Potent GCN2 Inhibitor Capable of Reversing MDSC-Driven T Cell Suppression Demonstrates In Vivo Efficacy as a Single Agent and in Combination with Anti-Angiogenesis Therapy.

General control nonderepressible 2 (GCN2) protein kinase is a cellular stress sensor within the tumor microenvironment (TME), whose signaling cascade has been proposed to contribute to immune escape in tumors. Herein, we report the discovery of cell-potent GCN2 inhibitors with excellent selectivity against its closely related Integrated Stress Response (ISR) family members heme-regulated inhibitor kinase (HRI), protein kinase R (PKR), and (PKR)-like endoplasmic reticulum kinase (PERK), as well as good kinome-wide selectivity and favorable PK. In mice, compound 39 engages GCN2 at levels ≥80% with an oral dose of 15 mg/kg BID. We also demonstrate the ability of compound 39 to alleviate MDSC-related T cell suppression and restore T cell proliferation, similar to the effect seen in MDSCs from GCN2 knockout mice. In the LL2 syngeneic mouse model, compound 39 demonstrates significant tumor growth inhibition (TGI) as a single agent. Furthermore, TGI mediated by anti-VEGFR was enhanced by treatment with compound 39 demonstrating the complementarity of these two mechanisms.

Optimization of LpxC Inhibitors for Antibacterial Activity and Cardiovascular Safety.

UDP-3-O-(R-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a Zn2+ deacetylase that is essential for the survival of most pathogenic Gram-negative bacteria. ACHN-975 (N-((S)-3-amino-1-(hydroxyamino)-3-methyl-1-oxobutan-2-yl)-4-(((1R,2R)-2-(hydroxymethyl)cyclopropyl)buta-1,3-diyn-1-yl)benzamide) was the first LpxC inhibitor to reach human clinical testing and was discovered to have a dose-limiting cardiovascular toxicity of transient hypotension without compensatory tachycardia. Herein we report the effort beyond ACHN-975 to discover LpxC inhibitors optimized for enzyme potency, antibacterial activity, pharmacokinetics, and cardiovascular safety. Based on its overall profile, compound 26 (LPXC-516, (S)-N-(2-(hydroxyamino)-1-(3-methoxy-1,1-dioxidothietan-3-yl)-2-oxoethyl)-4-(6-hydroxyhexa-1,3-diyn-1-yl)benzamide) was chosen for further development. A phosphate prodrug of 26 was developed that provided a solubility of >30 mg mL-1 for parenteral administration and conversion into the active drug with a t1/2 of approximately two minutes. Unexpectedly, and despite our optimization efforts, the prodrug of 26 still possesses a therapeutic window insufficient to support further clinical development.

Coelenterazine analogues emit red-shifted bioluminescence with NanoLuc.

We report the synthesis and characterization of novel coelenterazine analogues that demonstrate a red-shift in their bioluminescent emission with NanoLuc luciferase. These coelenterazines can be tuned to shift the bioluminescent emission from blue light in the native system. In particular, direct attachment of an aryl moiety to the imidazopyrazinone core of furimazine at the C8 position provides a significant red-shift while maintaining reasonable light output. In addition, modification of the C6 aryl moiety provided additive red-shifts, and by combining the most promising modifications we report a coelenterazine with a maximum emission near 600 nm with NanoLuc. Finally, we show that this new bioluminescent system is capable of efficient BRET to far-red fluorophores. We anticipate these new principles of NanoLuc substrate design will impact applications that depend on shifting the colour of emission to the red, most notably in vivo bioluminescent imaging.

Three Efficient Methods for Preparation of Coelenterazine Analogues.

The growing popularity of bioluminescent assays has highlighted the need for coelenterazine analogues possessing properties tuned for specific applications. However, the structural diversity of known coelenterazine analogues has been limited by current syntheses. Known routes for the preparation of coelenterazine analogues employ harsh reaction conditions that limit access to many substituents and functional groups. Novel synthetic routes reported here establish simple and robust methods for synthesis and investigation of structurally diverse marine luciferase substrates. Specifically, these new routes allow synthesis of coelenterazine analogues containing various heterocyclic motifs and substituted aromatic groups with diverse electronic substituents at the R(2) position. Interesting analogues described herein were characterized by their physicochemical properties, bioluminescent half-life, light output, polarity and cytotoxicity. Some of the analogues represent leads that can be utilized in the development of improved bioluminescent systems.

Preparation of fluorinated RNA nucleotide analogs potentially stable to enzymatic hydrolysis in RNA and DNA polymerase assays.

Analogs of ribonucleotides (RNA) stable to enzymatic hydrolysis were prepared and characterized. Computational investigations revealed that this class of compounds with a modified triphosphate exhibits the correct polarity and minimal steric effects compared to the natural molecule. Non-hydrolysable properties as well as the ability of the modified nucleotide to be recognized by enzymes were probed by performing single-turnover gap filling assays with T7 RNA polymerase and DNA polymerase ß.

BF3-H2O catalyzed hydroxyalkylation of aromatics with aromatic aldehydes and dicarboxaldehydes: efficient synthesis of triarylmethanes, diarylmethylbenzaldehydes, and anthracene derivatives.

BF(3)-monohydrate is found to be an efficient and strong acid catalyst as well as an effective protosolvating medium suitable for the hydroxyalkylation of arenes with aromatic aldehydes. This reaction has been extended to aromatic dialdehydes, such as terephthalic dicarboxaldehyde and isoterephthalic dicarboxaldehyde, for the efficient synthesis of diarylmethylbenzaldehydes, which are useful synthons for various organic transformations. Further, successful one step convergent synthesis of various synthetically useful anthracene derivatives from phthalaldehyde was also achieved. BF(3)-H(2)O is less expensive and acts as an efficient substitute for nonoxidizing strong protic acids/superacids.