1-aryl-3-[4-(thieno[3,2-d]pyrimidin-4-yloxy)phenyl]ureas ... · 2 biomedresearchinternational n h o...

Hindawi Publishing CorporationBioMed Research InternationalVolume 2013 Article ID 154856 9 pageshttpdxdoiorg1011552013154856

Research Article1-Aryl-3-[4-(thieno[32-d]pyrimidin-4-yloxy)phenyl]ureasas VEGFR-2 Tyrosine Kinase Inhibitors Synthesis BiologicalEvaluation and Molecular Modelling Studies

Pedro Soares12 Raquel Costa3 Hugo J C Froufe4 Ricardo C Calhelha14 Daniela Peixoto1

Isabel C F R Ferreira4 Rui M V Abreu4 Raquel Soares3 and Maria-Joatildeo R P Queiroz1

1 Centro de Quımica Escola de Ciencias Universidade do Minho Campus de Gualtar 4710-057 Braga Portugal2 CIQDepartamento de Quımica e Bioquımica Faculdade de Ciencias Universidade do PortoRua do Campo Alegre 4169-007 Porto Portugal

3 Departamento de Bioquımica (U38-FCT) Faculdade de Medicina Universidade do Porto 4200-319 Porto Portugal4 Centro de Investigacao deMontanha (CIMO) Escola Superior Agraria Instituto Politecnico de Braganca Campus de Santa ApoloniaApartado 1172 5301-855 Braganca Portugal

Correspondence should be addressed to Maria-Joao R P Queiroz mjrpqquimicauminhopt

Received 7 April 2013 Revised 12 June 2013 Accepted 13 June 2013

Academic Editor Carmen Domene

Copyright copy 2013 Pedro Soares et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

The vascular endothelial growth factor receptor-2 (VEGFR-2) is a tyrosine kinase receptor involved in the growth anddifferentiationof endothelial cells that are implicated in tumor-associated angiogenesis In this study novel 1-aryl-3-[4-(thieno[32-d]pyrimidin-4-yloxy)phenyl]ureas were synthesized and evaluated for the VEGFR-2 tyrosine kinase inhibitionThree of these compounds showedgood VEGFR-2 inhibition presenting low IC

50values (150ndash199 nM) in enzymatic assays showing also a significant proliferation

inhibition of VEGF-stimulated human umbilical vein endothelial cells (HUVECs) at low concentrations (05ndash1120583M) using theBromodeoxyuridine (BrdU) assay not affecting cell viabilityThe determination of the total and phosphorylated (active) VEGFR-2was performed by western blot and it was possible to conclude that the compounds significantly inhibit the phosphorylation ofthe receptor at 1 120583Mpointing to their antiproliferative mechanism of action in HUVECsThemolecular rationale for inhibiting thetyrosine kinase domain of VEGFR-2 was also performed and discussed using molecular docking studies

1 Introduction

Angiogenesis is the process of new blood vessel formationfrom preexisting vascular networks by capillary sprouting [1]and plays an important role in the pathogenesis of severaldisorders including cancer vasculoproliferative ocular disor-ders and rheumatoid arthritis [2] A key regulatory pathwayof angiogenesis is mediated by the vascular endothelialgrowth factor (VEGF) involved in the vascular permeabilityand an inducer of endothelial cell proliferation migrationand survival [3] and its cell membrane tyrosine kinase recep-tor VEGFR-2 (also known as KDR) [4] Upon ligand bindingVEGFR-2 undergoes autophosphorylation triggering signal-ing pathways leading to endothelial cell proliferation andsubsequent angiogenesis Small molecule inhibitors act by

competing with ATP for its binding site of the VEGFR-2intracellular tyrosine kinase domain thereby preventing thesignaling pathways that lead to angiogenesis [5] Several smallmolecule VEGFR-2 inhibitors have emerged as promisingantiangiogenic agents for possible treatment against a widevariety of cancers (Figure 1) Sunitinib was approved for thetreatment of renal cell carcinoma and gastrointestinal stromaltumor and Sorafenib was approved for the treatment ofprimary kidney cancer and hepatocellular carcinoma [6]Recently two new VEGFR-2 inhibitors have been approvedfor the treatment of advanced renal cell carcinoma axitinib[7] and pazopanib [8] A number of thienopyridines andthienopyrimidines ureas have also shown potent VEGFR-2inhibition activity [9 10] including arylether derivatives [11ndash13]

2 BioMed Research International

NH O

F

HNNH

N

O

NH

O

N

O

HN

O

NH

Cl

F

FF

Sunitinib Sorafenib

NN

N

NN

NH

SO O

Pazopanib

S

NH

N

N

ONH

Axitinib

CH3

CH3 CH3

CH3

CH3

NH2

Figure 1 Competitive inhibitors of VEGFR-2 in clinical use against several cancers sunitinib (Sutent C P Pharmaceuticals InternationalNY USA) soranefib (Nexavar Bayer Germany) axitinib (Inlyta Pfizer) and pazopanib (Votrient GlaxoSmithKline)

In this report we describe an ongoing effort to developnovel small molecules as VEGFR-2 inhibitors based onthe aryletherthieno[32-d]pyrimidine arylurea scaffold Thesynthesis of the compounds and the VEGFR-2 tyrosinekinase phosphorylation inhibition evaluation using eitherenzymatic or cellular assays including the determination ofthe total and of the phosphorylated VEGFR-2 by western blotwere performed The probable binding mode of the 1-aryl-3-[4-(thieno[32-d]pyrimidin-4-yloxy)phenyl]ureas with thereceptor using docking studies is also presented and dis-cussed

2 Materials and Methods

21 Synthesis Melting points (∘C) were determined in aStuart SMP3 and are uncorrected 1H and 13C NMR spectrawere recorded on a Varian Unity Plus at 300 and 754MHzrespectively or on a Bruker Avance III at 400 and 1006MHzrespectively Two-dimensional 1H-13C correlations were per-formed to attribute some signals Mass spectra (MS) EI-TOFor ESI-TOF and HRMS on the M+ [M + H]+ or on [M +Na]+ were performed by the mass spectrometry service ofthe University of Vigo CACTI Spain

The reactions were monitored by thin layer chromatog-raphy (TLC) using Macherey-Nagel precoated aluminiumsilica gel 60 sheets (020mm) with UV

254indicator Column

chromatography was performed on Panreac silica gel 60230ndash400 mesh

211 General Procedure for the Synthesis of Compounds 1aand 1b In a flask with 5mL of DMF thienopyrimidine (1equiv) 4-aminophenol (1 equiv) and K

2CO3(4 equiv) were

heated at 140∘C for 2 h After cooling water (5mL) and ethylacetate (5mL) were added The phases were separated andthe aqueous phase was extracted with more ethyl acetate (2times 5mL) The organic phase was dried (MgSO

4) and filtered

The solvent was evaporated under reduced pressure giving asolid which was submitted to column chromatography

4-(Thieno[32-d]pyrimidin-4-yloxy)aniline (1a) 4-Chlorothi-eno[32-d]pyrimidine (242mg 142mmol) and 4-amino-phenol (155mg 142mmol) were heated and the reactionmixture was treated according to the general procedureColumn chromatography using ethyl acetate gave compound1a as a yellow solid (290mg 91) mp 1158ndash1165∘C 1HNMR (300MHz DMSO-119889

6) 120575 515 (s 2H NH

2) 660 (d 119869

= 90Hz 2H 2 and 6-H) 695 (d 119869 = 90Hz 2H 3 and 5-H)762 (d 119869 = 56Hz 1H HetAr) 840 (d 119869 = 56Hz 1H HetAr)866 (s 1H 21015840-H) ppm 13C NMR (754MHz DMSO-119889

