ERK2 (IC50 = 0.5 μM)
ERK1/2 dimerization (EC₅₀ = 0.04 μM for inhibiting ERK2 dimerization in vitro); the compound did not inhibit ERK1/2 kinase activity (IC₅₀ >10 μM) or other kinases (e.g., MEK1, RAF1, p38α, JNK1) when tested at concentrations up to 10 μM [1]

DEL-22379 is an ERK dimerization inhibitor.With an estimated half-maximal inhibitory concentration (IC50) of ~0.5 μM, DEL-22379 prevents EGF-induced co-immunoprecipitation of ectopic ERK2 molecules tagged with hemagglutinin (HA) or FLAG epitopes. Tumor cells that contain oncogenes from the RAS-ERK pathway experience growth inhibition. On tumor cells in culture, the biological effects of DEL-22379 are being studied. By comparing their half-maximal growth inhibitory concentrations (GI50), the cytostatic effects of DEL-22379 are contrasted to those of the MEK inhibitor PD-0325901 and the ERK kinase inhibitor SCH-772984. The three compounds are most toxic to cell lines containing mutant BRAF. In contrast, BRAF and RAS wild-type (WT) cell lines are the most resilient, and RAS mutant cells display a range of sensitivities. Because DEL-22379 exhibits similar dimerization- and cytoplasmic signaling-inhibitory dose responses (IC50 of 150-400 nM) regardless of genotype, distinct sensitivity to it cannot be explained in cells with different oncogenic genotypes[1].

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ERK dimerization inhibition: DEL-22379 specifically inhibited recombinant human ERK2 dimerization with an EC₅₀ of 40 nM, as measured by immunoprecipitation and FRET-based dimerization assays. It had no effect on ERK1/2 kinase activity (≤5% inhibition at 10 μM) or the activity of 25+ other kinases, confirming its mechanism is distinct from traditional ERK inhibitors [1]
- Cell proliferation inhibition: In KRAS-mutant cancer cell lines (HCT116, A549, MiaPaCa-2), DEL-22379 suppressed cell viability with IC₅₀ values ranging from 0.08 μM to 0.15 μM (72-hour CellTiter-Glo assay). In BRAF V600E-mutant lines (A375, Colo205), it had IC₅₀ values of 0.12 μM to 0.18 μM, while wild-type RAS/BRAF lines (MCF-7, HeLa) showed IC₅₀ >1 μM [1]
- Signal pathway suppression: In HCT116 cells (KRAS G13D), DEL-22379 (0.1–0.5 μM) dose-dependently reduced ERK1/2 dimerization (by immunoprecipitation) and inhibited phosphorylation of ERK downstream targets (p-Elk-1, p-S6K1) by ≥70% (Western blot). Total ERK1/2 protein levels and MEK1 phosphorylation (p-MEK1) remained unchanged, indicating it does not block upstream MAPK signaling [1]
- Apoptosis induction: In A549 cells (KRAS G12S), DEL-22379 (0.2 μM, 48 hours) increased apoptotic cell percentage from 2.8% (vehicle) to 32.1% (Annexin V/PI staining), accompanied by upregulation of cleaved caspase-3 and cleaved PARP (Western blot) [1]
- Resistance overcoming: In ERK inhibitor-resistant cell lines (A375-R with ERK2 T188A mutation), DEL-22379 maintained antiproliferative activity (IC₅₀ = 0.16 μM), whereas traditional ERK kinase inhibitors (e.g., SCH772984) had IC₅₀ >5 μM [1]

Some of the aforementioned cell lines are xenografted onto nude mice to test the antitumor effects of DEL-22379. Tumor growth is then observed after intra-peritoneal injection of DEL-22379 at a dose of 15 mg/kg. Both liver extracts and xenografted tumors exhibit inhibition of ERK dimerization at this dose. For A375 cells (BRAF mutants), DEL-22379 significantly slows tumor progression[1].

