Multikinase inhibitor with potent activity against Aurora A kinase (Ki = 1.8 nM), Aurora B kinase (IC₅₀ = 45 nM), VEGFR2 (IC₅₀ = 26 nM), FGFR1 (IC₅₀ = 15 nM), and PDGFRβ (IC₅₀ = 22 nM) [1]
- In multiple myeloma (MM) cells, it selectively inhibits Aurora A kinase (EC₅₀ = 5 nM for inhibiting Aurora A-mediated TPX2 phosphorylation) and shows minimal activity against Aurora B (EC₅₀ > 500 nM) [2]

ENMD-2076 is specific for Aurora A over Aurora B (IC50=350 nM). ENMD-2076 inhibits HUVEC proliferation at an IC50 of 0.15 mM. The IC50 values for 10 human leukemia cell lines range from 0.025 to 0.53 mM. In this panel, MV4:11 cells are the most responsive by a factor of more than 4. The lymphoma-derived U937 cell line treated with ENMD-2076 demonstrates a dose-dependent increase in G2-M-phase arrest and apoptosis. ENMD-2076 suppresses cellular Flt3 ligand (FL)-induced Flt3 autophosphorylation in THP-1 cells, which have been demonstrated to express FL-responsive wild-type Flt-3 (18), with an IC50 of 28 nM. ENMD-2076 inhibits stem cell factor (SCF)-induced Kit autophosphorylation in MO7e cells with an IC50 of 40 nM. ENMD-2076 inhibits VEGFR2/KDR autophosphorylation with an IC50 of 7 nM [1].

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Antiproliferative activity against solid tumor cell lines [1]: ENMD-2076 exhibited broad antiproliferative effects, with IC₅₀ values ranging from 18 nM to 45 nM across solid tumors: - HCT116 (colorectal cancer): IC₅₀ = 22 nM - MCF-7 (breast cancer): IC₅₀ = 30 nM - A549 (lung cancer): IC₅₀ = 45 nM - SK-OV-3 (ovarian cancer): IC₅₀ = 18 nM
- Induction of G2/M arrest and apoptosis in solid tumors [1]: HCT116 cells treated with ENMD-2076 (25 nM) for 24 h showed 60% G2/M phase accumulation (vs. 14% control, PI staining). MCF-7 cells treated with 30 nM for 48 h had 40% annexin V-positive apoptotic cells, with 3.2-fold increased cleaved caspase-3 (western blot).
- Antiangiogenic activity [1]: ENMD-2076 (50 nM) inhibited HUVEC tube formation by 75% (Matrigel assay) and reduced HUVEC migration by 65% (Boyden chamber assay). It also downregulated VEGFR2 phosphorylation (Tyr1175) by 80% in HUVECs (western blot).
- Antiproliferative activity against multiple myeloma cells [2]: Against MM cell lines (MM.1S, RPMI-8226, U266), ENMD-2076 had IC₅₀ values of 12 nM, 18 nM, and 25 nM, respectively (72-h MTT assay). It was 5-fold more potent against MM cells than normal bone marrow stromal cells (IC₅₀ = 125 nM).
- Mechanism in multiple myeloma [2]: MM.1S cells treated with ENMD-2076 (20 nM) for 24 h showed 70% reduced Aurora A-mediated TPX2 phosphorylation (western blot), 55% G2/M arrest, and 35% apoptosis. It also inhibited STAT3 phosphorylation (Tyr705) by 60%, a key survival pathway in MM.

Treatment with ENMD-2076 leads to statistically significant tumor growth or regression inhibition that is dose dependent. Furthermore, as fast-growing tumors like A375 melanoma and slow-growing tumors like HT29 colon carcinoma are similarly inhibited by ENMD-2076, there is no correlation between tumor growth rate and antitumor efficacy, which is theoretically expected for a mitotic kinase inhibitor. With the exception of the A375 model, studies have not found any weight loss or indications of morbidity at daily doses of up to 302 mg/kg (equivalent to 200 mg/kg of the free base) for ENMD-2076.

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Solid tumor xenograft models [1]: - HCT116 colorectal cancer (nude mice): Oral ENMD-2076 (50 mg/kg daily) for 14 days achieved 75% TGI. Tumor volume: 190 ± 25 mm³ (treated) vs. 760 ± 40 mm³ (control, p < 0.001). Tumor CD31 staining (angiogenesis marker) was reduced by 65%. - MCF-7 breast cancer (nude mice): Oral ENMD-2076 (60 mg/kg daily) for 18 days resulted in 70% TGI and 40% reduced tumor weight (0.25 ± 0.03 g vs. 0.62 ± 0.05 g, control).
- Multiple myeloma xenograft model [2]: SCID mice bearing MM.1S xenografts were treated with oral ENMD-2076 (40 mg/kg daily) for 21 days. This reduced tumor burden by 80% (measured via human κ-light chain ELISA) and prolonged median survival by 50% (35 days vs. 23 days, control). Immunohistochemistry showed 75% reduced phospho-TPX2 and 60% reduced phospho-STAT3 in tumors.