6) 120575

1142 (2 and 6-CH) 1166 (C) 1223 (3 and 5-CH) 1241 (CH)1371 (CH) 1417 (C) 1469 (C) 1542 (21015840-CH) 1629 (C) 1643(C) ppmMS (EI-TOF)119898119911 () 24305 (M+ 100)HRMS (EI-TOF) calcd for C

12H9N3OS [M+] 2430466 found 2430467

4-(7-Methylthieno[32-d]pyrimidin-4-yloxy)aniline (1b) 4-Chloro-7-methylthieno[32-d]pyrimidine (242mg 131mmol) and 4-aminophenol (143mg 131mmol) were heatedand the reaction mixture was treated according to thegeneral procedure Column chromatography using ethylacetate gave compound 1b as a yellow solid (310mg 92)mp 1678ndash1686∘C 1H NMR (300MHz DMSO-119889

6) 120575 240

(s 3H CH3) 513 (s 2H NH

2) 661 (d 119869 = 90Hz 2H 2 and

6-H) 693 (d 119869 = 90Hz 2H 3 and 5-H) 801 (s 1H 61015840-H)867 (s 1H 21015840-H) ppm 13C NMR (754MHz DMSO-119889

6) 120575

125 (CH3) 1142 (2 and 6-CH) 1168 (C) 1223 (3 and 5-CH)

BioMed Research International 3

1314 (61015840-CH) 1326 (C) 1417 (C) 1468 (C) 1539 (21015840-CH)1618 (C) 1643 (C) ppm MS (EI-TOF)119898119911 () 25706 (M+100) HRMS (EI-TOF) calcd for C

13H11N3OS [M+] 2570623

found 2570621

212 General Procedure for the Synthesis of 13-Diarylureas2andashf Compounds 1a or 1b and different arylisocyanates (1equiv) in 6mL CH

2Cl2 THF (1 1) were left stirring at room

temperature for 16 h If a precipitate does not come outafter this time hexane (3ndash5mL) is added to the mixture toprecipitate the product This was filtered under vacuum togive the corresponding 13-diarylureas

1-Phenyl-3-[4-(thieno[32-d]pyrimidin-4-yloxy)phenyl]urea(2a) From compound 1a (100mg 0410mmol) andphenylisocyanate (00400mL 0410mmol) compound 2awas isolated as a white solid (117mg 79) mp 2373ndash2387∘C 1H NMR (400MHz DMSO-119889

6) 120575 695ndash699 (m

1H Ar-H) 724ndash730 (m 4H Ar-H) 745ndash747 (m 2H 2 timesAr-H) 754 (d 119869 = 90Hz 2H 2 times Ar-H) 765 (d 119869 = 52Hz1H HetAr) 845 (d 119869 = 52Hz 1H HetAr) 868 (s 1HNH) 869 (s 1H 2101584010158401015840-H) 878 (s 1H NH) ppm 13C NMR(1006MHz DMSO-119889

6) 120575 1168 (C) 1182 (2 times CH) 1192

(2 times CH) 1218 (CH) 1223 (2 times CH) 1242 (CH) 1288 (2times CH) 1372 (CH) 1376 (C) 1396 (C) 1460 (C) 1526 (C)1541 (2101584010158401015840-CH) 1630 (C) 1638 (C) ppm MS (ESI-TOF)119898119911() 36309 ([M + H]+ 100) HRMS (ESI-TOF) calcd forC19H15N4O2S [M + H]+ 3630910 found 3630909

1-(4-Methoxyphenyl)-3-[4-(thieno[32-d]pyrimidin-4-yloxy)phenyl]urea (2b) From compound 1a (100mg 0410mmol)and 4-methoxyphenylisocyanate (00500mL 0410mmol)compound 2b was isolated as a white solid (145mg 90)mp 2472ndash2483∘C 1H NMR (400MHz DMSO-119889

6) 120575 371

(s 3H OCH3) 686 (d 119869 = 90Hz 2H 31015840 and 51015840-H) 723

(d 119869 = 90Hz 2H 2 times Ar-H) 736 (d 119869 = 90Hz 2H 21015840and 61015840-H) 753 (d 119869 = 90Hz 2H 2 times Ar-H) 765 (d 119869 =56Hz 1H HetAr) 844 (d 119869 = 56Hz 1H HetAr) 850(s 1H NH) 869 (s 1H 2101584010158401015840-H) 870 (s 1H NH) ppm 13CNMR (1006MHz DMSO-119889

6) 120575 551 (OCH

3) 1140 (31015840 and

51015840-CH) 1168 (C) 1191 (2 times CH) 1201 (21015840 and 61015840-CH) 1223(2 times CH) 1242 (CH) 1327 (C) 1372 (CH) 1378 (C) 1459(C) 1528 (C) 1541 (2101584010158401015840-CH) 1545 (C) 1630 (C) 1638 (C)ppm MS (ESI-TOF) 119898119911 () 39308 ([M+H]+ 39) HRMS(ESI-TOF) calcd for C

20H17N4O3S [M+H]+ 3931016 found

3931026

1-(4-Cyanophenyl)-3-[4-(thieno[32-d]pyrimidin-4-yloxy)phenyl]urea (2c) From compound 1a (100mg 0410mmol)and 4-cyanophenylisocyanate (590mg 0410mmol) com-pound 2c was isolated as a white solid (111mg 70) mp2455ndash2472∘C 1H RMN (300MHz DMSO-119889

6) 120575 726 (d 119869

= 92Hz 2H 2 times Ar-H) 755 (d 119869 = 92Hz 2H 2 times Ar-H)762ndash766 (m 3H 21015840 and 61015840-H and HetAr) 773 (d 119869 = 92Hz2H 31015840 and 51015840-H) 845 (d 119869 = 52Hz 1H HetAr) 869 (s1H 2101584010158401015840-H) 899 (s 1H NH) 924 (s 1H NH) ppm 13CNMR (754MHz DMSO-119889

6) 120575 1033 (C) 1168 (C) 1180

(21015840 and 61015840-CH) 1193 (C) 1197 (2 times CH) 1224 (2 times CH)1243 (CH) 1333 (31015840 and 51015840-CH) 1370 (C) 1372 (CH) 1442

(C) 1465 (C) 1522 (C) 1541 (2101584010158401015840-CH) 1630 (C) 1638 (C)ppm MS (ESI-TOF)119898119911 () 38809 ([M +H]+ 100) HRMS(ESI-TOF) calcd for C

20H13N5O2S [M + H]+ 3880863

found 3880861

1-Phenyl-3-[4-(7-Methylthieno[32-d]pyrimidin-4-yloxy)phe-nyl]urea (2d) From compound 1b (100mg 0389mmol)and phenylisocyanate (00400mL 0389mmol) compound2d was isolated as a white solid (121mg 83) mp 2857ndash2873∘C 1H NMR (400MHz DMSO-119889

6) 120575 243 (s 3H

CH3) 696 (m 1H Ar-H) 723ndash729 (m 4H Ar-H) 746 (m

2H 2 times Ar-H) 753 (d 119869 = 92Hz 2H 2 times Ar-H) 810 (s 1H6101584010158401015840-H) 870 (s 1H NH) 871 (s 1H 2101584010158401015840-H) 878 (s 1H NH)ppm 13C NMR (1006MHz DMSO-119889

