Previous Lead Compound Research Articles

Novel inhibitors of PARP1 and PARP14: design, synthesis, and potentiation of cisplatin efficacy in cancer.

Poly(ADP-ribose) polymerase (PARP) is a superfamily of enzymes involved in cell survival. Both PARP1 and PARP14 are overexpressed in malignancies. No clinically approved PARP14 inhibitors are available, and PARP1 inhibitors are generally nonspecific, resulting in a need for a more diverse library of selective PARP1 and PARP14 inhibitors. Based on the previous lead compounds 1 and 2, 26 novel compounds were designed, synthesized, and screened against PARP1 and PARP14. Compounds with the best in vitro inhibitory results were further screened against PARP2, PARP3, PARP5a, PARP7, and PARP15. The 26 novel compounds demonstrated a lesser inhibitory effect than the lead compounds. Compounds 1 and 2 were further investigated using in vitro cell viability assays, which revealed that cells treated with either lead PARP inhibitor and cisplatin in combination had significantly lower survival rates than those treated with cisplatin alone. At 10 µM, the combination showed more significant cell survival reduction, suggesting greater inhibition of PARP increases lethality, particularly in HeLa and PC-3 cell lines at 96h and beyond.

Identification of Spiro[chromene-2,4'-piperidine]s as Potent, Selective, and Gq-Biased 5-HT2C Receptor Partial Agonists.

A series of spiropiperidines was designed and synthesized by structural modifications based on our previous lead compound 1 and evaluated with cellular signaling assays for the discovery of 5-HT2C receptor (5-HT2CR) selective agonists with a Gq bias. Structure-activity relationship (SAR) studies of spiropiperidines uncovered spiro[chromene-2,4'-piperidine]s as a novel chemotype of 5-HT2CR selective agonists. Among this new series, the 7-chloro analogue 8 was identified as the most potent and selective 5-HT2CR partial agonist (Emax = 71.09%) with an EC50 value of 121.5 nM and no observed activity toward 5-HT2AR or 5-HT2BR. Moreover, compound 8 exhibited no recruitment activity for β-arrestin and showed low inhibition of hERG at 10 μM. These findings may pave the way to develop more potent Gq-biased 5-HT2CR partial agonists as useful pharmacological tool compounds or potential drug candidates.

Design, synthesis and evaluation of 2-pyrimidinylindole derivatives as anti-obesity agents by regulating lipid metabolism

Obesity, a global pandemic posing a growing threat to human health, necessitates the development of effective and safe anti-obesity agents. Our previous studies highlighted the lipid-lowering effects of indolylquinazoline Bouchardatine and its derivatives. In this study, we employed scaffold hopping and simplification strategies to design and synthesize two new series derivatives by modifying the D ring. Extensive discussions have been conducted regarding the structure-activity relationship between lipid-lowering activity and the new compounds. These discussions have resulted in the discovery of 2-pyrimidinylindole derivatives as a promising scaffold for anti-obesity treatment. The new 2-pyrimidinylindole derivatives exhibited comparable lipid-lowering activity to the previously reported indolylquinazoline derivatives, including SYSU-3d and R17, with reduced toxicity. The most potent compound, 5a, demonstrated a larger therapeutic index, improved aqueous solubility and oral bioavailability compared to the previous lead compounds. In vivo evaluation indicated that 5a effectively reduced lipid accumulation in adipose tissue, improved glucose tolerance, and mitigated insulin resistance and liver function damage caused by a high-fat and high-cholesterol diet. Mechanism studies indicated that 5a may regulate lipid metabolism through the modulation of the PPARγ signaling pathway. Overall, our study has identified a highly active compound 5a, and provided the basis for further development of 2-pyrimidinylindole as a promising scaffold for obesity treatment.

Discovery of N-(4-(Aminomethyl)phenyl)-5-methylpyrimidin-2-amine Derivatives as Potent and Selective JAK2 Inhibitors.

