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Homozygous deletion of the tumor suppressor gene CDKN2A and the neighboring MTAP gene located at chr9p21 occurs in 10-15% of human cancers. Deletion of MTAP, an enzyme in methionine and adenine salvage pathways, results in accumulation of its substrate MTA, which is structurally similar to SAM, the substrate methyl donor for the type II methyltransferase PRMT5. In MTAP deleted cells, MTA competes with SAM for binding to PRMT5, placing PRMT5 in a partially inhibited or hypormorphic state. Multiple studies using shRNAi knockdown have shown that tumor cell lines harboring MTAP deletions are vulnerable to PRMT5 inhibition. PRMT5 inhibitors that have advanced to clinical studies do not selectively target the MTA-bound form of PRMT5, and the preclinical activity of these molecules is not enriched in MTAP-deleted tumor cells lines. Moreover, the therapeutic window of these molecules is narrow, presumably due to the inhibition of PRMT5 in normal cells. We set out to identify PRMT5 inhibitors that bind cooperatively with MTA, with the goal of selectively targeting PRMT5 in MTAP-deleted tumors. A DNA encoded library screen was conducted to identify small molecules that preferentially bind to PRMT5 in the presence of MTA. The subsequent optimization of screening hits to improve potency, MTA-cooperativity, and pharmacokinetic properties led to the identification of AM-9747. The nature of the MTA cooperativity of AM-9747 was interrogated by multiple biophysical methods and structural biology experiments. Following treatment with AM-9747, the levels of SDMA marks were lower in HCT116 MTAP-deleted cells (IC50 = 0.0002 μM) compared to HCT116 MTAP-WT cells (IC50 = 0.050 μM). AM-9747 selectively inhibited the proliferation of HCT116 MTAP-deleted cells (IC50 = 0.027 μM) compared to HCT116 MTAP-WT cells (IC50 = 0.63 μM). The profiling of AM-9747 in an expanded panel of tumor cell lines demonstrated that AM-9747 inhibited the proliferation of most MTAP-deleted cells, with minimal effects on MTAP-WT cells. In vitro mechanism of action studies demonstrated that treatment with AM-9747 induces DNA damage, as illustrated by increased phosphorylation of H2AX, and an arrest in the G2/M phase of the cell cycle in MTAP-deleted cells. In vivo, oral administration of AM-9747 selectively inhibits SDMA and tumor growth in HCT116 MTAP-deleted tumor xenografts, compared to HCT116 MTAP-WT xenografts. Furthermore, treatment with AM-9747 inhibits the growth of multiple MTAP-deleted tumor xenograft models, including BXPC3 (PDAC) and DOHH2 (DLBCL). AM-9747 was profiled against a panel of over twenty PDX models, with greater than 50% tumor growth inhibition observed in the majority of PDX models harboring deletion of the MTAP gene. Our data with AM-9747 indicates that PRMT5 inhibitors that selectively target PRMT5 in cooperation with MTA may represent a novel and compelling therapeutic strategy for the treatment of MTAP-deleted cancers. Citation Format: Brian Belmontes, Antonia Policheni, Siyuan Liu, Katherine Slemmons, Jodi Moriguchi, Hayley Ma, Daniel Aiello, Yajing Yang, Mikkel Vestergaard, Sanne Cowland, Jan Anderson, Ian Sarvary, Nuria Tamayo, Liping Pettus, Susmith Mukund, Leszek Pope, Jennifer R. Allen, Sanne Glad, Matthew Bourbeau, Paul E. Hughes. The discovery and preclinical characterization of the MTA cooperative PRMT5 inhibitor AM-9747 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1807.

The synthetic approach to a functionalized hydrochrysene skeleton is described. The route is highlighted by a Birch reduction–alkylation sequence and a 6-exo-trig radical cyclization.

Fragmentation reactions of keto iodolactones 4 provide access to butenolides 5 , 2-alkyl-4-hydroxy-2-cyclohexen-1-ones 6 , and butyrolactones 9 . Δ α, β -Butenolides 5e and 5f were converted to heterocycles 14–16 by way of intramolecular cycloaddition reactions.

