Spirocyclic thienopyrimidines: synthesis, new rearrangement under Vilsmeier conditions and in silico prediction of anticancer activity

© 2020 A. V. Kovtun et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited UDС 547.834.22 : 547.859


Introduction
Over the past few decades, the survival of cancer patients has improved significantly due to the progress in early detection and treatment [1].
The human genome encodes 538 kinases of proteins that transfer the γ-phosphate group with ATP to the residues of serine, threonine and tyrosine. Some of these kinases are responsible for signal transduction during many cellular processes including cell proliferation associated with human cancer. In recent years, the treatment of various types of cancer with low weight kinase inhibitors gained a success and became an effective in-clinic therapy. A survey of literature revealed some EGFR, CK2, FGFR1 and B-Raf protein kinase inhibitors to be useful as anti-cancer drugs.
Thus, first generation EGFR inhibitors are low weight tyrosine kinase inhibitors (TKI) such as erlotinib [2] and gefitinib [3], which bind to the adenosine triphosphatase (ATP) receptor site to inhibit the intracellular tyrosine kinase domain (TKD) of the receptor.
Casein kinase CK2 has been proven to play an important role in the pathogenesis of cancer [4].
FGFR1 critically affects the development of cancer cells by enhancing point mutations or translocations. It has also been reported that an increase or activation of FGFR1 is associated with many types of cancer [5].
The activation of serine/threonine kinase B-Raf and, accordingly, of the mitogenic signal along the MAPK / ERK pathway leads to skin melanoma in most cases.
For wide range of human tumors, a constant relationship between increased severity and a decreased response to chemotherapy was ob-served in the case of oncogenic mutations of the B-Raf gene [6,7].
Consequently, direct therapeutic inhibition of the oncogenic activity of B-Raf kinase provides a perspective for the treatment of these tumors. Because the majority of all melanomas contain an erroneous easily activated mutation (V600E) in the B-Raf oncogene [6], the targeted inhibition of the gene product of V599E is a reasonable therapeutic target in the treatment of the tumor.
Taking into account the role of the kinases in the development and progression of cancer diseases, we choose them as molecular targets for receptor-oriented screening in the series of thieno [2,3-d]pyrimidines.

Ligand and Receptor Structure Preparation
Ligand structures were prepared using MGL Tools 1.5.6 software (MGL Tools 1.5.6 (Scrips Research Institute) http://mgltools.scripps.edu/) and Vega ZZ [8]. Receptor-based virtual screening was used to analyze the binding of the compound collection. Docking was performed at ATP-binding sites of protein kinases CK2 (database code RCSB 4GRB -2.15 Å), FGFR1 (database code RCSB 3GQI -2.50 Å), EGFR WT (database code PDB: 4HJO -2.75 Å), V599E B-RAF(database code PDB: 1UWJ -3.5 Å ) using Autodock 4.2.6. The structures taken for docking were kinase domains in an active state. Water molecules, ions and ligands were removed from the PDB file. The receptor structures were prepared using MGL Tools and AutoGrid. Hydrogen atoms were removed from nonpolar atoms. The incoming formats of receptor and ligands data were converted into PDBQT-format with Vega ZZ in AUTODOCK force field.
Flexible docking. Autodock 4.2.6 programs package was used for the receptor-based flexible docking [9].

Visual analysis
A visual analysis of the molecular docking results (interaction of compounds with the amino acid residues of CK2 and FGFR1 ATPbinding site) was carried out using Discovery Studio Visualizer 4.0 (http://accelrys.com/).

Chemical synthesis
1 Н and 13 C NMR spectra were recorded on a Bruker Avance II 400 spectrometer (400 and 100 MHz, respectively) in DMSO-d 6 or DMSO-d 6 -CF 3 CO 2 D (10:1) using TMS as an internal standard. Mass spectra (EI ionization, 70 eV) for compound 8 was recorded on a МХ1321 apparatus with direct sample injection at 200 °С ionization chamber temperature 200 °С. Mass spectra (FAB ionization) were registered on a VG-7070 spectrometer. Ion desorption from m-nitrobenzyl alcohol was done by a beam of argon atoms with an energy of 8 keV. Elemental analysis was performed on LECO CHN-900 Elemental analyzer. Melting points were determined on Electrothermal 9100 Digital apparatus. The reaction progress and purity of compounds were monitored by TLC on Silicagel gel 60 F 254 (Merck) plates, eluent CHCl 3 −i-PrOH (10:1), visualization in the iodine chamber.
Synthesis of spirocyclic compounds 3a-c: To the mixture of compound 1 or 2 (1 mmol) and the corresponding cyclic ketone (1 mmol), in EtOH (5 mL), a solution of NaOH (1 mmol) in EtOH (5 mL) was added dropwise under stirring, and the reaction mixture was heated under reflux for 3 h. The reaction mixture allowed cooling to room temperature. The resulting precipitate was collected by filtration. The crude products were recrystallized from i-PrOH to afford pure compounds 3a-c. Synthesis of Vilsmeier-type rearranged products 10, 11. Methods A: The Vilsmeier-Haack reagent was prepared by mixing POCl 3 (2 ml, 22 mmol) and DMF (5 ml, 66 mmol) in an ice bath. The corresponding spiro compounds 3a or 8 (7.2 mmol) were added to the prepared Vilsmeier reagent. The reaction mixture was heated in water bath at 80°С for 2 h, then cooled to 10°С, and treated with 15 % aqueous NaOH (15 ml). The precipitate was filtered off, washed with H 2 O, and resulted compounds were purified by recrystallization from MeCN.
Method B: Compound 3a (2 g, 0.007 mol) was mixed with DMF (1 ml) and the slurry obtained was added portion wise to the cold Vilsmeier-Haack reagent prepared from DMF (5 ml) and POCl 3 (2 ml, 0.022 mol). The mixture was left to warm to room temperature for 0.5 h and maintained for 4 days at room temperature. Then it was poured onto ice and neutralized with 10 % NaOH solution to weakly basic reaction. The product was filtered off.  1 , 2 , 3 , 4 , 7 , 8 , 9 , 1 0

