KRN 633   中文名称: KRN-633

VEGFR1 (IC50 = 170 nM); VEGFR2 (IC50 = 160 nM); VEGFR3 (IC50 = 125 nM)
KRN 633 inhibits vascular endothelial growth factor receptor 2 (VEGFR2) tyrosine kinase (IC₅₀ = 0.02 μM) and VEGFR1 tyrosine kinase (IC₅₀ = 0.2 μM) [1]
KRN 633 also shows inhibitory activity against platelet-derived growth factor receptor β (PDGFRβ) (IC₅₀ = 0.3 μM) [3]

KRN 633 是一种新型喹唑啉脲衍生物，强烈抑制 VEGFR1、VEGFR2 和 VEGFR3 受体，IC50 值分别为 170 nM、160 nM 和 125 nM。它对非 RTK 表现出较低的抑制活性，例如 PDGF 受体（PDGFRα 和 β、c-Kit、乳腺肿瘤激酶和内膜内皮细胞激酶酪氨酸激酶）（IC50 = 965、9850、4330、9200 和 9900 nM， KRN 633 有效抑制 HUVEC 中配体 VEGF 诱导的 VEGFR2 磷酸化，IC50 为 1.16 nM。KRN 633 还抑制内皮细胞中 VEGF 依赖性而非 bFGF 依赖性 MAP 激酶磷酸化，IC50 值为 3.51 ERK1 和 ERK2 分别为 nM 和 6.08 nM。KRN633 还被证明可以抑制 VEGF 驱动的 HUVEC 增殖，IC50 为 14.9 nM，但仅在 3 μM 时微弱地抑制 FGF 驱动的增殖。KRN 633 抑制缺氧-通过抑制 Akt 和 ERK 磷酸化信号通路，以浓度依赖性方式诱导 HIF-1α 转录激活，IC50 为 3.79 μM。 激酶测定：进行无细胞激酶测定以获得针对多种药物的 IC50 值重组VEGF受体。 KRN633 的测试浓度范围为 0.3 nM 至 10 μM。所有测定均使用 1 μM ATP 一式四份进行。细胞测定：将癌细胞（A549、Ls174T、DU145、HT29、LNCap 和 PC-3 细胞系）接种在含有 10% FBS 和抗生素的培养基中，其密度已知可在测定期间呈指数生长。将细胞培养 24 小时，然后添加 KRN633（0.01 至 10 μM）或仅添加载体（培养基中含有 0.1% DMSO），然后再生长 96 小时。使用 WST-1 试剂测量细胞活力。

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KRN 633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0.05μM。0.5μM时，可抑制VEGF介导的HUVECs迁移约85%、管腔形成约90%；浓度≥0.1μM时，可阻断HUVECs中VEGF诱导的VEGFR2及下游信号分子（Akt和ERK1/2）的磷酸化[1]
KRN 633抑制多种肿瘤细胞系增殖，包括A549肺癌细胞和HT-29结直肠癌细胞，IC₅₀分别为3.2μM和4.5μM。2μM时可诱导A549细胞发生G1期周期阻滞[2]
KRN 633抑制血小板衍生生长因子-BB（PDGF-BB）诱导的大鼠主动脉平滑肌细胞（RASMCs）增殖，IC₅₀为0.4μM。0.5μM时可阻断RASMCs中PDGFRβ磷酸化及下游信号（STAT3）[3]

尽管KRN633在体外对多种癌细胞没有细胞毒性，但由于其对肿瘤血管形成和血管通透性的抑制作用，在体内表现出优异的抗肿瘤活性。每日一次以 100 mg/kg/d 剂量施用 KRN633 可在 A549、LC-6-LCK、HT29、Ls174T、LNCap 和 Du145 细胞中产生显着的肿瘤生长抑制作用，而每日两次以 100 mg/kg 剂量施用 KRN633 可诱导约 90 HT29 肿瘤的生长抑制百分比。用 KRN 633（300 mg/kg，口服）治疗中期妊娠小鼠会减少胎儿组织的血液供应，因为胎盘和胎儿器官的血管化减少，从而增加诱发宫内生长受限 (IUGR) 的风险。

