1. Synthesis and Evaluation of Novel Heterocyclic
Compounds as Antitumorals and/or Antiangiogenics
Maria João R. P. Queiroz

2. Thieno[3,2-b]pyridine derivatives synthesized
by Pd or Pd/Cu-catalyzed couplings

3. Most promising C-N Buchwald- Hartwig C-N coupling product
It was effective at low GI50 values (4-7 µM) at inhibiting human tumor cell lines growth, breast adenocarcinoma (MCF-7), melanoma (A375-C5) and non-small cell lung cancer (NCI-H460), using the sulforhodamine assay .
M.-J.R. P. Queiroz et al.
Eur. J. Med. Chem. 2010, 45,

4. Most promising Suzuki C-C coupling products
Several compounds of this series presented GI50 values below 15 µM against breast adenocarcinoma (MCF-7), melanoma (A375-C5) and non-small cell lung cancer (NCI-H460) human cell lines, using the sulforhodamine assay. The o-anilino derivative was selective against MCF-7 and NCI-H460 cell lines with very low GI50 values ( µM ) while the bithiophene derivative was active against the three cell lines with low GI50 values ( µM). These two compounds interfered with normal cell cycle distribition in NCI-460 cell line.
M.-J.R.P. Queiroz et al.
Eur. J. Med. Chem. 2010, 45,

5. Most promising Sonogashira C-C coupling products
The p-OMePhenyl derivative presented GI μM for MCF7 and 0.32 μM in NCI-H460, and was not active for A375-C5. An increase of cells in G0/G1 phases of the cell cycle of NCI-460 at its GI50 was observed.
The o-anilino derivative presented GI μM for MCF7, 3.1 μM for A375-C5 and 5.7 μM for NCI-H460. An increase of cells was observed in G2/M in the cell cycle of NCI-H460 at its GI50, and induction of apoptosis was significatly increased
The p-anilino derivative presented GI μM forMCF7, 4.8 μM for A375-C5 and 8.9 μM for NCI-H460. An increase of cells in G0/G1 phases of the cell cycle of NCI-460 at its GI50 , was observed.

6. M.-J.R.P. Queiroz et al. Eur. J. Med. Chem. 2011, 46, 236-240

7. Angiogenesis & Tumor Angiogenesis
Multistep process characterized by a combination of sprouting of new vessels from the sides and ends of pre-existing ones
Tumor angiogenesis
Tumor blood vessel formation is characterized by the ability to supply oxygen, growth factors and nutrients to the tumor being a requisite for the maintenance and progression of many solid tumors
involved in the capacity of tumor cells to detach from the primary tumor and disseminate to distant organs giving rise to new secondary tumors
Metastasis
one of the most serious problems for the final
outcome of cancer
Antiangiogenic therapy is a promising strategy for treatment of cancer
Carmeliet P. Nature Medicine, 2003, 9:
Weis, S.M. and Cheresh, D.A. Nature Reviews, 2011, 17:

8. Adapted from http://www.angioworld.com/DominiqueGarrel.htmln
Tumor Angiogenesis
Adapted from

9. Adapted from http://www.angioworld.com/DominiqueGarrel.html
Cellular growth factors and transmembrane tyrosine kinase growth factor receptors (RTKs)
Título
Adapted from
EGF and EGFR- Epidermal Growth Factor Receptor
VEGF and VEGFR – Vascular Endothelium Growth Factor Receptor (VEGFR2 the most
related with angiogenesis)

10. Cellular growth factors and transmembrane tyrosine kinase growth factor receptors (RTKs)
RTKs’ structure consists of three different parts
1 - extracellular ligand-binding domain
2 - a single transmembrane region
3 - cytoplasmic portion with a conserved protein tyrosine kinase domain.
The binding of specific growth factors to the extracellular domain leads to receptor dimerization, resulting in autophosphorylation of the cytoplasmic region, a process that is crucial for the activation of tyrosine kinase residues, thus initiating a cascade of downstream signaling pathways.

11. VEGFR2 activation by VEGF produced by the tumor
The use of Tyrosine kinase Inhibitors (TKIs) against VEGFR-2 is an important antiangiogenic /anticancer therapeutic approach

12. Types of TKIs according to their interactions with the receptors
Type I
Several small molecules has been approved by FDA
for treating cancer and/or angiogenesis
Type of binding
Binding site
ATP-competitive
Selectivity
Type I
Reversible
ATP site
Yes
Low
Type II
ATP site and DFG pocket No
High
Type III
Allosteric (by ATP pocket)
Very high
Type IV
Allosteric (substrate binding domain)
Covalent
Irreversible
Covalent
Type II