-O c t a h y d r o [ 1 ] benzothieno[2,3-b]quinolin-11-amine (12):
A mixture of compound 3a (2.0 g, 0,013 mmol) and POCl 3 (20 ml) was refluxed in absolute toluene (70 ml) for 2 h. The toluene was decanted off and the dark-brown, viscous residue was dissolved in MeOH. Concentrated ammonia was added to adjust the mixture to alkaline reaction, and the product was extracted with CH 2 Cl 2 (30 ml). The extract was dried over Na 2 SO 4 , and the solvent was evaporated.
For the receptor-oriented flexible screening, we used the Autodock 4 and the AutoDock Vina software packages [43,44]. Additionally, the molecular modeling studies were performed in order to rationalize the anticancer activities of the proposed compounds. All the synthesized thieno[2,3-d]pyrimidine derivatives were subjected to the docking study together with the internal ligand, erlotinib, as a reference molecule to explore their calculated binding modes with the EGFR wild type receptor (EGFR WT , PDB: 4HJO) [45]. The ATP binding pocket of EGFR WT consists of five main parts; sugar pocket, two hydrophobic regions, adenine binding pocket, and phosphate binding region.
The results of the docking studies against the EGFR WT revealed that the synthesized compounds have similar orientations inside the ATP binding site. The designed compounds have good binding energies ranging from -8.4 to -10.2 kcal/mol ( Table 1).
As seen from Fig. 1, the oxygen atom of compound 8 forms a hydrogen bond with Lys721 amino acid residue with a distance of 2.30 Å.
The results of the compounds verification as inhibitors of protein kinase CK2 are presented in the Table 2. Among the studied compounds, compound 7 revealed hydrogen bonding interaction with Val66 amino acid residue and showed the best binding energy value (Fig. 2).
All spiro compounds showed the interaction with Val66 and Ile174 key amino acid residues for CK2 to form a π-σ and π-alkyl contacts, respectively. Noteworthy, such contacts are necessary for selective inhibition of protein kinase CK2 [46]. The compounds 7 and 9 were joined via hydrophobic interactions at the CK2 ATP-acceptor site with the amino acid residues Val53, Val66, Met1163, Ile174 and by a hydrogen bond with Val116 (2.67 Å), which was located in the hinge re-gion of the kinase. There was a π-Sulfur link between the thiophene cycle and the amino acid residue Met163.
When the FGFR1 kinase inhibitors were tested, none of the studied compounds formed any hydrogen bonds with the ATP-acceptor site FGFR1. The binding energies against FGFR1 are presented in Table 3.
The previous generations of B-Raf inhibitors showed Raf inhibitory activity at the concentrations as low as nanomolar. However, the therapeutic effects of such inhibitors were complicated by the lack of bioavailability and a number of non-specific targets that are also affected inhibition [47]. Noteworthy, thienopyrimidine ligands have not yet been studied as inhibitors of B-Raf kinase. The binding energies are given in the Table 4.
In addition to the various visible van der Waals interactions with B-RAF inhibitors, the hydrogen bonds with the active sites of the protein Glu500 and Cys531 also contributed to the binding of a ligand. Thus, a carbonyl oxygen atom of the compound 7 increased the affinity compared to the carbon atom and formed a hydrogen bond with the main nitrogen atom of the Cys531 residue in the region of the interdomain hinge. Compounds 7 and 8 were found to form a π-π interaction between their thiophene rings and benzene ring of Phe594 (Fig 3).
The results of molecular docking studies of compounds 10-12 are presented in the Table 5. Thienopyrimidine 10 was found to form a hydrogen bond with the amino acid residue Val66 (1.83 Å, Fig. 4) showin the best result with a binding energy of -10.4 kcal/mol and therefore might be considered as perspective inhibitor of CK2 kinase. However, we have to admit that the rearrangement products often do not form hydrogen bonds with kinase amino acid residues while revealing moderate binding energies. The absence of binding with H-bonds does not allow us to consider the rearranged products as perspective targets for further screening.
The inhibition activities of novel compounds as potential inhibitors of EGFR, CK2, FGFR1 and B-raf kinases have been examined. Spiro-fused thieno [2,3-d]pyrimidines were recognized as perspective targets for futher screening. According to the docking studies, in most cases the rearranged products do not tend to form any hydrogen bonds with kinase amino acid residues, but showed moderate binding energies. The lack of hydrogen bonding allowed us to consider the products of Vilsmeier-type rearrangement as unsuitable for further studies as kinase inhibitors.