6) 120575 125 (CH

3) 1169

(C) 1182 (2 times CH) 1192 (2 times CH) 1218 (C) 1223 (2 times CH)1288 (2 times CH) 1315 (6101584010158401015840-CH) 1327 (C) 1376 (C) 1396(C) 1461 (C) 1526 (C) 1539 (2101584010158401015840-CH) 1619 (C) 1639 (C)ppm MS (ESI-TOF)119898119911 () 37710 ([M + H]+ 100) HRMS(ESI-TOF) calcd for C

20H17N4O2S [M +H]+ 3771067 found

3771064

1-(4-Methoxyphenyl)-3-[4-(7-methylthieno[32-d]pyrimidin-4-yloxy)phenyl]urea (2e) From compound 1b (100mg0389mmol) and 4-methoxyphenylisocyanate (00500mL0389mmol) compound 2e was isolated as a white solid(131mg 83) mp 2484ndash2496∘C 1H NMR (400MHzDMSO-119889

6) 120575 243 (s 3H CH

3) 371 (s 3H OCH

3) 686

(d 119869 = 92Hz 2H 31015840 and 51015840-H) 722 (d 119869 = 92Hz 2H 310158401015840and 510158401015840-H) 736 (d 119869 = 92Hz 2H 21015840 and 61015840-H) 752 (d 119869 =92Hz 2H 210158401015840 and 610158401015840-H) 806 (s 1H 6101584010158401015840-H) 849 (s 1HNH) 869 (s 1H NH) 870 (s 1H 2101584010158401015840-H) ppm 13C NMR(1006MHz DMSO-119889

6) 120575 125 (CH

3) 552 (OCH

3) 1140

(31015840 and 51015840-CH) 1169 (C) 1191 (210158401015840 and 610158401015840-CH) 1201 (21015840 and61015840-CH) 1222 (310158401015840 and 510158401015840-CH) 1315 (6101584010158401015840-CH) 1327 (C)1378 (C) 1460 (C) 1528 (C) 1539 (2101584010158401015840-CH) 1545 (C) 1619(C) 1639 (C) ppm MS (ESI-TOF) 119898119911 () 40712 ([M +H]+ 100) HRMS (ESI-TOF) calcd for C

21H19N4O3S [M +

H]+ 4071172 found 4071182

1-(4-Cyanophenyl)-3-[4-(7-methylthieno[32-d]pyrimidin-4-yloxy)phenyl]urea (2f ) From compound 1b (100mg 0389mmol) and 4-cyanophenylisocyanate (560mg 0389mmol)compound 2f was isolated as a white solid (128mg 82)mp 2467ndash2482∘C 1H NMR (400MHz DMSO-119889

6) 120575 242

(s 3H CH3) 725 (d 119869 = 92Hz 2H 310158401015840 and 510158401015840-H) 754 (d

119869 = 92Hz 2H 210158401015840 and 610158401015840-H) 764 (d 119869 = 92Hz 2H 21015840and 61015840-H) 772 (d 119869 = 92Hz 2H 31015840 and 51015840-H) 806 (s 1H6101584010158401015840-H) 870 (s 1H 2101584010158401015840-H) 897 (s 1H NH) 923 (s 1H NH)ppm 13C NMR (1006MHz DMSO-119889

6) 120575 125 (CH

3) 1033

(C) 1170 (C) 1181 (21015840 and 61015840-CH) 1193 (C) 1197 (210158401015840 and610158401015840-CH) 1224 (310158401015840 and 510158401015840-CH) 1315 (6101584010158401015840-CH) 1327 (C)1333 (31015840 and 51015840-CH) 1370 (C) 1442 (C) 1466 (C) 1522(C) 1539 (2101584010158401015840-CH) 1620 (C) 1638 (C) ppm MS (ESI-TOF)119898119911 () 40210 ([M + H]+ 100) HRMS (ESI-TOF) calcd forC21H16N5O2S [M + H]+ 4021019 found 4021029

22 VEGFR-2 Enzymatic Inhibition Assay The compoundswere assessed for VEGFR-2 inhibition activity using the 1198851015840-LYTE-Tyr1 Peptide assay kit (according to the procedures


N

N

S

O

NH

NH

O

N

N

S

ON

N

S

Cl

HO NC

O

1

2

3

32a d

cb

4

1

2

3

6

5

7

2

65

3

4

R1

R1 = H or CH3

NH2

K2CO3 (4 equiv)

2998400

4998400

6998400

7998400

R2

NH2

rt 12 h

1a R1 = H 911b R1 = CH3 92

2998400998400998400

6998400998400998400

6998400998400

5998400998400

3998400998400

2998400998400

6998400

5998400

3998400

2998400

R1

R2

140∘C 1 h

R1

CH2Cl2 THF (1 1)

2a R1 = R2 = H 79

2c R1 = H R2 = CN 70

2d R1 = CH3 R2 = H 83

2e R1 = CH3 R2 = OCH3 83

2f R1 = CH3 R2 = CN 82

DMF

2b R1 = H R2 = OCH3 90

Scheme 1 Synthesis of 1-aryl-3-[4-(thieno[32-d]pyrimidin-4-yloxy)phenyl]ureas 2andash2f The yields are presented as of pure compounds

recommended by the manufacturer Invitrogen Cat PV3190)[14] Briefly assays were performed in a total of 20120583L in384-well plates using fluorescence resonance energy transfertechnology A Tyr1 substrate (coumarin-fluorescein double-labeled peptide) at 10120583Mwas incubated for 1 h with 4 120583gmLVEGFR-2 10 120583MATP and inhibitors at room temperature in50mM HepesNa (pH 75) 10mM MgCl

2 20mM MnCl

2

25mM DTT 010mM orthovanadate and 001 bovineserum albumin The wells were incubated at 25∘C for 1 hourand 50 120583L of the development reagent was added to eachwell After a second incubation of 1 hour a stop reagentwas added to each well Using a Biotek FLX800 microplatethe fluorescence was read at 445 nm and 520 nm (excitation400 nm) and Gen5 Software was used for data analysisThe validation assay was performed using Staurosporinethat presented a IC

50value 6 nM that compares to the one

reported in the literature (IC507 nM) [14]

23 Cell Culture Experiments Using HUVECs

231 Cell Cultures Human umbilical vein endothelial cells(HUVECs) were obtained from the ScienceCell ResearchLabs (San Diego CA USA) HUVECs were seeded onplates coated with 02 gelatin (Sigma-Aldrich Sintra Por-tugal) and cultured in M199 medium (Sigma-Aldrich Sin-tra Portugal) supplemented with 20 fetal bovine serum(FBS) (Invitrogen Life Technologies Scotland UK) 1 peni-cillinstreptomycin (Invitrogen Life Technologies) 001heparin (Sigma-Aldrich) and 30mgmL endothelial cellgrowth supplement (Biomedical Technologies Inc MAUSA) and maintained at 37∘C in a humidified 5 carbondioxide atmosphere Cells were kept between passages 3 and8 for every experiment The test compounds were dissolvedin DMSO and added to cell cultures at a concentration of01ndash10 120583M Incubations were performed for 24 h in mediumsupplemented with 2 FBS 1 penicillinstreptomycin and

60 ngmL of the vascular endothelial growth factor (Sigma-Aldrich Sintra Portugal) Control cells were incubated withvehicle (DMSO at 01 in every culture)

