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.

Novel Piperidinyl-Azetidines as Potent and Selective CCR4 Antagonists Elicit Antitumor Response as a Single Agent and in Combination with Checkpoint Inhibitors.

The C-C chemokine receptor 4 (CCR4) is broadly expressed on regulatory T cells (Treg) as well as other circulating and tissue-resident T cells. Treg can be recruited to the tumor microenvironment (TME) through the C-C chemokines CCL17 and CCL22. Treg accumulation in the TME has been shown to dampen the antitumor immune response and is thought to be an important driver in tumor immune evasion. Preclinical and clinical data suggest that reducing the Treg population in the TME can potentiate the antitumor immune response of checkpoint inhibitors. We have developed small-molecule antagonists of CCR4, featuring a novel piperidinyl-azetidine motif, that inhibit the recruitment of Treg into the TME and elicit antitumor responses as a single agent or in combination with an immune checkpoint blockade. The discovery of these potent, selective, and orally bioavailable CCR4 antagonists, and their activity in in vitro and in vivo models, is described herein.

Discovery of Potent, Selective, and Orally Bioavailable Inhibitors of USP7 with In Vivo Antitumor Activity.

USP7 is a promising target for cancer therapy as its inhibition is expected to decrease function of oncogenes, increase tumor suppressor function, and enhance immune function. Using a structure-based drug design strategy, a new class of reversible USP7 inhibitors has been identified that is highly potent in biochemical and cellular assays and extremely selective for USP7 over other deubiquitinases. The succinimide was identified as a key potency-driving motif, forming two strong hydrogen bonds to the allosteric pocket of USP7. Redesign of an initial benzofuran-amide scaffold yielded a simplified ether series of inhibitors, utilizing acyclic conformational control to achieve proper amine placement. Further improvements were realized upon replacing the ether-linked amines with carbon-linked morpholines, a modification motivated by free energy perturbation (FEP+) calculations. This led to the discovery of compound 41, a highly potent, selective, and orally bioavailable USP7 inhibitor. In xenograft studies, compound 41 demonstrated tumor growth inhibition in both p53 wildtype and p53 mutant cancer cell lines, demonstrating that USP7 inhibitors can suppress tumor growth through multiple different pathways.

Discovery of a Potent and Selective CCR4 Antagonist That Inhibits Treg Trafficking into the Tumor Microenvironment.

Recruitment of suppressive CD4+ FOXP3+ regulatory T cells (Treg) to the tumor microenvironment (TME) has the potential to weaken the antitumor response in patients receiving treatment with immuno-oncology (IO) agents. Human Treg express CCR4 and can be recruited to the TME through the CC chemokine ligands CCL17 and CCL22. In some cancers, Treg accumulation correlates with poor patient prognosis. Preclinical data suggests that preventing the recruitment of Treg and increasing the population of activated effector T cells (Teff) in the TME can potentiate antitumor immune responses. We developed a novel series of potent, orally bioavailable small molecule antagonists of CCR4. From this series, several compounds exhibited high potency in distinct functional assays in addition to good in vitro and in vivo ADME properties. The design, synthesis, and SAR of this series and confirmation of its in vivo activity are reported.

Discovery of a novel potent GABA(B) receptor agonist.

Structure-activity studies have led to a discovery of 3-(4-pyridyl)methyl ether derivative 9d that has 25- to 50-fold greater functional potency than R-baclofen at human and rodent GABA(B) receptors in vitro. Mouse hypothermia studies confirm that this compound crosses the blood-brain barrier and is approximately 50-fold more potent after systemic administration.

Oxadiazolone bioisosteres of pregabalin and gabapentin.

A series of oxadiazolone bioisosteres of pregabalin 1 and gabapentin 2 were prepared, and several were found to exhibit similar potency for the alpha(2)-delta subunit of voltage-gated calcium channels. Oxadiazolone 9 derived from 2 achieved low brain uptake but was nevertheless active in models of osteoarthritis. The high clearance associated with compound 9 was postulated to be a consequence of efflux by OAT and/or OCT, and was attenuated on co-administration with cimetidine or probenecid.

