Synthesis and Evaluation of Novel Heterocyclic Compounds as Antitumorals and/or Antiangiogenics Maria João R. P. Queiroz mjrpq@quimica.uminho.pt

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

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, 5732-5738

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 (0.7-1.0 µM ) while the bithiophene derivative was active against the three cell lines with low GI50 values (2.5-4.2 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, 5628-5634.

Most promising Sonogashira C-C coupling products The p-OMePhenyl derivative presented GI50 0.46 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 GI50 4.1 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 GI50 2.6 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.

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

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, 9:653-660 Weis, S.M. and Cheresh, D.A. Nature Reviews, 2011, 17:1359-1370.

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

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

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.

VEGFR2 activation by VEGF produced by the tumor http://www.avastin-info.com/content/dam/avastin/en/vegf-biology-and-avastin-moa/vegfr_2.jpg The use of Tyrosine kinase Inhibitors (TKIs) against VEGFR-2 is an important antiangiogenic /anticancer therapeutic approach

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

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

(enzymatic kit control) Earlier in the research group Enzymatic assays Compound VEGFR-2; IC50 a (using 10 µM of ATP) 3a >100000 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

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.

(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

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 .

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: 6497-6509.

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: 6497-6509. 2.5 , 5 and 10 µM low cytotoxic effect

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: 6497-6509.

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: 6497-6509.

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: 6497-6509.

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: 6497-6509.

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: 6497-6509.

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

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: 2791-2799

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: 2791-2799

… 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: 2791-2799

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: 2791-2799

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: 2791-2799

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: 2791-2799

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 10-28 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.

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

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/UI0686/2011-2012 Pest-C/QUI/UI0686/2013-2014 UID/0686/2016 PTNMR 2010-2016 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