The JAK2V617F mutation leads to JAK2 autophosphorylation and activation of downstream pathways, eventually resulting in myeloproliferative neoplasms (MPNs). Selective inhibitors showed advantages in terms of side effects; therefore, there is an urgent need to develop novel selective JAK2 inhibitors for treating MPNs. In this study, we described a series of N-(4-(aminomethyl)phenyl)pyrimidin-2-amine derivatives as selective JAK2 inhibitors. Systematic exploration through opening the tetrahydroisoquinoline based on the previous lead compound 13ac led to the discovery of the optimal compound A8. Compound A8 showed excellent potency on JAK2 kinase, with an IC50 value of 5 nM, and inhibited the phosphorylation of JAK2 and its downstream signaling pathway. Moreover, A8 exhibited 38.6-, 54.6-, and 41.2-fold selectivity for JAK1, JAK3, and TYK2, respectively. Compared to the lead compound, A8 demonstrated much better metabolic stabilities, with a bioavailability of 41.1%. These findings suggest that A8 is a relatively selective JAK2 inhibitor, deserving to be developed for treating MPNs.

Balancing Nonsense Mutation Readthrough and Toxicity of Designer Aminoglycosides for Treatment of Genetic Diseases.

New derivatives of aminoglycosides with a side chain 1,2-aminoalcohol at the 5" position of ring III were designed, synthesized, and biologically evaluated. The novel lead structure (compound 6), exhibiting substantially enhanced selectivity toward eukaryotic versus prokaryotic ribosome, high readthrough activity, and considerably lower toxicity than the previous lead compounds, was discovered. Balanced readthrough activity and toxicity of 6 were demonstrated in three different nonsense DNA-constructs underlying the genetic diseases, cystic fibrosis and Usher syndrome, and in two different cell lines, baby hamster kidney and human embryonic kidney cells. Molecular dynamics simulations within the A site of the 80S yeast ribosome demonstrated a remarkable kinetic stability of 6, which potentially determines its high readthrough activity.

Discovery of N,4-Di(1H-pyrazol-4-yl)pyrimidin-2-amine-Derived CDK2 Inhibitors as Potential Anticancer Agents: Design, Synthesis, and Evaluation.

Cyclin-dependent kinase 2 (CDK2) has been garnering considerable interest as a target to develop new cancer treatments and to ameliorate resistance to CDK4/6 inhibitors. However, a selective CDK2 inhibitor has yet to be clinically approved. With the desire to discover novel, potent, and selective CDK2 inhibitors, the phenylsulfonamide moiety of our previous lead compound 1 was bioisosterically replaced with pyrazole derivatives, affording a novel series of N,4-di(1H-pyrazol-4-yl)pyrimidin-2-amines that exhibited potent CDK2 inhibitory activity. Among them, 15 was the most potent CDK2 inhibitor (Ki = 0.005 µM) with a degree of selectivity over other CDKs tested. Meanwhile, this compound displayed sub-micromolar antiproliferative activity against a panel of 13 cancer cell lines (GI50 = 0.127-0.560 μM). Mechanistic studies in ovarian cancer cells revealed that 15 reduced the phosphorylation of retinoblastoma at Thr821, arrested cells at the S and G2/M phases, and induced apoptosis. These results accentuate the potential of the N,4-di(1H-pyrazol-4-yl)pyrimidin-2-amine scaffold to be developed into potent and selective CDK2 inhibitors for the treatment of cancer.

Validation of Recombinant Heparan Sulphate Reagents for CNS Repair.

Therapies that target the multicellular pathology of central nervous system (CNS) disease/injury are urgently required. Modified non-anticoagulant heparins mimic the heparan sulphate (HS) glycan family and have been proposed as therapeutics for CNS repair since they are effective regulators of numerous cellular processes. Our in vitro studies have demonstrated that low-sulphated modified heparan sulphate mimetics (LS-mHeps) drive CNS repair. However, LS-mHeps are derived from pharmaceutical heparin purified from pig intestines, in a supply chain at risk of shortages and contamination. Alternatively, cellular synthesis of heparin and HS can be achieved using mammalian cell multiplex genome engineering, providing an alternative source of recombinant HS mimetics (rHS). TEGA Therapeutics (San Diego) have manufactured rHS reagents with varying degrees of sulphation and we have validated their ability to promote repair in vitro using models that mimic CNS injury, making comparisons to LS-mHep7, a previous lead compound. We have shown that like LS-mHep7, low-sulphated rHS compounds promote remyelination and reduce features of astrocytosis, and in contrast, highly sulphated rHS drive neurite outgrowth. Cellular production of heparin mimetics may, therefore, offer potential clinical benefits for CNS repair.

