VEGFR1 (IC50 = 170 nM); VEGFR2 (IC50 = 160 nM); VEGFR3 (IC50 = 125 nM)

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]

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]

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.

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.

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 mm³), 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 mm³ or 103 to 260 mm³. 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]

[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.

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)