Synthesis and biological studies of new quinazolines with ether functions in position 2

A series of new quinazolines linked to triazoles through an ether chain in position 2 has been designed and synthetized through a flexible route. Cytotoxicity assays on selected cancer cell lines and inhibition studies toward a panel of representative mammalian kinases have been performed on these molecules


Introduction
Many quinazoline alkaloids and derivatives have been isolated already, and the antimalarial (+)-Febrifugine 1 and the cytotoxic Luotonin A 2 are representative examples of this family of natural products (Figure 1). 1 From a structural point of view, almost all of them possess a quinazoline-4(3H)-one ring system.3][4] Representative examples are indicated in Figure 1 and it is worth noting that all compounds 3-8, which are potent kinase inhibitors used as anticancer drugs, have an aniline-type substituent in position 4 of this structure.Some other quinazolines, like Doxasocine 9 and Prazosin 10 (these two derivatives are used against hypertension) have both a primary amine in position 4 and a substituted piperazine in position 2. Further, extensive literature search shows that much less is known about type-A molecules with X-R 1 chains in position 2 (X being heteroatom like O, S and N and R 1 = alkyl or substituted alkyl chains, aromatics, heteroaromatics…..) (Figure 2).Therefore, we embarked on a program involving the synthesis and biological evaluation of such derivatives.The goal of this paper is to report the first part of this research, dealing with the synthesis of a focused library of oxygen-linked molecules 11 with a short linker and triazoles in terminal position.The choice of the triazole nucleus is based upon the fact that this core structure is found also in many biologically active molecules. 5

Chemical synthesis
7] In our case we favored the approach through two C-N bond disconnections and starting from an ortho-aminobenzaldehyde derivative 13 (Scheme 1).Reaction with urea gave in good yield quinazoline 14 which was further treated by POCl3 to give chloroquinazoline 14a.This intermediate was reacted with propargyl alcohol to isolate in 65% yield propargyl ether 15.Final click-type reactions, [8][9][10][11] with various azido derivatives 16a-j, 12 afforded the target molecules 11a-j in good yields (Table1).All intermediates and final products have spectral and analytical data in agreement with their structures.

Biological studies
Based on the known biological properties of molecules with quinazoline scaffolds, we performed a few primary biological screenings of the ten molecules 11a-11j.First, their cytotoxicity has been checked on seven representative cancer cell lines (HuH7, CaCo-2, MDA-MB231, HCT116, PC3, NCI-H727, MCF7).All compounds were found to be devoid of any significant cytotoxicity at 10 M concentration (See Supplementary Material).
Their activity was also screened against a panel of eight representative kinases (HsCDK5/p25, HsCDK9/CyclinT, HsPIM1, MmCLK1, RnDYRK1A, HsHASPIN, HsGSK3β, HsCK1ε) and the results are reported in Table 2. Interestingly only one of these kinases (MmCLK1) was found to be responding to these derivatives.A few molecules (11a, 11c, 11d, 11e, 11f, 11j) exhibited a moderate inhibition of this kinase at 10M.On the other hand, these molecules were found to be devoid of any significant activity at 10M concentration on the other kinases.Percentages of residual kinase activity were determined at 1 and 10 M concentration for each compound.Kinase activities were assayed in duplicate.

Conclusions
In summary, we designed a short and convergent strategy toward the desired quinazolines linked to triazoles through an ether chain in position 2 of the quinazoline.Some of these derivatives exhibit a moderate, but significant and selective, activity against the MmCLK1 kinase.Extension of this approach to other quinazolinederived derivatives is under active study and will be reported in due course.

Chemical Synthesis
General.All anhydrous reactions were performed in heat gun-dried round-bottomed flasks under a dry argon or nitrogen atmosphere.Air and moisture-sensitive compounds were introduced via syringes or cannula, using standard inert atmosphere techniques.In addition, the gas stream was passed through glass cylinder filled with P2O5 to remove any traces of residual moisture.Reactions were monitored by thin layer chromatography (TLC) using E. Merck silica gel plates and components were visualized by illumination with short wavelength UV light and/or staining (Ninhydrin or basic KMnO4).THF and Et2O were dried over sodium-benzophenone and distilled prior to use.Anhydrous CH2Cl2 was prepared by refluxing in the presence of CaH2 and distilled right before use unless otherwise noted.Infrared spectra have been recorded on a Bruker alpha II FTIR spectrometer. 1 H NMR spectra were recorded at 300 and 400 MHz, and 13 C NMR spectra at 75 and 100 MHz,in CDCl3 or DMSO-d6 using tetramethylsilane (TMS) as an internal standard on Bruker spectrometers (Avance 300III and Avance 300I and Avance III 400).Assignments were made using standard 2D NMR techniques (COSY, HMQC/HSQC, HMBC).High resolution mass spectra were performed using a time of flight Maxis 4G (Bruker Daltonik Gmbh, Bremen, Germany) in Electrospray positive ionization mode.LC-MS analyses were carried out on a Shimadzu [LCMS-2020], SHIMPAK, XR ODS-II column (50 x 2 mm) utilizing the following method.Solvent A = Acetonitrile, B = 0.1% TFA in water; Initial 95% of solvent B, then run gradient, which should reach 90% solvent A within 10 min and hold 90% solvent A for another 10 min.Flow Rate: 0.2 ml/min.