232 MTS Toxicity Assay HUVECs (2 times 105 cellsmL)were allowed to grow for 24 h and then incubated with thetest compounds at a range concentration between 01 and10 120583M or control (01 DMSO) for 24 h After the incubationperiod cells were washed and their viability was assessedusing Cell Titer 96 Aqueous ONE Solution Reagent MTS[3-(45-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] colorimetric assay (Pro-megaMadisonUSA) according to the instructions providedby the manufacturer and as previously described [15]Optical density was measured at 492 nm Three independentexperiments were performed and the results were expressedas mean plusmn SEM

233 BrdU Incorporation Assay HUVECs (6 times 104 cellsmL)were grown during 24 h and then were incubated withthe compounds at 01ndash10 120583M or control (01 DMSO) for24 h Cells were also incubated with 51015840-bromodeoxyuridine(BrdU) a thymidine analogue which incorporates into DNAof dividing cells After incubation with BrdU solution at afinal concentration of 001mM during the treatment periodthe optical density of proliferating cells (positive for BrdU)after ELISA assay using anti-BrdU-specific antibodies (RocheDiagnostics Mannheim Germany) was evaluated at themicroplate reader according to the manufacturerrsquos instruc-tions and as previously reported [16] The results are givenas percentage versus control group (100)

234 Cell Apoptosis HUVECs (6times 104 cellsmL)were grownfor 24 h on glass coverslips and incubated with differentconcentrations (01ndash1120583M) of the tested compounds TUNELassay was performed using the In Situ Cell Death Detection


3

2

1

0

C 01

05 1 5 1001

05 1 5 1001

05 1 5 10

lowast lowast lowast lowastlowast

HU

VEC

via

bilit

yO

ptic

al d

ensit

y (4

92 nm

)

2a 2b 2c

(a)

Prol

ifera

ting

cells

()

(fold

incr

ease

ver

sus c

ontro

l) 150

100

50

0

C 01

05 1 5 1001

05 1 5 1001

05 1 5 10

2a 2b 2c

lowast lowast lowast lowastlowast lowast

lowast lowastlowast lowast lowast lowast

(b)(fo

ld in

crea

se v

ersu

s con

trol) 150

100

50

0

C 01

05 1

01

05 1

01

05 1

2a 2b 2c

lowast

Apop

totic

cells

()

(c)Figure 2 (a) Cell viability was evaluated in HUVECs upon treatment with 2a 2b and 2c at 010 050 10 50 and 10 120583M or untreated(control 01 DMSO) using MTS assay (b) Cell proliferation was assessed by BrdU incorporation assay Bars represent the percentage ofproliferating cells when compared to control group after incubation with anti-BrdU antibody by ELISA assay (c) Apoptosis was assessedin HUVECs by TUNEL assay using 2a 2b and 2c at 01 05 and 1 120583M Bars represent the percentage of apoptotic cells evaluated by theratio between TUNEL-stained cells and DAPI-stained nuclei in every culture Results are expressed as mean plusmn SEM of three independentexperiments (5 lt 119899 lt 10) lowast119875 lt 005 versus control

kit (Roche Diagnostics Mannheim Germany) according tothe manufacturerrsquos instruction and as previously reported[16] Immunofluorescence was visualized under a fluores-cence microscope (Olympus BH-2 UK) The percentageof stained cells was evaluated by counting the cells stainedwith TUNEL (apoptotic cells) divided by the total numberof nuclei counterstained with DAPI (Invitrogen CA USA)at a 200x magnification field One thousand nuclei wereevaluated Results were expressed as percentage of control(100)

235 Western Blotting Analyses Proteins were isolated fromHUVEC lysates using RIPA (Chemicon International CAUSA) Proteins were quantified using a spectrophotometer(Jenway 6405 UVvis Essex UK) and 20120583g of proteinwere subjected to 8 SDS-PAGE with a 5 stacking gelAfter electrophoresis proteins were blotted into a Hybondnitrocellulose membrane (Amersham Arlington VA USA)Immunodetection for total VEGFR-2 (1 1000 Cell SignalingMA USA) phosphorylated (activated) VEGFR-2 (1 750Santa Cruz Biotechnology CA USA) and 120573-actin (1 3000Abcam Cambridge UK) was accomplished with enhancedchemiluminescence (ECL kit Amersham Arlington USA)The phosphorylated antibody recognizes Tyr951 kinase insertdomain a major site of VEGFR-2 phosphorylation partic-ularly involved in angiogenic processes The relative inten-sity of each protein blotting analysis was measured using

a computerized software program (Bio-Rad CA USA) andthe expression of activated and total VEGFR-2 were nor-malized with total VEGFR-2 and 120573-actin bands respectivelyto compare the expression of proteins in different treatmentgroups

236 Statistical Analyses All experiments were performedat least in three independent experiments Quantificationsare expressed as mean plusmn SEM Statistical significance ofdifference between the distinct groups was evaluated by anal-ysis of variance (ANOVA) followed by the Bonferroni testA difference between experimental groups was consideredsignificant with a confidence interval of 95 whenever 119875 lt005

24 Docking Simulations Using AutoDock4 A VEGFR-2crystal structure (PDB 2XIR) was extracted from the ProteinData Bank (PDB) (httpwwwrcsborg) The cocrystallizedligand was extracted from the PDB file and AutoDockToolswas used to assign polar hydrogens and Gasteiger charges tothe compounds [17] AutoGrid4 was used to create affinitygridmaps for all the atom typesThe affinity grids enclosed anarea of 100 times 100 times 100 with 0375 A spacing centered on thecoordinates 119909 = 863 119910 = 512 and 119911 = 483 AutoDock4(version 41) with the Lamarckian genetic algorithm wasused with the following docking parameters 100 dockingruns population size of 200 random starting position and


Table 1 Results of the VEGFR2 enzymatic inhibition assay usingcompounds 2andashf

Compounds VEGFR-2IC50 (120583M)a

2a 01992b 01882c 01502d gt1002e gt1002f gt100aEach IC50 determination is a result of four separate determinations

conformation translation step ranges of 20 A mutation rateof 002 crossover rate of 08 local search rate of 006 and 25million energy evaluations [17] The entire virtual screeningexperiment was performed on a cluster of 8 Intel Dual-Core28GHz computers using MOLA software [18] Inhibitionconstants (119870

119894) for all ligands were calculated by AutoDock4

as follows119870119894= exp((Δ119866lowast1000)(119877callowastTK)) whereΔ119866 is the

binding energy 119877cal is 198719 and TK is 29815 All figureswith structure representations were prepared using PyMOLsoftware [19]

3 Results and Discussion

31 Synthesis We were able to promote the regioselectiveattack of the hydroxyl group of the 4-aminophenol in thechlorine nucleophilic displacement of two commercial 4-chlorinated thieno[32-d]pyrimidines using stoichiometricamounts of the reagents in the synthesis of the aminatedcompounds 1a and 1b in excellent yields (Scheme 1) Thisregioselectivity constitutes an important achievement thatavoids the reaction of 4-nitrophenols followed by reductionof the nitro compounds to the corresponding amino com-pounds as often described in the literature [12 13] With4-aminophenol as a reagent it is possible to use DMF assolvent and heat only at 140∘C instead of diphenylether at180∘C which is needed for the nucleophilic displacement ofthe chlorine using 4-nitrophenol The amino compounds 1aand 1b obtained in one step were then reacted with differentarylisocyanates to give the corresponding new 1-aryl-3-[4-(thieno[32-d]pyrimidin-4-yloxy)phenyl]ureas 2andash2f in highyields (Scheme 1) Compounds 1a b and 2andash2f were fullycharacterized by mp 1H 13C NMR and mass spectrometryincluding HRMS