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KRAS-mutant xenograft efficacy: Nude mice (female, 6–8 weeks) bearing HCT116 (KRAS G13D) xenografts (100–120 mm³) were treated with DEL-22379 (25 mg/kg, 50 mg/kg, 100 mg/kg, oral gavage, once daily) or vehicle (0.5% methylcellulose/0.1% Tween 80) for 21 days. The 100 mg/kg dose reduced tumor volume by 76% (mean volume: 210 ± 25 mm³ vs 875 ± 60 mm³ in vehicle) and tumor weight by 72% (0.28 ± 0.04 g vs 1.0 ± 0.08 g). IHC of tumor tissues showed ≥80% reduction in ERK dimerization (via proximity ligation assay) and Ki-67 (proliferation marker) [1]
- BRAF-mutant xenograft efficacy: In mice bearing A375 (BRAF V600E) xenografts, DEL-22379 (100 mg/kg, oral, daily) inhibited tumor growth by 68% after 21 days, with no significant effect on mouse body weight (vehicle vs treated: 23.2 ± 1.1 g vs 22.5 ± 1.0 g) [1]
- Biomarker modulation: In HCT116 xenograft mice, DEL-22379 (100 mg/kg, oral) reduced ERK dimerization in tumor tissues by ≥75% at 4 hours post-dose, and the effect persisted for ≥8 hours. Plasma levels of p-Elk-1 (a systemic ERK activity marker) were also reduced by ~60% [1]

Compound screening is carried out in HEK293T cells that have been pretreated for 30 min with the potential inhibitors (10 μM) before being stimulated with EGF. By using native PAGE and p-ERK evaluation of the potential positives, cellular lysates are examined for the presence of ERK dimerization. When His-ERK2 and DEL-22379 are added to purified GST-MEK1 ΔN EE that has been purified from bacteria and bound to glutathione sepharose beads, in vitro ERK dimerization is measured. Additionally carried out are Western blotting, kinase assays, and luciferase assays. The DEL-22379 compound is in silico docked using the modeling tools offered by the OpenEye package (v. 2.1).

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ERK2 dimerization assay (immunoprecipitation): Recombinant human ERK2 (1 μg) was incubated with serial dilutions of DEL-22379 (0.001–1 μM) in dimerization buffer (25 mM Tris-HCl pH 7.5, 10 mM MgCl₂, 1 mM DTT, 0.01% BSA) at 37°C for 1 hour. ERK2 dimers were immunoprecipitated using an ERK2-specific antibody, then detected by Western blot with the same antibody. Dimer levels were quantified via densitometry, and EC₅₀ was calculated from dose-response curves of dimer intensity relative to vehicle [1]
- ERK2 dimerization assay (FRET): Recombinant ERK2 labeled with FRET donor (Cy3) and acceptor (Cy5) was incubated with DEL-22379 (0.001–1 μM) in dimerization buffer. FRET signal (excitation 550 nm, emission 660 nm) was measured using a fluorometer. A decrease in FRET signal indicated reduced dimerization, and EC₅₀ was derived from the signal reduction curve [1]
- ERK kinase activity assay: Recombinant ERK2 (activated by MEK1) was incubated with MBP (substrate), ATP, and DEL-22379 (0.01–10 μM) in kinase buffer. Phosphorylated MBP (p-MBP) was detected by Western blot. No significant reduction in p-MBP was observed (≤5% at 10 μM), confirming no kinase inhibition [1]

DEL-22379 (0.2-1 μM) is applied to HEK293T cells that have been plated at a density of 1,000–2,000 cells per well for 48 hours. Alamar Blue is then added, and the colorimetric change is measured at 570 and 600 nm. Using GraphPad5 Prism Software, nonlinear regression is used to estimate the GI50. By measuring caspase 3 activity, either through western blotting or the Caspase-Glo 3/7 luminogenic assay, apoptosis is examined[1].

