Discovery of Potent VEGFR-2 Inhibitors based on Furopyrimidine and Thienopyrimidne Scaffolds as Cancer Targeting Agents

Vascular endothelial growth factor receptor-2 (VEGFR-2) plays a crucial role in cancer angiogenesis. In this study, a series of novel furo[2,3-d]pyrimidine and thieno[2,3-d]pyrimidine based-derivatives were designed and synthesized as VEGFR-2 inhibitors, in accordance to the structure activity relationship (SAR) studies of known type II VEGFR-2 inhibitors. The synthesized compounds were evaluated for their ability to in vitro inhibit VEGFR-2 kinase enzyme. Seven compounds (15b, 16c, 16e, 21a, 21b, 21c and 21e) demonstrated highly potent dose-related VEGFR-2 inhibition with IC50 values in nanomolar range, of which the thieno[2,3-d]pyrimidine based-derivatives (21b, 21c and 21e) exhibited IC50 values of 33.4, 47.0 and 21 nM respectively. Moreover, furo[2,3-d]pyrimidine-based derivative (15b) showed the strongest inhibition of human umbilical vein endothelial cells (HUVEC) proliferation with 99.5% inhibition at 10 μM concentration. Consistent with our in vitro findings, compounds (21b and 21e) orally administered at 5 and 10 mg/kg/day for 8 consecutive days demonstrated potent anticancer activity in Erhlich ascites carcinoma (EAC) solid tumor murine model. Such compounds blunted angiogenesis in EAC as evidenced by reduced percent microvessel via decreasing VEGFR-2 phosphorylation with subsequent induction of apoptotic machinery. Furthermore, Miles vascular permeability assay confirmed their antiangiogenic effects in vivo. Intriguingly, such compounds showed no obvious toxicity.


Biological Evaluation assay In vitro VEGFR-2 tyrosine kinase activity Assay protocol
The assay was performed using Kinase-Glo Plus luminescence kinase assay kit (Promega). It measures kinase activity by quantitating the amount of ATP remaining in solution following a kinase reaction. The luminescent signal from the assay is correlated with the amount of ATP present and is inversely correlated with the amount of kinase activity. The compounds were diluted to 100 µl in 10% DMSO and 5 µl of the dilution was added to a 50 µl reaction so that the final concentration of DMSO is 1% in all of reactions. All of the enzymatic reactions were was added to each reaction and incubate the plate for 5 minutes at room temperature.
Luminescence signal was measured using a BioTek Synergy 2 microplate reader. VEGFR activity assays were performed in duplicate at each concentration.

Data analysis
The luminescence data were analyzed using the computer software, Graphpad Prism. The

In vitro multiple-kinase inhibition assay Assay protocol
The assay was performed using the same conditions for VEGFR-2 activity assay (section 5.2.1.1). The assays for c-Kit was an exception, it was performed using ADP-Glo Kinase assay reagents (Promega). It measures kinase activity by quantitating the ADP amount produced from the enzymatic reaction. The luminescent signal from the assay is correlated with the amount of ADP present and is directly correlated with the amount of kinase activity. Other conditions are same to the previous section except the final reaction volume was reduced to 25 µl. After the 40 min kinase reaction at 30 o C, 25 µl of ADP-Glo reagent was added and incubated for 45 min at room temperature followed by another 50 min incubation with 50 µl of Kinase detection mixture.
Luminescence signal was measured using a BioTek Synergy 2 microplate reader

Data analysis
The luminescence data were analyzed using the same computer software and the same protocol used for VEGFR-2 activity assay (section 5.2.1.2) In vitro HUVEC Anti-proliferative assay Assay protocol Doxorubicin, a known cancer treatment, is used as control compound. Doxorubicin concentration is 0.0033-33.3 µM, 3 folds dilutions of the tested compounds. HUVEC cells were cultured in Medium 200 with 2% LVES and 1% Pen-strep. To perform the proliferation assay, HUVEC cells were seeded at 5000 cells/ 50l /well in a 96-well black clear-bottom tissue culture plate. Cells were incubated at 37C and 5% CO 2 overnight to allow them to recover and reattach.
Dilutions are made in growth medium so that the final concentration of DMSO is 0.1% in all treatments.
Next day cells were treated with test compounds for 72 hours. After treatment, cell proliferation was measured by Fluorescent quantitation of alamarBlue reagent. The alamarBlue assay incorporates a fluorometric/colorimetric growth indicator based on detection of metabolic activity. Specifically, resazurin, the active ingredient in the alamarBlue reagent, is blue in color and virtually non-fluorescent. Upon entering cells, resazurin is reduced to resorufin, a compound that is red in color and highly fluorescent. Continued cell growth maintains a reduced environment, therefore increasing the overall fluorescence and color of the media surrounding cells. The fluorescence intensity of alamarBlue reagent is shown to be directly proportional to cell number. To perform the alamarBlue assay, 10 l of alamarBlue reagent was added to each well and the plate was incubated at 37C for an additional 2 hours. Fluorescence intensity was measured at an excitation of 530 nm and an emission of 590 nm using a BioTek Synergy TM 2 microplate reader.