The highly diastereoselective potassium in ammonia reduction−ethylation (EtI) of the chiral 2-(trimethylsilyl)benzamide 1b to give 1,4-cyclohexadiene 3 is the key step in asymmetric syntheses of (−)-eburnamonine (4) and (−)-aspidospermidine (5). Cyclohexadiene 3 was converted to cyclohexanone 7, which provided the trimethylsilyl-substituted butyrolactone 9 utilized for the synthesis of 4 and butyrolactone 13 required for the synthesis of 5. The preparation of 9 depended upon the completely regioselective silicon-directed Baeyer−Villiger oxidation 7 → 8; Baeyer−Villiger oxidation of the cyclohexenone 10 also was regioselective to give the desired enol lactone 11 in 92% yield. Remarkable diastereoselectivity was observed for the kinetically controlled cyclization of the acyl imminium ion derived from the vinyl-substituted carboxaldehyde 16b; treatment of 16b with 5 equiv of CF3CO2H in CH2Cl2 at −55 °C gave an 18:1 mixture of 17 and its C(3) β-epimer in 93% yield. The oxidation of alcohol 18 containing sensi...

Birch reduction-alkylation of the chiral 2-(trimethylsilyl)benzamide 4 provides 1,4-cyclohexadienes 5b-5e with diastereomer ratios of >100:1. The conversions of 5b-5e to the 4,4-disubstituted-2-cyclohexen-1-ones 8b-8e are described.

Birch reduction-alkylation of the chiral 2-(trimethylsilyl)benzamide 4 provides 1,4-cyclohexadienes 5b-5e with diastereomer ratios of >100:1. The conversions of 5b-5e to the 4,4-disubstituted-2-cyclohexen-1-ones 8b-8e are described.

The highly diastereoselective potassium in ammonia reduction−ethylation (EtI) of the chiral 2-(trimethylsilyl)benzamide 1b to give 1,4-cyclohexadiene 3 is the key step in asymmetric syntheses of (−)-eburnamonine (4) and (−)-aspidospermidine (5). Cyclohexadiene 3 was converted to cyclohexanone 7, which provided the trimethylsilyl-substituted butyrolactone 9 utilized for the synthesis of 4 and butyrolactone 13 required for the synthesis of 5. The preparation of 9 depended upon the completely regioselective silicon-directed Baeyer−Villiger oxidation 7 → 8; Baeyer−Villiger oxidation of the cyclohexenone 10 also was regioselective to give the desired enol lactone 11 in 92% yield. Remarkable diastereoselectivity was observed for the kinetically controlled cyclization of the acyl imminium ion derived from the vinyl-substituted carboxaldehyde 16b; treatment of 16b with 5 equiv of CF3CO2H in CH2Cl2 at −55 °C gave an 18:1 mixture of 17 and its C(3) β-epimer in 93% yield. The oxidation of alcohol 18 containing sensi...

2-Alkyl- and 2,4-dialkyl-3-iodo-1-oxocyclohexan-2,4-carbolactones undergo lithium hydroxide- and lithium alkoxide-induced fragmentation reactions to provide butenolides, gamma-hydroxycyclohexenones, and/or gamma-butyrolactones. In general, product distribution is governed by two factors: (1) the nature of nucleophiles and (2) the steric bulkiness of the substituents at C-2 and C-4 of the cyclohexanones. Lithium hydroxide-induced fragmentation provides butenolides and gamma-hydroxycyclohexenones. In contrast, lithium alkoxide-promoted fragmentation results in predominantly 5-substituted gamma-butyrolactones along with a small amount of butenolides in limited cases. Fragmentation products induced by lithium hydroxide are largely influenced by the steric bulkiness of the substituents at C-2 and C-4 of the cyclohexanone ring. The bulky substituents render the exclusive formation of butenolides.