Fragment-Based Discovery of a Novel, Brain Penetrant, Orally Active HDAC2 Inhibitor.

Fragment-based ligand discovery was successfully applied to histone deacetylase HDAC2. In addition to the anticipated hydroxamic acid- and benzamide-based fragment screening hits, a low affinity (∼1 mM) α-amino-amide zinc binding fragment was identified, as well as fragments binding to other regions of the catalytic site. This alternative zinc-binding fragment was further optimized, guided by the structural information from protein-ligand complex X-ray structures, into a sub-µM, brain penetrant, HDAC2 inhibitor (17) capable of modulating histone acetylation levels in vivo.

Discovery of ASTX029, A Clinical Candidate Which Modulates the Phosphorylation and Catalytic Activity of ERK1/2.

Aberrant activation of the mitogen-activated protein kinase pathway frequently drives tumor growth, and the ERK1/2 kinases are positioned at a key node in this pathway, making them important targets for therapeutic intervention. Recently, a number of ERK1/2 inhibitors have been advanced to investigational clinical trials in patients with activating mutations in B-Raf proto-oncogene or Ras. Here, we describe the discovery of the clinical candidate ASTX029 (15) through structure-guided optimization of our previously published isoindolinone lead (7). The medicinal chemistry campaign focused on addressing CYP3A4-mediated metabolism and maintaining favorable physicochemical properties. These efforts led to the identification of ASTX029, which showed the desired pharmacological profile combining ERK1/2 inhibition with suppression of phospho-ERK1/2 (pERK) levels, and in addition, it possesses suitable preclinical pharmacokinetic properties predictive of once daily dosing in humans. ASTX029 is currently in a phase I-II clinical trial in patients with advanced solid tumors.

Structure-Based Design of Potent and Orally Active Isoindolinone Inhibitors of MDM2-p53 Protein-Protein Interaction.

Inhibition of murine double minute 2 (MDM2)-p53 protein-protein interaction with small molecules has been shown to reactivate p53 and inhibit tumor growth. Here, we describe rational, structure-guided, design of novel isoindolinone-based MDM2 inhibitors. MDM2 X-ray crystallography, quantum mechanics ligand-based design, and metabolite identification all contributed toward the discovery of potent in vitro and in vivo inhibitors of the MDM2-p53 interaction with representative compounds inducing cytostasis in an SJSA-1 osteosarcoma xenograft model following once-daily oral administration.

A Fragment-Derived Clinical Candidate for Antagonism of X-Linked and Cellular Inhibitor of Apoptosis Proteins: 1-(6-[(4-Fluorophenyl)methyl]-5-(hydroxymethyl)-3,3-dimethyl-1 H,2 H,3 H-pyrrolo[3,2- b]pyridin-1-yl)-2-[(2 R,5 R)-5-methyl-2-([(3R)-3-methylmorpholin-4-yl]methyl)piperazin-1-yl]ethan-1-one (ASTX660).

Inhibitor of apoptosis proteins (IAPs) are promising anticancer targets, given their roles in the evasion of apoptosis. Several peptidomimetic IAP antagonists, with inherent selectivity for cellular IAP (cIAP) over X-linked IAP (XIAP), have been tested in the clinic. A fragment screening approach followed by structure-based optimization has previously been reported that resulted in a low-nanomolar cIAP1 and XIAP antagonist lead molecule with a more balanced cIAP-XIAP profile. We now report the further structure-guided optimization of the lead, with a view to improving the metabolic stability and cardiac safety profile, to give the nonpeptidomimetic antagonist clinical candidate 27 (ASTX660), currently being tested in a phase 1/2 clinical trial (NCT02503423).

Fragment-Based Discovery of a Potent, Orally Bioavailable Inhibitor That Modulates the Phosphorylation and Catalytic Activity of ERK1/2.

Aberrant activation of the MAPK pathway drives cell proliferation in multiple cancers. Inhibitors of BRAF and MEK kinases are approved for the treatment of BRAF mutant melanoma, but resistance frequently emerges, often mediated by increased signaling through ERK1/2. Here, we describe the fragment-based generation of ERK1/2 inhibitors that block catalytic phosphorylation of downstream substrates such as RSK but also modulate phosphorylation of ERK1/2 by MEK without directly inhibiting MEK. X-ray crystallographic and biophysical fragment screening followed by structure-guided optimization and growth from the hinge into a pocket proximal to the C-α helix afforded highly potent ERK1/2 inhibitors with excellent kinome selectivity. In BRAF mutant cells, the lead compound suppresses pRSK and pERK levels and inhibits proliferation at low nanomolar concentrations. The lead exhibits tumor regression upon oral dosing in BRAF mutant xenograft models, providing a promising basis for further optimization toward clinical pERK1/2 modulating ERK1/2 inhibitors.

