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H3B-5942 in combination with palbocyclib shows synergy in vitro and in vivo

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Supplementary Figure from Covalent ERα Antagonist H3B-6545 Demonstrates Encouraging Preclinical Activity in Therapy-Resistant Breast Cancer

Supplementary Data from Covalent ERα Antagonist H3B-6545 Demonstrates Encouraging Preclinical Activity in Therapy-Resistant Breast Cancer

Enriched GSEA pathways

Nearly 30% of patients with relapsed breast cancer present activating mutations in estrogen receptor alpha (ERα) that confer partial resistance to existing endocrine-based therapies. We previously reported the development of H3B-5942, a covalent ERα antagonist that engages cysteine-530 (C530) to achieve potency against both wild-type (ERαWT) and mutant ERα (ERαMUT). Anticipating that the emergence of C530 mutations could promote resistance to H3B-5942, we applied structure-based drug design to improve the potency of the core scaffold to further enhance the antagonistic activity in addition to covalent engagement. This effort led to the development of the clinical candidate H3B-6545, a covalent antagonist that is potent against both ERαWT/MUT, and maintains potency even in the context of ERα C530 mutations. H3B-6545 demonstrates significant activity and superiority over standard-of-care fulvestrant across a panel of ERαWT and ERαMUT palbociclib sensitive and resistant models. In summary, the compelling preclinical activity of H3B-6545 supports its further development for the potential treatment of endocrine therapy–resistant ERα+ breast cancer harboring wild-type or mutant ESR1, as demonstrated by the ongoing clinical trials (NCT03250676, NCT04568902, NCT04288089). Summary: H3B-6545 is an ERα covalent antagonist that exhibits encouraging preclinical activity against CDK4/6i naïve and resistant ERαWT and ERαMUT tumors.

Mutations in estrogen receptor alpha (ERα) are detected in up to 30% of breast cancer patients who have relapsed during endocrine therapy. ERα mutations functionally confer resistance to existing classes of endocrine therapies, likely through gaining constitutive activity. The fact that current ER-directed therapies are only partially effective in the ERα mutant setting, and that a significant proportion of resistant breast cancer metastases continue to remain dependent on ERα signaling for growth/survival, highlights the critical need to develop the next generation of ERα antagonists that can overcome aberrant ERα activity. Using structure-based drug design approaches we have identified a novel class of ERα antagonist referred to as Selective ERα Covalent Antagonist (SERCA) that inactivate both wild-type and mutant ERα by targeting a unique cysteine residue that is not conserved among other steroid hormone receptors. Biophysical, biochemical and cellular analyses confirm the covalent mechanism of action, specific binding to ER and selective inhibition of ERα-dependent transcription of SERCAs. H3B-6545 is a highly selective SERCA that potently antagonizes wild-type and mutant ERα in biochemical and cell based assays demonstrating increased potency over standard of care and other experimental agents. In vivo, H3B-6545 shows superior efficacy to fulvestrant in the MCF-7 xenograft model with once daily oral dosing, achieving maximal antitumor activity at doses >10x below the maximum tolerated dose in mice. In addition, H3B-6545 shows superior antitumor activity to both tamoxifen and fulvestrant in patient derived xenograft models of breast cancer carrying estrogen receptor mutations. In summary, H3B-6545 is a first-in-class, orally available and selective ER covalent antagonist with a compelling pre-clinical profile that is being developed for the treatment of ERα positive breast cancer. Citation Format: Korpal M, Puyang X, Furman C, Zheng GZ, Banka D, Wu J, Zhang Z, Thomas M, Mackenzie C, Yao H, Rimkunas V, Kumar P, Caleb B, Karr C, Subramanian V, Irwin S, Larsen N, Vaillancourt F, Nguyen T-V, Davis A, Chan B, Hao MH, O'Shea M, Prajapati S, Agoulnik S, Kuznetsov G, Kumar N, Yu Y, Lai G, Hart A, Eckley S, Fekkes P, Bowser T, Joshi JJ, Selvaraj A, Wardell S, Norris J, Smith S, Reynolds D, Mitchell L, Wang J, Yu L, Kim A, Rioux N, Sahmoud T, Warmuth M, Smith PG, Zhu P. Development of a first-in-class oral selective ERα covalent antagonist (SERCA) for the treatment of ERαWT and ERαMUT breast cancer [abstract]. In: Proceedings of the 2017 San Antonio Breast Cancer Symposium; 2017 Dec 5-9; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2018;78(4 Suppl):Abstract nr P1-10-08.