Data analysis
Cell proliferation assays were performed in triplicate at each concentration. The fluorescent intensity data were analyzed using the computer software, Graphpad Prism. In the absence of the compound, the fluorescent intensity (F t ) in each data set was defined as 100%. In the absence of cells, the fluorescent intensity (F b ) in each data set was defined as 0%. The percent cell in the presence of each compound was calculated according to the following equation: %cell = (F-

Histopathological Examination
Autopsy samples were taken from subcutaneously transplanted EAC, liver, kidney, heart and brain of mice in different groups and fixed in 10% formol saline for 24 hours. Washing was carried out with tap water then serial dilutions of alcohol (methyl, ethyl and absolute ethyl) were used for dehydration. Specimens were cleared in xylene and embedded in paraffin at 56°C in hot air oven for 24 hours. Paraffin beeswax tissue blocks were prepared for sectioning at 4 µm thickness by slidge microtome. The obtained tissue sections were collected on glass slides, deparaffinized, stained by hematoxylin & eosin stain for routine examination by light electric microscopy. 1

Immunohistochemistry
Paraffin embedded EAC sections of 3 μm thickness were first rehydrated in xylene and then in graded ethanol solutions. The slides were then blocked with 5% bovine serum albumin (BSA) in tris buffered saline (TBS) for 2 h. The sections were then immunostained with primary antibodies specific to phospho-VEGFR-2 (Tyr951), CD31, CD34 and active caspase 3 at a concentration of 1 μg/ml containing 5% BSA in TBS and incubated overnight at 4 °C. After washing the slides with TBS, the sections were incubated with goat anti-rabbit secondary antibody. Sections were then washed with TBS and incubated for 5-10 min in a solution of 0.02% diaminobenzidine containing 0.01% H 2 O 2 . Counter staining was performed using hematoxylin, and slides were visualized using a light microscope. Fraction of DAB-positive immunoreactive area was automatically calculated in high power fields (100x) as area percentage of immunopositive cells to the total area of the microscopic field using a digital video camera mounted on a light microscope (CX21, OLYMPUS, JAPAN). Percent of microvessel density (% MVD) was obtained by automatically calculating fraction of CD31 and CD34 DAB-positive immunoreactive area in high power fields (100x) as area percentage of immunopositive cells to the total area of the microscopic field. All steps for immunohistochemical evaluation were carried out using image analysis software (Image J, 1.46a, NIH, USA) according to the method described by Mezei et al. 2 In vitro Anti-proliferative activity against 60 cell line panel

Assay protocol
The human tumor cell lines of the cancer screening panel are grown in RPMI 1640 medium containing 5% fetal bovine serum and 2 mM L-glutamine. For a typical screening experiment, cells are inoculated into 96 well microtiter plates in 100 µL at plating densities ranging from 5,000 to 40,000 cells/well depending on the doubling time of individual cell lines. After cell inoculation, the microtiter plates are incubated at 37° C, 5 % CO 2 , 95 % air and 100 % relative humidity for 24 hours prior to addition of experimental drugs. After 24 hours, two plates of each cell line are fixed in situ with TCA, to represent a measurement of the cell population for each cell line at the time of drug addition (Tz). Experimental drugs are solubilized in dimethyl sulfoxide at 400-fold the desired final maximum test concentration and stored frozen prior to use.
At the time of drug addition, an aliquot of frozen concentrate is thawed and diluted to twice the desired final maximum test concentration with complete medium containing 50 µg/mL gentamicin. Additional four, 10-fold or ½ log serial dilutions are made to provide a total of five drug concentrations plus control. Aliquots of 100 µl of these different drug dilutions are added to the appropriate microtiter wells already containing 100 µl of medium, resulting in the required final drug concentrations. Following drug addition, the plates are incubated for an additional 48 hrs at 37°C, 5 % CO 2 , 95 % air, and 100 % relative humidity. For adherent cells, the assay is terminated by the addition of cold TCA. Cells are fixed in situ by the gentle addition of 50 µl of cold 50 % (w/v) TCA (final concentration, 10 % TCA) and incubated for 60 minutes at 4°C. The supernatant is discarded, and the plates are washed five times with tap water and air dried.
Sulforhodamine B (SRB) solution (100 µl) at 0.4 % (w/v) in 1 % acetic acid is added to each well, and plates are incubated for 10 minutes at room temperature. After staining, unbound dye is removed by washing five times with 1 % acetic acid and the plates are air dried. Bound stain is subsequently solubilized with 10 mM trizma base, and the absorbance is read on an automated plate reader at a wavelength of 515 nm. For suspension cells, the methodology is the same except that the assay is terminated by fixing settled cells at the bottom of the wells by gently adding 50 µl of 80 % TCA (final concentration, 16 % TCA).

Data analysis
Using the seven absorbance measurements [time zero, (Tz), control growth, (C), and test growth in the presence of drug at the five concentration levels (Ti)], the percentage growth is calculated at each of the drug concentrations levels. Percentage growth inhibition is calculated as:[(Ti-Tz)/(C-Tz)] x 100 for concentrations for whichTi>/ = Tz and [(Ti -Tz)/Tz] x 100 for concentrations for which Ti < Tz. 3