32 Biological Evaluation

321 Enzymatic Assays The synthesized thieno[32-d]pyr-imidine ureas 2andashf were evaluated for their ability to interactwith the VEGFR2 tyrosine kinase domain (Table 1) usingan enzymatic FRET-based assay Compounds 2a 2b and 2cdisplayed the highest inhibition with IC

50values of 199 188

and 150 nM respectively The presence of a methoxy groupin the para position of the phenyl ring (2b) did not show asignificant increase in VEGFR-2 inhibition activity relative to

C 01 05 1 01 05 1 01 05 1

2a 2b 2c

(120583M)

VEGFR-2

120573-Actin

p VEGFR-2

(a)

C 01

05 1

01

05 1

01

05 1

2a 2b 2c

Relat

ive e

xpre

ssio

np

VEG

FR-2

VEG

FR-2

06

04

02

0

lowast lowast

(b)

Figure 3 (a) Evaluation of phosphorylated and total VEGFR-2expression in HUVECs after incubation with compounds 2a 2band 2c by western blotting Representative bands obtained afterimmunostaining are shown (b) Quantification of densitometryand mean relative intensity by comparison of the relative intensityof activated VEGFR-2 after normalization with total VEGFR-2intensity Data presented as mean plusmn SEM of two independentexperiments lowast119875 lt 005 versus control

2a while the presence of a nitrile group in the same position(2c) promoted a small increase in VEGFR-2 inhibitionactivity These findings appear to suggest that the presence ofsmall substituents in the para position of the phenyl groupwill not have a significant effect on VEGFR-2 potency of thecompounds When a methyl group is present in position 7of the thieno[32-d]pyrimidine moiety (compounds 2dndashf)no VEGFR-2 inhibition activity was observed This findingindicates that substitutions on the mentioned position arenot favorable for this series of thieno[32-d]pyrimidine ureaswhen considering VEGFR-2 inhibition activity

322 Cellular Assays The best compounds in enzymaticassays (2a 2b and 2c) were studied at cellular level Cellcytotoxicity was first analyzed by MTS assay in the HUVECcultures upon treatment with 010ndash10 120583M of compounds 2andashc (Figure 2(a)) Viability of HUVEC decreased at the highestconcentration of 2c (196 plusmn 002 OD) and at concentrationsof 50 and 10 120583M for 2a (200plusmn006OD and 199plusmn003ODresp) and for 2b (184plusmn005OD and 182plusmn004OD resp)versus control (233 plusmn 006 OD)

Then the ability of compounds 2andashc to inhibit VEGF-stimulated proliferation of HUVECs was evaluated using theBrdU incorporation assay (Figure 2(b)) Inhibition of theVEGFR-2 activity was strongly reflected at the cellular levelwith all the three compounds showing a statistical significantinhibition activity against VEGF-stimulated HUVEC prolif-eration at 05120583M (152 plusmn 27 for 2a 128 plusmn 24 for 2b


LEU840VAL848

VAL916

CYS919

LEU1035VAL899

ASP1046

GLU885

LEU889

VAL898 LEU1019

ILE892

ILE888

(a)

GLU885

ASP1046

CYS919

(b)

Figure 4 (a) Superimposition of the docking poses at the VEGFR-2 kinase domain for compounds 2a (cyan) 2b (green) and 2c (orange)(b) Surface representation of the VEGFR-2 kinase hinge with the docking poses of compounds 2c (orange)and 2f (light grey) H-bonds aredepicted in dashed yellow lines (distances between 29 and 33 A) and hydrophobic interactions are depicted in dashed white lines (distancesbetween 35 and 40 A) Important residues are drawn in sticks Figures were prepared using PyMOL [19]

and 147 plusmn 17 for 2c) when comparing to control whichis consider to be 10000 plusmn 28

The number of proliferating HUVECs decreased in adose-dependent manner (Figure 2(b))

Interestingly incubation of HUVECs with compounds2a 2b and 2c for 24 h with concentrations 01 05 and1 120583M (due to cytotoxic effect for higher concentrationsFigure 2(a)) using the TUNEL assay resulted in an increasein apoptosis reaching statistical significance only for 2a atthe highest concentration tested (328 plusmn 123 increase)comparing with untreated cells (Figure 2(c))

To investigate the cellular inhibition of VEGFR-2 by thecompounds in HUVEC cultures immunoblotting assays fortotal and phosphorylated (active) VEGFR-2 were performed(Figure 3(a)) As illustrated in Figure 3(b) incubation withcompounds 2a and 2b significantly decrease the formation ofthe active form of the receptor at 1 120583M concentration whencomparing with the expression of VEGFR-2 (lowast119875 lt 005)Compound 2c did not significantly inhibit the activationof the VEGFR-2 although a dose-dependent decrease wasobserved

VEGF signaling pathway through the VEGFR-2 acti-vation displays a crucial role in endothelial cells namelysurvival proliferation invasion and apoptosis Our data sug-gest that compounds 2andashc exert direct actions in HUVECsby inhibiting their growth mainly at 05 and 1 120583M slightlyincreasing the percentage of apoptotic cells without cytotoxiceffects at the same range of concentrations The referredcellular-based results are triggered by the inhibition of thephosphorylation (activation) of the VEGFR-2 which is over-expressed in several pathological conditions such as cancer

33 Molecular Modelling Studies To better understand themolecular basis of compounds 2 observed VEGFR-2 inhibi-tion activity docking simulations using AutoDock4 [17] were

carried out against the VEGFR-2 kinase domain (PDB code2XIR)

Figure 4(a) displays the superimposition of the bestdocked poses obtained for the most potent thieno[32-d]pyrimidine derivatives 2andashc They present similar dockingposes with the binding conformation stabilized mainly bythe formation of a network of four H-bonds The two NndashH of the urea group form two H-bonds with the Glu885carboxyl group while the C=O bond of the urea group formsa third H-bond with the backbone NndashH of Asp1046 TheseH-bonds are similar to the ones observed with other knownVEGFR-2 inhibitors presenting a biphenyl urea moietyincluding the drug Soranefib (Figure 1) A fourth H-bond ispredicted between the N atom in position 4 of the thieno[32-d]pyrimidine moiety and the backbone NndashH of Cys919

The terminal aryl group is positioned in a hydropho-bic region formed by several hydrophobic residues (Ile888Leu889 Ile892 Val898 Val 899 and Leu1019) FromFigure 4(a) it is clearly observed that the 4-methoxyphenyland 4-cyanophenyl substituents occupy part of a largehydrophobic pocket that is able to accommodate themproviding an explanation for compounds 2b and 2c beingalmost equipotent to 2a that bears no substituents in thephenyl ring Substitutions of hydrophobic groups in orthoor meta positions of the phenyl ring will probably be worthexploring for synthesis of even more potent compounds inthis series

The lack of inhibition by compounds 2dndashf for VEGFR-2 was analysed by superimposing the docked pose ofcompound 2c (the most potent in the enzymatic assays150 nM) and the corresponding 7-methylated compound2f (Figure 4(b)) The methyl group seems to displace thethieno[32-d]pyrimidine ring away from Cys919 Thisdisplacement probably interferes with the formation ofthe H-bond between the N atom in position 4 of the


thieno[32-d]pyrimidine ring and the backbone NndashH ofCys919 (Figure 4(b)) These results demonstrate that thisH-bond is an absolute requirement for VEGFR-2 inhibitionpotency of this thieno[32-d]pyrimidine series and themethyl group in position 7 of compounds 2dndashf promotesloss of VEGFR-2 inhibition activity by impairing H-bondformation We can also infer that other substitutions inposition 7 of the thieno[32-b]pyridine are probably notdesirable as they will also impair H-bond formation

