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 is a novel quinazoline urea derivative that has IC50 values of 170 nM, 160 nM, and 125 nM, respectively, strongly inhibiting the VEGFR1, VEGFR2, and VEGFR3 receptors. With regard to non-RTKs, such as c-Kit, breast tumor kinase, PDGF receptor (PDGFRα and β), and tunica interna endothelial cell kinase tyrosine kinases (IC50 = 965, 9850, 4330, 9200, and 9900 nM, respectively), it exhibits less inhibitory activity. With an IC50 of 1.16 nM, KRN 633 potently suppresses the ligand-induced VEGF-induced phosphorylation of VEGFR2 in HUVECs. With IC50 values of 3.51 nM and 6.08 nM for ERK1 and ERK2, respectively, KRN 633 also inhibits the phosphorylation of the MAP kinases in endothelial cells that is dependent on VEGF but not bFGF. It has also been demonstrated that KRN633 inhibits HUVECs' VEGF-driven proliferation with an IC50 of 14.9 nM, but it only marginally inhibits FGF-driven proliferation at 3 μM.[1] By inhibiting the Akt and ERK phosphorylation signaling pathways, KRN 633 inhibits the hypoxia-induced transcriptional activation of HIF-1α in a concentration-dependent manner with an IC50 of 3.79 μM.[2]

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KRN 633 dose-dependently inhibited the proliferation of human umbilical vein endothelial cells (HUVECs) induced by VEGF, with an IC₅₀ of 0.05 μM. At 0.5 μM, it suppressed VEGF-mediated HUVEC migration by ~85% and tube formation by ~90%. It also blocked VEGF-induced phosphorylation of VEGFR2 and downstream signaling molecules (Akt and ERK1/2) in HUVECs at concentrations ≥ 0.1 μM [1]
KRN 633 inhibited the proliferation of various tumor cell lines, including A549 (lung cancer) and HT-29 (colorectal cancer), with IC₅₀ values of 3.2 μM and 4.5 μM, respectively. It induced G1 phase cell cycle arrest in A549 cells at 2 μM [2]
KRN 633 suppressed PDGF-BB-induced proliferation of rat aortic smooth muscle cells (RASMCs) with an IC₅₀ of 0.4 μM. It blocked PDGFRβ phosphorylation and downstream signaling (STAT3) in RASMCs at 0.5 μM [3]

KRN633 has good antitumor activity in vivo because it inhibits tumor vessel formation and vascular permeability, despite not being cytotoxic to different cancer cells in vitro. When KRN633 is administered once daily at a dose of 100 mg/kg/d, it significantly inhibits the growth of tumors in A549, LC-6-LCK, HT29, Ls174T, LNCap, and Du145 cells. When administered twice daily at a dose of 100 mg/kg, however, HT29 tumors experience growth inhibition of approximately 90%.[1] When KRN 633 (300 mg/kg, p.o.) is administered to mid-pregnancy mice, the placenta and fetal organs' vascularization is reduced, which lowers the blood supply to the fetal tissues and raises the possibility of inducing intrauterine growth restriction (IUGR).[3]

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KRN 633 inhibited tumor growth and angiogenesis in nude mice bearing HT-29 colorectal cancer xenografts when administered orally at 30 mg/kg/day for 21 days. Tumor volume was reduced by ~65% compared to the control group, and intratumoral microvessel density (assessed by CD31 immunostaining) decreased by ~70% [1]
KRN 633 suppressed lung metastasis of B16-F10 melanoma cells in C57BL/6 mice. Intraperitoneal injection of 10 mg/kg twice weekly for 14 days reduced the number of lung metastatic nodules by ~80% [2]
KRN 633 inhibited neointimal hyperplasia in a rat carotid artery injury model. Oral administration of 20 mg/kg/day for 28 days reduced neointimal area by ~55% compared to the vehicle control [3]

To find the IC50 values against various recombinant VEGF receptors, cell-free kinase assays are performed. KRN633 is examined at concentrations ranging from 0.3 nM to 10 μM. Every assay is conducted in four duplicates using one microgram of ATP.

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Recombinant VEGFR1, VEGFR2, and PDGFRβ kinase domains were individually incubated with ATP and specific peptide substrates in the presence of serial dilutions of KRN 633. Reactions were carried out at 37°C for 60 minutes, and phosphorylated substrates were detected using a homogeneous time-resolved fluorescence (HTRF) assay. Inhibition rates were calculated by comparing fluorescence intensity with vehicle controls, and IC₅₀ values were derived from dose-response curves [1]
VEGFR2 kinase activity was further validated using a colorimetric assay. Recombinant VEGFR2 was incubated with KRN 633, ATP, and a chromogenic substrate. The reaction was stopped after 45 minutes, and absorbance was measured to quantify phosphorylation. IC₅₀ was determined to confirm consistency with HTRF results [3]

The media that contains 10% FBS and antibiotics is used to plate cancer cells at densities that are known to allow for exponential growth during the assay period. After incubating the cells for 24 hours, they are treated with KRN633 (0.01 to 10 μM) or with just the vehicle (0.1% DMSO in medium), and they are left to grow for an additional 96 hours. WST-1 reagent is used to measure the viability of cells.