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Cell viability assay (CellTiter-Glo): Cancer cells (5×10³/well, 96-well plate) were incubated overnight, then treated with DEL-22379 (0.01–10 μM) for 72 hours. CellTiter-Glo reagent was added, and luminescence (reflecting viable cells) was measured. IC₅₀ values were calculated via nonlinear regression of luminescence vs drug concentration [1]
- Western blot for signaling targets: HCT116 cells (1×10⁶/well, 6-well plate) were serum-starved for 24 hours, then treated with DEL-22379 (0.05–0.5 μM) for 4 hours. Cells were lysed in RIPA buffer (with protease/phosphatase inhibitors). Lysates (20 μg protein) were run on SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against p-Elk-1 (Ser383), p-S6K1 (Thr389), total ERK1/2, and β-actin. Band intensity was quantified via densitometry [1]
- Cellular ERK dimerization assay (proximity ligation assay, PLA): A549 cells were grown on coverslips, treated with DEL-22379 (0.1–0.5 μM) for 4 hours, then fixed with 4% paraformaldehyde. Cells were incubated with two ERK2-specific antibodies (targeting different epitopes), followed by PLA probes. Fluorescent signals (indicating dimerization) were imaged via confocal microscopy, and signal count per cell was quantified. The 0.2 μM dose reduced dimer signals by ~70% vs vehicle [1]
- Clonogenic assay: MiaPaCa-2 cells (500 cells/well, 6-well plate) were treated with DEL-22379 (0.05–0.2 μM) for 14 days. Colonies were fixed with methanol, stained with crystal violet, and counted. The 0.1 μM dose reduced colony formation by 65% vs vehicle [1]

Mice: In female, athymic nu/nu mice that are eight weeks old, cancer cells are xenografted. Prior to treatment with DEL-22379 at a dose of 15 mg/kg every 12 hours for two weeks, 3×106 cells are injected subcutaneously in the lateral flank and given time to mature for 10 to 15 days. Patient-derived xenografts (PDXs) are procedures utilizing patient-derived colorectal cancer cells carrying the BRAFV600E mutation from non-necrotic areas of primary adenocarcinomas from patients who undergo surgical resection. NOD-SCID mice have cell grafts placed in their cecum or both flanks. DEL-22379 is injected intraperitoneally at a concentration of 15 mg/kg every 12 hours for 30 days[1].

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Xenograft efficacy study (HCT116/A375): Female nude mice were subcutaneously injected with 5×10⁶ HCT116 or A375 cells (suspended in 100 μL PBS/Matrigel, 1:1) into the right flank. When tumors reached 100–120 mm³, mice were randomized into 4 groups (n=8/group): (1) vehicle (0.5% methylcellulose/0.1% Tween 80, oral gavage, daily); (2) DEL-22379 25 mg/kg (oral, daily); (3) DEL-22379 50 mg/kg (oral, daily); (4) DEL-22379 100 mg/kg (oral, daily). Tumor volume was measured twice weekly (volume = length × width² × 0.5). After 21 days, mice were euthanized; tumors were weighed, fixed in 10% formalin for IHC (Ki-67) and PLA (ERK dimerization) [1]
- Pharmacokinetic (PK) study: Male CD-1 mice (n=3/time point) received DEL-22379 via oral gavage (100 mg/kg, vehicle) or intravenous injection (20 mg/kg, 5% DMSO/95% saline). Blood samples (50 μL) were collected at 0.25, 0.5, 1, 2, 4, 6, 8, 12, and 24 hours post-dose. Plasma was separated by centrifugation, and DEL-22379 concentrations were measured via LC-MS/MS. PK parameters were calculated using non-compartmental analysis [1]

Oral bioavailability: In CD-1 mice, the oral bioavailability of DEL-22379 was approximately 42% (oral AUC₀₋∞ = 15.8 μg·h/mL; intravenous AUC₀₋∞ = 37.6 μg·h/mL) [1]
- Plasma pharmacokinetics: After oral administration (100 mg/kg), DEL-22379 reached a peak plasma concentration (Cmax) of 3.2 μg/mL at 1.5 hours (Tmax), and the terminal half-life (T₁/₂) was approximately 3.8 hours. Following intravenous injection (20 mg/kg), Cmax was 9.5 μg/mL, and T₁/₂ was approximately 3.2 hours [1]
- Tissue distribution: In HCT116 xenograft mice, the tumor/plasma concentration ratio of oral administration of DEL-22379 (100 mg/kg) was 3.1 (2 hours after administration). It was moderately distributed in the liver (liver/plasma = 2.4) and spleen (spleen/plasma = 1.8), but less distributed in brain tissue (brain/plasma = 0.12) [1]
- Metabolism: In human liver microsomes, DEL-22379 is primarily metabolized by CYP2D6 (≥55% of total metabolism) and CYP3A4 (approximately 30%). No inhibitory effect on CYP1A2, 2C9, or 2C19 was observed [1]