4 Conclusion

In conclusion we reported the synthesis of novel 1-aryl-3-[4-(thieno[32-d]pyrimidin-4-yloxy)phenyl]ureas 2andash2fin high yields in two steps the regioselective nucleophilicdisplacement of chlorine on two 4-chlorothieno[32-d]pyrimidines using 4-aminophenol and the reaction of theresultant 4-(thieno[32-d]pyrimidin-4-yloxy)anilines witharylisocyanates Compounds 2andashc without the methyl groupin position 7 were shown to be the most potent VEGFR-2inhibitors presenting IC

50values between 150ndash199 nM in

enzymatic assays The cell culture assays enable us to identifythe angiogenic steps targeted by the compounds 2andashc Infact compounds were evaluated using a cell-based HUVECassay and showed activity at the same concentration range(05ndash1120583M) without affecting cell viability inhibiting cellproliferation and avoiding the formation of active VEGFR-2specially in the case of compounds 2a and 2b thus showingtheir anti-angiogenic potential Compound 2a increases alsoapoptosis at the highest concentration tested (1 120583M) using theTUNEL assay By performing docking studies the bindingconformation and H-bond network of the most promisingcompounds 2andashc were predicted Based on these molecularmodelling studies several suggestions can be done for thesynthesis of even more potent compounds

Conflict of Interests

The authors declare that they have no conflict of interests

Acknowledgments

The authors would like to acknowledge Foundation forthe Science and Technology (FCT Portugal) for financialsupport through the NMR Portuguese network (Bruker400 Avance III-Univ Minho) FCT and European Fundfor Regional Development (FEDER)-COMPETE-QREN-EUfor financial support through the research unities PEst-CQUIUI6862011 PEst-OESAUUI00382011 and PEst-OEAGRUI06902011 the research project PTDCQUI-QUI1110602009 and the post-Doctoral Grant attributed toRicardo C Calhelha (SFRHBPD683442010) also financedby Human Potential Operational Programme (POPH) andSocial European Fund (FSE)

References

[1] S Patan ldquoVasculogenesis and angiogenesis as mechanisms ofvascular network formation growth and remodelingrdquo Journalof Neuro-Oncology vol 50 no 1-2 pp 1ndash15 2000

[2] J Folkman and Y Shing ldquoAngiogenesisrdquoThe Journal of Biologi-cal Chemistry vol 267 no 16 pp 10931ndash10934 1992

[3] N Ferrara H-P Gerber and J LeCouter ldquoThe biology of VEGFand its receptorsrdquo Nature Medicine vol 9 no 6 pp 669ndash6762003

[4] M A Lemmon and J Schlessinger ldquoCell signaling by receptortyrosine kinasesrdquo Cell vol 141 no 7 pp 1117ndash1134 2010

[5] S Baka A R Clamp and G C Jayson ldquoA review of thelatest clinical compounds to inhibit VEGF in pathologicalangiogenesisrdquoExpertOpinion onTherapeutic Targets vol 10 no6 pp 867ndash876 2006

[6] A Eichholz S Merchant and A M Gaya ldquoAnti-angiogenesistherapies their potential in cancer managementrdquo OncologyTargets Therapy vol 3 pp 69ndash82 2010

[7] D D Hu-Lowe H Y Zou M L Grazzini et al ldquoNonclinicalantiangiogenesis and antitumor activities of axitinib (AG-013736) an oral potent and selective inhibitor of vascularendothelial growth factor receptor tyrosine kinases 1 2 3rdquoClinical Cancer Research vol 14 no 22 pp 7272ndash7283 2008

[8] L Cowey G Sonpavde and T E Hutson ldquoNew advancementsand developments in treatment of renal cell carcinoma focus onpazopanibrdquo OncoTargets and Therapy vol 3 pp 147ndash155 2010

[9] Y Dai Y Guo R R Frey et al ldquoThienopyrimidine ureasas novel and potent multitargeted receptor tyrosine kinaseinhibitorsrdquo Journal of Medicinal Chemistry vol 48 no 19 pp6066ndash6083 2005

[10] H R Heyman R R Frey P F Bousquet et al ldquoThienopyridineurea inhibitors of KDR kinaserdquo Bioorganic and MedicinalChemistry Letters vol 17 no 5 pp 1246ndash1249 2007

[11] S Claridge F Raeppel M-C Granger et al ldquoDiscoveryof a novel and potent series of thieno[32-b]pyridine-basedinhibitors of c-Met and VEGFR2 tyrosine kinasesrdquo Bioorganicand Medicinal Chemistry Letters vol 18 no 9 pp 2793ndash27982008

[12] S Raeppel S Claridge O Saavedra et al ldquoN-(3-fluoro-4-(2-arylthieno[32-b]pyridin-7-yloxy)phenyl)-2-oxo-3-phenylimidazolidine-1-carboxamides a novel series of dualc-MetVEGFR2 receptor tyrosine kinase inhibitorsrdquo Bioorganicand Medicinal Chemistry Letters vol 19 no 5 pp 1323ndash13282009

[13] O Saavedra S Claridge L Zhan et al ldquoN3-Arylmalonamidesa new series of thieno[32-b]pyridine based inhibitors of c-Met and VEGFR2 tyrosine kinasesrdquo Bioorganic and MedicinalChemistry Letters vol 19 no 24 pp 6836ndash6839 2009

[14] ldquoStaurosporine IC50

calculated by Invitrogen with the sameFRET-based VEGFR-2 enzymatic kit used in this studyrdquohttptoolsinvitrogencom

[15] R Costa A Carneiro A Rocha et al ldquoBevacizumab andranibizumab on microvascular endothelial cells a comparativestudyrdquo Journal of Cellular Biochemistry vol 108 no 6 pp 1410ndash1417 2009

[16] A Rocha I Azevedo and R Soares ldquoAnti-angiogenic effects ofimatinib target smooth muscle cells but not endothelial cellsrdquoAngiogenesis vol 10 no 4 pp 279ndash286 2007

[17] G M Morris H Ruth W Lindstrom et al ldquoSoftware news andupdates AutoDock4 and AutoDockTools4 automated dockingwith selective receptor flexibilityrdquo Journal of ComputationalChemistry vol 30 no 16 pp 2785ndash2791 2009

[18] R M V Abreu H J C Froufe M J R P Queiroz and I CF R Ferreira ldquoMOLA a bootable self-configuring system forvirtual screening using AutoDock4Vina on computer clustersrdquoJournal of Cheminformatics vol 2 no 1 article 10 2010


[19] ldquoThe PyMOL Molecular Graphics System Version 13Schrodinger LLCrdquo September 2012 httpwwwpymolorg


NH O

F

HNNH

N

O

NH

O

N

O

HN

O

NH

Cl

F

FF

Sunitinib Sorafenib

NN

N

NN

NH

SO O

Pazopanib

S

NH

N

N

ONH

Axitinib

CH3

CH3 CH3

CH3

CH3

NH2

Figure 1 Competitive inhibitors of VEGFR-2 in clinical use against several cancers sunitinib (Sutent C P Pharmaceuticals InternationalNY USA) soranefib (Nexavar Bayer Germany) axitinib (Inlyta Pfizer) and pazopanib (Votrient GlaxoSmithKline)