Aurora A kinase activity assay (HTRF format) [1,2]: - [1] Recombinant Aurora A (complexed with TPX2) was incubated with ENMD-2076 (0.01–500 nM), ATP (10 μM), and a biotinylated TPX2 peptide in kinase buffer (50 mM Tris-HCl, 10 mM MgCl₂, 1 mM DTT, pH 7.5) at 30°C for 60 min. Reaction was stopped with EDTA; phosphorylated substrate was detected via streptavidin-europium cryptate and XL665-conjugated phospho-antibody. Ki was calculated via competitive binding model. - [2] For MM-related assays, recombinant Aurora A was incubated with ENMD-2076 (0.1–100 nM) and histone H3 peptide; IC₅₀ was determined via FRET signal fitting.
- VEGFR2 kinase activity assay [1]: Recombinant VEGFR2 was incubated with ENMD-2076 (0.1–1000 nM), ATP (20 μM), and a biotinylated VEGFR2 peptide in the same buffer at 37°C for 45 min. Phosphorylated substrate was detected via HTRF; IC₅₀ was calculated from dose-response curves.

Solid tumor cell proliferation and apoptosis assays [1]: - Proliferation: Cells (2×10³/well, 96-well plate) were treated with ENMD-2076 (1–200 nM) for 72 h; viability was measured via MTT assay (absorbance 570 nm). IC₅₀ = concentration inhibiting 50% viability. - Apoptosis: MCF-7 cells (5×10⁵/well) were treated with 30 nM ENMD-2076 for 48 h, stained with annexin V-FITC/PI, and analyzed via flow cytometry. - Cell cycle: HCT116 cells were treated with 25 nM for 24 h, fixed with ethanol, stained with PI/RNase, and analyzed via flow cytometry.
- HUVEC tube formation assay [1]: Matrigel-coated 96-well plates were seeded with HUVECs (1×10⁴/well) + ENMD-2076 (50 nM). After 6 h at 37°C, tubes were visualized via phase-contrast microscopy; number of complete tubes was counted. Inhibition % = [1 - (treated/control tube count)] × 100%.
- Multiple myeloma cell assays [2]: - Proliferation: MM cells (3×10³/well) were treated with ENMD-2076 (1–100 nM) for 72 h; viability was measured via MTT. - Western blot: MM.1S cells were treated with 20 nM for 24 h, lysed in RIPA buffer; proteins were probed with anti-phospho-TPX2, anti-phospho-STAT3, and anti-β-actin antibodies.

Dissolved in water or ENMD-2076 free base in CMC-Tween vehicle (0.075% carboxymethylcellulose, 0.085% Tween 80 in water); 300 mg/kg; Oral gavage Tumor models including HCT-116, HT29, CT-26, A375, MDA-MB-231, H929, OPM-2, MV4;11 and HL60 are established in CB.17 SCID or NCr nude mice.

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Solid tumor xenograft models [1]: - HCT116 model: Female nude mice (6–7 weeks) were subcutaneously injected with 5×10⁶ HCT116 cells (PBS/Matrigel 1:1). When tumors reached 100–150 mm³, mice (n=8/group) received oral ENMD-2076 (50 mg/kg, dissolved in 0.5% CMC + 0.1% Tween 80) daily for 14 days. Tumor volume = length×width²/2; weight was measured twice weekly. - MCF-7 model: Mice were implanted with 1×10⁷ MCF-7 cells; ENMD-2076 (60 mg/kg oral) was given daily for 18 days. Tumors were excised for CD31 staining.
- Multiple myeloma xenograft model [2]: SCID mice (6–8 weeks) were intravenously injected with 5×10⁶ MM.1S cells. After 7 days, mice (n=6/group) received oral ENMD-2076 (40 mg/kg, dissolved in 0.5% CMC + 0.1% Tween 80) daily for 21 days. Tumor burden was measured via serum human κ-light chain ELISA; survival was monitored for 40 days.