Characterization of N-(adamantan-1-ylmethyl)-5-[(3R-amino-pyrrolidin-1-yl)methyl]-2-chloro-benzamide, a P2X7 antagonist in animal models of pain and inflammation.

Recent evidence suggests that the P2X(7) receptor may play a role in the pathophysiology of preclinical models of pain and inflammation. Therefore, pharmacological agents that target this receptor may potentially have clinical utility as anti-inflammatory and analgesic therapy. We investigated and characterized the previously reported P2X(7) antagonist N-(adamantan-1-ylmethyl)-5-[(3R-amino-pyrrolidin-1-yl)methyl]-2-chloro-benzamide, hydrochloride salt (AACBA; GSK314181A). In vitro, AACBA was a relatively potent inhibitor of both human P2X(7)-mediated calcium flux and quinolinium,4-[(3-methyl-2(3H)-benzoxazolylidene)methyl]-1-[3-(triemethylammonio)propyl]-diiodide (YO-PRO-1) uptake assays, with IC(50) values of approximately 18 and 85 nM, respectively. Compared with the human receptor, AACBA was less potent at the rat P2X(7) receptor, with IC(50) values of 29 and 980 nM in the calcium flux and YO-PRO-1 assays, respectively. In acute in vivo models of pain and inflammation, AACBA dose-dependently reduced lipopolysaccharide-induced plasma interleukin-6 release and prevented or reversed carrageenan-induced paw edema and mechanical hypersensitivity. In chronic in vivo models of pain and inflammation, AACBA produced a prophylactic, but not therapeutic-like, prevention of the clinical signs and histopathological damage of collagen-induced arthritis. Finally, AACBA could not reverse L(5) spinal nerve ligation-induced tactile allodynia when given therapeutically. Consistent with previous literature, these results suggest that P2X(7) receptors do play a role in animal models of pain and inflammation. Further study of P2X(7) antagonists both in preclinical and clinical studies will help elucidate the role of the P2X(7) receptor in pain and inflammatory mechanisms and may help identify potential clinical benefits of such molecules.

1-Benzylbenzimidazoles: the discovery of a novel series of bradykinin B(1) receptor antagonists.

The design, synthesis, and structure-activity studies of a novel series of BK B(1) receptor antagonists based on a 1-benzylbenzimidazole chemotype are described. A number of compounds, for example, 38g, with excellent affinity for the cynomolgus macaque and rat bradykinin B(1) receptor were discovered.

Aminopyrazine CB1 receptor inverse agonists.

A series of 5,6-diaryl-2-amino-pyrazines were prepared and found to have antagonist-like properties at the CB1 receptor. Subsequent SAR studies optimized both receptor potency and drug-like properties including solubility and Cytochrome-P450 inhibition potential. Optimized compounds were demonstrated to be inverse agonists and compared in vivo with rimonabant for their ability to inhibit food intake, to occupy central CB1 receptors and to influence hormonal markers associated with obesity.

Carboxylate bioisosteres of pregabalin.

Several beta-amino tetrazole analogs of gabapentin 1 and pregabalin 2 were prepared by one of two convergent, highly efficient routes, and their affinity for the alpha(2)-delta protein examined. Two select compounds with potent affinity for alpha(2)-delta, 8a and 16a, were subsequently tested in vivo in an audiogenic seizure model and found to elicit protective effects.

Carboxylate bioisosteres of gabapentin.

A series of carboxylate bioisosteres of structures related to gabapentin 1 have been prepared. When the carboxylate was replaced by a tetrazole, this group was recognized by the alpha2-delta protein. Further characterization of alpha2-delta binding compounds 14a and 14b revealed a similar pattern of functional in vitro and in vivo activity to gabapentin 1.

Heteroaromatic side-chain analogs of pregabalin.