Development of sulfonamide-based NLRP3 inhibitors: Further modifications and optimization through structure-activity relationship studies

NLRP3 inflammasome dysregulation has been observed in many human diseases including neurodegenerative disorders. Thus, development of small molecule inhibitors targeting this protein complex represents a promising strategy to achieve disease intervention. In our continuing efforts to develop NLRP3 inhibitors, a recently identified lead inhibitor, YQ128, was further modified and optimized. The structure-activity relationship studies of this lead compound suggested its flexibility for structural modifications while the sulfonamide and benzyl moiety demonstrated being important for selectivity. Additionally, the systematic SAR studies also provided insights for designing NLRC4 and AIM2 inflammasome inhibitors. A new lead inhibitor, 19, was identified with improved potency (IC50: 0.12 ± 0.01 μM) and binding affinity (KD: 84 nM). Further characterization of this lead compound using wild type and nlrp3−/− mice confirmed its in vivo selective target engagement. PET studies using a radiotracer based on the structure of 19 also demonstrated its improved brain penetration compared to previous lead compounds. These results strongly encourage further testing of 19 in disease models.

Synthesis and evaluation of dual fatty acid amide hydrolase-monoacylglycerol lipase inhibition and antinociceptive activities of 4-methylsulfonylaniline-derived semicarbazones

Fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL) are promising targets for neuropathic pain and other CNS disorders. Based on our previous lead compound SIH 3, we designed and synthesized a series of 4-methylsulfonylphenyl semicarbazones and evaluated for FAAH and MAGL inhibition properties. Most of the compounds showed potency towards both enzymes with leading FAAH selectivity. Compound (Z)-2-(2,6-dichlorobenzylidene)-N-(4-(methylsulfonyl)phenyl)hydrazine-1-carboxamide emerged as the lead inhibitor against both FAAH (IC50 = 11 nM) and MAGL (IC50 = 36 nM). The lead inhibitor inhibited FAAH by non-competitive mode, but showed a mixed-type inhibition against MAGL. Molecular docking study unveiled that the docked ligands bind favorably to the active sites of FAAH and MAGL. The lead inhibitor interacted with FAAH and MAGL via π–π stacking via phenyl ring and hydrogen bonding through sulfonyl oxygen atoms or amide NH. Moreover, the stability of docked complexes was rationalized by molecular simulation studies. PAMPA assay revealed that the lead compound is suitable for blood–brain penetration. The lead compound showed better cell viability in lipopolysaccharide-induced neurotoxicity assay in SH-SY5Y cell lines. Further, in-vivo experiments unveiled that dual inhibitor was safe up to 2000 mg/kg with no hepatotoxicity. The dual FAAH-MAGL inhibitor produced significant anti-nociceptive effect in the CCI model of neuropathic pain without altering locomotion activity. Lastly, the lead compound exhibited promising ex-vivo FAAH/MAGL inhibition activity at the dose of 10 mg/kg and 20 mg/kg. Thus, these findings suggest that the semicarbazone-based lead compound can be a potential template for the development of agents for neuropathic pain.

Synthesis and evaluation of cyclic diamino benzamide based D3 receptor ligands

Dopamine (1) is a key neurotransmitter whose impact on pharmacological processes is mediated by a family of dopamine receptors designated D1, D2, D3, D4, and D5. Various diseases and conditions such as schizophrenia, drug abuse, depression, restless leg syndrome, Parkinson’s disease (PD), and inflammatory diseases have been linked to aberrant D3 activity. Herein, we report a series of novel D3 ligands with improved solubility over our previous lead compound, MC25-41 (2).

In Silico Structure-Guided Optimization and Molecular Simulation Studies of 3-Phenoxy-4-(3-trifluoromethylphenyl)pyridazines as Potent Phytoene Desaturase Inhibitors.

A series of novel 3-phenoxy-4-(3-trifluoromethylphenyl)pyridazines 2–5 were designed, based on the structure of our previous lead compound 1 through the in silico structure-guided optimization approach. The results showed that some of these new compounds showed a good herbicidal activity at the rate of 750 g ai/ha by both pre- and post-emergence applications, especially compound 2a, which displayed a comparable pre-emergence herbicidal activity to diflufenican at 300–750 g ai/ha, and a higher post-emergence herbicidal activity than diflufenican at the rates of 300–750 g ai/ha. Additionally, 2a was safe to wheat by both pre- and post-emergence applications at 300 g ai/ha, showing the compound’s potential for weed control in wheat fields. Our molecular simulation studies revealed the important factors involved in the interaction between 2a and Synechococcus PDS. This work provided a lead compound for weed control in wheat fields.