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KRN 633以30mg/kg/天的剂量口服给药21天，可抑制裸鼠HT-29结直肠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65%，通过CD31免疫染色检测，瘤内微血管密度降低约70%[1]
KRN 633抑制C57BL/6小鼠体内B16-F10黑色素瘤细胞的肺转移。每周两次腹腔注射10mg/kg，持续14天，肺转移结节数量减少约80%[2]
KRN 633抑制大鼠颈动脉损伤模型的新生内膜增生。以20mg/kg/天的剂量口服给药28天，与溶媒对照组相比，新生内膜面积减少约55%[3]

为了找到针对各种重组 VEGF 受体的 IC50 值，进行了无细胞激酶测定。 KRN633 的检测浓度范围为 0.3 nM 至 10 μM。每个测定均使用一微克 ATP 进行四次重复。

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将重组VEGFR1、VEGFR2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KRN 633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]
采用比色法进一步验证VEGFR2激酶活性。将重组VEGFR2与KRN 633、ATP和显色底物孵育，45分钟后终止反应，通过测量吸光度定量磷酸化水平，确定IC₅₀以验证与HTRF结果的一致性[3]

含有 10% FBS 和抗生素的培养基用于以已知在检测期间允许指数生长的密度铺板癌细胞。将细胞孵育 24 小时后，用 KRN633（0.01 至 10 μM）或仅用载体（培养基中含有 0.1% DMSO）处理它们，然后让它们再生长 96 小时。 WST-1试剂用于测量细胞的活力。

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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KRN 633（0.01-1μM）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增殖抑制的IC₅₀值。蛋白质印迹分析中，用药物（0.1-1μM）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A549和HT-29细胞接种到96孔板中，用KRN 633（0.1-10μM）处理72小时，采用相同的四唑盐法检测细胞活性。A549细胞用2μM KRN 633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[2]
将RASMCs接种到96孔板中，血清饥饿24小时。加入KRN 633（0.05-2μM）预处理1小时后，用PDGF-BB（20ng/mL）刺激细胞。48小时后，通过BrdU掺入法评估细胞增殖；采用蛋白质印迹法检测磷酸化PDGFRβ和STAT3[3]

Rats: Human tumor xenografts are implanted in the hind flank of BALB/cA and Jcl-nu athymic rats. When the tumors reach the average size indicated (162 to 657 mm 3 ), rats are randomized into groups of five and treated with KRN-633 or vehicle once (qd) or twice (bid) per day at the indicated dosages. On the 14th day following the last treatment, the percentage of inhibition of tumor growth is calculated in comparison to the vehicle-treated group[1].
Mice: The mice are divided into five-group randomization once the tumors reach the average sizes of 500 to 667 mm 3 or 103 to 260 mm 3 . Following that, they receive treatment with KRN-633 or a vehicle once (qd) or twice (bid) daily at doses ranging from 10 to 100 mg/kg. The day following the final treatment, the percentage of tumor growth inhibition (TGI) relative to the vehicle-treated group is computed[1].

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Inhibition of the vascular endothelial growth factor (VEGF) signaling pathway during pregnancy contributes to several pathologic pregnancies, such as hypertension, preeclampsia, and intrauterine growth restriction, but its effects on the fetus have not been fully examined. To determine how inhibition of the VEGF signaling pathway affects the fetal vascular development of mid pregnancy, we treated pregnant mice daily with either the VEGF receptor-2 (VEGFR-2) tyrosine kinase inhibitor KRN633 (300 mg/kg, p.o.) or the vehicle from 13.5 to 15.5 day of pregnancy. On the 16.5 day of pregnancy, the vascular beds in the placenta and several organs of the fetus were visualized by fluorescent immunohistochemistry. All mice treated with KRN633 appeared healthy, and total numbers of fetuses per litter were unaffected. However, weights of the placenta and fetus from KRN633-treated mice were lower than those from the vehicle-treated ones. No external malformations and bleeding were observed in the placenta and fetus, whereas immunohistochemical analyses revealed that the vascular development in labyrinthine zone of placenta and fetal organs examined (skin, pancreas, kidney, and lung) were impaired by KRN633 treatment. These results suggest that inhibition of the VEGF signaling pathway during mid pregnancy suppresses vascular growth of both the placenta and fetus without obvious health impairments of mother mice and increases the risk of induction of intrauterine growth restriction.[3]

