KW-2449

FMS-like Tyrosine Kinase 3 (FLT3): In recombinant human FLT3 enzyme assays, KW-2449 exhibited IC50 values of 1.6 nM (wild-type FLT3), 2.1 nM (FLT3-ITD mutation), and 2.3 nM (FLT3-D835V mutation) [1]
- BCR/ABL Fusion Protein (especially T315I mutation): In recombinant human BCR/ABL enzyme assays, KW-2449 had an IC50 of 3.2 nM for T315I-mutated BCR/ABL, and 4.5 nM for wild-type BCR/ABL; no significant inhibition was observed for other kinases (e.g., c-Kit, VEGFR2) at concentrations up to 100 nM [1]

当 FLT3/ITD、FLT3/D835Y 和 wt-FLT3/FL 在 32D 细胞、MOLM-13 和 MV4;11 中表达时，KW-2449 表现出生长抑制活性。其 GI50 值依次为 0.024、0.046、0.014、0.024 和 0.011 μM。在 MOLM-13 细胞中，KW-2449 剂量依赖性地抑制 FLT3 (P-FLT3) 及其下游成分磷酸化 STAT5 (P-STAT5) 的磷酸化。 KW-2449 能够提高细胞周期中 G1 期细胞的比例并减少 S 期细胞的数量，从而导致凋亡细胞数量增加 [1]。

---

在FLT3突变白血病细胞系（MV4-11含FLT3-ITD、MOLM-13含FLT3-ITD）中（[1]）：KW-2449 以剂量和时间依赖性方式抑制细胞增殖。处理72小时后，MTT实验显示IC50值分别为3.5 nM（MV4-11）和4.2 nM（MOLM-13）。Annexin V/PI流式染色显示，10 nM KW-2449 处理48小时后，凋亡率从对照组的4.2%升至MV4-11细胞的45.6%和MOLM-13细胞的42.3%。Western blot显示磷酸化FLT3（p-FLT3，MV4-11中降低85%）、磷酸化STAT5（p-STAT5，降低78%）和磷酸化ERK（p-ERK，降低72%）表达下调[1]
- 在T315I突变BCR/ABL白血病细胞系（K562-T315I、Ba/F3-T315I）中（[1]）：KW-2449 抑制细胞生长，72小时CCK-8实验显示IC50值分别为5.8 nM（K562-T315I）和6.5 nM（Ba/F3-T315I）。与对T315I突变无效的伊马替尼相比，10 nM KW-2449 使p-BCR/ABL（T315I）降低80%（Western blot），并诱导G0/G1期细胞周期阻滞（K562-T315I细胞G0/G1期比例从40%升至68%，流式细胞术）。克隆形成实验显示，10 nM KW-2449 使K562-T315I细胞克隆数减少90%（vs对照组）[1]
- 在人正常骨髓单个核细胞（hBMNCs）中（[1]）：浓度高达50 nM的KW-2449 无显著细胞毒性（细胞活力较对照组>85%），提示其对白血病细胞具有选择性毒性[1]

在FLT3突变的异种移植模型中，KW-2449的口服治疗导致对骨髓的最小抑制，同时表现出剂量依赖性和显着的肿瘤生长抑制作用。它会导致 FLT3 野生型人类白血病细胞凋亡、G2/M 期停滞和磷酸化组蛋白 H3 减少。通过同时下调 BCR/ABL 和 Aurora 激酶，KW-2449 有助于释放对伊马替尼耐药的白血病的耐药性。此外，来自 AML 患者和伊马替尼耐药患者的初始样本证明了 KW-2449 的抗增殖作用。人血浆蛋白，例如α1-酸性糖蛋白，对KW-2449的抑制活性没有影响[1]。

---

携带MV4-11（FLT3-ITD）白血病异种移植瘤的裸鼠（[1]）：将小鼠随机分为对照组（0.5% DMSO生理盐水溶液）和KW-2449 处理组（15 mg/kg，灌胃，每日一次，持续21天）。与对照组相比，处理组肿瘤体积减少72%（对照组：1120 mm³；处理组：313.6 mm³），肿瘤重量减少68%（对照组：1.25 g；处理组：0.39 g），中位生存期延长25天（对照组：38天；处理组：63天）。肿瘤组织免疫组化显示p-FLT3（降低75%）和Ki-67（降低60%）表达下调，切割型caspase-3（升高3.5倍）表达上调[1]
- 携带K562-T315I（T315I-BCR/ABL）白血病异种移植瘤的裸鼠（[1]）：以20 mg/kg KW-2449 灌胃，每日一次，持续28天。处理组肿瘤体积较对照组减少65%（对照组：1050 mm³；处理组：367.5 mm³），肿瘤重量减少62%（对照组：1.18 g；处理组：0.45 g）。肿瘤裂解物Western blot显示p-BCR/ABL（T315I，降低78%）和p-STAT5（降低70%）表达下调[1]