Discovery of a Potent Thieno[2,3-d]pyrimidine-2,4-dione-Based Protoporphyrinogen IX Oxidase Inhibitor through an In Silico Structure-Guided Optimization Approach.

A key objective for herbicide research is to develop new compounds with improved bioactivity. Protoporphyrinogen IX oxidase (PPO) is an essential target for herbicide discovery. Here, we report using an in silico structure-guided optimization approach of our previous lead compound 1 and designed and synthesized a new series of compounds 2-6. Systematic bioassays led to the discovery of a highly potent compound 6g, 1-methyl-3-(2,2,7-trifluoro-3-oxo-4-(prop-2-yn-1-yl)-3,4-dihydro-2H-benzo[b][1,4]oxazin-6-yl)thieno[2,3-d]pyrimidine-2,4(1H,3H)-dione, which exhibited an excellent and wide spectrum of weed control at the rates of 30-75 g ai/ha by the postemergence application and is relatively safe on maize at 75 g ai/ha. Additionally, the Ki value of 6g to Nicotiana tabacum PPO (NtPPO) was found to be 2.5 nM, showing 3-, 12-, and 18-fold higher potency relative to compound 1 (Ki = 7.4 nM), trifludimoxazin (Ki = 31 nM), and flumioxazin (Ki = 46 nM), respectively. Furthermore, molecular simulations further suggested that the thieno[2,3-d]pyrimidine-2,4-dione moiety of 6g could form a more favorable π-π stacking interaction with the Phe392 of NtPPO than the heterocyclic moiety of compound 1. This study provides an effective strategy to obtain enzyme inhibitors with improved performance through molecular simulation and structure-guided optimization.

Design and development of trifluoromethylated enaminone derivatives as potential anticonvulsants

Enaminone derivatives have been widely studied as potential anticonvulsant agents to treat epilepsy. Our research group is focused on evaluating the anticonvulsant activities of cyclic enaminones connected to a substituted aromatic ring via a sp2 hybridized linker. We herein report our lead optimization efforts and the subsequent synthesis of the target fluorinated N-benzamide enaminone analog N-(3-oxo-5-(trifluoromethyl) cyclohex-1-en-1-yl)-4-(trifluoromethyl) benzamide (compound 7) from fluorinated intermediates, and its bioevaluation in acute seizure rodent models. The synthesis of the fluorinated cycloalkyl intermediates, 5-(trifluoromethyl) cyclohexane-1,3-dione (compound 5) and 3-amino-5-(trifluoromethyl) cyclohex-2-en-1-one (compound 6) were derived from an improve method of a one-pot hydrolysis and acid catalyzed decarboxylation of 4-carbo-tert-butoxy-5-trifluoromethylcyclohexane-1,3-dione (compound 4). Compound 7 produced pharmacological response at a higher dose of 300 mg/kg with only 25% protection observed, when compared to our previous lead compound THA40 in the 6 Hz 44 mA animal model. In the maximal electroshock (MES) model, compound 7 showed moderate activity at 100 mg/kg with 25% after 2 h. At a higher dose of 300 mg/kg, 25% of the animals were protected both at 0.5 h and 2 h. Although compound 7 did not produce the expected results, it allowed for a new avenue to investigate during our lead optimization studies.

A drug repurposing screen identifies hepatitis C antivirals as inhibitors of the SARS-CoV2 main protease.

Effective SARS-CoV-2 antiviral drugs are desperately needed. The SARS-CoV-2 main protease (Mpro) appears as an attractive target for drug development. We show that the existing pharmacopeia contains many drugs with potential for therapeutic repurposing as selective and potent inhibitors of SARS-CoV-2 Mpro. We screened a collection of ~6,070 drugs with a previous history of use in humans for compounds that inhibit the activity of Mpro in vitro and found ~50 compounds with activity against Mpro. Subsequent dose validation studies demonstrated 8 dose responsive hits with an IC50 ≤ 50 μM. Hits from our screen are enriched with hepatitis C NS3/4A protease targeting drugs including boceprevir, ciluprevir. narlaprevir, and telaprevir. This work suggests previous large-scale commercial drug development initiatives targeting hepatitis C NS3/4A viral protease should be revisited because some previous lead compounds may be more potent against SARS-CoV-2 Mpro than boceprevir and suitable for rapid repurposing.

