Intermediate for PROTAC EZH2 degrader

Traditional EZH2 inhibitors are developed to suppress the enzymatic methylation activity, and they may have therapeutic limitations due to the nonenzymatic functions of EZH2 in cancer development. Here, we report proteolysis-target chimera (PROTAC)-based EZH2 degraders to target the whole EZH2 in lymphoma. Two series of EZH2 degraders were designed and synthesized to hijack E3 ligase systems containing either von Hippel-Lindau (VHL) or cereblon (CRBN), and some VHL-based compounds were able to mediate EZH2 degradation. Two best degraders, YM181 and YM281, induced robust cell viability inhibition in diffuse large B-cell lymphoma (DLBCL) and other subtypes of lymphomas, outperforming a clinically used EZH2 inhibitor EPZ6438 (tazemetostat) that was only effective against DLBCL. The EZH2 degraders displayed promising antitumor activities in lymphoma xenografts and patient-derived primary lymphoma cells. Our study demonstrates that EZH2 degraders have better therapeutic activity than EZH2 inhibitors, which may provide a potential anticancer strategy to treat lymphoma [1].

[1]. Design, Synthesis, and Evaluation of VHL-Based EZH2 Degraders to Enhance Therapeutic Activity against Lymphoma. J Med Chem. 2021 Jul 22;64(14):10167-10184.

In this work, the therapeutic efficacy of EZH2 inhibitors was limited to DLBCL cell lines among the tested lymphoma cell lines (Figure 1A). We wonder whether the direct EZH2 degradation via PROTAC technology could be developed to improve their targeting capacity to the other types of lymphoma cells. In our current study, we developed PROTAC-based EZH2 degraders and investigated their efficiency of EZH2 degradation and therapeutic efficacy in various types of lymphoma in vitro and in vivo. Our study revealed that only VHL-targeting compounds enabled the EZH2 degradation with an appropriate linker at 7 or 9 atoms length. Compared to the parental EZH2 inhibitor EPZ6438, our two best EZH2 degraders YM181 and YM281 selectively degraded EZH2 over EZH1, and they exhibited effective antiproliferative activity both in DLBCL and other types of lymphoma cell lines. Furthermore, the EZH2 degrader showed an apparent advantage to prevent in vivo tumor growth in lymphoma xenografts without obvious toxicity at the efficacious doses.
However, the incomplete EZH2 degradation and the modest cellular potencies for YM281 and YM181 in the inhibition of cell viability at low concentrations suggest that there is still room for further optimization. In the future, a structure and activity relationship study on different linker scaffoldings with the same linking length as YM181 and YM281 may be warranted to obtain more potent EZH2 degraders. Meanwhile, cancer cells that may not depend on EZH2 for their tumorigenesis, for example, pancreatic cancer cell AsPC1 and lung cancer cell NCI-H460, are not sensitive to YM181 and YM181, although the compounds were able to decrease their EZH2 levels (Figure S6). By measuring cell permeability with Caco-2 cells, both YM181 and YM281 showed their apparent permeability ability largely compromised compared to the parental EHZ2 inhibitor EPZ6438 (Table S2), indicating the importance to improve their oral bioavailability through further structural optimization. Overall, our results demonstrate that EZH2 degraders may have better therapeutic potential than EZH2 inhibitors against lymphomas. The exact mechanism of action and the application of our EZH2 degraders in other cancers are under investigation.[1]

C30H35N3O5

517.616008043289

517.257

2685873-44-1

165437231

Off-white to yellow solid powder

4.2

3

6

8

38

946

0

MIEXIPQREXYLRA-UHFFFAOYSA-N

InChI=1S/C30H35N3O5/c1-5-33(24-10-12-38-13-11-24)27-16-23(21-6-8-22(9-7-21)30(36)37)15-25(20(27)4)28(34)31-17-26-18(2)14-19(3)32-29(26)35/h6-9,14-16,24H,5,10-13,17H2,1-4H3,(H,31,34)(H,32,35)(H,36,37)

4-[3-[(4,6-dimethyl-2-oxo-1H-pyridin-3-yl)methylcarbamoyl]-5-[ethyl(oxan-4-yl)amino]-4-methylphenyl]benzoic acid

Tazemetostat de(methyl morpholine)-COOH; 2685873-44-1; CHEMBL5398431; BDBM50633529;

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO :~220 mg/mL (~425.02 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)