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 study, the therapeutic effect of EZH2 inhibitors was limited to the diffuse large B-cell lymphoma (DLBCL) cell line tested (Figure 1A). We wanted to explore whether a method for directly degrading EZH2 using PROTAC technology could be developed to enhance its targeting ability against other types of lymphoma cells. In this study, we developed PROTAC-based EZH2 degraders and investigated their EZH2 degradation efficiency and therapeutic effects against different types of lymphoma in vitro and in vivo. Our study showed that only VHL-targeting compounds could degrade EZH2 through suitable linker groups of 7 or 9 atoms in length. Compared to the parent EZH2 inhibitor EPZ6438, our two optimal EZH2 degraders, YM181 and YM281, showed higher selectivity for EZH2 than EZH1 and exhibited potent antiproliferative activity in both DLBCL and other types of lymphoma cell lines. Furthermore, at effective doses, the EZH2 degraders significantly inhibited the in vivo growth of lymphoma xenografts without significant toxicity. However, YM281 and YM181 exhibit incomplete EZH2 degradation at low concentrations and limited efficacy in inhibiting cell viability, indicating room for further optimization. Future research may necessitate structure-activity relationship studies on different linker backbones with similar linker lengths to YM181 and YM281 to obtain more effective EZH2 degraders. Furthermore, some EZH2-independent tumorigenesis cancer cells, such as pancreatic cancer cells AsPC1 and lung cancer cells NCI-H460, are insensitive to YM181 and YM281, despite these compounds reducing their EZH2 levels (Figure S6). Cell permeability measurements using Caco-2 cells showed that the apparent permeability of both YM181 and YM281 was significantly lower than that of their parent EZH2 inhibitor EPZ6438 (Table S2), suggesting the need for further structural optimization to improve their oral bioavailability. Overall, our results suggest that EZH2 degraders may have better therapeutic potential than EZH2 inhibitors in treating lymphoma. We are investigating the exact mechanism of action of EZH2 degraders and their application in other cancers. [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.)