagulants (DOACs), which includes thrombin and factor Xa inhibitors, have emerged as the preferred therapeutics for thrombotic disorders, penetrating a market previously dominated by warfarin and heparin. This article describes the discovery and profiling of a novel series of N-acylpyrazoles, which act as selective, covalent, reversible, non-competitive inhibitors of thrombin. We describe in vitro stability issues associated with this chemotype and, importantly, demonstrate that N-acylpyrazoles successfully act in vivo as anticoagulants in basic thrombotic animal models. Crucially, this anticoagulant nature is unaccompanied by the higher bleeding risk profile that has become an undesirable characteristic of the DTIs and factor Xa inhibitors. We propose that the N-acylpyrazole chemotype shows intriguing promise as next-generation oral anticoagulants.

Discovery of MDV6058 (PF-06952229), a selective and potent TGFßR1 inhibitor: Design, synthesis and optimization.

Compound 1 is a potent TGF-ß receptor type-1 (TGFßR1 or ALK5) inhibitor but is metabolically unstable. A solvent-exposed part of this molecule was used to analogue and modulate cell activity, liver microsome stability and mouse pharmacokinetics. The evolution of SAR that led to the selection of 2 (MDV6058 / PF-06952229) as a preclinical lead compound is described.

Design, synthesis and optimization of bis-amide derivatives as CSF1R inhibitors.

Signaling via the receptor tyrosine kinase CSF1R is thought to play an important role in recruitment and differentiation of tumor-associated macrophages (TAMs). TAMs play pro-tumorigenic roles, including the suppression of anti-tumor immune response, promotion of angiogenesis and tumor cell metastasis. Because of the role of this signaling pathway in the tumor microenvironment, several small molecule CSF1R kinase inhibitors are undergoing clinical evaluation for cancer therapy, either as a single agent or in combination with other cancer therapies, including immune checkpoint inhibitors. Herein we describe our lead optimization effort that resulted in the identification of a potent, cellular active and orally bioavailable bis-amide CSF1R inhibitor. Docking and biochemical analysis allowed the removal of a metabolically labile and poorly permeable methyl piperazine group from an early lead compound. Optimization led to improved metabolic stability and Caco2 permeability, which in turn resulted in good oral bioavailability in mice.

Dual Inhibition of Bruton's Tyrosine Kinase and Phosphoinositide-3-Kinase p110δ as a Therapeutic Approach to Treat Non-Hodgkin's B Cell Malignancies.

Although new targeted therapies, such as ibrutinib and idelalisib, have made a large impact on non-Hodgkin's lymphoma (NHL) patients, the disease is often fatal because patients are initially resistant to these targeted therapies, or because they eventually develop resistance. New drugs and treatments are necessary for these patients. One attractive approach is to inhibit multiple parallel pathways that drive the growth of these hematologic tumors, possibly prolonging the duration of the response and reducing resistance. Early clinical trials have tested this approach by dosing two drugs in combination in NHL patients. We discovered a single molecule, MDVN1003 (1-(5-amino-2,3-dihydro-1H-inden-2-yl)-3-(8-fluoro-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine), that inhibits Bruton's tyrosine kinase and phosphatidylinositol-3-kinase δ, two proteins regulated by the B cell receptor that drive the growth of many NHLs. In this report, we show that this dual inhibitor prevents the activation of B cells and inhibits the phosphorylation of protein kinase B and extracellular signal-regulated kinase 1/2, two downstream mediators that are important for this process. Additionally, MDVN1003 induces cell death in a B cell lymphoma cell line but not in an irrelevant erythroblast cell line. Importantly, we found that this orally bioavailable dual inhibitor reduced tumor growth in a B cell lymphoma xenograft model more effectively than either ibrutinib or idelalisib. Taken together, these results suggest that dual inhibition of these two key pathways by a single molecule could be a viable approach for treatment of NHL patients.

Discovery of pyrazolopyrimidine derivatives as novel inhibitors of ataxia telangiectasia and rad3 related protein (ATR).

The ATR pathway is a critical mediator of the replication stress response in cells. In aberrantly proliferating cancer cells, this pathway can help maintain sufficient genomic integrity for cancer cell progression. Herein we describe the discovery of 19, a pyrazolopyrimidine-containing inhibitor of ATR via a strategic repurposing of compounds targeting PI3K.

Novel 3-methylindoline inhibitors of EZH2: Design, synthesis and SAR.

