Discovery of Novel TLR7 Agonists as Systemic Agent for Combination With aPD1 for Use in Immuno-oncology

Abstract

We have designed and developed novel and selective TLR7 agonists that exhibited potent receptor activity in a cellbased reporter assay. In vitro, these agonists significantly induced secretion of cytokines IL-6, IL-1β, IL-10, TNFa, IFNa, and IP-10 in human and mouse whole blood. Pharmacokinetic and pharmacodynamic studies in mice showed a significant secretion of IFNα and TNFα cytokines. When combined with aPD1 in a CT-26 tumor model, the lead compound showed strong synergistic antitumor activity with complete tumor regression in 8/10 mice dosed using the intravenous route. Structure−activity relationship studies enabled by structure-based designs of TLR7 agonists are disclosed.

Identification and Optimization of Small Molecule Pyrazolopyrimidine TLR7 Agonists for Applications in Immuno-oncology

“…In the preceding article, we disclosed a medicinal chemistry campaign which identified compound 1 as a novel and selective TLR7 agonist that showed potent activity in vitro (Table ). On the basis of these initial findings, we embarked on a second round of SAR optimization where our primary objective was to design a compound that (1) is structurally distinct from compound 1 , (2) has a favorable ADME profile including good metabolic stability for advancing to in vivo studies, and (3) displays minimal off-target activities including hERG, CYP inhibition, and T-cell toxicity.…”

“…We subsequently shifted our SAR efforts toward modifications at the C-7 position of the core, as well as the piperidine side chain (Table ) to boost mTLR7 assay potency. We have previously demonstrated that addition of a branched ( S )-3-aminohexan-1-ol side chain at the C-7 position provided a favorable combination of potency and physicochemical properties . Indeed, incorporation of this aminohexanol side chain in this piperidine series ( 9 ) afforded improvements across a range of in vitro parameters without significantly compromising TLR7 activity when compared with compound 4 .…”

“…First, compounds 11 , 13 , and 14 were evaluated in a single-dose (1 mg/kg) PK/PD study in Balb/C female mice and type I interferon levels were measured at 2 h postadministration (see Supporting Information). Compound 14 was found to be the most effective at promoting IFNα secretion and was progressed to PK/PD evaluation with compound 27 , which is disclosed in the preceding manuscript (Figure a).…”

“…The nitrogen atom of this pyridyl group also engages in a water-mediated hydrogen bond with N-2 of the pyrazolo core. As anticipated, the N–H group at the C-7 position and the methoxy group forms an intramolecular hydrogen bond, where a similar interaction was found in the cocrystal structure of compound 27 and monkey TLR7 protein . Binding of the basic piperidine amine to Gln354 is also conserved between the two crystal structures.…”

“…Subsequent design iterations including incorporation of a pyridyl group and piperidine capping motifs resulted in the discovery of TLR7 agonist 14 that demonstrated nanomolar potency and excellent metabolic stability and was clean in CYP and hERG panels. Importantly, compound 14 demonstrated superior in vivo PK/PD properties compared to lead compound 27 , which was disclosed in the preceding manuscript …”