Discovery of a Potent Nonpeptidomimetic, Small-Molecule Antagonist of Cellular Inhibitor of Apoptosis Protein 1 (cIAP1) and X-Linked Inhibitor of Apoptosis Protein (XIAP).

XIAP and cIAP1 are members of the inhibitor of apoptosis protein (IAP) family and are key regulators of anti-apoptotic and pro-survival signaling pathways. Overexpression of IAPs occurs in various cancers and has been associated with tumor progression and resistance to treatment. Structure-based drug design (SBDD) guided by structural information from X-ray crystallography, computational studies, and NMR solution conformational analysis was successfully applied to a fragment-derived lead resulting in AT-IAP, a potent, orally bioavailable, dual antagonist of XIAP and cIAP1 and a structurally novel chemical probe for IAP biology.

Fragment-Based Discovery of Potent and Selective DDR1/2 Inhibitors.

The DDR1 and DDR2 receptor tyrosine kinases are activated by extracellular collagen and have been implicated in a number of human diseases including cancer. We performed a fragment-based screen against DDR1 and identified fragments that bound either at the hinge or in the back pocket associated with the DFG-out conformation of the kinase. Modeling based on crystal structures of potent kinase inhibitors facilitated the "back-to-front" design of potent DDR1/2 inhibitors that incorporated one of the DFG-out fragments. Further optimization led to low nanomolar, orally bioavailable inhibitors that were selective for DDR1 and DDR2. The inhibitors were shown to potently inhibit DDR2 activity in cells but in contrast to unselective inhibitors such as dasatinib, they did not inhibit proliferation of mutant DDR2 lung SCC cell lines.

Fragment-Based Drug Discovery Targeting Inhibitor of Apoptosis Proteins: Discovery of a Non-Alanine Lead Series with Dual Activity Against cIAP1 and XIAP.

Inhibitor of apoptosis proteins (IAPs) are important regulators of apoptosis and pro-survival signaling pathways whose deregulation is often associated with tumor genesis and tumor growth. IAPs have been proposed as targets for anticancer therapy, and a number of peptidomimetic IAP antagonists have entered clinical trials. Using our fragment-based screening approach, we identified nonpeptidic fragments binding with millimolar affinities to both cellular inhibitor of apoptosis protein 1 (cIAP1) and X-linked inhibitor of apoptosis protein (XIAP). Structure-based hit optimization together with an analysis of protein-ligand electrostatic potential complementarity allowed us to significantly increase binding affinity of the starting hits. Subsequent optimization gave a potent nonalanine IAP antagonist structurally distinct from all IAP antagonists previously reported. The lead compound had activity in cell-based assays and in a mouse xenograft efficacy model and represents a highly promising start point for further optimization.

Discovery and characterization of novel, potent, non-peptide parathyroid hormone-1 receptor antagonists.

A 1,3,4-benzotriazepine was identified as a suitable lead in our effort toward obtaining a non-peptide parathyroid hormone-1 receptor (PTH1R) antagonist. A process of optimization afforded derivatives displaying nanomolar PTH1R affinity, a representative example of which behaved as a PTH1R antagonist in cell-based cyclic adenosine monophosphate (cAMP) assays, with selectivity over PTH2 receptors.

Optimization of 1,3,4-benzotriazepine-based CCK(2) antagonists to obtain potent, orally active inhibitors of gastrin-mediated gastric acid secretion.

Starting from a novel, achiral 1,3,4-benzotriazepine-based CCK2 receptor antagonist, a process of optimization has afforded further compounds of this type that maintain the nanomolar affinity for recombinant, human CCK2 receptors and high selectivity over CCK1 receptors observed in the initial lead but display more potent inhibition of pentagastrin-stimulated gastric acid secretion in vivo. Moreover, this has largely been achieved without altering their potency at wild-type canine and rat receptors, as judged by their displacement of [125I]-BH-CCK-8S in a radioligand binding assay and by their activity in an isolated, perfused rat stomach bioassay, respectively. 2-(5-Cyclohexyl-1-(2-cyclopentyl-2-oxo-ethyl)-2-oxo-1,2-dihydro-3H-1,3,4-benzotriazepin-3-yl)-N-(3-(5-oxo-2,5-dihydro- [1,2,4]oxadiazol-3-yl)-phenyl)-acetamide (47) was identified as the most effective compound stemming from this approach, proving to be a potent inhibitor of pentagastrin-stimulated gastric acid secretion in rats and dogs by intravenous bolus as well as by enteral administration.