A total synthesis of the natural product 6-deoxypladienolide D (1) has been achieved. Two noteworthy attributes of the synthesis are (1) a late-stage allylic oxidation which proceeds with full chemo-, regio-, and diastereoselectivity and (2) the development of a scalable and cost-effective synthetic route to support drug discovery efforts. 6-Deoxypladienolide D (1) demonstrates potent growth inhibition in a mutant SF3B1 cancer cell line, high binding affinity to the SF3b complex, and inhibition of pre-mRNA splicing.

A series of 1,2,3,6-tetrahydropyridyl-4-carboxamides, exemplified by 6, have been synthesized and evaluated for in vitro TRPV1 antagonist activity, and in vivo analgesic activity in animal pain models. The tetrahydropyridine 6 is a novel TRPV1 receptor antagonist that potently inhibits receptor-mediated Ca2+ influx in vitro induced by several agonists, including capsaicin, N-arachidonoyldopamine (NADA), and low pH. This compound penetrates the CNS and shows potent anti-nociceptive effects in a broad range of animal pain models upon oral dosing due in part to its ability to antagonize both central and peripheral TRPV1 receptors. The SAR leading to the discovery of 6 is presented in this report.

The synthesis and structure-activity relationship of 1-(aryl)-3-(4-(amino)benzyl)urea transient receptor potential vanilloid 1 (TRPV1) antagonists are described. A variety of cyclic amine substituents are well tolerated at the 4-position of the benzyl group on compounds containing either an isoquinoline or indazole heterocyclic core. These compounds are potent antagonists of capsaicin activation of the TRPV1 receptor in vitro. Analogues, such as compound 45, have been identified that have good in vivo activity in animal models of pain. Further optimization of 45 resulted in compound 58 with substantially improved microsome stability and oral bioavailability, as well as in vivo activity.

Fatty acid amide hydrolase (FAAH) is the primary regulator of several bioactive lipid amides including anandamide. Inhibitors of FAAH are potentially useful for the treatment of pain, anxiety, depression, and other nervous system disorders. However, FAAH inhibitors must display selectivity for this enzyme relative to the numerous other serine hydrolases present in the human proteome in order to be therapeutically acceptable. Here we employed activity-based protein profiling (ABPP) to assess the selectivity of FAAH inhibitors in multiple rat and human tissues. We discovered that some inhibitors, including carbamate compounds SA-47 and SA-72, and AM404 are exceptionally selective while others, like URB597, BMS-1, OL-135, and LY2077855 are less selective, displaying multiple off-targets. Since proteins around 60kDa constitute the major off-targets for URB597 and several other FAAH inhibitors with different chemical structures, we employed the multi-dimensional protein identification technology (MudPIT) approach to analyze their identities. We identified multiple carboxylesterase isozymes as bona fide off-targets of FAAH inhibitors. Consistently, enzymatic assay confirmed inhibition of carboxylesterase activities in rat liver by FAAH inhibitors. Since carboxylesterases hydrolyze a variety of ester-containing drugs and prodrugs, we speculate that certain FAAH inhibitors, by inhibiting carboxylesterases, might have drug-drug interactions with other medicines if developed as therapeutic agents.

We have discovered a novel, potent, and selective triazafluorenone series of metabotropic glutamate receptor 1 (mGluR1) antagonists with efficacy in various rat pain models. Pharmacokinetic and pharmacodynamic profiles of these triazafluorenone analogs revealed that brain/plasma ratios of these mGluR1 antagonists were important to achieve efficacy in neuropathic pain models. This correlation could be used to guide our in vivo SAR (structure–activity relationship) modification. For example, compound 4a has a brain/plasma ratio of 0.34, demonstrating only moderate efficacy in neuropathic pain models. On the other hand, antagonist 4b with a brain/plasma ratio of 2.70 was fully efficacious in neuropathic pain models.

Novel 5,6-fused heteroaromatic ureas were synthesized and evaluated for their activity as TRPV1 antagonists. It was found that 4-aminoindoles and indazoles are the preferential cores for the attachment of ureas. Bulky electron-withdrawing groups in the para-position of the aromatic ring of the urea substituents imparted the best in vitro potency at TRPV1. The most potent derivatives were assessed in in vivo inflammatory and neuropathic pain models. Compound 46, containing the indazole core and a 3,4-dichlorophenyl group appended to it via a urea linker, demonstrated in vivo analgesic activity upon oral administration. This derivative also showed selectivity versus other receptors in the CEREP screen and exhibited acceptable cardiovascular safety at levels exceeding the therapeutic dose.

