Identification of 4-methoxythieno [ 2 , 3-d ] pyrimidines as FGFR 1 Inhibitors

I. M. Kotey, M. V. Protopopov, S. A. Starosyla © 2019 I. M. Kotey 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 UDC 577.322


Introduction
Fibroblast growth factor receptor 1 (FGFR1) is a receptor tyrosine kinase.Normally this enzyme is involved in tissue repair, hematopoiesis, angiogenesis and embryonic development.However, due to the FGFR role in regulating cell growth and survival, amplification and/or mutation of this protein kinase drives a number of cancer types, including gastric cancer, multiple myeloma, breast cancer and nonsmall cell lung cancer (NSCLC).Such inhibitors of FGFR1 as AZD4547, E3810, G141 and E7090 showed anticancer potential and now are in clinical trials.This means that FGFR1 is an appealing target for the drug development [1][2][3][4].

Molecular docking
Preparation of ligands and receptor molecules.Autodock 4.2.6 programs package was used for the receptor-based flexible docking [11].
Ligands were prepared by Vega ZZ (command line) [12] and MGL Tools 1.5.6 [11].The incoming formats of receptor and ligands data were converted into PDBQT-format with Vega ZZ using AUTODOCK force field.This format contains the coordinates of the atoms and partial charges.Hydrogen atoms were removed from nonpolar atoms.The receptor was prepared using MGL Tools and AutoGrid [11].
We have used docking parameters reported earlier [13].
Flexible docking.The catalytic subunit of protein kinase FGFR1 complex with inhibitor (PDB code 3GQI) was used for molecular docking via Autodock [14].Water molecules, ions, and ligands were removed from the PDB file.

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

In vitro testing
The FGFR1 kinase assays with recombinant cytoplasmic domain of the FGFR1 tyrosine kinase (Millipore, Cat.N. 14-582) were performed in a total volume of 30 μl containing 10 mM MOPS (рН 7.2), 0.1 mM sodium orthovanadate, 0.2 mM EDTA, 0.002 % Brij 35, 0.2 mg/ml BSA, 0.02 % β-mercaptoethanol, 250 μM of peptide substrate (KKKSPGEYVNIEFG, GenScript), various concentrations of inhibitor dissolved in DMSO (final DMSO concentration in probe less than 1 %) and 10 mU of enzyme.The reaction was initiated by the addition of ATP (50 μM ATP, 25 mM MgAc containing 0.1 μCi of [γ-32 P] ATP per probe) and samples incubated at 30°C for 25 min.The reaction was terminated by the addition of 5 % phosphoric acid and the precipitation of material onto phosphocellulose filters "Whatman P81".Filters were washed three times with 0.75 % phosphoric acid and the incorporation of [ 32 P] into the peptide substrate was determined by counting the radioactivity retained on the filters in a PerkinElmer scintillation counter.As negative control an equal volume of dimethyl sulfoxide (DMSO) was added to the reaction mixture.Inhibition percentage was calculated as a ratio of the substrate-incorporated radioactivity in the presence of inhibitor to the radioactivity incorporated in control reactions, i.e. in the absence of inhibitor.The concentration of compound that inhibited enzymatic activity by 50 % (IC50) was determined graphically.

Chemistry
The thieno [2,3-d]pyrimidine derivatives were synthesized from corresponding carbonyl compounds according to Scheme 1. Initially substituted 2-amino-3-carbethoxythiofenes (2) were obtained by Gewald reaction (in case of acetophenone [the] synthesis was carried out in two stages: firstly ethyl cyanoacetate was condensed with the corresponding acetophenone and then the product was cyclized with sulfur) (method 1).Then thienopyrimidinones (3) were obtained by condensation of 2-amino-3-carbethoxythiofenes with formamide (method 2).The various 4-chloro-thieno [2,3-d]pyrimidines (4) were synthesized after interaction of thienopyrimidinones with PCl 5 (method 3) and used as a building block for further formation of combinatorial rows.The final compounds were obtained by chlorine substitution in previous molecules with corresponding phenols (method 4a) or protected aminophenols (method 4b).