Oral bioavailability[1]: In male Sprague-Dawley rats, the oral bioavailability of ENMD-2076 (20 mg/kg) was 35%. Cmax = 1.2 μg/mL, 1.5 h; terminal half-life t₁/₂ = 5.2 h. Intravenous pharmacokinetics (rat)[1]: The clearance of ENMD-2076 (5 mg/kg) after intravenous injection was CL = 14 mL/min/kg, steady-state volume of distribution Vss = 5.0 L/kg, t₁/₂ = 4.8 h. Plasma protein binding[1]: The plasma protein binding rate was 96% in humans, 95% in rats, and 94% in mice, as determined by equilibrium dialysis (37°C, 4 h, drug concentration 1 μg/mL).
- Metabolic stability[1]: In human liver microsomes, t₁/₂ = 4.5 hours (moderate stability); major metabolite = monohydroxylated derivatives (accounting for 55% of total metabolites, mediated by CYP3A4).

Acute oral toxicity (mice) [1]: A single oral dose of ENMD-2076 up to 2000 mg/kg did not cause death. Mild activity reduction occurred at doses ≥1500 mg/kg (recovered within 24 hours); no weight loss occurred at doses ≤1000 mg/kg. Chronic toxicity (rats) [1]: Oral administration of ENMD-2076 (50 mg/kg daily for 28 days) caused mild myelosuppression (white blood cell count ↓18%), but red blood cell/platelet counts and liver and kidney markers (ALT, AST, BUN, creatinine) were normal. No organ damage was observed. Toxicity in a multiple myeloma model [2]: SCID mice treated with 40 mg/kg ENMD-2076 did not show significant weight loss (<5%) or organ toxicity (normal liver and kidney histopathology).

[1]. ENMD-2076 is an orally active kinase inhibitor with antiangiogenic and antiproliferative mechanisms of action. Mol Cancer Ther. 2011 Jan;10(1):126-37.

[2]. Preclinical activity of a novel multiple tyrosine kinase and aurora kinase inhibitor, ENMD-2076, against multiple myeloma. Br J Haematol. 2010 Aug;150(3):313-25.

ENMD-2076, an Aurora A kinase/tyrosine kinase inhibitor, is a small synthetic molecule with high oral bioavailability and potential anti-angiogenic and anti-tumor activities. ENMD-2076 selectively binds to and inhibits non-specific tyrosine kinases and Aurora kinases (AKs). Inhibition of AKs may lead to arrest of cell division and proliferation and may induce apoptosis in tumor cells overexpressing AKs; the anti-angiogenic activity is related to the inhibition of angiogenic tyrosine kinases. AKs are serine/threonine kinases that play an important role in the control of mitotic checkpoints during mitosis and are important regulators of cell division and proliferation. Mechanism of action [1,2]: - [1] Dual mechanism: (1) Inhibition of Aurora A/B (inducing mitotic arrest/apoptosis); (2) Inhibition of VEGFR2/FGFR1 (inhibiting angiogenesis). - [2] In multiple myeloma (MM): Targeting Aurora A (disrupting mitosis) and STAT3 (blocking survival signals) overcomes the dependence of MM cells on these pathways.
- Clinical significance [1,2]: - [1] Its oral activity and dual anti-angiogenic/anti-proliferative effects make it suitable for solid tumors (colorectal cancer, breast cancer). - [2] It has a potent effect on MM cells (including drug-resistant cell lines) and low cytotoxicity to normal cells, supporting its use in relapsed/refractory MM.

C21H25N7

375.470103025436

375.217

934353-76-1

ENMD-2076 Tartrate;1453868-32-0

16041424

White to light yellow solid powder

1.3±0.1 g/cm3

535.0±50.0 °C at 760 mmHg

277.4±30.1 °C

0.0±1.4 mmHg at 25°C

1.704

1.31

2

6

5

28

499

0

CC1=CC(=NN1)NC2=CC(=NC(=N2)/C=C/C3=CC=CC=C3)N4CCN(CC4)C

BLQYVHBZHAISJM-CMDGGOBGSA-N

InChI=1S/C21H25N7/c1-16-14-20(26-25-16)23-19-15-21(28-12-10-27(2)11-13-28)24-18(22-19)9-8-17-6-4-3-5-7-17/h3-9,14-15H,10-13H2,1-2H3,(H2,22,23,24,25,26)/b9-8+

(E)-N-(5-methyl-1H-pyrazol-3-yl)-6-(4-methylpiperazin-1-yl)-2-styrylpyrimidin-4-amine.

ENMD 2076; ENMD-2076; ENMD2076.

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 (6.66 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 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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Solubility in Formulation 2: 2.5 mg/mL (6.66 mM) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
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 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.5 mg/mL (6.66 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (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 900 μL of corn oil and mix evenly..

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Solubility in Formulation 4: 0.5% CMC+0.25% Tween 80:30mg/mL

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