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pound 10 as a yellow solid (60 mg, 32%): mp: 2488C; 1H NMR
(300 MHz, MeOD): d=2.71 (s, 3H), 3.79 (s, 3H), 3.86 (s, 3H), 6.48 (d,
Tube formation assays:[39,40] The effect of 1, 5a, and 10 on the pro-
pensity of stimulated Ea.hy926 cells to form vascular-like tubular
networks in vitro was assessed by growing the cells (0.5ꢂ106 mL)
on thin matrigel (BD Biosciences) layers for 12 h and then treating
them with DMSO (control) or 25 nm of the test compounds. Docu-
mentation by light microscopy after 6 h and 24 h (10ꢂ magnifica-
tion, Axiovert 135, AxioCam MRc 5, ZEISS). MTT was additionally
added to each well after 24 h to ensure that more than 80% of the
remaining cells are vital.
3
3J=3.1 Hz, 1H), 6.74 (d, J=8.6 Hz, 1H), 6.9–7.0 (m, 1H), 7.14 (dd,
3J=8.6 Hz, 4J=2.2 Hz, 1H), 7.30 (d, 3J=3.1 Hz, 1H), 7.5–7.6 ppm
(m, 4H); 13C NMR (75.5 MHz, MeOD): d=23.8, 33.3, 45.0, 102.7,
112.6, 119.3, 120.4, 122.5, 126.7, 128.7, 131.0, 132.7, 135.1, 137.5,
139.3, 158.2, 162.7 ppm; ATR-IR (neat): n˜max =3639, 3343, 2948,
1623, 1608, 1588, 1569, 1528, 1488, 1424, 1389, 1366, 1338, 1271,
1242, 1196, 1162, 1108, 1084, 996, 829, 761, 737, 686 cmÀ1; MS (EI,
70 eV): m/z (%): 302 (100) [M]+, 301 (89), 286 (10), 159 (37), 144
(45), 130 (22), 102 (38), 77 (14).
Chorioallantoic membrane (CAM) assays:[41] Fertilized white leg horn
chicken eggs (SPF eggs, VALO Biomedia) were incubated (378C,
50–60% humidity) and opened on day six by cutting a window of
2–3 cm diameter into the eggshell at the more rounded pole.
Rings of silicon foil (Ø 5 mm) were placed on the developing CAM
vessels, the windows were sealed with tape and the eggs were in-
cubated for a further 12–18 h. 1 nmol or 0.1 nmol (10 mL of
a 100 mm or 10 mm solution in ddH2O) of 1, 5a, 10, or vehicle
(DMSO) were pipetted inside the silicon ring. The effects were
documented after 0 h, 6 h, and 24 h post application with a micro-
scope (60ꢂ magnification, Traveller).
Molecular docking studies
Coordinate files of the ligand structures were generated by the
c.uk/prodrg/submit.html).[43] Molecular docking calculations were
carried out with the AutoDock Vina software.[34] and Gasteiger par-
tial charges[44] were calculated on ligand atoms using AutoDock
Tools. The X-ray structure of the crystallized tubulin–colchicine
complex (PDB ID: 1SA0) was downloaded from the Protein Data
the protein and Gasteiger partial charges were calculated. Water
molecules, heteroatoms, and ligands were removed from the struc-
ture prior to docking calculations. Residues Lys-b254, Lys-b352,
Asn-a101, Val-b318, and Ile-b378 were treated as flexible residues.
Simulation boxes were centered on the originally crystallized
ligand colchicine. A 17ꢂ23ꢂ19 ꢃ simulation box and an exhaus-
tiveness option of 1,000 were used in the docking calculations. Fig-
ures were prepared with the program PyMOL.[45]
Animal studies: The vascular-disrupting activity of 10 was studied
on the established model of highly vascularized 1411HP xenograft
tumors previously described.[17] This study was approved by the
Laboratory Animal Care Committee of Sachsen-Anhalt, Germany.
