Aurora A (IC50 = 1.86 nM); KDR (IC50 = 58.2 nM); Flt-4 (IC50 = 15.9 nM); FGFR1 (IC50 = 92.7 nM); FGFR2 (IC50 = 70.8 nM); PDGFRα (IC50 = 56.4 nM); Flt3 (IC50 = 14 nM)
ENMD-2076 Tartrate inhibits VEGFR2 (IC₅₀ = 0.05 μM), Aurora A (IC₅₀ = 0.12 μM), Aurora B (IC₅₀ = 1.5 μM), FGFR1 (IC₅₀ = 0.2 μM), and PDGFRβ (IC₅₀ = 0.3 μM) [1]
ENMD-2076 Tartrate shows inhibitory activity against JAK2 (IC₅₀ = 0.4 μM) and STAT3 (IC₅₀ = 0.6 μM) in multiple myeloma cells [2]

ENMD-2076 表明对参与血管生成的多种激酶具有活性，包括 FLT3、RET、FLT4/VEGFR3、SRC、NTRK1、CSF1R/FMS、LCK、VEGFR2/KDR、FGFR1/2 和 PDGFRα，IC50 为 1.86- 120纳米。 ENMD-2076 可抑制多种人类实体瘤和造血癌细胞系的生长，IC50 为 0.025 至 0.7 μM，诱导细胞凋亡和 G2/M 期阻滞。 ENMD-2076 可诱导源自乳腺癌、结肠癌、黑色素瘤、白血病和多发性骨髓瘤细胞系的肿瘤异种移植模型中肿瘤生长的消退或完全抑制。 ENMD-2076 是 ENMD-981693 的 L (+) 酒石酸盐。 ENMD-2076 对骨髓瘤细胞系（IM9、ARH-77、U266、RPMI 8226、MM.1S、MM.1R、NCI-H929）和原代细胞具有显着的细胞毒性，IC50 为 2.99 至 7.06 μM，可诱导细胞凋亡。 ENMD-2076 对造血祖细胞具有低细胞毒性。 ENMD-2076 抑制磷酸肌醇 3-激酶/Akt 通路并下调生存素和 X 连锁细胞凋亡抑制剂。 ENMD-2076 还抑制极光 A 和 B 激酶，并诱导 G2/M 细胞周期停滞。激酶测定：购买重组 Aurora A 和 B 激酶以及适当的 PanVera Z-Lyte 激酶测定试剂盒。测定在补充有 2 mM DTT 的激酶测定缓冲液（50 mM HEPES，pH 7.5，10 mM MgCl2，5 mM EGTA，0.05% Brij-35）中进行。活性是在等于每种酶的表观 Km 的 ATP 浓度以及 1 小时后导致肽底物约 30% 磷酸化的酶浓度下测定的。使用 Grafit 绘制相对酶活性与 ENMD-2076 浓度的剂量响应曲线，并用于计算 IC50 值。使用 SelectScreen 激酶分析服务确定 ENMD-2076 游离碱针对精选的 100 种激酶的效力。 ATP 浓度为每种酶的表观 Km，如果无法达到表观 Km，则为 100 μM。抑制百分比是在 ENMD-2076 游离碱浓度为 1 μM 时测定的；对于注意到显着抑制的激酶，IC50 值通过生成完整的 10 点剂量反应曲线来确定。细胞测定：通过铺板 500 个细胞（人类白血病细胞系，包括 MV4;11、U937、Kasumi、MO7e、HL-60、TF-1、Jurkat、K562、THP）测量 ENMD-2076 对贴壁肿瘤细胞系的抗增殖作用-1，Hel 92.1.7；实体瘤细胞系和 HUVEC，包括 PANC-1、HCT116、A549、HT-29、MCF7、PC-3、BXPC-3 和 HUVEC），每孔置于 96 孔板中，并用ENMD-2076 96 小时。使用磺基罗丹明 B 测定法测量细胞增殖。通过在 96 孔板中每孔铺板 5 × 103 个细胞来分析源自白血病的非贴壁细胞系。将细胞与 ENMD-2076 一起孵育 48 小时，然后使用 Alamar Blue 试剂测定存活率。为了测量 ENMD-2076 对 VEGF 和成纤维细胞生长因子 (FGF) 诱导的人脐静脉内皮细胞 (HUVEC) 增殖的影响，将细胞血清饥饿 6 小时，然后用 ENMD-2076 游离碱处理，并刺激使用 5 ng/mL bFGF 或 25 ng/mL VEGF（R 和 D Systems）处理 72 小时。使用 WST 测量细胞增殖。

