Discovery of Potent KIFC1 Inhibitors Using a Method of Integrated High-Throughput Synthesis and Screening

KIFC1 (HSET), a member of the kinesin-14 family of motor proteins, plays an essential role in centrosomal bundling in cancer cells, but its function is not required for normal diploid cell division. To explore the potential of KIFC1 as a therapeutic target for human cancers, a series of potent KIFC1 inhibitors featuring a phenylalanine scaffold was developed from hits identified through high-throughput screening (HTS). Optimization of the initial hits combined both design–synthesis–test cycles and an integrated high-throughput synthesis and biochemical screening method. An important aspect of this integrated method was the utilization of DMSO stock solutions of compounds registered in the corporate compound collection as synthetic reactants. Using this method, over 1500 compounds selected for structural diversity were quickly assembled in assay-ready 384-well plates and were directly tested after the necessary dilutions. Our efforts led to the discovery of a potent KIFC1 inhibitor, <b>AZ82</b>, which demonstrated the desired centrosome declustering mode of action in cell studies

Identification and Optimization of Benzimidazole Sulfonamides as Orally Bioavailable Sphingosine 1‑Phosphate Receptor 1 Antagonists with in Vivo Activity

We report here a novel series of benzimidazole sulfonamides that act as antagonists of the S1P₁ receptor, identified by exploiting an understanding of the pharmacophore of a high throughput screening (HTS)-derived series of compounds described previously. Lead compound <b>2</b> potently inhibits S1P-induced receptor internalization in a cell-based assay (EC₅₀ = 0.05 μM), but has poor physical properties and metabolic stability. Evolution of this compound through structure–activity relationship development and property optimization led to <i>in vivo</i> probes such as <b>4</b>. However, this compound was unexpectedly found to be a potent CYP3A inducer in human hepatocytes, and thus further chemistry efforts were directed at addressing this liability. By employing a pregnane X receptor (PXR) reporter gene assay to prioritize compounds for further testing in human hepatocytes, we identified lipophilicity as a key molecular property influencing the likelihood of P450 induction. Ultimately, we have identified compounds such as <b>46</b> and <b>47</b>, which demonstrate the desired S1P₁ antagonist activity while having greatly reduced risk of CYP3A induction in humans. These compounds have excellent oral bioavailability in preclinical species and exhibit pharmacodynamic effects of S1P₁ antagonism in several <i>in vivo</i> models following oral dosing. Relatively modest antitumor activity was observed in multiple xenograft models, however, suggesting that selective S1P₁ antagonists would have limited utility as anticancer therapeutics as single agents

Identification and Optimization of Benzimidazole Sulfonamides as Orally Bioavailable Sphingosine 1‑Phosphate Receptor 1 Antagonists with in Vivo Activity

We report here a novel series of benzimidazole sulfonamides that act as antagonists of the S1P₁ receptor, identified by exploiting an understanding of the pharmacophore of a high throughput screening (HTS)-derived series of compounds described previously. Lead compound <b>2</b> potently inhibits S1P-induced receptor internalization in a cell-based assay (EC₅₀ = 0.05 μM), but has poor physical properties and metabolic stability. Evolution of this compound through structure–activity relationship development and property optimization led to <i>in vivo</i> probes such as <b>4</b>. However, this compound was unexpectedly found to be a potent CYP3A inducer in human hepatocytes, and thus further chemistry efforts were directed at addressing this liability. By employing a pregnane X receptor (PXR) reporter gene assay to prioritize compounds for further testing in human hepatocytes, we identified lipophilicity as a key molecular property influencing the likelihood of P450 induction. Ultimately, we have identified compounds such as <b>46</b> and <b>47</b>, which demonstrate the desired S1P₁ antagonist activity while having greatly reduced risk of CYP3A induction in humans. These compounds have excellent oral bioavailability in preclinical species and exhibit pharmacodynamic effects of S1P₁ antagonism in several <i>in vivo</i> models following oral dosing. Relatively modest antitumor activity was observed in multiple xenograft models, however, suggesting that selective S1P₁ antagonists would have limited utility as anticancer therapeutics as single agents

Discovery of a Novel Class of Dimeric Smac Mimetics as Potent IAP Antagonists Resulting in a Clinical Candidate for the Treatment of Cancer (AZD5582)

A series of dimeric compounds based on the AVPI motif of Smac were designed and prepared as antagonists of the inhibitor of apoptosis proteins (IAPs). Optimization of cellular potency, physical properties, and pharmacokinetic parameters led to the identification of compound <b>14</b> (AZD5582), which binds potently to the BIR3 domains of cIAP1, cIAP2, and XIAP (IC₅₀ = 15, 21, and 15 nM, respectively). This compound causes cIAP1 degradation and induces apoptosis in the MDA-MB-231 breast cancer cell line at subnanomolar concentrations in vitro. When administered intravenously to MDA-MB-231 xenograft-bearing mice, <b>14</b> results in cIAP1 degradation and caspase-3 cleavage within tumor cells and causes substantial tumor regressions following two weekly doses of 3.0 mg/kg. Antiproliferative effects are observed with <b>14</b> in only a small subset of the over 200 cancer cell lines examined, consistent with other published IAP inhibitors. As a result of its in vitro and in vivo profile, <b>14</b> was nominated as a candidate for clinical development