Phenylthiazoles with nitrogenous side chain: An approach to overcome molecular obesity

A novel series of phenylthiazoles bearing cyclic amines at the phenyl-4 position was prepared with the objective of decreasing lipophilicity and improving the overall physicochemical properties and pharmacokinetic profile of the compounds. Briefly, the piperidine ring (compounds 10 and 12) provided the best ring size in terms of antibacterial activity when tested against 16 multidrug-resistant clinical isolates. Both compounds were superior to vancomycin in the ability to eliminate methicillin-resistant Staphylococcus aureus (MRSA), residing within infected macrophages and to disrupt mature MRSA biofilm. Additionally, compounds 10 and 12 exhibited a fast-bactericidal mode of action in vitro. Furthermore, the new derivatives were 160-times more soluble in water than the previous lead compound 1b. Consequently, compound 10 was orally bioavailable with a highly-acceptable pharmacokinetic profile in vivo that exhibited a half-life of 4 h and achieved a maximum plasma concentration that exceeded the minimum inhibitory concentration (MIC) values against all tested bacterial isolates.

Exploring small molecules with pan-genotypic inhibitory activities against hepatitis C virus NS3/4A serine protease.

Among the many Hepatitis C virus (HCV) genotypes and subtypes, genotypes 1b and 3a are most prevalent in United States and Asia, respectively. A total of 132 commercially available analogs of a previous lead compound were initially investigated against wild-type HCV genotype 1b NS3/4A protease. Ten compounds showed inhibitory activities (IC50 values) below 10 µM with comparable direct binding affinities (KD values) determined by surface plasmon resonance (SPR). To identify pan-genotypic inhibitors, these ten selected compounds were tested against four additional genotypes (1a, 2a, 3a, and 4) and three drug-resistant mutants (A156S, R155K, and V36M). Four new analogs have been identified with better activities against all five tested genotypes than the prior lead compound. Further, the original lead compound did not show activity against genotype 3a NS3/4A, whereas four newly identified compounds exhibited IC50 values below 33 µM against genotype 3a NS3/4A. Encouragingly, the best new compound F1813-0710 possessed promising activity toward genotype 3a, which is a huge improvement over the previous lead compound that had no effect on genotype 3a. This intriguing observation was further analyzed by molecular docking and molecular dynamics (MD) simulations to understand their different binding interactions, which should benefit future pan-genotypic inhibitor design and drug discovery.

Benzimidazole-based DGAT1 inhibitors with a [3.1.0] bicyclohexane carboxylic acid moiety

Previously disclosed benzimidazole-based DGAT1 inhibitors containing a cyclohexane carboxylic acid moiety suffer from isomerization at the alpha position of the carboxylic acid group, generating active metabolites which exhibit DGAT1 inhibition comparable to the corresponding parent compounds. In this report, we describe the design, synthesis and profiling of benzimidazole-based DGAT1 inhibitors with a [3.1.0] bicyclohexane carboxylic acid moiety. Our results show that single isomer 3A maintains in vitro and in vivo inhibition against DGAT1. In contrast to previous lead compounds, 3A does not undergo isomerization during in vitro hepatocyte incubation study or in vivo mouse study.

Characterization and structure-activity relationships of indenoisoquinoline-derived topoisomerase I inhibitors in unsilencing the dormant Ube3a gene associated with Angelman syndrome