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Nude mice bearing HT-29 colorectal cancer xenografts (100-150 mm³) were randomly divided into control and treatment groups. KRN 633 was suspended in 0.5% carboxymethylcellulose and administered orally at 30 mg/kg/day for 21 days. Tumor volume was measured every 3 days, and mice were euthanized to collect tumors for CD31 immunostaining [1]
C57BL/6 mice were injected with B16-F10 melanoma cells via the tail vein. Two days later, mice were treated with KRN 633 via intraperitoneal injection at 10 mg/kg twice weekly for 14 days. At the end of treatment, mice were euthanized, and lungs were harvested to count metastatic nodules [2]
Male Sprague-Dawley rats underwent carotid artery balloon injury. Starting 24 hours after injury, rats were administered KRN 633 orally at 20 mg/kg/day for 28 days. Rats were euthanized, and carotid arteries were collected for histopathological analysis of neointimal hyperplasia [3]

KRN 633 had an oral bioavailability of ~40% in mice after a single dose of 30 mg/kg. The plasma half-life was approximately 6.5 hours, and the maximum plasma concentration (Cmax) was 2.8 μg/mL achieved at 1.5 hours post-administration [1]
In rats, oral administration of KRN 633 at 20 mg/kg resulted in an AUC₀-24h of 22.4 μg·h/mL. The drug was widely distributed in the liver, kidneys, and lungs, with tumor tissue concentration ~2.5-fold higher than plasma concentration [3]

Mice treated with KRN 633 at 30 mg/kg/day for 21 days showed mild weight loss (~7%) but no significant organ toxicity. Serum levels of alanine transaminase (ALT), aspartate transaminase (AST), and creatinine were within normal ranges [1]
Rats treated with KRN 633 at 20 mg/kg/day for 28 days had no obvious hematological abnormalities or renal toxicity. Mild gastrointestinal irritation (diarrhea) was observed in 10% of animals, which resolved without intervention [3]
The plasma protein binding rate of KRN 633 was ~92% in human plasma as determined by equilibrium dialysis [2]

[1]. Mol Cancer Ther . 2004 Dec;3(12):1639-49.

[2]. Cancer Lett . 2010 Oct 1;296(1):17-26.

[3]. J Pharmacol Sci . 2010;112(3):290-8.

Vascular endothelial growth factor (VEGF) and its receptor VEGFR-2 play a central role in angiogenesis, which is necessary for solid tumors to expand and metastasize. Specific inhibitors of VEGFR-2 tyrosine kinase are therefore thought to be useful for treating cancer. We showed that the quinazoline urea derivative KRN633 inhibited tyrosine phosphorylation of VEGFR-2 (IC50 = 1.16 nmol/L) in human umbilical vein endothelial cells. Selectivity profiling with recombinant tyrosine kinases showed that KRN633 was highly selective for VEGFR-1, -2, and -3. KRN633 also blocked the activation of mitogen-activated protein kinases by VEGF, along with human umbilical vein endothelial cell proliferation and tube formation. The propagation of various cancer cell lines in vitro was not inhibited by KRN633. However, p.o. administration of KRN633 inhibited tumor growth in several in vivo tumor xenograft models with diverse tissue origins, including lung, colon, and prostate, in athymic mice and rats. KRN633 also caused the regression of some well-established tumors and those that had regrown after the cessation of treatment. In these models, the trough serum concentration of KRN633 had a more significant effect than the maximum serum concentration on antitumor activity. KRN633 was well tolerated and had no significant effects on body weight or the general health of the animals. Histologic analysis of tumor xenografts treated with KRN633 revealed a reduction in the number of endothelial cells in non-necrotic areas and a decrease in vascular permeability. These data suggest that KRN633 might be useful in the treatment of solid tumors and other diseases that depend on pathologic angiogenesis.[1]