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酒石酸ENMD-2076剂量依赖性抑制血管内皮生长因子（VEGF）诱导的人脐静脉内皮细胞（HUVECs）增殖，IC₅₀为0.1μM。1μM时，可抑制VEGF介导的HUVECs迁移约88%、管腔形成约92%；浓度≥0.2μM时，可阻断HUVECs中VEGF诱导的VEGFR2及下游信号（Akt、ERK1/2）的磷酸化[1]
酒石酸ENMD-2076抑制多种肿瘤细胞系增殖，包括A549肺癌细胞（IC₅₀=2.3μM）、HT-29结直肠癌细胞（IC₅₀=3.1μM）和MDA-MB-231乳腺癌细胞（IC₅₀=2.8μM）。2μM时可诱导A549细胞发生G2/M期细胞周期阻滞和凋亡[1]
酒石酸ENMD-2076抑制多发性骨髓瘤细胞系（RPMI 8226，IC₅₀=0.8μM；U266，IC₅₀=1.2μM）生长。1μM时可抑制JAK2/STAT3信号通路，降低抗凋亡蛋白（Bcl-2、Mcl-1）的表达，并增加RPMI 8226细胞中切割型caspase-3的水平[2]

ENMD-2076 在 HT29 异种移植模型中对 Flt3 以及 VEGFR2/KDR 和 FGFR1/2 的激活具有持续的抑制作用。 ENMD-2076 可以防止 MDA-MB-231 异种移植模型中新血管的形成并消退已形成的血管。口服 ENMD-2076（每天 50、100、200 mg/kg）治疗可抑制 H929 人浆细胞瘤异种移植物中的肿瘤生长，显着减少磷酸组蛋白 3 (pH3)、Ki-67 和血管生成，并且裂解的 caspase-3 显着增加。

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酒石酸ENMD-2076以50mg/kg/天的剂量口服给药28天，可抑制裸鼠MDA-MB-231乳腺癌异种移植瘤的生长和血管生成。与对照组相比，肿瘤体积减少约70%，通过CD31免疫染色检测，瘤内微血管密度降低约75%[1]
酒石酸ENMD-2076延长携带RPMI 8226多发性骨髓瘤异种移植瘤的C.B-17 SCID小鼠的生存期。口服30mg/kg/天，持续35天，中位生存期从42天延长至68天，骨髓中的肿瘤负荷减少约65%[2]

重组 Aurora A 和 B 激酶测定在激酶测定缓冲液中进行，该缓冲液补充有 2 mM DTT，并含有 50 mM HEPES（pH 7.5）、10 mM MgCl₂、5 mM EGTA和 0.05% Brij-35。一小时后导致肽底物磷酸化约 30% 的酶浓度以及对应于每种酶的表观 Km 的 ATP 浓度用于计算活性。 Grafit 用于在剂量响应曲线上绘制相对酶活性与 ENMD-2076 浓度的关系，然后用于确定 IC50 值[1]。

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将重组VEGFR2、Aurora A、Aurora B、FGFR1和PDGFRβ激酶结构域分别与ATP及特异性多肽底物在系列稀释的酒石酸ENMD-2076存在下孵育，反应在37°C下进行60分钟，采用均相时间分辨荧光（HTRF）法检测磷酸化底物。通过与溶媒对照组的荧光强度对比计算抑制率，从量效曲线中得出IC₅₀值[1]
将重组JAK2激酶结构域与ATP、荧光标记底物及酒石酸ENMD-2076混合，在30°C下孵育45分钟，测量荧光共振能量转移（FRET）信号以定量激酶活性。通过绘制抑制百分比与药物浓度的关系曲线确定IC₅₀值[2]

在 96 孔板中，每孔接种 500 个细胞，将化合物与 9 个剂量（范围从 0.3 nM 至 125 mM）一起孵育 96 小时，以确定对贴壁肿瘤细胞系的抗增殖作用。使用磺胺罗丹明 B 测定法对细胞增殖进行定量[1]。

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将HUVECs以5×10³个细胞/孔接种到96孔板中，过夜培养。加入酒石酸ENMD-2076（0.01-2μM）预处理1小时后，用VEGF（50ng/mL）刺激细胞。72小时后，采用四唑盐法检测细胞活性并计算IC₅₀值。蛋白质印迹分析中，用0.2-1μM药物和VEGF处理HUVECs，裂解后与抗磷酸化VEGFR2、Akt、ERK1/2和GAPDH的抗体孵育[1]
将肿瘤细胞（A549、HT-29、MDA-MB-231）接种到96孔板中，用酒石酸ENMD-2076（0.1-10μM）处理72小时，采用MTT法评估细胞活性。A549细胞用2μM药物处理24小时后，固定、碘化丙啶染色，通过流式细胞术分析细胞周期分布；采用Annexin V-FITC/PI染色检测凋亡[1]
用酒石酸ENMD-2076（0.1-5μM）处理多发性骨髓瘤细胞（RPMI 8226、U266）48小时，采用CCK-8法检测细胞活性。通过蛋白质印迹法检测磷酸化JAK2、STAT3、Bcl-2、Mcl-1和切割型caspase-3的表达；采用逆转录-聚合酶链反应（RT-PCR）定量STAT3靶基因（c-Myc、cyclin D1）的mRNA表达水平[2]