Synthesis of 2-amino-3-methoxybenzaldehyde (13).
To a stirred solution of 3-methoxy-2-nitrobenzaldehyde (12) (10 g, 54 mmol) in a mixture of methanol (100 mL) and glacial acetic acid (100 mL) and water (50 mL) was added reduced iron powder (16 g, 144 mmol) in lot wise manner.The resulting suspension was stirred at 30-35 o C for 2h.The reaction mixture was filtered on Celite bed and washed with methanol (50 mL).The filtrate was concentrated to remove volatiles and it was partitioned between sat.NaHCO3 (150 mL) and ethyl acetate (200 mL).The basic layer was further extracted with ethyl acetate (100 mL).The combined organic extracts were washed with brine (50 mL), dried over Na2SO4 and concentrated to dryness and purified by chromatography on silica gel eluting with 20% EtOAc in hexane giving a compound 13 (6.5 g, 78% Yield) as a off white solid.Synthesis of 8-methoxyquinazolin-2-ol (14).2-Amino-3-methoxybenzaldehyde (13) (5 g, 33.1 mmol), urea (20 g, 336.3 mmol) and cat.NH4OAc (10 mg) were thoroughly mixed together in a round bottom flask.The solid mixture was heated to 160 o C and the solids quickly melted and stirring was continued for 15 min.A solid started to precipitate from the hot solution, NMP (25 mL) was added to dissolve the solids, the reaction was heated with stirring for an additional 2 h at 155-160 o C. The hot reaction mixture was poured into vigorously stirred ice water (150 mL).The reaction mixture was filtered, washed with water (50 mL) and ethyl acetate (15 mL) giving a brown color solid which was dried to give compound 14 (4.9 g, 82% yield) as a off white solid. 1  General procedure for preparation of compounds, 11a-j.To the stirred solution of compound 15 (0.1 mmol, 1.0 eq.) and compound 16a-j [prepared using general protocol, 12 (0.1 mmol, 1 eq.) in a 1:1 mixture of t-butanol and water (0.56 mL) was added copper sulfate pentahydrate (0.01 mmol, 0.1 eq.) and sodium ascorbate (0.03 mmol, 0.3 eq.) at room temperature.The resulting mixture was stirred for overnight.After completion of the reaction, it was diluted with a mixture of ethyl acetate and water (7.5 ml in a 1:1 ratio), the organic layer was separated and washed with 5% ammonium hydroxide solution (0.5 mL) and followed by brine (0.5 mL).The combined organic layers were dried over Na2SO4.The solvents were evaporated under reduced pressure to get the crude product, which was purified by chromatography on silica gel affording target compounds 11a-j (65-75% yield) as light yellow to brown solids.Cytotoxic assay.Chemicals are solubilized in DMSO at a concentration of 10 mM (stock solution) and diluted in culture medium to the desired final concentrations.The dose effect cytotoxic assay of chemical is performed at 25 µM.Cells are plated in 96 wells plates (4000 cells/well).Twenty-four hours after seeding, cells are exposed to chemicals.After 48h of treatment, cells are washed in PBS and fixed in cooled 90% ethanol/5% acetic acid for 20 minutes and the nuclei are stained with Hoechst 33342 (B2261 Sigma).Image acquisition and analysis are performed using a Cellomics ArrayScan VTI/HCS Reader (ThermoScientific).The survival percentages are calculated as the percentage of cell number after compound treatment over cell number after DMSO treatment.

Protein kinase assays
Kinase enzymatic activities were assayed in 384-well plates using the ADP-Glo assay kit (Promega, Madison, WI) according to the recommendations of the manufacturer.This assay is a luminescent ADP detection assay that provides a homogeneous and high-throughput screening method to measure kinase activity by quantifying the amount of ADP produced during a kinase reaction.Briefly, the reactions were carried out in a

Figure 1 .
Figure 1.Representative examples of bioactive natural products and drugs with quinazoline cores.

Table 1 .
Synthesis of our target molecules

Table 2 .
Study of the inhibition of quinazolines 11 against a representative panel of mammalian kinases mL, 38.8 mmol) in acetonitrile (80 mL) was stirred at room temperature for 10 min.POCl3 (22 mL, 222 mmol) was added to reaction mixture at 0 o C and the reaction mixture was heated for 10 h at reflux.The mixture was concentrated to dryness.The obtained residue was partitioned between EtOAc (200 mL) and sat.Propargyl alcohol (518 mg, 9.24 mmol) was added to a stirred suspension of Cs2CO3 (3g, 9.24 mmol) and DMF (10 mL), after stirring 15 minutes at 30 o C compound 14a (600 mg, 3.08 mmol) was added.After stirring at 100 o C for 16h, the reaction mixture was quenched with water (30 mL) and extracted with DCM (2x25 mL).The combined organic extracts were washed with brine (5%, 15 mL), dried over Na2SO4 and concentrated to dryness.Purification by chromatography on silica gel (elution with EtOAc in hexane) gave compound 15 (450 mg, 65% yield) as a off white solid.