Diversity-oriented synthesis of amide derivatives of tricyclic thieno[2,3-d]pyrimidin-4(3H)-ones and evaluation of their influence on melanin synthesis in murine B16 cells

Synthesis

The synthetic route of the target compounds (Scheme 1) follows our previous procedure [5]. An intermediate product 1 was prepared from diethyl oxalate via the Grignard reaction. Application of the Gewald reaction to compound 1, cyanoacetate and sulfur in the presence of triethylamine at room temperature produced compound 2 in high yield. Then, ethyl thieno[2,3-d]pyrimidine carboxylates 3–5 were prepared through the condensation of compound 2 and cyclic lactams in the presence of phosphorus oxychloride in dichloromethane at 45°C. Hydrolysis of esters 3–5 was completed in a mixture of water and methanol in the presence of lithium hydroxide to yield the acids 6–8. In order to obtain the amidation products in good yields, the optimization reactions with acid 6 under different conditions using a mixture of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide and hydroxybenzotriazole (EDCI and HOBt), (1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexafluorophosphate (HATU) and oxalyl chloride as activating reagents were conducted. n-Butylamine and 1-(4-aminophenyl)ethan-1-one were selected to represent aliphatic and aromatic amines, respectively. Using the coupling reagents EDCI and HOBt, the yields were only moderate for both aliphatic amine (53%) and aromatic amine (65%). HATU provided a good yield for the reaction of aliphatic amine (88%), slightly better than for aromatic amine (74%). the conversion of acid 6 to carboxylic chloride and then condensation with amine provided excellent yields with the aromatic amine (90%), much better than for the aliphatic n-butylamine. Therefore, it was decided to synthesize the target compounds 9a–e, 10a–e, 11a–e bearing the aliphatic amino moiety in the presence of HATU and to prepare compounds 9f–q, 10f–q, 11f–q with aromatic amine moiety from carboxylic acid chloride.

Scheme 1

Synthetic route to compounds 9a–q, 10a–q and 11a–q.

Melanin synthesis evaluation

All the tricyclic thieno[2,3-d]pyrimidin-4(3H)-one derivatives 9a–q, 10a–q, 11a–q were tested for their effect on melanin synthesis in murine B16 cells, following a previously published method [20]. As shown in Table 1, the reference drug 8-MOP enhances the melanin synthesis by 126.1±3.2% at 50 μm as compared to the blank control. The effectiveness of the synthetic compounds on melanin synthesis varies from 40.6±11.4 to 234.5±5.9%. It can be noticed that the five-membered ring compounds (9a–q, n=1) showed decreased melanin synthesis, no matter what kind of substituent group is present on the amide moiety. Among these 17 compounds, only compounds 9d and 9i showed higher values than 8-MOP. Several compounds fused with a seven-membered ring (11, n=3) possess better activities to promote melanin content. Some six-membered derivatives (10, n=2) show high potency. Most of them (11 out of 17) increase the synthesis of melanin. It can be concluded that the size of fused rings of the synthetic compounds dramatically affects their ability to influence melanin synthesis.

Substituents on the amide moiety also contribute to the biological effectiveness. For example, compounds with n-octylamino group 9d, 10d, 11d exhibit an increased potency in comparison to the derivatives substituted with an n-butylamino group 9a, 10a, 11a. Compound 11n is the lead candidate showing the highest potency on melanin synthesis in murine B16 cells, with a value of 234.5±5.9%, which is 1.86-fold greater compared to that of 8-MOP. Another hit compound is 10h which contains electron-donating substituent (methoxy group) attached to position 4 of the aromatic ring and increases the melanin content by 227.6±4.3%. Interestingly, activities of compounds with a 2,6-dichlorophenyl fragment in amide linker increase with the increase in size of the saturated ring 9n<10n<11n.

General procedure for the synthesis of compounds 9a–q, 10a–q and 11a–q

Method A To a solution of 2-methyl-4-oxo-thieno[2,3-d]pyrimidine-3-carboxylic acid (6–8, 1.0 mmol) and HATU (1.2 mmol) in dichloromethane (20 mL) was added N,N-diisopropylethylamine (DIPEA, 0.27 mL, 1.5 mmol) and an aliphatic amine (see Scheme 1, 1.5 mmol) dropwise at 0–5°C. The mixture was stirred at room temperature overnight and then concentrated under reduced pressure. The residue was extracted with dichloromethane (2×30 mL). The extract was washed with brine, dried over anhydrous MgSO₄, filtered and concentrated under reduced pressure. The residue was purified by silica gel chromatography to produce the pure compounds 9a–e, 10a–e, 11a–e.

Method B A solution of 2-methyl-4-oxo-thieno[2,3-d]pyrimidine-3-carboxylic acid (6–8, 1.0 mmol) in dichloromethane (10 mL) was treated with oxalyl chloride (3 mmol) and the mixture was stirred at room temperature for 3 h. The solvent and the excess of oxalyl chloride were removed under reduced pressure and the residue was treated with an aromatic amine (see Scheme 1, 1.1 mmol) and triethylamine (1.2 mmol). The mixture was stirred at room temperature for 8 h, then washed with water and concentrated under reduced pressure. The residue was purified by silica gel chromatography and eluted with petroleum ether/ethyl acetate to give the pure compounds 9f–q, 10f–q, 11f–q.

Cell culture

Murine B16 melanoma cell lines (B16F10) were obtained from the Chinese Academy of Sciences. The cells were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) (GIBICO, USA) supplemented with 10% heat-inactivated fetal bovine serum (GIBICO, USA), 100 U/mL penicillin and 100 mg/mL streptomycin (GIBCO, USA) in a humidified atmosphere with 5% CO₂ at 37°C.

Melanin content assay

Exponentially growing cells were seeded into 6-well plates at a concentration of 5×10⁵ cells per well. After 24 h of incubation at 37°C, the culture medium was removed and replaced with fresh medium containing the candidate compounds at different concentrations. The cells were incubated for another 48 h, washed with ice-cold phosphate-buffered saline (PBS), followed by lysis with radioimmunoprecipitation assay (RIPA) buffer for 40 min on ice, and the lysates were centrifuged at 10 000 g for 20 min. Supernatants containing protein were subjected to protein assay, and the pellets with intracellular melanin were solubilized in 200 μL of 1 m NaOH for 2 h at 60°C. The melanin amount was determined spectrophotometrically at 405 nm using a multi-plate reader. The melanin amount was calculated by normalizing the total melanin values with protein content (abs melanin/L g protein).