In this report we describe an ongoing effort to developnovel small molecules as VEGFR-2 inhibitors based onthe aryletherthieno[32-d]pyrimidine arylurea scaffold Thesynthesis of the compounds and the VEGFR-2 tyrosinekinase phosphorylation inhibition evaluation using eitherenzymatic or cellular assays including the determination ofthe total and of the phosphorylated VEGFR-2 by western blotwere performed The probable binding mode of the 1-aryl-3-[4-(thieno[32-d]pyrimidin-4-yloxy)phenyl]ureas with thereceptor using docking studies is also presented and dis-cussed

2 Materials and Methods

21 Synthesis Melting points (∘C) were determined in aStuart SMP3 and are uncorrected 1H and 13C NMR spectrawere recorded on a Varian Unity Plus at 300 and 754MHzrespectively or on a Bruker Avance III at 400 and 1006MHzrespectively Two-dimensional 1H-13C correlations were per-formed to attribute some signals Mass spectra (MS) EI-TOFor ESI-TOF and HRMS on the M+ [M + H]+ or on [M +Na]+ were performed by the mass spectrometry service ofthe University of Vigo CACTI Spain

The reactions were monitored by thin layer chromatog-raphy (TLC) using Macherey-Nagel precoated aluminiumsilica gel 60 sheets (020mm) with UV

254indicator Column

chromatography was performed on Panreac silica gel 60230ndash400 mesh

211 General Procedure for the Synthesis of Compounds 1aand 1b In a flask with 5mL of DMF thienopyrimidine (1equiv) 4-aminophenol (1 equiv) and K

2CO3(4 equiv) were

heated at 140∘C for 2 h After cooling water (5mL) and ethylacetate (5mL) were added The phases were separated andthe aqueous phase was extracted with more ethyl acetate (2times 5mL) The organic phase was dried (MgSO

4) and filtered

The solvent was evaporated under reduced pressure giving asolid which was submitted to column chromatography

4-(Thieno[32-d]pyrimidin-4-yloxy)aniline (1a) 4-Chlorothi-eno[32-d]pyrimidine (242mg 142mmol) and 4-amino-phenol (155mg 142mmol) were heated and the reactionmixture was treated according to the general procedureColumn chromatography using ethyl acetate gave compound1a as a yellow solid (290mg 91) mp 1158ndash1165∘C 1HNMR (300MHz DMSO-119889

6) 120575 515 (s 2H NH

2) 660 (d 119869

= 90Hz 2H 2 and 6-H) 695 (d 119869 = 90Hz 2H 3 and 5-H)762 (d 119869 = 56Hz 1H HetAr) 840 (d 119869 = 56Hz 1H HetAr)866 (s 1H 21015840-H) ppm 13C NMR (754MHz DMSO-119889

6) 120575

1142 (2 and 6-CH) 1166 (C) 1223 (3 and 5-CH) 1241 (CH)1371 (CH) 1417 (C) 1469 (C) 1542 (21015840-CH) 1629 (C) 1643(C) ppmMS (EI-TOF)119898119911 () 24305 (M+ 100)HRMS (EI-TOF) calcd for C

12H9N3OS [M+] 2430466 found 2430467

4-(7-Methylthieno[32-d]pyrimidin-4-yloxy)aniline (1b) 4-Chloro-7-methylthieno[32-d]pyrimidine (242mg 131mmol) and 4-aminophenol (143mg 131mmol) were heatedand the reaction mixture was treated according to thegeneral procedure Column chromatography using ethylacetate gave compound 1b as a yellow solid (310mg 92)mp 1678ndash1686∘C 1H NMR (300MHz DMSO-119889

6) 120575 240

(s 3H CH3) 513 (s 2H NH

2) 661 (d 119869 = 90Hz 2H 2 and

6-H) 693 (d 119869 = 90Hz 2H 3 and 5-H) 801 (s 1H 61015840-H)867 (s 1H 21015840-H) ppm 13C NMR (754MHz DMSO-119889

6) 120575

125 (CH3) 1142 (2 and 6-CH) 1168 (C) 1223 (3 and 5-CH)



13H11N3OS [M+] 2570623

found 2570621





6) 120575 695ndash699 (m


6) 120575 1168 (C) 1182 (2 times CH) 1192



6) 120575 371

(s 3H OCH3) 686 (d 119869 = 90Hz 2H 31015840 and 51015840-H) 723


6) 120575 551 (OCH

3) 1140 (31015840 and


20H17N4O3S [M+H]+ 3931016 found

3931026


6) 120575 726 (d 119869


6) 120575 1033 (C) 1168 (C) 1180



20H13N5O2S [M + H]+ 3880863

found 3880861


6) 120575 243 (s 3H



6) 120575 125 (CH

3) 1169


20H17N4O2S [M +H]+ 3771067 found

3771064


6) 120575 243 (s 3H CH

3) 371 (s 3H OCH

3) 686


6) 120575 125 (CH

3) 552 (OCH

3) 1140


21H19N4O3S [M +

H]+ 4071172 found 4071182


6) 120575 242

(s 3H CH3) 725 (d 119869 = 92Hz 2H 310158401015840 and 510158401015840-H) 754 (d


6) 120575 125 (CH

3) 1033




N

N

S

O

NH

NH

O

N

N

S

ON

N

S

Cl

HO NC

O

1

2

3

32a d

cb

4

1

2

3

6

5

7

2

65

3

4

R1

R1 = H or CH3

NH2

K2CO3 (4 equiv)

2998400

4998400

6998400

7998400

R2

NH2

rt 12 h

1a R1 = H 911b R1 = CH3 92

2998400998400998400

6998400998400998400

6998400998400

5998400998400

3998400998400

2998400998400

6998400

5998400

3998400

2998400

R1

R2

140∘C 1 h

R1

CH2Cl2 THF (1 1)

2a R1 = R2 = H 79

2c R1 = H R2 = CN 70

2d R1 = CH3 R2 = H 83

2e R1 = CH3 R2 = OCH3 83

2f R1 = CH3 R2 = CN 82

DMF

2b R1 = H R2 = OCH3 90



2 20mM MnCl

2











3

2

1

0

C 01

05 1 5 1001

05 1 5 1001

05 1 5 10


HU

VEC

via

bilit

yO

ptic

al d

ensit

y (4

92 nm

)

2a 2b 2c

(a)

Prol

ifera

ting

cells

()

(fold

incr

ease

ver

sus c

ontro

l) 150

100

50

0

C 01

05 1 5 1001

05 1 5 1001

05 1 5 10

2a 2b 2c



(b)(fo

ld in

crea

se v

ersu

s con

trol) 150

100

50

0

C 01

05 1

01

05 1

01

05 1

2a 2b 2c

lowast

Apop

totic

cells

()



















50values of 199 188


C 01 05 1 01 05 1 01 05 1

2a 2b 2c

(120583M)

VEGFR-2

120573-Actin

p VEGFR-2

(a)

C 01

05 1

01

05 1

01

05 1

2a 2b 2c

Relat

ive e

xpre

ssio

np

VEG

FR-2

VEG

FR-2

06

04

02

0

lowast lowast

(b)






LEU840VAL848

VAL916

CYS919

LEU1035VAL899

ASP1046

GLU885

LEU889

VAL898 LEU1019

ILE892

ILE888

(a)

GLU885

ASP1046

CYS919

(b)