13. Aims of this study
The bibliographic search and the rational design using PDB co-crystalizeed TK domain of VEGFR2, prompted us to synthesize novel thieno[3,2-b]pyridine 1,3-diarylureas derivatives as type II VEGFR-2 inhibitors, bearing a S-linker in the ortho, meta and para- positions and different substituents in the terminal phenyl ring.
Synthesis of 1-aryl-3-[(thieno[3,2-b]pyridine-7-ylthio)phenyl]ureas as type II VEGFR-2 inhibitors and study of their molecular docking poses
Enzymatic TK VEGFR-2 inhibition assays
Evaluation of VEGFR-2 inhibitory potential and their antiangiogenic effects in human endothelial cells
Evaluation of the antitumor potential in human breast cancer cells of the most promising antiangiogenic compounds and study of their toxicity using non-neoplastic human breast epithelial cells

14. (enzymatic kit control)
Earlier in the research group
Enzymatic assays
Compound
VEGFR-2; IC50 a
(using 10 µM of ATP) 3a
> nM 3b 3c 4a
94 nM 4b
107 nM 4c
206 nM 5a
1160 nM 5b
1390 nM 5c
8360 nM
Staurosporineb
(enzymatic kit control)
6 nM
a Each IC50 determination is a result of at least four separate determinations.
b Staurosporine experimental VEGFR-2 IC50 = 7 nM

15. Following rational design
In an attempt to obtain more potent VEGFR-2 inhibitors and based on rational design,
compounds with hydrophobic groups (present in active drugs, like Sorafenib) in the phenyl terminal ring
4d-4h and 5d and 5e were prepared.

16. (enzymatic kit control)
Enzymatic assays
Compound
VEGFR-2; IC50 a
(using 10 µM of ATP) 4d
10 nM 4e
28 nM 4f
11 nM 4g
15 nM 4h
16 nM 5d
988 nM 5e
1947 nM
Staurosporineb
(enzymatic kit control)
6 nM
a Each IC50 determination is a result of at least four separate
determinations.
b Staurosporine experimental VEGFR-2 IC50 = 7 nM

17. Molecular docking poses
Docking pose superimposition of: (A) 5a (green) and (B) 4d and 4f (green) with the
co-crystallized pyrrolo[3,2-d]pyrimidine derivative (cyan) (IC50 = 6.2 nM)* in VEGFR-2
Pyrrolo[3,2-d]pyrimidine derivative (IC50 = 6.2 nM)*
* Oguro et al. Bioorg. Med. Chem. 2010, 18, 7260.
The docking simulations were performed with AutoDock4 using a VEGFR-2 kinase domain crystal structure
(PDB code 3VHE) to acess the binding mode of compounds. A partial surface representation of the VEGFR-2
kinase binding site is depicted to outline the relevant pockets. Pictures were prepared using PyMOL. Dashed red lines
are H bonds .

18. Biological Assays in HUVECs
Best compounds in enzymatic assays were studied in Human Umbilical Vein Endothelial Cells (HUVECs)
Sorafenib was used as a positive control
A classic model system to study many aspects of endothelial function and diseases
Similarity of the hydrophobic chemical pattern substitution
Queiroz M.-J. R. P.et al. Bioorganic & Med. Chem. 2015, 23:

19. Effect of compounds on HUVECs viability using MTS assay
generally by each compound in a dose-dependent manner
5.0 and 10 µM cytotoxic effect
Queiroz M.-J R. P. et al. Bioorganic & Medicinal Chemistry, 2015, 23:
2.5 , 5 and 10 µM low
cytotoxic effect

20. Effects on HUVECs Proliferation by BrdU assay
Significant decrease at 0.5 µM
All compounds inhibit cell proliferation without high cytotoxic effect and compounds 4g and 4h at lower concentration (2,5 µM) than Sorafenib
Queiroz M.-J. R. P.et al. Bioorganic & Med. Chem. 2015, 23:

21. Effects on HUVECs Apoptosis by TUNEL assay
Increase in apoptosis
Higher effect than Sorafenib
Queiroz M.-J. R. P.et al. Bioorganic & Med. Chem. 2015, 23:

22. Formation of capillary-like structures
Effects on HUVECs tube formation by Matrigel
Formation of capillary-like structures
Significant decrease after treatment with compounds
This imply the important antiangiogenic role of these compounds
Queiroz M.-J. R. P.et al. Bioorganic & Med. Chem. 2015, 23:

23. Inhibition of migration
Effects on HUVECs Migration by Wound-healing assay
Essential feature for ECs in angiogenesis
Inhibition of migration
by all compounds
4d-h
Compound 4h is
more effective than Sorafenib
Queiroz M.-J. R. P.et al. Bioorganic & Med. Chem. 2015, 23:

24. Effects on VEGFR-2 phosphorylation by Western blotting
Active form of VEGFR-2
VEGFR-2 phosphorylation
Presenting a better effect than Sorafenib
Queiroz M.-J. R. P.et al. Bioorganic & Med. Chem. 2015, 23:

25. Queiroz M.-J. R. P.et al. Bioorganic & Med. Chem. 2015, 23: 6497-6509.

26. Antitumor activity of the most promising antiangiogenic compounds in human breast cancer cell lines – MCF-7 and MDA-MB-231
Cancer is progressively considered a global problem and breast cancer is one of the most recurrently diagnosed in women throughout the world
Breast cancer prevails and is the leading cause of female cancer death in many developed countries
Discovering novel compounds with low toxicity, which efficiently inhibit the growth, migration, and invasion is an important research area to suppress cancer progression and metastasis and thus reducing mortality
MCF-7 – ER positive
Two different human breast cancer cell lines
Representative of two types of tumors
MDA-MB-231 – triple negative (ER, PR, HER2 negative)
Soares, R.; Queiroz M.-J. R.P. et al. J. Cell. Biochem. 2016, 117:

27. different susceptibilities to compounds
Cytotoxicity of compounds in MCF-7 and MDA-MB-231
different susceptibilities to compounds
Most effective
2.5 µM led to a reduction of more than 50% of cell viability
More significant effect in this aggressive cell line
MCF-7
MDA-MB-231
Soares, R.; Queiroz M.-J. R.P. et al. J. Cell. Biochem. 2016, 117:

28. … and in MCF-10A cells – non-neoplastic human breast epithelial cell line
Compounds in study did not show cytotoxicity to the human mammary epithelial cell line,
except for 4f-h at 10 µM
We next explored the antitumoral potential of these compounds in the breast cancer cells
Soares, R.; Queiroz M.-J. R.P. et al. J. of Cell. Biochem. 2016, 117:

29. Antiproliferative effects using BrdU
MCF-7
Compounds 4g and 4f are comparable
with Sorafenib in MCF-7
MDA-MB-231
Better antiproliferative effects than sorafenib in MDA-MB-231
Soares, R.; Queiroz M.-J. R.P. et al. Journal of Cellular Biochemistry, 2016, 117:

30. Effects of compounds in cell migration
a key step of tumour metastasis
The in vitro wound healing assay was used to investigate the effect of compounds 4d-h on both cell lines.
Cell migration for both breast cancer cell lines are severely affected by administration of compounds
Soares, R.; Queiroz M.-J. R.P. et al. Journal of Cellular Biochemistry, 2016, 117:

31. dose-dependent manner
Effects of compounds on apoptosis by TUNEL assay
Apoptosis
dose-dependent manner
Highly-metastatic human breast cancer cell line
Apoptosis
Inhibition of the cell proliferation and migration is not mainly due to extensive cell death, in this case.
Soares, R.; Queiroz M.-J. R.P. et al. Journal of Cell. Biochem.
2016, 117:

32. Antiangiogenic/Antitumor compounds
Novel 1-aryl-3-[3-(thieno[3,2-b]pyridine-7-ylthio)phenyl]ureas 4 with the arylurea in meta-position relative to the S-linker and bearing hydrophobic groups in the terminal phenyl ring 4d-h (Me, p-F, m-F m-CF3, m-CF3 /p-Cl) were synthesized following the predictions of rational design as new Type II TK VEGFR2 inhibitors . The latter compounds showed to be the most potent in enzymatic assays against
TK domain of VEGFR2 (IC50 values between nM)
Compounds 4 in general inhibited VEGFR-2 phosphorylation and affected various steps of angiogenesis, such as proliferation, migration and tube formation as evaluated in HUVECs
The most promising antiangiogenic compounds, 4d-h, showed anticancer effects at low concentrations in breast cancer cell lines, particularly in MDA-MB-231 cells.
Our findings support the assumption that compounds 4d-h may be potent antiangiogenic and antitumor agents exhibiting a dual effect at low concentrations and without toxicity.

33. Soares, R. ; Queiroz M. -J. R. P. et al. J. Cell. Biochem

34. Ricardo Calhelha SFRH/BD/29274/2006 SFRH/BPD/68344/2010
University of Minho
School of Sciences
University of Porto
Faculty of Medicine
Dept. of Biochemistry
Ricardo Calhelha SFRH/BD/29274/2006
SFRH/BPD/68344/2010
Vera Machado SFRH/BD/77373/2011
Daniela Peixoto BI and MSc
PTDC/QUI-QUI/11160/2009
Pest-C/QUI/UI0686/
Pest-C/QUI/UI0686/
UID/0686/2016
PTNMR and 2017
Raquel Soares
Raquel Costa BI
Vera Machado
SFRH/BD/77373/2011
Isabel Ferreira
Rui Abreu
Hugo Froufe BI
Ricardo Calhelha
SFRH/BPD/68344/2010