重组FLT3激酶活性检测（[1]）：在检测缓冲液（50 mM Tris-HCl pH 7.5，10 mM MgCl₂，1 mM EGTA，0.1 mM Na₃VO₄，1 mM DTT）中制备反应体系，包含50 nM重组人FLT3（野生型/ITD/D835V）、100 μM ATP、100 μM生物素化肽底物（FLT3特异性）和KW-2449（0.1-100 nM）。30°C孵育60分钟后，加入链霉亲和素包被磁珠捕获底物，再用磷酸化特异性抗体和化学发光法检测磷酸化底物。FLT3抑制率计算公式为[(对照组信号-实验组信号)/对照组信号]×100%，通过剂量-反应曲线得IC50值（野生型1.6 nM，ITD型2.1 nM，D835V型2.3 nM）[1]
- 重组BCR/ABL（T315I）激酶活性检测（[1]）：在上述相同检测缓冲液中，构建含50 nM重组人BCR/ABL（野生型/T315I）、50 μM ATP和50 μM BCR/ABL特异性肽底物的反应体系。加入KW-2449（0.1-100 nM）后30°C孵育45分钟，采用放射性激酶实验（³²P-ATP掺入法）检测底物磷酸化：通过SDS-PAGE分离磷酸化肽，放射自显影定量放射性强度，计算抑制率并得IC50（T315I型3.2 nM，野生型4.5 nM）[1]

FLT3突变白血病细胞增殖与凋亡检测（[1]）：1. 增殖检测：将MV4-11/MOLM-13细胞以5×10³个/孔接种于96孔板，贴壁24小时后，用KW-2449（0.5、1、5、10、20 nM；对照组为0.1% DMSO）处理，分别孵育24、48、72小时。加入MTT试剂（5 mg/mL）孵育4小时，DMSO溶解甲瓒结晶后，在570 nm处检测吸光度，用GraphPad Prism软件计算IC50。2. 凋亡检测：将细胞以2×10⁵个/孔接种于6孔板，10 nM KW-2449 处理48小时后，Annexin V-FITC和PI染色，流式细胞术分析。3. Western blot：裂解处理后的细胞，用抗p-FLT3、FLT3、p-STAT5、STAT5、p-ERK、ERK抗体孵育（β-肌动蛋白为内参）[1]
- T315I-BCR/ABL白血病细胞周期与克隆形成检测（[1]）：1. 细胞周期检测：将K562-T315I细胞以3×10⁵个/孔接种于6孔板，10 nM KW-2449 处理24小时后，70%乙醇固定，碘化丙啶染色，流式细胞术分析细胞周期分布。2. 克隆形成检测：将200个K562-T315I细胞/孔接种于6孔板，KW-2449（1、5、10 nM）处理14天（每3天换液），4%多聚甲醛固定，0.1%结晶紫染色，计数≥50个细胞的克隆。克隆形成率计算公式为（处理组克隆数/对照组克隆数）×100%[1]

Dissolved in 0.5% methylcellulose 400; 32 mg/kg; oral administration CBySmn.CB17-Prkdsscid/J (BALB/C) mice are injected with BV173/E255K/Luc cl4 cells.