Plasma protein binding: DEL-22379 has a plasma protein binding rate of approximately 96% in human plasma (as determined by balanced dialysis) [1]
- Acute toxicity: In CD-1 mice, single oral doses of up to 300 mg/kg of DEL-22379 did not cause death or clinical symptoms (e.g., lethargy, weight loss). Serum biochemical parameters (ALT, AST, BUN, creatinine) and hematological parameters (leukocytes, erythrocytes, platelets) were within normal ranges 24 hours after administration [1]
- Chronic toxicity: A 28-day repeated-dose study in mice (25–100 mg/kg, orally, once daily) showed no significant organ toxicity (histopathology of liver, kidneys, spleen, and heart) at any dose. No treatment-related changes in body weight or food intake were observed [1]

[1]. Small Molecule Inhibition of ERK Dimerization Prevents Tumorigenesis by RAS-ERK Pathway Oncogenes. Cancer Cell. 2015 Aug 10;28(2):170-82.

DEL-22379 is an indole ketone compound with the structure 5-aminoindole ketone, in which the 5-amino group undergoes a condensation reaction with the carboxyl group of 3-(piperidin-1-yl)propionic acid to generate the corresponding carboxamide, and the hydrogen atom at the 3 position is replaced by (5-methoxy-1H-indole-3-yl)methylene (Z configuration). It is an extracellular signal-regulated kinase (ERK) dimer inhibitor. It is both an ERK dimer inhibitor and an anti-tumor drug. It belongs to the piperidine, indole ketone, enamide and secondary carboxamide compounds. Mechanism of action: DEL-22379 is the first ERK1/2 dimer inhibitor. It binds to the ERK dimerization interface and prevents the formation of active ERK dimers required for downstream signal transduction (such as MEF2C activation, ERK nuclear translocation). This mechanism avoids the MEK/RAF feedback activation common in traditional ERK kinase inhibitors [1].
- Research significance: This compound overcomes a key limitation of traditional MAPK pathway inhibitors (such as MEK/ERK kinase inhibitors), namely, overcomes acquired resistance caused by ERK mutations (such as T188A) or upstream kinase feedback activation. It is a promising tool compound for studying ERK dimerization-dependent biology [1].
- Clinical development: As of the date of publication, DEL-22379 has not entered clinical trials; it is primarily used as a preclinical research tool to validate the possibility of ERK dimerization as a therapeutic target [1].

C26H28N4O3

444.53

444.216

C, 70.25; H, 6.35; N, 12.60; O, 10.80

181223-80-3

181223-80-3;DEL22379 HCl;

11224574

Yellow to orange solid

1.3±0.1 g/cm3

763.9±60.0 °C at 760 mmHg

415.8±32.9 °C

0.0±2.6 mmHg at 25°C

1.697

3.13

3

4

6

33

750

0

O=C(C([H])([H])C([H])([H])N1C([H])([H])C([H])([H])C([H])([H])C([H])([H])C1([H])[H])N([H])C1C([H])=C([H])C2=C(C=1[H])/C(/C(N2[H])=O)=C(/[H])\C1=C([H])N([H])C2C([H])=C([H])C(=C([H])C1=2)OC([H])([H])[H]

INQUULPXCZAKMS-XKZIYDEJSA-N

InChI=1S/C26H28N4O3/c1-33-19-6-8-23-20(15-19)17(16-27-23)13-22-21-14-18(5-7-24(21)29-26(22)32)28-25(31)9-12-30-10-3-2-4-11-30/h5-8,13-16,27H,2-4,9-12H2,1H3,(H,28,31)(H,29,32)/b22-13-

N-[(3Z)-3-[(5-methoxy-1H-indol-3-yl)methylidene]-2-oxo-1H-indol-5-yl]-3-piperidin-1-ylpropanamide

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)

Solubility in Formulation 1: ≥ 2.5 mg/mL (5.62 mM) (saturation unknown) in 10% DMSO + 40% PEG300 +5% Tween-80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 25.0 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 + to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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