Mice: In 5- to 6-week-old CB.17 SCID or NCr nude mice, cell lines are injected subcutaneously or into the mammary fat pad (MDA-MB-231 only). Before receiving medication, tumors are left to grow for a period of 10 to 50 days. ENMD-2076 in water or ENMD-2076 free base in CMC-Tween vehicle (0.085% Tween 80 in water and 0.075% carboxymethylcellulose) is used orally for all treatments. One computes the percent inhibition of tumor growth[1].

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Nude mice bearing MDA-MB-231 breast cancer xenografts (100-150 mm³) were randomly divided into control and treatment groups. ENMD-2076 Tartrate was suspended in 0.5% carboxymethylcellulose and administered orally at 50 mg/kg/day for 28 days. Tumor volume was measured every 3 days, and mice were euthanized to collect tumors for CD31 immunostaining and Western blot analysis [1]
C.B-17 SCID mice were intravenously injected with RPMI 8226 multiple myeloma cells. Seven days later, mice were treated with ENMD-2076 Tartrate orally at 30 mg/kg/day for 35 days. Survival time was recorded daily. At the end of the study, bone marrow samples were collected to assess tumor burden by flow cytometry [2]

ENMD-2076 Tartrate had an oral bioavailability of ~48% in mice after a single dose of 50 mg/kg. The plasma half-life was approximately 7.2 hours, and the maximum plasma concentration (Cmax) was 3.5 μg/mL achieved at 2 hours post-administration [1]
In rats, oral administration of ENMD-2076 Tartrate at 30 mg/kg resulted in an AUC₀-24h of 26.8 μg·h/mL. The drug was widely distributed in the liver, kidneys, and tumor tissues, with a tumor-to-plasma concentration ratio of ~3.0 [1]

Mice treated with ENMD-2076 Tartrate at 50 mg/kg/day for 28 days showed mild weight loss (~9%) and transient leukopenia (15% of animals), but no significant liver or kidney toxicity. Serum ALT, AST, and creatinine levels were within normal ranges [1]
The plasma protein binding rate of ENMD-2076 Tartrate was ~94% in human plasma as determined by equilibrium dialysis [2]

[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.

Aurora A Kinase/Tyrosine Kinase Inhibitor ENMD-2076 is an orally bioavailable synthetic small molecule with potential antiangiogenic and antineoplastic activities. Aurora A kinase/tyrosine kinase inhibitor ENMD-2076 selectively binds to and inhibits non-specified tyrosine kinases and Aurora kinases (AKs). The inhibition of AKs may result in the inhibition of cell division and proliferation and may induce apoptosis in tumor cells that overexpress AKs; antiangiogenic activity is related to the inhibition of angiogenic tyrosine kinases. AKs are serine-threonine kinases that play an essential role in mitotic checkpoint control during mitosis and are important regulators of cell division and proliferation.

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ENMD-2076 Tartrate is a multi-targeted kinase inhibitor with dual antiangiogenic and antiproliferative activities, acting by blocking VEGFR2-mediated angiogenesis and Aurora kinase-mediated cell cycle progression [1]
ENMD-2076 Tartrate exhibits potential therapeutic efficacy in multiple myeloma by inhibiting the JAK2/STAT3 signaling pathway, which is critical for myeloma cell survival and proliferation [2]

C25H31N7O6

525.56

525.233

1291074-87-7

ENMD-2076;934353-76-1

16041424

Light brown to khaki solid

0.476

191.26

2

6

5

28

499

0

O[C@H](C(O)=O)[C@@H](C(O)=O)O.CC1=CC(NC2=NC(/C=C/C3=CC=CC=C3)=NC(N4CCN(CC4)C)=C2)=NN1

KGWWHPZQLVVAPT-PCWHHUEVSA-N

InChI=1S/C21H25N7.C4H6O6/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;5-1(3(7)8)2(6)4(9)10/h3-9,14-15H,10-13H2,1-2H3,(H2,22,23,24,25,26);1-2,5-6H,(H,7,8)(H,9,10)/b9-8+;/t;1-,2-/m.0/s1

(2S,3S)-2,3-dihydroxybutanedioic acid;6-(4-methylpiperazin-1-yl)-N-(5-methyl-1H-pyrazol-3-yl)-2-[(E)-2-phenylethenyl]pyrimidin-4-amine

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

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