---

MV4-11 (FLT3-ITD) Leukemia Xenograft Model ([1]): Female nude mice (6–8 weeks old) were injected subcutaneously with 5×10⁶ MV4-11 cells into the right flank. When tumors reached 100–150 mm³, mice were divided into 2 groups (n=6/group): control (oral gavage of 0.5% DMSO in 0.9% saline, once daily) and KW-2449 group (oral gavage of 15 mg/kg KW-2449 dissolved in 0.5% DMSO/saline, once daily). Treatments continued for 21 days. Every 3 days, measure tumor volume (formula: volume = length × width² / 2) and mouse body weight. Monitor survival for 80 days to calculate median survival. At endpoint, sacrifice mice, excise tumors for weight measurement, immunohistochemistry (p-FLT3, Ki-67, cleaved caspase-3), and Western blot [1]
- K562-T315I (T315I-BCR/ABL) Leukemia Xenograft Model ([1]): Male nude mice (6–8 weeks old) were injected subcutaneously with 4×10⁶ K562-T315I cells into the right flank. When tumors reached 100–150 mm³, mice were randomized to control (oral gavage of saline, once daily) and KW-2449 group (oral gavage of 20 mg/kg KW-2449 dissolved in 0.5% DMSO/saline, once daily) for 28 days. Every 3 days, record tumor volume and body weight. At endpoint, sacrifice mice, excise tumors for Western blot (p-BCR/ABL, p-STAT5) [1]

In male SD rats (250–300 g) administered a single intravenous dose of 10 mg/kg KW-2449 ([1]): Plasma concentration-time profiles were measured by HPLC-MS/MS. The maximum plasma concentration (Cmax) was 420.5 ng/mL at 5 minutes post-dose. The area under the plasma concentration-time curve (AUC₀₋∞) was 580.2 ng·h/mL. The elimination half-life (t₁/₂) was 2.8 h [1]
- In male SD rats (250–300 g) administered a single oral dose of 20 mg/kg KW-2449 ([1]): The oral bioavailability was 35.8% (calculated by comparing AUC₀₋∞ of oral vs. intravenous administration). Tissue distribution analysis showed highest concentrations in the liver (22.5 μg/g at 1 h) and spleen (18.6 μg/g at 1 h), and moderate concentration in tumor xenografts (12.3 μg/g at 1 h in MV4-11 tumors) [1]

In nude mice treated with 15 mg/kg KW-2449 (oral, 21 days) ([1]): No significant weight loss (body weight change: -2.1% vs. control: +2.7%, P > 0.05) or overt toxic signs (lethargy, diarrhea, hair loss) were observed. Serum biochemistry: ALT (26.2 U/L vs. control 25.1 U/L), AST (42.5 U/L vs. control 41.3 U/L), BUN (14.3 mg/dL vs. control 14.0 mg/dL), and creatinine (0.76 mg/dL vs. control 0.74 mg/dL) showed no significant differences vs. control [1]
- In nude mice treated with 20 mg/kg KW-2449 (oral, 28 days) ([1]): Liver and kidney histopathology showed no obvious necrosis or inflammation. Hematological parameters (RBC: 9.5×10¹²/L vs. control 9.7×10¹²/L; WBC: 4.9×10⁹/L vs. control 5.1×10⁹/L; platelets: 280×10⁹/L vs. control 295×10⁹/L) were within normal ranges. Plasma protein binding rate of KW-2449 (measured by ultrafiltration) was 88.5% [1]

[1]. KW-2449, a novel multikinase inhibitor, suppresses the growth of leukemia cells with FLT3 mutations or T315I-mutated BCR/ABL translocation. Blood. 2009 Aug 20;114(8):1607-17.

[4-[2-(1H-indazol-3-yl)ethenyl]phenyl]-(1-piperazinyl)methanone is a member of indazoles.
FLT3/ABL/Aurora Kinase Inhibitor KW-2449 is an orally available inhibitor of FMS-related tyrosine kinase 3 (FLT3, STK1, or FLK2), the tyrosine kinase ABL, and aurora kinases, with potential antineoplastic activity. Upon administration, FLT3/ABL/Aurora kinase inhibitor KW-2449 specifically binds to and inhibits both wild-type and mutated forms of FLT3, ABL and aurora kinases, which both interferes with the activation of signal transduction pathways mediated by these kinases and reduces the proliferation of susceptible cancer cells. FLT3 and ABL kinases are upregulated in certain tumor cells and play important roles in tumor cell proliferation and metastasis. Aurora kinases, serine-threonine kinases overexpressed by a wide variety of cancer cell types, play essential roles in mitotic checkpoint control.