BackgroundAngelman syndrome (AS) is a severe neurodevelopmental disorder lacking effective therapies. AS is caused by mutations in ubiquitin protein ligase E3A (UBE3A), which is genomically imprinted such that only the maternally inherited copy is expressed in neurons. We previously demonstrated that topoisomerase I (Top1) inhibitors could successfully reactivate the dormant paternal allele of Ube3a in neurons of a mouse model of AS. We also previously showed that one such Top1 inhibitor, topotecan, could unsilence paternal UBE3A in induced pluripotent stem cell-derived neurons from individuals with AS. Although topotecan has been well-studied and is FDA-approved for cancer therapy, its limited CNS bioavailability will likely restrict the therapeutic use of topotecan in AS. The goal of this study was to identify additional Top1 inhibitors with similar efficacy as topotecan, with the expectation that these could be tested in the future for safety and CNS bioavailability to assess their potential as AS therapeutics.MethodsWe tested 13 indenoisoquinoline-derived Top1 inhibitors to identify compounds that unsilence the paternal allele of Ube3a in mouse neurons. Primary cortical neurons were isolated from embryonic day 14.5 (E14.5) mice with a Ube3a-YFP fluorescent tag on the paternal allele (Ube3am+/pYFP mice) or mice that lack the maternal Ube3a allele and hence model AS (Ube3am−/p+ mice). Neurons were cultured for 7 days, treated with drug for 72 h, and examined for paternal UBE3A protein expression by Western blot or fluorescence immunostaining. Dose responses of the compounds were determined across a log range of drug treatments, and cytotoxicity was tested using a luciferase-based assay.ResultsAll 13 indenoisoquinoline-derived Top1 inhibitors unsilenced paternal Ube3a. Several compounds exhibited favorable paternal Ube3a unsilencing properties, similar to topotecan, and of these, indotecan (LMP400) was the most effective based on estimated Emax (maximum response of unsilencing paternal Ube3a) and EC50 (half maximal effective concentration).ConclusionsWe provide pharmacological profiles of indenoisoquinoline-derived Top1 inhibitors as paternal Ube3a unsilencers. All 13 tested compounds were effective at unsilencing paternal Ube3a, although with variable efficacy and potency. Indotecan (LMP400) demonstrated a better pharmacological profile of Ube3a unsilencing compared to our previous lead compound, topotecan. Taken together, indotecan and its structural analogues are potential AS therapeutics whose translational potential in AS treatment should be further assessed.

Novel Highly Potent and Metabolically Resistant Oxoeicosanoid (OXE) Receptor Antagonists That Block the Actions of the Granulocyte Chemoattractant 5-Oxo-6,8,11,14-Eicosatetraenoic Acid (5-oxo-ETE).

5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a potent lipid mediator that induces tissue eosinophilia via the selective OXE receptor (OXE-R), which is an attractive therapeutic target in eosinophilic diseases. We previously identified indole OXE-R antagonists that block 5-oxo-ETE-induced primate eosinophil activation. Although these compounds possess good oral absorption, their plasma levels decline rapidly due to extensive oxidation of their hexyl side chain. We have now succeeded in dramatically increasing antagonist potency and resistance to metabolism by replacing the hexyl group with phenylpentyl or phenylhexyl side chains. Compared with our previous lead compound S-230, our most potent antagonist, S-C025, has an IC50 (120 pM) over 80 times lower and a substantially longer plasma half-life. A single major metabolite, which retains antagonist activity (IC50, 690 pM) and has a prolonged lifetime in plasma was observed. These new highly potent OXE-R antagonists may provide a novel strategy for the treatment of eosinophilic disorders like asthma.

Optimization of Orally Bioavailable Enhancer of Zeste Homolog 2 (EZH2) Inhibitors Using Ligand and Property-Based Design Strategies: Identification of Development Candidate (R)-5,8-Dichloro-7-(methoxy(oxetan-3-yl)methyl)-2-((4-methoxy-6-methyl-2-oxo-1,2-dihydropyridin-3-yl)methyl)-3,4-dihydroisoquinolin-1(2H)-one (PF-06821497)

A new series of lactam-derived EZH2 inhibitors was designed via ligand-based and physicochemical-property-based strategies to address metabolic stability and thermodynamic solubility issues associated with previous lead compound 1. The new inhibitors incorporated an sp3 hybridized carbon atom at the 7-position of the lactam moiety present in lead compound 1 as a replacement for a dimethylisoxazole group. This transformation enabled optimization of the physicochemical properties and potency compared to compound 1. Analysis of relationships between calculated logD (clogD) values and in vitro metabolic stability and permeability parameters identified a clogD range that afforded an increased probability of achieving favorable ADME data in a single molecule. Compound 23a exhibited the best overlap of potency and pharmaceutical properties as well as robust tumor growth inhibition in vivo and was therefore advanced as a development candidate (PF-06821497). A crystal structure of 23a in complex with the three-protein PRC2 complex enabled understanding of the key structural features required for optimal binding.