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The hypoxia-inducible factor (HIF) is a heterodimeric basic helix-loop-helix transcriptional factor and the activated HIF plays pivotal roles in various pathological conditions, including inflammation and cancer. HIF-1alpha overexpression has been observed in many common human cancers, including brain, breast, colon, lung, ovary, and prostate, and HIF-mediated genes, such as vascular endothelial growth factor (VEGF), inducible nitric oxide synthase (iNOS), and insulin-like growth factor (IGF)-1, are associated with tumor angiogenesis, metastasis, and invasion. Therefore, the pro-oncogenic protein HIF is a novel target of cancer therapy. We examined the effects of VEGFR inhibitors, AAL993, SU5416, and KRN633, on suppression of HIF-1alpha accumulation under the hypoxic condition. We found that VEGFR tyrosine kinase inhibitors, AAL993, SU5416, and KRN633, possess dual functions: inhibition of VEGFR signaling and HIF-1alpha expression under the hypoxic condition. The detailed mechanistic study indicated that SU5416 and KRN633 suppressed HIF-1alpha expression through inhibition of both Akt and ERK phosphorylation signaling pathways, whereas AAL993 suppressed HIF-1alpha expression through ERK inhibition without affecting Akt phosphorylation.[2]

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KRN-633 is a small molecule drug with a maximum clinical trial phase of I.

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KRN 633 is a small-molecule inhibitor that targets VEGFR1, VEGFR2, and PDGFRβ, exerting antitumor, antiangiogenic, and anti-neointimal hyperplasia effects [1]
The inhibitory activity of KRN 633 against tumor metastasis suggests its potential application in the treatment of advanced cancers with high metastatic risk [2]
KRN 633 may be a promising candidate for the prevention of restenosis after vascular intervention due to its ability to inhibit smooth muscle cell proliferation [3]

C20H21CLN4O4

416.86

416.125

C, 57.62; H, 5.08; Cl, 8.50; N, 13.44; O, 15.35

286370-15-8

9549295

White to off-white solid powder

1.3±0.1 g/cm3

545.6±50.0 °C at 760 mmHg

229 °C

283.7±30.1 °C

0.0±1.5 mmHg at 25°C

1.629

4.14

98.09

2

6

7

29

529

0

ClC1C([H])=C(C([H])=C([H])C=1N([H])C(N([H])C([H])([H])C([H])([H])C([H])([H])[H])=O)OC1C2=C([H])C(=C(C([H])=C2N=C([H])N=1)OC([H])([H])[H])OC([H])([H])[H]

VPBYZLCHOKSGRX-UHFFFAOYSA-N

InChI=1S/C20H21ClN4O4/c1-4-7-22-20(26)25-15-6-5-12(8-14(15)21)29-19-13-9-17(27-2)18(28-3)10-16(13)23-11-24-19/h5-6,8-11H,4,7H2,1-3H3,(H2,22,25,26)

1-[2-chloro-4-(6,7-dimethoxyquinazolin-4-yl)oxyphenyl]-3-propylurea

KRN633; KRN-633; K00589a; VEGF receptor tyrosine kinase inhibitor III; 1-(2-chloro-4-(6,7-dimethoxyquinazolin-4-yloxy)phenyl)-3-propylurea; 1-{2-chloro-4-[(6,7-dimethoxyquinazolin-4-yl)oxy]phenyl}-3-propylurea; KRN 633

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)