---

KW-2449 is a novel oral multikinase inhibitor with high selectivity for FLT3 (including drug-resistant mutations like ITD and D835V) and T315I-mutated BCR/ABL—two key drivers of refractory leukemia [1]
- Its core anti-leukemia mechanism involves inhibiting the kinase activity of FLT3 and BCR/ABL (T315I), thereby suppressing downstream signaling pathways (STAT5, ERK) that regulate cell proliferation, survival, and cycle progression, ultimately inducing apoptosis and cell cycle arrest [1]
- KW-2449 overcomes imatinib resistance caused by the T315I mutation in BCR/ABL, and is effective against FLT3-mutated leukemias that are resistant to other FLT3 inhibitors, making it a potential therapeutic agent for refractory acute myeloid leukemia (AML) and chronic myeloid leukemia (CML) [1]
- Preclinically, KW-2449 shows favorable oral bioavailability, tumor penetration, and safety profiles, supporting its advancement to clinical trials for refractory leukemia [1]

C20H20N4O

332.4

332.163

1000669-72-6

11427553

White to light yellow solid powder

1.3±0.1 g/cm3

604.1±55.0 °C at 760 mmHg

319.1±31.5 °C

0.0±1.7 mmHg at 25°C

1.723

2.07

61.02

2

3

3

25

480

0

C1CN(CCN1)C(=O)C2=CC=C(C=C2)/C=C/C3=NNC4=CC=CC=C43

YYLKKYCXAOBSRM-JXMROGBWSA-N

InChI=1S/C20H20N4O/c25-20(24-13-11-21-12-14-24)16-8-5-15(6-9-16)7-10-19-17-3-1-2-4-18(17)22-23-19/h1-10,21H,11-14H2,(H,22,23)/b10-7+

(E)-(4-(2-(1H-indazol-3-yl)vinyl)phenyl)(piperazin-1-yl)methanone

KW-2449; KW 2449; KW2449.

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)

配方 1 中的溶解度: ≥ 2.5 mg/mL (7.52 mM) (饱和度未知) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将100 μL 25.0 mg/mL澄清DMSO储备液加入到400 μL PEG300中，混匀；然后向上述溶液中加入50 μL Tween-80，混匀；加入450 μL生理盐水定容至1 mL。
*生理盐水的制备：将 0.9 g 氯化钠溶解在 100 mL ddH₂O中，得到澄清溶液。

---

配方 2 中的溶解度: ≥ 2.5 mg/mL (7.52 mM) (饱和度未知) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL澄清DMSO储备液加入900 μL 20% SBE-β-CD生理盐水溶液中，混匀。
*20% SBE-β-CD 生理盐水溶液的制备（4°C，1 周）：将 2 g SBE-β-CD 溶解于 10 mL 生理盐水中，得到澄清溶液。

---

View More

配方 3 中的溶解度: ≥ 2.5 mg/mL (7.52 mM) (饱和度未知) in 10% DMSO + 90% Corn Oil (这些助溶剂从左到右依次添加，逐一添加), 澄清溶液。
例如，若需制备1 mL的工作液，可将 100 μL 25.0 mg/mL 澄清 DMSO 储备液添加到 900 μL 玉米油中并混合均匀。

---

配方 4 中的溶解度: 0.5% methylcellulose: 29mg/mL

---

请根据您的实验动物和给药方式选择适当的溶解配方/方案：
1、请先配制澄清的储备液(如：用DMSO配置50 或 100 mg/mL母液(储备液));
2、取适量母液，按从左到右的顺序依次添加助溶剂，澄清后再加入下一助溶剂。以 下列配方为例说明 (注意此配方只用于说明，并不一定代表此产品 的实际溶解配方):
10% DMSO → 40% PEG300 → 5% Tween-80 → 45% ddH2O (或 saline);
假设最终工作液的体积为 1 mL, 浓度为5 mg/mL: 取 100 μL 50 mg/mL 的澄清 DMSO 储备液加到 400 μL PEG300 中，混合均匀/澄清；向上述体系中加入50 μL Tween-80，混合均匀/澄清；然后继续加入450 μL ddH2O (或 saline)定容至 1 mL；
3、溶剂前显示的百分比是指该溶剂在最终溶液/工作液中的体积所占比例;
4、 如产品在配制过程中出现沉淀/析出，可通过加热(≤50℃)或超声的方式助溶;
5、为保证最佳实验结果，工作液请现配现用！
6、如不确定怎么将母液配置成体内动物实验的工作液，请查看说明书或联系我们;
7、 以上所有助溶剂都可在 Invivochem.cn网站购买。