Discovery of 5,7-Dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-ones as Highly Selective CDK2 Inhibitors.

pubs.acs.org/acsmedchemlett Letter Discovery of 5,7-Dihydro‑6H‑pyrrolo[2,3‑d]pyrimidin-6-ones as Highly Selective CDK2 Inhibitors Alexander Sokolsky,* Sarah Winterton, Keith Kennedy, Katherine Drake, Kristine Stump, Lu Huo, Yvonne Lo, Min Ye, Maryanne Covington, Sharon Diamond, Yan-ou Yang, Sunkyu Kim, Swamy Yeleswaram, Liangxing Wu, and Wenqing Yao Cite This: ACS Med. Chem. Lett. 2022, 13, 1797−1804 ACCESS Metrics & More Read Online Article Recommendations sı Supporting Information * ABSTRACT: A series of exceptionally selective CDK2 inhibitors are described. Starting from an HTS hit, we successfully scaffold hopped to a 5,7-dihydro-6H-pyrrolo[2,3-d]pyrimidin-6-one core structure, which imparted a promising initial selectivity within the CDK family. Extensive further SAR identified additional factors that drove selectivity to above 200× for CDKs 1/4/6/7/9. General kinome selectivity was also greatly improved. Finally, use of in vivo metabolite identification allowed us to pinpoint sulfonamide dealkylation as the primary metabolite, which was ameliorated through the deuterium effect. KEYWORDS: CDK2, metabolism, kinase, biotransformation T he cyclin-dependent kinases (CDKs) comprise a large family of serine/threonine kinases involved in the regulation of cell cycle progression via the binding of specific cyclins. The promise of inhibiting cell division has made the CDKs a long-standing target in oncology.1,2 Early work led to the identification of numerous pan-selective inhibitors, with several compounds progressing into clinical trials (Figure 1).3−6 However, toxicities associated with certain CDK family members, in particular CDK17 and CDK9, led to the search for more selective inhibitors. Blockade of CDK4/6 has proven to be particularly successful; e.g., palbociclib (Ibrance), ribociclib (Kisqali), and abemaciclib (Verzenio) are all FDA approved Figure 2. Initial hit and scaffold merging design. drugs to treat HR+/HER2- metastatic breast cancer.8 Recent research indicates that resistance to CDK4/6-based therapies frequently proceeds through a compensatory pathway involving CDK2.9,10 Therefore, selective inhibition of CDK2 represents a promising potential therapeutic option for patients progressing after CDK4/6 therapy. In addition, a number of cancers are driven by CDK2 hyperactivity11 or amplification of CCNE1,12−14 the gene responsible for producing the cyclin partner to CDK2, cyclin E. Although a number of preclinical compounds with varying degrees of CDK2 selectivity have been reported,15,16 at the time we started this work, there were no reports of a CDK2 Received: August 30, 2022 Accepted: October 3, 2022 Published: October 6, 2022 Figure 1. Pan-selective CDK inhibitors in clinical trials. © 2022 American Chemical Society 1797 https://doi.org/10.1021/acsmedchemlett.2c00408 ACS Med. Chem. Lett. 2022, 13, 1797−1804 ACS Medicinal Chemistry Letters pubs.acs.org/acsmedchemlett Letter The program started with a high throughput screen of our in house compound collection. Compounds were screened in a CDK2/cyclin E1 homogeneous time-resolved fluorescence (HTRF) binding assay using eIF4E-binding protein-1 peptide as substrate, at 1 mM ATP concentration. A number of structurally distinct hits were identified. Two in particular are shown in Figure 2. Pyrimidine pyrazole 1a was the parent compound of a series of related congeners with varying substitution at the 1-position of the pyrazole. Given the tolerance for diverse pyrazole substitution, we reasoned that the combination of the aryl sulfonamide and an appropriately placed lipophilic substituent off of the 4-position of the pyrimidine were responsible for the potency of 1a. Compound 1b gave credence to this hypothesis. Albeit less potent, the cyclohexyl substituent was located in a similar area of space as the pyrazole, likely enforced by the rigid framework of the tricycle. In an effort to access distinct chemical space, we merged these two scaffolds as shown in Figure 2, in essence opening the pyrrole ring of 1b and replacing it with the 4aminobenzenesulfonamide of 1a.20,21 We then set about identifying a bicyclic scaffold that would allow us to project an appropriate substituent to the pocket shared by 1a and 1b. Select examples of our heterocycle screen can be seen in Table 1, with CDK1 serving as an initial surrogate of CDK family selectivity. For synthetic ease, we chose to use the unsubstituted sulfonamide as a starting point. Cyclic lactam 2a was an initial indication that our hypothesis had potential, with reasonable potency against CDK2 and promising levels of selectivity against CDK1. However, it was reversing the lactam that provided us with our first significant breakthrough. Potency was greatly enhanced for both the γ- and δ-lactam. But it was the selectivity profile that proved truly surprising: the γ-lactam was 10× less active against CDK1! The source of this selectivity, especially versus the δ-lactam, remains unclear, but it supports our design hypothesis that conformational rigidity would be beneficial to distinguishing minor differences in CDK family binding pockets. Table 1. Bicyclic Core Screen a Measured at 1 mM [ATP]. Figure 3. 1-Methanesulfonyl-4-aminopiperdine 3a as a route to improved hWB potency. inhibitor with excellent potency and selectivity across the CDK family.17−19 We thus set out to identify a single-digit nanomolar CDK2 inhibitor possessing excellent (>100×) selectivity against CDK1, CDK4, CDK6, CDK7, and CDK9 in the primary enzyme assay, to test in vivo effects of CDK2 inhibition independently from CDK4/6. Table 2. Initial SAR on the Sulfonylpiperidine-Substituted Pyrimidine Lactam Series a Measured at 1 mM [ATP]. 1798 https://doi.org/10.1021/acsmedchemlett.2c00408 ACS Med. Chem. Lett. 2022, 13, 1797−1804 ACS Medicinal Chemistry Letters pubs.acs.org/acsmedchemlett Letter Table 3. Optimization of the Pyrimidine Lactam (R2) and Benzenesulfonamide (R1) Enhances hWB Potency and Selectivity a Measured at 1 mM [ATP]. heterocycloalkyl replacements proved to be significantly less active and/or completely devoid of selectivity. In fact, of the groups surveyed, only 1-methanesulfonyl-4-aminopiperdine23 (3a, Figure 3) showed a promising level of selectivity, albeit the cutoff for our CDK1 assay precluded the exact number from being determined. In spite of the diminished potency (∼20× relative to the aminobenzenesulfonamide), 3a showed considerable promise. We reasoned that the lack of a primary sulfonamide would provide improved translation from the cellular to the whole blood setting if sufficient potency could be achieved. We were also drawn to the aminopiperidine as a potential source of general kinome selectivity, as sp3 substitution is rarely tolerated in the hinge region of kinases. We first elected to keep the 1-methylsulfonylpiperdine ' constant and explored the SAR on the lactam (R3, R3 ) (Table 2, entries 2−4). With even small modifications (e.g (...truncated)