Experimental section
Starting materials and solvents were purchased from commercial suppliers and were used without further purification.The progress of the reaction and purity of obtained compounds were controlled by TLC on "Kieselgel 60 F 254 " plates (Merck).The structures of synthesized compounds were confirmed by 1H NMR spectrometry and liquid chromatography-mass spectra (LC-MS) analysis.Nuclear magnetic resonance spectra were recorded on a Varian Mercury VRX-400 spectrometer using DMSO-d 6 as solvent and tetramethylsilane as internal standard.Chemical shift values (δ) are quoted in ppm and coupling constants (J) in Hz.Liquid chromatography-mass spectra (LC-MS) analyses were performed using the Agilent 1100 LC/MSD SL (Agilent Technologies) separations module and Mass Quad G1956B mass detector with electrospray ionization (Agilent Technologies).HPLC is performed using Zorbax SB-C18 (Agilent Technologies), Rapid Resolution HT Cartridge 4.6x30 mm 1.8-µ (Agilent Technologies P/N:823975-902) i.d.column, at a temperature of 40 o C with gradient elution of 0-100 % CH 3 CN (with 1 mL/L HCOOH): H 2 O (with 1 mL/L HCOOH) at a flow rate of 3 mL/min and a run time of 2.8 min.Compounds were detected at 215 nm using a Diode Array G1315B detector.All tested compounds had ≥ 90 % purity as determined by this method.All purified synthetic intermediates had ≥ 95 % purity as determined by this method.
Method 1.A mixture of 107 mL (1 mol) of ethyl cyanoacetate, 32 g (1 mol) of elemental sulfur and 1 mol of the corresponding ketone in 200 ml of ethanol was stirred at room temperature.80 ml of diethylamine were added to this mixture for 12 h.The reaction mixture was left overnight; the precipitate was filtered, washed by aqueous alcohol (1:1) and dried in air [15].Yield was 50-88 %.Method 2. 2 mol of formamide were added to 1 mol 2-aminothiophene and the mixture was heated to 160-170 o C for 24 h.The hot mixture was diluted with isopropyl alcohol and cooled.The solid product was collected by filtration, washed with isopropyl alcohol and water and dried at 60 o C [16].Yield was 85-90 %.Method 3. A mixture of 0.01 mol of thienopyrimidinone, 11 ml of POCl 3 and 1.5 g PCl 5 was refluxed for 5-6 hours.The solvent was carefully evaporated to dryness.The dry product was dissolved in methylene chloride and poured to a cold 0.5 N NaOH solution.The organic phase was separated and dried with anhydrous Na 2 SO 4 , then solvent was evaporated in vacuum.The residue was recrystallized from isopropanol [17].Yield was 70-80 %.
Method 4A.NaH (60 % dispersion in mineral oil) (0.5g, 0.012 mol) was added to solution of 0.011 mol of corresponding phenol in 10 ml of dimethylformamide at 0 o C, after 30 minutes at ambient temperature 0.01 mol of substituted 4-chloro-thieno[2,3-d]pyrimidine was added and the mixture was stirred at 80 o C for 8 hours.The content of the flask was diluted with water.Precipitate was filtered off and dried at room temperature.The resulting solid was recrystallized from ethanol.Yield was 60-80 %.
To understand the activity of the compounds and make some conclusions about SAR we have performed molecular docking and analyzed the complexes of studied compounds with FGFR1 ATP-binding pocket to propose their binding mode.The complex of the most active found compound with the ATP-acceptor site of FGFR1 is shown in Fig. 1.
Proposed binding mode of the studied compounds belongs to the type I protein kinases inhibitors.The thieno [2,3-d] The Structure Activity Relationship (SAR) study showed that R 1 of the most active compounds is phenyl.Mostly it is a hydrophobic 4-substituted phenyl.The compound 10 with 4-methylphenyl (more hydrophobic) at R 1 has two-fold higher activity than compound 12 with 4-methoxyphenyl at R 1 .IC 50 values were 2.5 and 5.6 µM respectively.But the compound 8 with 4-chlorophenyl (more hydrophobic) at R 1 has lower activity than compound 13 with 4-methylphenyl.IC 50 values were 2.14 and 0.9 µM, respectively.Therefore, the order of potency for the substituents at R 1 could be proposed as following: 4-methylphenyl >4-chlorophenyl >4-methoxyphenyl.
SAR study of substituents at R 3 -R 6 showed that the most active compounds have hydrogen bond acceptor at positions R 4 and/or R 5 .Compounds 8 and 13 contain dioxymethan and have IC 50 values of 2.14 and 0.9 µM, respectively.

Conclusions
Nine new inhibitors of FGFR1 were discovered among 23 4-methoxythieno [2,3-d]py-rimidine derivatives.SAR study showed that the most profitable substituent at R 1 is 4-methylphenyl and hydrogen bond acceptors at R 4 and/or R 5 are important for revealing the inhibitory activity toward FGFR1.The most potent compound in this series showed the IC 50 value of 0.9 µM.The studied compounds are good candidates for the further development of effective FGFR1 inhibitors and potential anti-cancer drugs.

Table 1 .
Structure, in vitro inhibitory

activity toward FGFR1 and IC 50 of studied compounds.
6 pyrimidine core is located in adenine binding region and forms The complex of compound 13 with ATP-acceptor site of FGFR1 obtained with Autodock 4.2.6.H-bonds are shown by green dashed lines, hydrophobic interactions -purple dashed lines.