EZH2 (enhancer of zeste homologue 2) is the catalytic subunit of the polycomb repressive complex 2 (PRC2) that catalyzes the methylation of lysine 27 of histone H3 (H3K27). Dysregulation of EZH2 activity is associated with several human cancers and therefore EZH2 inhibition has emerged as a promising therapeutic target. Several small molecule EZH2 inhibitors with different chemotypes have been reported in the literature, many of which use a bicyclic heteroaryl core. Herein, we report the design and synthesis of EZH2 inhibitors containing an indoline core. Partial saturation of an indole to an indoline provided lead compounds with nanomolar activity against EZH2, while also improving solubility and oxidative metabolic stability.

Discovery of Pyrazolopyrimidine Derivatives as Novel Dual Inhibitors of BTK and PI3Kδ.

The aberrant activation of B-cells has been implicated in several types of cancers and hematological disorders. BTK and PI3Kδ are kinases responsible for B-cell signal transduction, and inhibitors of these enzymes have demonstrated clinical benefit in certain types of lymphoma. Simultaneous inhibition of these pathways could result in more robust responses or overcome resistance as observed in single agent use. We report a series of novel compounds that have low nanomolar potency against both BTK and PI3Kδ as well as acceptable PK properties that could be useful in the development of treatments against B-cell related diseases.

Temozolomide analogs with improved brain/plasma ratios - Exploring the possibility of enhancing the therapeutic index of temozolomide.

Temozolomide is a chemotherapeutic agent that is used in the treatment of glioblastoma and other malignant gliomas. It acts through DNA alkylation, but treatment is limited by its systemic toxicity and neutralization of DNA alkylation by upregulation of the O6-methylguanine-DNA methyltransferase gene. Both of these limiting factors can be addressed by achieving higher concentrations of TMZ in the brain. Our research has led to the discovery of new analogs of temozolomide with improved brain:plasma ratios when dosed in vivo in rats. These compounds are imidazotetrazine analogs, expected to act through the same mechanism as temozolomide. With reduced systemic exposure, these new agents have the potential to improve efficacy and therapeutic index in the treatment of glioblastoma.

Design, validation, and implementation of a rapid-injection NMR system.

A Rapid Injection NMR (RINMR) apparatus has been designed and constructed to allow the observation of fast chemical reactions in real time by NMR spectroscopy. The instrument was designed to allow the rapid (<2 s) injection and mixing of a metered volume of a reagent into a spinning NMR tube followed by rapid acquisition of the data resulting from the evolution of the chemical process. The various design criteria for this universal system included the ability to deliver any chemical reagent at any temperature and allow for the observation of any nucleus. The various challenges associated with the construction and implementation of this instrument are documented along with the validation of the accuracy of the apparatus with respect to volume and temperature. Finally, the ultimate validation and reproducibility of the technique is presented in the form of three case studies that used the instrument to elucidate various aspects of organic reaction mechanisms. The authors urge interested parties to not embark on the construction of their own instrument and invite those whose research problems might be amenable to this kind of analysis to contact the corresponding author for access to the apparatus described herein.

Chiral phosphoramide-catalyzed aldol additions of ketone trichlorosilyl enolates. Mechanistic aspects.

The mechanism of the catalytic, enantioselective addition of trichlorosilyl enolates to aldehydes has been investigated. Kinetic studies using ReactIR and rapid injection NMR (RINMR) spectroscopy have confirmed the simultaneous operation of dual mechanistic pathways involving either one or two phosphoramides bound to a siliconium ion organizational center. This mechanistic dichotomy was initially postulated on the basis of catalyst loading studies and nonlinear effects studies. This duality explains the difference in reactivity and stereoselectivity of various classes of phosphoramides. Determination of Arrhenius activation parameters revealed that aldol addition occurs through the reversible albeit unfavorable formation of an activated complex, and natural-abundance 13C NMR kinetic isotope effect (KIE) studies have determined that the turnover limiting step is the aldol addition. A thorough examination of a range of phosphoramides has established empirical structure-activity selectivity relationships. In addition, the effects of catalyst loading, rate of addition, solvents, and additives have been studied and together allow the formulation of a unified mechanistic picture for the aldol addition.

Lewis base catalyzed aldol additions of chiral trichlorosilyl enolates and silyl enol ethers.