Novel, achiral 1,3,4-benzotriazepine analogues of 1,4-benzodiazepine-based CCK(2) antagonists that display high selectivity over CCK(1) receptors.

A series of 1,3,4-benzotriazepine-based CCK(2) antagonists have been devised by consideration of the structural features that govern CCK receptor affinity and the receptor subtype selectivity of 1,4-benzodiazepine-based CCK(2) antagonists. In contrast to the latter compounds, these novel 1,3,4-benzotriazepines are achiral, yet they display similar affinity for CCK(2) receptors to the earlier molecules and are highly selective over CCK(1) receptors.

Scaffold hopping with molecular field points: identification of a cholecystokinin-2 (CCK2) receptor pharmacophore and its use in the design of a prototypical series of pyrrole- and imidazole-based CCK2 antagonists.

A new molecular modeling approach has been used to derive a pharmacophore of the potent and selective cholecystokinin-2 (CCK(2)) receptor antagonist 5 (JB93182), based on features shared with two related series. The technique uses "field points" as simple and effective descriptions of the electrostatic and van der Waals maxima and minima surrounding a molecule equipped with XED (extended electron distribution) charges. Problems associated with the high levels of biliary elimination of 5 in vivo required us to design a compound with significantly lower molecular weight without sacrificing its nanomolar levels of in vitro activity. Two new series of compounds were designed to mimic the arrangement of field points present in the pharmacophore rather than its structural elements. In a formal sense, two of the three amides in 5 were replaced with either a simple pyrrole or imidazole, while some features thought to be essential for the high levels of in vitro activity of the parent compounds were retained and others deleted. These compounds maintained activity and selectivity for this receptor over CCK(1). In addition, the reduction in molecular weight coupled with lower polarities greatly reduced levels of biliary elimination associated with 5. This makes them good lead compounds for development of drug candidates whose structures are not obviously related to those of the parents and represents the first example of scaffold hopping using molecular field points.

Optimization of the in vitro and in vivo properties of a novel series of 2,4,5-trisubstituted imidazoles as potent cholecystokinin-2 (CCK2) antagonists.

The systematic optimization of the structure of a novel 2,4,5-trisubstituted imidazole-based cholecystokinin-2 (CCK(2)) receptor antagonist afforded analogues with nanomolar receptor affinity. These compounds were now comparable in their potency to the bicyclic heteroaromatic-based compounds 5 (JB93182) and 6 (JB95008), from which the initial examples were designed using a field-point based molecular modeling approach. They were also orally active as judged by their inhibition of pentagastrin stimulated acid secretion in conscious dogs, in contrast to the bicyclic heteroaromatic-based compounds, which were ineffective because of biliary elimination. Increasing the hydrophilicity through replacement of a particular methylene group with an ether oxygen, as in 3-{[5-(adamantan-1-yloxymethyl)-2-cyclohexyl-1H-imidazole-4-carbonyl]amino}benzoic acid (53), had little effect on the receptor affinity but significantly increased the oral potency. Comparison of the plasma pharmacokinetics and the inhibition of pentagastrin-stimulated acid output following bolus intraduodenal administration of both 53 and 6 indicated that 53 was well absorbed, had a longer half-life, and was not subject to the elimination pathways of the earlier series.

CCK(2) receptor antagonists containing the conformationally constrained phenylalanine derivatives, including the new amino acid Xic.

The conformationally constrained analogues of phenylalanine, tetrahydroisoquinoline-3-carboxylic acid (Tic), Sic, Hic and Nic, and the new amino acid Xic have been incorporated into a potent and highly selective cholecystokinin-2 (CCK(2)) receptor antagonist (2) in place of the phenylalanine residue, producing compounds 15a-e. High selectivities for CCK(2) over CCK(1) were observed for compounds 15a-e. The in vitro profile of the analogue containing the Nic residue (15d) was identical to that of compound 2, whereas the alternative conformational constraints resulted in a significant loss of affinity. The apparent advantage of Nic in the context of these CCK(2) ligands was subsequently demonstrated to be statistically significant.

omega-(Imidazol-4-yl)alkane-1-sulfonamides: a new series of potent histamine H(3) receptor antagonists.

omega-(1H-Imidazol-4-yl)alkane-1-sulfonamides were prepared and found to be potent histamine H(3) receptor antagonists. High receptor affinity and a low difference in the data between the bioassays were achieved with 5-(1H-imidazol-4-yl)pentane-1-sulfonic acid 4-chlorobenzylamide (16). Good in vitro profiles were also obtained for 2-hydroxysulfonamide and vinylsulfonamide analogues. This complements and completes the existing set of imidazole-based sulfonamides and sulfamides.