A series of heteroaromatic analogs of pregabalin has been identified that possess anticonvulsant activity in the DBA/2 mouse model. The methods of synthesis and preliminary pharmacology are discussed herein.

The design and synthesis of human branched-chain amino acid aminotransferase inhibitors for treatment of neurodegenerative diseases.

The inhibition of the cytosolic isoenzyme BCAT that is expressed specifically in neuronal tissue is likely to be useful for the treatment of neurodegenerative and other neurological disorders where glutamatergic mechanisms are implicated. Compound 2 exhibited an IC50 of 0.8 microM in the hBCATc assays; it is an active and selective inhibitor. Inhibitor 2 also blocked calcium influx into neuronal cells following inhibition of glutamate uptake, and demonstrated neuroprotective efficacy in vivo. SAR, pharmacology, and the crystal structure of hBCATc with inhibitor 2 are described.

Structure-activity relationships of pregabalin and analogues that target the alpha(2)-delta protein.

Pregabalin exhibits robust activity in preclinical assays indicative of potential antiepileptic, anxiolytic, and antihyperalgesic clinical efficacy. It binds with high affinity to the alpha(2)-delta subunit of voltage-gated calcium channels and is a substrate of the system L neutral amino acid transporter. A series of pregabalin analogues were prepared and evaluated for their alpha(2)-delta binding affinity as demonstrated by their ability to inhibit binding of [(3)H]gabapentin to pig brain membranes and for their potency to inhibit the uptake of [(3)H]leucine into CHO cells, a measure of their ability to compete with the endogenous substrate at the system L transporter. Compounds were also assessed in vivo for their ability to promote anxiolytic, analgesic, and anticonvulsant actions. These studies suggest that distinct structure activity relationships exist for alpha(2)-delta binding and system L transport inhibition. However, both interactions appear to play an important role in the in vivo profile of these compounds.

Novel cyclopropyl beta-amino acid analogues of pregabalin and gabapentin that target the alpha2-delta protein.

As part of a program aimed at generating compounds with affinity for the alpha(2)-delta subunit of voltage-gated calcium channels, several novel beta-amino acids were prepared using an efficient nitroalkane-mediated cyclopropanation as a key step. Depending on the ester that was chosen, the target amino acids could be prepared in as few as three steps. The cyclopropyl amino acids derived from ketones proved to be potent binders of the alpha(2)-delta subunit of voltage-gated calcium channels, but did not interact with the large neutral amino acid system L (leucine) transporter. Anticonvulsant effects were observed in vivo with compound 34 but only after intracerebroventricular (icv) administration, presumably due to inadequate brain concentrations of the drug being achieved following oral dosing. However, pregabalin 1 was active in the DBA/2 model after oral (and icv) dosing, supporting a hypothesis that active transport is a prerequisite for such zwitterionic species to cross the blood-brain barrier.

Orally active analogues of the dopaminergic prodrug 6-(N,N-di-n-propylamino)-3,4,5,6,7,8-hexahydro-2H-naphthalen-1-one: synthesis and pharmacological activity.

A series of analogues of 6-(N,N-di-n-propylamino)-3,4,5,6,7,8-hexahydro-2H-naphthalen-1-one (6), an enone prodrug of the mixed DA D(1)/D(2) agonist 5,6-diOH-DPAT (2), was synthesized. The pharmacological profiles of these new enones and their in vivo pharmacological activities were investigated in the Ungerstedt rat rotation model for Parkinson's disease. At 0.1 mg kg(-1) po, the N-methyl-N-n-propyl (12) and the N-ethyl-N-propyl (13) analogues induced pronounced and long lasting pharmacological effects. The pharmacological profile of enone 12 was found to be similar to that of 6, while enone 13 was significantly more potent than 6 (p < 0.01). Analyses of rat brains after the administration of (-)-6 and 13 indicated the presence of hydroxylated metabolites of the parent enones. It is speculated that such metabolites are alpha'-hydroxylated enones that may constitute the first step in the formation of the corresponding catechols.