[structures: see text] The consequences of double diastereodifferentiation in chiral Lewis base catalyzed aldol additions using chiral enoxysilanes derived from lactate, 3-hydroxyisobutyrate, and 3-hydroxybutyrate have been investigated. Trichlorosilyl enolates derived from the chiral methyl and ethyl ketones were subjected to aldolization in the presence of phosphoramides, and the intrinsic selectivity of these enolates and the external stereoinduction from chiral catalyst were studied. In the reactions with the lactate derived enolate, the strong internal stereoinduction dominated the stereochemical outcome of the aldol addition. For the 3-hydroxyisobutyrate- and 3-hydroxybutyrate derived enolates, the catalyst-controlled diastereoselectivities were observed, and the resident stereogenic centers exerted marginal influence. The corresponding trimethylsilyl enol ethers were employed in SiCl4/bisphosphoramide catalyzed aldol additions, and the effect of double diastereodifferentiation was also investigated. The overall diastereoselection of the process was again controlled by the strong external influence of the catalyst.

Multicomponent cycloadditions: the four-component [5+1+2+1] cycloaddition of vinylcyclopropanes, alkynes, and CO.

Prompted by the view that intermediates of transition metal-catalyzed reactions could be intercepted by one or more additional components, studies in our laboratory have led to the design and development of new three-component [5+2+1], [4+2+1], and [2+2+1] cycloadditions. These continuing studies have now led to the identification of a fundamentally new four-component [5+1+2+1] cycloaddition reaction of vinylcyclopropanes, alkynes and CO, yielding hydroxyindanone products in generally good yields. Terminal alkynes bearing aryl or alkyl groups are tolerated well. Substitution at any position of the VCP leads predictably to substituted hydroxyindanone products. Using a bis-alkynyl substrate, the reaction can be carried out bi-directionally, forming 10 C-C bonds and four new rings from seven components in a single, operationally simple process.

High-resolution Micro-CT evaluation of mid- to long-term effects of estrogen deficiency on rat trabecular bone.

RATIONALE AND OBJECTIVES: Previous studies have shown dramatic short-term bone loss following ovariectomy. The purpose of this study was to evaluate mid- to long-term effects of estrogen deficiency on microarchitecture of tibial trabecular bone using a high-resolution microcomputed tomography (Micro-CT). MATERIALS AND METHODS: Twenty-eight female rats were divided into two equal groups: ovariectomized (OVX, n = 14) and sham-operated (SOVX, n = 14), which in turn were divided into two equal subgroups (euthanized either 6 or 16 weeks after surgery). The left tibia of each animal was scanned with a high-resolution micro-CT (Skyscan 1072 micro-CT system; SkyScan, Aartselaar, Belgium). The Micro-CT system used an X-ray CCD-camera with a cooled 1024 x 1024-pixel 12-bit sensor. RESULTS: Ovariectomy significantly decreased the ratio of bone volume:tissue volume (-42% at 6 weeks and -69% at 16 weeks) and trabecular thickness (-13% at 6 weeks and -30% at 16 weeks), while significantly increasing trabecular separation (+73% at 6 weeks and +100% at 16 weeks) and structure model index (+15% at 6 weeks and +48% at 16 weeks), when compared with SOVX, from 6 to 16 weeks. CONCLUSION: Based on the experimental results, there are alterations of trabecular microarcihtecture in the tibia following mid- and long-term estrogen deficiency. More rod-shaped trabeculae are formed with increasing duration of estrogen deficiency. The high-resolution micro-CT imaging system is useful to evaluate the mid- to long-term trabecular changes seen with estrogen deficiency or osteoporosis.

Stereoselective aldol additions of achiral ethyl ketone-derived trichlorosilyl enolates.

Methods for the preparation of geometrically defined enoxy(trichlorosilanes) derived from ethyl ketone enolates have been developed. The addition of enoxy(trichlorosilanes) (trichlorosilyl enolates) to aldehydes proceeds with good yields in the presence of catalytic amounts of chiral phosphoramides. The reaction of Z-trichlorosilyl enolates to aryl aldehydes affords aldol products with good to excellent diastereo- and enantioselectivities. Phosphoramide-catalyzed aldol additions lacked substrate generality providing modest selectivities with unsaturated and aliphatic aldehydes. In all cases, the phosphoramide-catalyzed aldol addition of E-trichlorosilyl enolates to aldehydes provided good yields with moderate to good stereoselectivities.