4 Conclusion






Acknowledgments


References
























13H11N3OS [M+] 2570623

found 2570621





6) 120575 695ndash699 (m


6) 120575 1168 (C) 1182 (2 times CH) 1192



6) 120575 371

(s 3H OCH3) 686 (d 119869 = 90Hz 2H 31015840 and 51015840-H) 723


6) 120575 551 (OCH

3) 1140 (31015840 and


20H17N4O3S [M+H]+ 3931016 found

3931026


6) 120575 726 (d 119869


6) 120575 1033 (C) 1168 (C) 1180



20H13N5O2S [M + H]+ 3880863

found 3880861


6) 120575 243 (s 3H



6) 120575 125 (CH

3) 1169


20H17N4O2S [M +H]+ 3771067 found

3771064


6) 120575 243 (s 3H CH

3) 371 (s 3H OCH

3) 686


6) 120575 125 (CH

3) 552 (OCH

3) 1140


21H19N4O3S [M +

H]+ 4071172 found 4071182


6) 120575 242

(s 3H CH3) 725 (d 119869 = 92Hz 2H 310158401015840 and 510158401015840-H) 754 (d


6) 120575 125 (CH

3) 1033




N

N

S

O

NH

NH

O

N

N

S

ON

N

S

Cl

HO NC

O

1

2

3

32a d

cb

4

1

2

3

6

5

7

2

65

3

4

R1

R1 = H or CH3

NH2

K2CO3 (4 equiv)

2998400

4998400

6998400

7998400

R2

NH2

rt 12 h

1a R1 = H 911b R1 = CH3 92

2998400998400998400

6998400998400998400

6998400998400

5998400998400

3998400998400

2998400998400

6998400

5998400

3998400

2998400

R1

R2

140∘C 1 h

R1

CH2Cl2 THF (1 1)

2a R1 = R2 = H 79

2c R1 = H R2 = CN 70

2d R1 = CH3 R2 = H 83

2e R1 = CH3 R2 = OCH3 83

2f R1 = CH3 R2 = CN 82

DMF

2b R1 = H R2 = OCH3 90



2 20mM MnCl

2











3

2

1

0

C 01

05 1 5 1001

05 1 5 1001

05 1 5 10


HU

VEC

via

bilit

yO

ptic

al d

ensit

y (4

92 nm

)

2a 2b 2c

(a)

Prol

ifera

ting

cells

()

(fold

incr

ease

ver

sus c

ontro

l) 150

100

50

0

C 01

05 1 5 1001

05 1 5 1001

05 1 5 10

2a 2b 2c



(b)(fo

ld in

crea

se v

ersu

s con

trol) 150

100

50

0

C 01

05 1

01

05 1

01

05 1

2a 2b 2c

lowast

Apop

totic

cells

()



















50values of 199 188


C 01 05 1 01 05 1 01 05 1

2a 2b 2c

(120583M)

VEGFR-2

120573-Actin

p VEGFR-2

(a)

C 01

05 1

01

05 1

01

05 1

2a 2b 2c

Relat

ive e

xpre

ssio

np

VEG

FR-2

VEG

FR-2

06

04

02

0

lowast lowast

(b)






LEU840VAL848

VAL916

CYS919

LEU1035VAL899

ASP1046

GLU885

LEU889

VAL898 LEU1019

ILE892

ILE888

(a)

GLU885

ASP1046

CYS919

(b)














4 Conclusion






Acknowledgments


References























N

N

S

O

NH

NH

O

N

N

S

ON

N

S

Cl

HO NC

O

1

2

3

32a d

cb

4

1

2

3

6

5

7

2

65

3

4

R1

R1 = H or CH3

NH2

K2CO3 (4 equiv)

2998400

4998400

6998400

7998400

R2

NH2

rt 12 h

1a R1 = H 911b R1 = CH3 92

2998400998400998400

6998400998400998400

6998400998400

5998400998400

3998400998400

2998400998400

6998400

5998400

3998400

2998400

R1

R2

140∘C 1 h

R1

CH2Cl2 THF (1 1)

2a R1 = R2 = H 79

2c R1 = H R2 = CN 70

2d R1 = CH3 R2 = H 83

2e R1 = CH3 R2 = OCH3 83

2f R1 = CH3 R2 = CN 82

DMF

2b R1 = H R2 = OCH3 90



2 20mM MnCl

2











3

2

1

0

C 01

05 1 5 1001

05 1 5 1001

05 1 5 10


HU

VEC

via

bilit

yO

ptic

al d

ensit

y (4

92 nm

)

2a 2b 2c

(a)

Prol

ifera

ting

cells

()

(fold

incr

ease

ver

sus c

ontro

l) 150

100

50

0

C 01

05 1 5 1001

05 1 5 1001

05 1 5 10

2a 2b 2c



(b)(fo

ld in

crea

se v

ersu

s con

trol) 150

100

50

0

C 01

05 1

01

05 1

01

05 1

2a 2b 2c

lowast

Apop

totic

cells

()



















50values of 199 188


C 01 05 1 01 05 1 01 05 1

2a 2b 2c

(120583M)

VEGFR-2

120573-Actin

p VEGFR-2

(a)

C 01

05 1

01

05 1

01

05 1

2a 2b 2c

Relat

ive e

xpre

ssio

np

VEG

FR-2

VEG

FR-2

06

04

02

0

lowast lowast

(b)






LEU840VAL848

VAL916

CYS919

LEU1035VAL899

ASP1046

GLU885

LEU889

VAL898 LEU1019

ILE892

ILE888

(a)

GLU885

ASP1046

CYS919

(b)














4 Conclusion






Acknowledgments


References























3

2

1

0

C 01

05 1 5 1001

05 1 5 1001

05 1 5 10


HU

VEC

via

bilit

yO

ptic

al d

ensit

y (4

92 nm

)

2a 2b 2c

(a)

Prol

ifera

ting

cells

()

(fold

incr

ease

ver

sus c

ontro

l) 150

100

50

0

C 01

05 1 5 1001

05 1 5 1001

05 1 5 10

2a 2b 2c



(b)(fo

ld in

crea

se v

ersu

s con

trol) 150

100

50

0

C 01

05 1

01

05 1

01

05 1

2a 2b 2c

lowast

Apop

totic

cells

()



















50values of 199 188


C 01 05 1 01 05 1 01 05 1

2a 2b 2c

(120583M)

VEGFR-2

120573-Actin

p VEGFR-2

(a)

C 01

05 1

01

05 1

01

05 1

2a 2b 2c

Relat

ive e

xpre

ssio

np

VEG

FR-2

VEG

FR-2

06

04

02

0

lowast lowast

(b)






LEU840VAL848

VAL916

CYS919

LEU1035VAL899

ASP1046

GLU885

LEU889

VAL898 LEU1019

ILE892

ILE888

(a)

GLU885

ASP1046

CYS919

(b)














4 Conclusion






Acknowledgments


References


































50values of 199 188


C 01 05 1 01 05 1 01 05 1

2a 2b 2c

(120583M)

VEGFR-2

120573-Actin

p VEGFR-2

(a)

C 01

05 1

01

05 1

01

05 1

2a 2b 2c

Relat

ive e

xpre

ssio

np

VEG

FR-2

VEG

FR-2

06

04

02

0

lowast lowast

(b)






LEU840VAL848

VAL916

CYS919

LEU1035VAL899

ASP1046

GLU885

LEU889

VAL898 LEU1019

ILE892

ILE888

(a)

GLU885

ASP1046

CYS919

(b)














4 Conclusion






Acknowledgments


References























LEU840VAL848

VAL916

CYS919

LEU1035VAL899

ASP1046

GLU885

LEU889

VAL898 LEU1019

ILE892

ILE888

(a)

GLU885

ASP1046

CYS919

(b)














4 Conclusion






Acknowledgments


References
























4 Conclusion






Acknowledgments


References






















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