SAR (structure-activity relationship) studies of triazafluorenone derivatives as potent mGluR1 antagonists are described. The triazafluorenone derivatives are non-amino acid derivatives and noncompetitive mGluR1 antagonists that bind at a putative allosteric recognition site located within the seven-transmembrane domain of the receptor. These triazafluorenone derivatives are potent, selective, and systemically active mGluR1 antagonists. Compound 1n, for example, was a very potent mGluR1 antagonist (IC50 = 3 nM) and demonstrated full efficacy in various in vivo animal pain models.

4-Amino-5,7-disubstituted pyridopyrimidines are potent, non-nucleoside inhibitors of adenosine kinase (AK). We recently identified a potent, orally efficacious analog, 4 containing a 7-pyridylmorpholine substituted ring system as the key structural element of this template. In this report, we disclose the pharmacologic effects of five- and six-membered heterocyclic ring replacements for the pyridine ring in 4. These replacements were found to have interesting effects on in vivo efficacy and genotoxicity as well as in vitro potency. We discovered that the nitrogen in the heterocyclic ring at C(7) is important for the modulation of mutagenic side effects (Ames assay).

We have discovered that polar 7-substituents of pyridopyrimidine derivatives affect not only whole cell AK inhibitory potency, but also selectivity in causing locomotor side effects in vivo animal models. We have identified compound, 1o, which has potent whole cell AK inhibitory potency, analgesic activity and minimal reduction of locomotor activity.

3-(Triphenylgermyl)propanoic acid was synthesized through several reaction steps from germanium tetrachloride. Aromatic GeC bonds in the compound can be cleaved selectively by bromine and the β-carboxylic functional group shows some unusual properties.

Tropinone (6) was deprotonated with lithium diisopropylamide and with chiral lithium amides (18–24) and the resulting enolates (two enantiomers) were treated with electrophiles. The aldol reaction ...

Tropinone (6) was deprotonated with lithium diisopropylamide and with chiral lithium amides (18–24) and the resulting enolates (two enantiomers) were treated with electrophiles. The aldol reaction with benzaldehyde and deuteration were both diastereoselective. The former yielded only one isomer (exo, anti) of the aldol 8a; the latter proceeded from the exo face. This selectivity permitted us to probe the deprotonation of tropinone with lithium amides; it was concluded that the reaction involves predominantly the exo axial protons. The reaction of tropinone enolate with ethyl chloroformate led, via a ring opening, to the cycloheptenone derivative 9. The reaction with methyl cyanoformate yielded, in the presence of silver acetate and acetic acid, the β-ketoester 8b; however, in the absence of these additives, and especially when 12-crown-4 was added to the enolate, a ring opening leading to the pyrrolidine derivative 10 occurred instead. Deprotonation of tropinone with chiral lithium amides proceeded with modest enantioselectivity. A synthesis of non-racemic anhydroecgonine via this strategy allowed establishing the absolute stereochemistry of deprotonation.

Novel transient receptor potential vanilloid 1 (TRPV1) receptor antagonists with various bicyclic heteroaromatic pharmacophores were synthesized, and their in vitro activity in blocking capsaicin activation of TRPV1 was assessed. On the basis of the contribution of these pharmacophores to the in vitro potency, they were ranked in the order of 5-isoquinoline > 8-quinoline = 8-quinazoline > 8-isoquinoline > or = cinnoline approximately phthalazine approximately quinoxaline approximately 5-quinoline. The 5-isoquinoline-containing compound 14a (hTRPV1 IC50 = 4 nM) exhibited 46% oral bioavailability and in vivo activity in animal models of visceral and inflammatory pain. Pharmacokinetic and pharmacological properties of 14a are substantial improvements over the profile of the high-throughput screening hit 1 (hTRPV1 IC50 = 22 nM), which was not efficacious in animal pain models and was not orally bioavailable.

The unambiguous structure of the title compound (which was formed in the reaction of the lithium enolate of tropinone with benzaldehyde) has been determined to be the exo-anti diastereoisomer. The piperidine ring adopts a flattened-chair conformation with the carbonyl group pushed away from the ethylene bridge. There is an intermolecular hydrogen bond between the hydroxyl group and the N atom which replaces the intramolecular O-H...N hydrogen bond indicated by solution NMR data

A novel series of pyridopyrimidine analogues 9 was identified as potent adenosine kinase inhibitors based on the SAR and computational studies. Substitution of the C7 position of the pyridopyrimidino core with C2' substituted pyridino moiety increased the in vivo potency and enhanced oral bioavailability of these adenosine kinase inhibitors.