EZM0414 is a potent, selective, orally bioavailable SETD2 inhibitor (IC50=18 nM in SETD2 biochemical assay; IC50=34 nM in cellular assay). EZM0414 is indicated for the study of relapsed or refractory multiple myeloma and diffuse large B-cell lymphoma.

Physicochemical Properties

Molecular Formula	C22H29FN4O2
Molecular Weight	400.49
Exact Mass	400.227
Elemental Analysis	C, 67.26; H, 7.76; F, 4.43; N, 13.07; O, 7.47
CAS #	2411748-50-8
Related CAS #	EZM0414 TFA;2411759-92-5
PubChem CID	146395245
Appearance	Off-white to light yellow solid powder
LogP	2.8
Hydrogen Bond Donor Count	2
Hydrogen Bond Acceptor Count	4
Rotatable Bond Count	3
Heavy Atom Count	29
Complexity	610
Defined Atom Stereocenter Count	2
SMILES	C1C=C(C2NC(=CC=2C=1F)C(N[C@@H]1CCC[C@H](N2CCN(CC2)C(=O)C)C1)=O)C
InChi Key	PGNLXEBQMQHFNK-SJORKVTESA-N
InChi Code	InChI=1S/C22H29FN4O2/c1-14-6-7-19(23)18-13-20(25-21(14)18)22(29)24-16-4-3-5-17(12-16)27-10-8-26(9-11-27)15(2)28/h6-7,13,16-17,25H,3-5,8-12H2,1-2H3,(H,24,29)/t16-,17+/m1/s1
Chemical Name	N-[(1R,3S)-3-(4-acetylpiperazin-1-yl)cyclohexyl]-4-fluoro-7-methyl-1H-indole-2-carboxamide
Synonyms	EZM0414; 2411748-50-8; KCY37T9RXU; EZM-0414; UNII-KCY37T9RXU; N-[(1R,3S)-3-(4-acetylpiperazin-1-yl)cyclohexyl]-4-fluoro-7-methyl-1H-indole-2-carboxamide; N-((1R,3S)-3-(4-Acetylpiperazin-1-yl)cyclohexyl)-4-fluoro-7-methyl-1H-indole-2-carboxamide; 1H-Indole-2-carboxamide, N-((1R,3S)-3-(4-acetyl-1-piperazinyl)cyclohexyl)-4-fluoro-7-methyl-;
HS Tariff Code	2934.99.9001
Storage	Powder-20°C 3 years 4°C 2 years In solvent -80°C 6 months -20°C 1 month Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture and light.
Shipping Condition	Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Biological Activity

Solubility Data

Targets	SETD2 (IC50=18 nM)
ln Vitro	EZM0414 suppresses the growth of a panel of MM and DLBCL cell lines with an IC50 of 0.24 μM for t(4;14) cells and 0.023 μM->10 μM for DLBCL cell lines [2]. Inhibition of SETD2 by EZM0414 results in potent anti-proliferative effects in a panel of MM and DLBCL cell lines. EZM0414 inhibited proliferation in both t(4;14) and non-t(4;14) MM cell lines, with higher anti-proliferative activity generally observed in the t(4;14) subset of MM cell lines. The median IC 50value for EZM0414 in t(4;14) cell lines was 0.24 μM as compared to 1.2 μM for non-t(4;14) MM cell lines. Additionally, inhibitory growth effects on DLBCL cell lines demonstrated a wide range of sensitivity with IC 50 values from 0.023 μM to >10 μM. [2] In vitro testing of EZM0414 in a safety panel consisting of 47 targets and a diversity panel of 72 kinases showed IC50 > 25 μM for all targets except D2 (IC50 = 13.0 μM, antagonist) and 5-HT1B (IC50 = 3.2 μM, agonist).[3]
ln Vivo	EZM0414 (15 and 30 mg/kg, po, BID, Daily) suppresses tumor growth in a NOD SCID cell xenograft model of the human KMS-11 and is well tolerated [3]. EZM0414 (50 mg/kg, exhibited bucally) had almost 100% bioavailability in the stent and mouse, with t1/2 of 1.8 hours (mouse) and 3.8 hours (mouse) [3]. EZM0414 resulted in statistically significant potent antitumor activity compared to the vehicle control in three MM and four DLBCL cell line-derived xenograft models. In the t(4;14) MM cell line-derived xenograft model, KMS-11, robust tumor growth regressions were observed at the top two doses with maximal TGI of 95%. In addition, two non-t(4;14) MM (RPMI-8226, MM.1S) and two DLBCL xenograft models (TMD8, KARPAS422) demonstrated > 75% TGI; with two additional DLBCL models (WSU-DLCL2, SU-DHL-10) exhibiting > 50% TGI in response to EZM0414. In all models tested, the antitumor effects observed correlated with reductions in intratumoral H3K36me3 levels demonstrating on-target inhibition of SETD2 methyltransferase activity in vivo. [2]
Enzyme Assay	SETD2 (1434-1711) Assay [3] The biochemical assay monitored the incorporation of the tritiated methyl group from S-adenosyl-methionine (SAM) into a biotinylated histone 3 peptide corresponding to residues 26-40. The sequence of the substrate peptide is biotin-Ahx-RKSAPATGGVKKPHR-NH2 and 3H-SAM was purchased from American Radiolabeled Chemicals, Inc. For the assay, 40 L of enzyme was incubated with 1 L of compound or DMSO for 30 minutes before initiating the reaction with 10 L of substrate solution in a 384- well assay plate. The assay was performed at room temperature in assay buffer composed of 25 mM bicine, pH 8.0, 7.5 mM -mercaptoethanol, 0.002 % Tween-20, and 0.01 % bovine skin gelatin (BSG). The reaction was quenched during the linear portion of product formation with 10 L of 1 mM S-adenosyl-homocysteine (SAH) and 1 mM SAM. From the quenched reaction, 50 L was transferred to a streptavidin-coated Flashplate (Perkin Elmer) and incubated for at least 2h before washing once with 0.1 % Tween-20. Signal from the 3H-labeled peptide captured by the streptavidin-coated plates was counted by a Topcount plate reader. Percent inhibition (%I) and IC50 values were calculated using equations 1 and 2 respectively. %	Solubility (In Vitro) DMSO : ~120 mg/mL (~299.63 mM) H2O : < 0.1 mg/mL Solubility (In Vivo) 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. Injection Formulations (e.g. IP/IV/IM/SC) 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). 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) Oral Formulations 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). 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 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.  (Please use freshly prepared in vivo formulations for optimal results.) Preparing Stock Solutions 1 mg 5 mg 10 mg 1 mM 2.4969 mL 12.4847 mL 24.9694 mL 5 mM 0.4994 mL 2.4969 mL 4.9939 mL 10 mM 0.2497 mL 1.2485 mL 2.4969 mL *Note: Please select an appropriate solvent for the preparation of stock solution based on your experiment needs. For most products, DMSO can be used for preparing stock solutions (e.g. 5 mM, 10 mM, or 20 mM concentration); some products with high aqueous solubility may be dissolved in water directly. Solubility information is available at the above Solubility Data section. Once the stock solution is prepared, aliquot it to routine usage volumes and store at -20°C or -80°C. Avoid repeated freeze and thaw cycles.