Nude mice (Harlan and Winkelmann, Borchen, Germany) received
5 mgkgÀ1 body weight of compound 10 by intraperitoneal injec-
tion and tumor discoloration was documented immediately and
after 48 h with a Canon IXUS 50. For histological examination the
tumors were explanted, fixed in 4% formalin, and embedded in
paraffin. Hematoxylin/eosin staining of the tissue slices was per-
formed according to standard protocols.
Biological studies
Cell-cycle analyses: Ea.hy926 cells (1ꢂ105 mL) grown on six-well
plates were treated with DMSO (control), 1, 5a, or 10 (10 nm,
24 h), fixed (70% EtOH, 1 h, 48C) and incubated with propidium
iodide (PI; Carl Roth) staining solution (50 mgmLÀ1 PI, 0.1% sodium
citrate, 50 mgmLÀ1 RNase A in PBS) for 30 min at 378C. The fluores-
cence intensity of 10,000 single cells at lem =620 nm (lex =488 nm
laser source) was recorded with a Beckman Coulter Cytomics FC
500 flow cytometer and analyzed for the distribution of single cells
(%) to G1, S, or G2/M phases as well as for the content of sub-G1
(apoptotic) events (CXP software, Beckman Coulter).
Supporting Information
Instruments used; syntheses, microanalytical and spectroscopic
data of all new compounds; MTT and SRB assays; tubulin polymeri-
zation assays.
Abbreviations
BOP, benzotriazol-1-yloxy-tris(dimethylamino)phosphonium hexa-
fluorophosphate; CA-4, combretastatin A-4; CAM, chorioallantoic
membrane; DAPI, 4’,6-diamidino-2-phenylindole; DBU, 1,8-
diazabicyclo[5.4.0]undec-7-ene; HE, hematoxylin-eosin; MTT, 3-(4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide; PI, propidi-
um iodide; SRB, sulforhodamine-B; VDA, vascular-disrupting agent.
Fluorescence labeling of microtubules and actin filaments: Ea.hy926
cells (1ꢂ105 mL) were grown on glass coverslips in 24-well plates,
treated with DMSO (control), 1, 5a, or 10 (10 nm) for 24 h, fixed
with 4% formaldehyde in PBS for 20 min at RT, and permeabilized
with 1% BSA, 0.1% Triton X-100 (in PBS) for 30 min. Nonmalignant
HF were treated with 100 nm of 1, 5a, or 10. To visualize F-actin,
coverslips were incubated with 1 U AlexaFluorꢀ-488-conjugated
phalloidin (Invitrogen) for 1 h at 378C. For microtubule staining,
fixed and permeabilized cells were treated with a primary antibody
against a-tubulin (anti-a-tubulin, mouse mAb, Invitrogen;
5 mgmLÀ1) for 1 h (378C, 5% CO2, 95% humidity) followed by incu-
bation with the secondary antibody conjugated to the fluorescent
AlexaFluorꢀ-488 dye (goat anti-mouse IgG-AlexaFluor-488 conju-
gate, Invitrogen; 4 mgmLÀ1) for 1 h at RT in the dark. The coverslips
were then mounted in Mowiol 4-88-based mounting medium con-
taining 2.5% (w/v) DABCO and 1 mgmLÀ1 DAPI (4’,6-diamidino-2-
phenylindole) for counterstaining the nuclei. Fluorescence micro-
scopic analysis of the effects on both cytoskeletal components was
performed using the ZEISS Axio Imager.A1 microscope.
Keywords: angiogenesis · antitumor agents · quinazolines ·
vascular-disrupting agents · verubulin
[2] D. J. Chaplin, G. J. Dougherty, Br. J. Cancer 1999, 80 (Suppl. 1), 57–64.
[7] G. R. Pettit, S. B. Singh, E. Hamel, C. M. Lin, D. S. Alberts, D. Garcia-Ken-
ꢁ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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