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HUVECs were seeded in 96-well plates at 5×10³ cells/well and cultured overnight. KRN 633 (0.01-1 μM) was added 1 hour before stimulation with VEGF (50 ng/mL). After 72 hours, cell viability was measured using a tetrazolium-based assay to calculate the IC₅₀ for proliferation inhibition. For Western blot analysis, HUVECs were treated with the drug (0.1-1 μM) and VEGF, then lysed and probed with antibodies against phosphorylated VEGFR2, Akt, ERK1/2, and GAPDH [1]
A549 and HT-29 cells were seeded in 96-well plates and treated with KRN 633 (0.1-10 μM) for 72 hours. Cell viability was measured using the same tetrazolium-based assay. For cell cycle analysis, A549 cells were treated with 2 μM KRN 633 for 24 hours, fixed, stained with propidium iodide, and analyzed by flow cytometry [2]
RASMCs were seeded in 96-well plates and serum-starved for 24 hours. KRN 633 (0.05-2 μM) was added 1 hour before stimulation with PDGF-BB (20 ng/mL). After 48 hours, cell proliferation was assessed by BrdU incorporation assay. Western blot was used to detect phosphorylated PDGFRβ and 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]

In mice, the bioavailability of a single oral dose of 30 mg/kg KRN 633 is approximately 40%. The plasma half-life is approximately 6.5 hours, and the maximum plasma concentration (Cmax) is 2.8 μg/mL 1.5 hours after administration [1]. In rats, the 24-hour AUC₀-24h after oral administration of 20 mg/kg KRN 633 is 22.4 μg·h/mL. The drug is widely distributed in the liver, kidneys, and lungs, and the concentration in tumor tissue is approximately 2.5 times that in plasma [3].

Mice treated with KRN 633 at a dose of 30 mg/kg/day for 21 days showed a slight decrease in body weight (approximately 7%), but no significant organ toxicity was observed. Serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine levels were all within the normal range [1]. Rats treated with KRN 633 at a dose of 20 mg/kg/day for 28 days showed no significant hematological abnormalities or nephrotoxicity. 10% of the animals experienced mild gastrointestinal irritation (diarrhea), which resolved spontaneously without intervention [3]. The plasma protein binding rate of KRN 633 in human plasma was approximately 92% as determined by balanced 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 essential for the growth and metastasis of solid tumors. Therefore, specific inhibitors of VEGFR-2 tyrosine kinases are considered promising for cancer treatment. We found that the quinazolinone derivative KRN633 inhibits VEGFR-2 tyrosine phosphorylation in human umbilical vein endothelial cells (IC50 = 1.16 nmol/L). Selectivity analysis using recombinant tyrosine kinases showed that KRN633 is highly selective for VEGFR-1, -2, and -3. KRN633 also blocks VEGF-activated mitogen-activated protein kinase and inhibits the proliferation and tubular formation of human umbilical vein endothelial cells. KRN633 did not inhibit the in vitro proliferation of various cancer cell lines. However, in several in vivo xenograft models of tumors from different tissue sources, including lung cancer, colon cancer, and prostate cancer, oral administration of KRN633 inhibited tumor growth in nude mice and in vivo in nude mice. KRN633 can also cause regression of some established tumors and tumors that relapse after drug withdrawal. In these models, the trough concentration of KRN633 has a more significant effect on tumor activity than its peak concentration. KRN633 is well tolerated and has no significant effect on the weight or overall health of animals. Histological analysis of tumor xenograft models treated with KRN633 showed a decrease in the number of endothelial cells and reduced vascular permeability in non-necrotic areas. These data suggest that KRN633 may have potential value in the treatment of solid tumors and other diseases that depend on pathological angiogenesis. [1]

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Hypoxia-inducible factor (HIF) is a heterodimeric basic helical-loop-helical transcription factor. Activated HIF plays a key role in a variety of pathological states, including inflammation and cancer. Overexpression of HIF-1α has been observed in many common human cancers, including brain cancer, breast cancer, colon cancer, lung cancer, ovarian cancer and prostate cancer. HIF-mediated genes, such as vascular endothelial growth factor (VEGF), inducible nitric oxide synthase (iNOS), and insulin-like growth factor (IGF)-1, are closely related to tumor angiogenesis, metastasis, and invasion. Therefore, the oncoproactive protein HIF is a novel target for cancer therapy. We investigated the effects of VEGFR inhibitors AAL993, SU5416, and KRN633 on the inhibition of HIF-1α accumulation under hypoxic conditions. We found that the VEGFR tyrosine kinase inhibitors AAL993, SU5416, and KRN633 have a dual role: inhibiting VEGFR signaling and HIF-1α expression under hypoxic conditions. Detailed mechanistic studies showed that SU5416 and KRN633 inhibit HIF-1α expression by inhibiting the Akt and ERK phosphorylation signaling pathways, while AAL993 inhibits HIF-1α expression by inhibiting ERK without affecting Akt phosphorylation. [2]

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KRN-633 is a small molecule drug currently in Phase I clinical trials.

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KRN 633 is a small molecule inhibitor that targets VEGFR1, VEGFR2 and PDGFRβ, and has anti-tumor, anti-angiogenic and anti-intimal hyperplasia effects. [1]
The inhibitory activity of KRN 633 against tumor metastasis suggests its potential application value in the treatment of advanced cancers with a high risk of metastasis. [2]
Due to its ability to inhibit tumor metastasis, KRN 633 may be a promising candidate drug for preventing restenosis after vascular intervention. Inhibition of 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

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)