Synthesis and Cytotoxic Activity of Some Novel Thieno[2,3-d:4,5-d’]Dipyrimidine Derivatives

Synthesis of some novel tricyclic compounds bearing thienodipyrimidine moiety by the reaction of ethyl 6-amino-1,3-dimethyl-2,4-dioxo-1,2,3,4-tetrahydrothieno[3,2-d]pyrimidine-7-carboxylate (1) with different types of aromatic amines to give the corresponding carboxamide derivatives 2–11, which cyclized with triethyl orthoformate in acetic anhydride to afford thieno[2,3-d:4,5-d’]dipyrimidine derivatives 12–15 is described. Similarly, thieno[2,3-d:4,5-d’]dipyrimidine derivative 17 was also prepared via the reaction of carboxamide derivative 2 with chloroacetyl chloride in chloroform to give thieno[3,2-d]pyrimidine-7-carboxamide derivative 16, followed by a cyclization in boiling acetic anhydride. The structure of these compounds was confirmed on the basis of their spectral and analytical data such as IR, 1H-NMR, 13C-NMR spectroscopy and mass spectral data. The synthesized compounds were screened for their cytotoxic activity.


Experimental
All melting points are uncorrected and were determined on Gallenkamp electric melting point device. Pre-coated Merck silica gel 60F-254 plates were utilized for thin-layer chromatography (TLC) and the spots were visualized under UV light (254 nm). The infrared (IR) spectra were recorded (KBr disk) on Pye Unicam SP 1000 IR spectrophotometer (Thermoelectron Co. Egelsbach, Germany. The 1 H-NMR spectra were obtained on Varian Gemini 400 MHz (Varian Co., Cairo university, Egypt), and 13 C-NMR on the same instrument at 100 MHz. Deuterated DMSO-d 6 was used as the solvent, tetramethylsilane (TMS) was used as the internal standard and chemical shifts were measured in δ ppm. Mass spectra were determined on a GC-MS.QP-100 EX Shimadzu (Japan). Elemental analyses were recorded on Perkin-Elmer 2400 Elemental analyzer at the Micro-analytical Center at Cairo University, Cairo, Egypt.

General Procedure for the Reaction of Compound 16 with Piperidine and Morpholine
To an equimolar amount of 16 (4.07 g, 0.01 mol) and piperidine (0.85 g, 0.01 mol) or morpholine (0.87 g, 0.01 mol) in dry xylene (20 mL) DMF (5 drops) was added. The reaction mixture was refluxed for 3 h, then poured onto iced water and the formed solid products, in each case, were collected by filtration and crystallized from EtOH/ DMF to give compounds 18 and 19, respectively.

1. Cytotoxic Activity
The potential cytotoxicity of the tested compounds was evaluated using the MTT assay. The cell lines were plated in 96-multiwell plate (10 4 cells/well) for 24 h before treatment with the prepared compounds to allow the attachment of cells to the wall of the plate. The tested compounds were dissolved in dimethylsulfoxide (DMSO) and diluted 1000-fold in the assay. Different concentrations of the tested compounds ranging from 1 to 50 µg/mL, were added to the cell monolayer. The monolayer cells were incubated with the compounds for 48 h at 37 ºC, in atmosphere of 5% CO 2 . After 48 h, cells were fixed, washed and stained for 30 min with 0.4% (wt/vol) sulforhodamine B (SRB). The excess stain was washed with 1% acetic acid and attached stain was recovered with base tris-(hydroxymethyl)aminomethane (tris-EDTA) buffer, color intensity was measured in a plate reader at 570 nm, the relation between surviving fraction and drug concentration was plotted to get the survival curve of tumor cell line and the IC 50 was calculated. The obtained IC 50 values are illustrated in Table 1.
Next, we aimed to prepare the tricyclic compounds bearing thienopyrimidine scaffolds, being the focus of great interest because of their pharmacological activities. Thus, treatment of carboxamide derivatives 2, 3, 5 and 6 with triethyl orthoformate in acetic anhydride afforded thieno[2,3-d:4,5-d']dipyrimidine derivatives 12-15. Structures 12-15 were established on the basis of elemental and spectral analyses. In general, the IR spectra showed absorption bands at 1667-1655 cm −1 due to carbonyl groups. Its 1 H-NMR spectra revealed singlet signals at 3.01 and 3.09 assignable to two N-CH 3 protons, in addition to a singlet signal at 8.51-8.56 ppm assignable to pyrimidine-H 7 . The mass spectra of these compounds showed the molecular ion peaks at m/z = 341 (M + ), 419 (M + ), 502 (M + ) and 450 (M + ) respectively, which are in agreement with their molecular formulas.
We studied the reactivity of N-ethoxymethino group with different types of amines. Thus, treatment of compound 23 with aniline gave ethyl 1,3-dimethyl-2,4-dioxo-6-(((phenylamino)methylene)amino)-1,2,3,4-tetra hydrothieno[3,2-d]pyrimidine-7-carboxylate (24). The assignment of structure 24 was supported by elemental analysis and spectral data. The IR spectrum showed an absorption band at 3285 cm −1 due to NH group, in addition to the stretching vibration of three carbonyl groups at 1701 and 1653 cm −1 . Its 1 H-NMR spectrum revealed a triplet signal at 3.00 ppm assignable to CH 3 protons, a quartet signal at 3.71 ppm assignable to CH 2 , two singlet signals at 3.01 and 3.09 ppm assignable to two N-CH 3 , a singlet signal at 8.44 ppm assignable to N=CH and a singlet signal at 10.47 ppm assignable to NH proton. Also, 13 C-NMR spectra showed signals at 18.3, 67.5 and 158 ppm due to OCH 2 CH 3 and N=CH. The mass spectrum showed the molecular ion peak at m/z = 386 (M + ), corresponding to the molecular formula C 18 H 18 N 4 O 4 S. We tried to obtain the cyclic compound 25 by refluxing the compound 24 in DMF and TEA but failed.
On the other hand, the compound 23 was treated with phenylhydrazine to give 1,3-dimethyl-8-(phenylamino)thieno[2,3-d:4,5-d']dipyrimidine-2,4,9(1H, 3H,8H)-trione (26). The assignment of structure 26 was supported by elemental analysis and spectral data. The IR spectrum showed absorption bands at 3283 and 1652 cm −1 due to NH group and carbonyl group. Its 1 H-NMR spectrum revealed two singlet signals at 3.01 and 3.09 ppm assignable to two N-CH 3 , a singlet signal at 8.45 ppm assign-Scheme 3 able to pyrimidine-H 7 and a singlet signal at 10.24 ppm assignable to NH proton. Also, 13 C-NMR spectra showed a signal at 148.6 ppm due to C7 of pyrimidine ring. The mass spectrum showed the molecular ion peak at m/z = 355 (M + ), corresponding to the molecular formula C 16 H 13 N 5 O 3 S.
Moreover, compound 23 reacted with hydrazine hydrate to afford ethyl 6-((hydrazinylmethylene)amino)-1,3dimethyl-2,4-dioxo-1,2,3,4-tetrahydrothieno[3,2-d]pyrimidine-7-carboxylate (27). The assignment of structure 27 was supported by elemental analysis and spectral data. The IR spectrum showed absorption bands at 3448, 3419 and 3285 cm −1 due to NH 2 and NH groups, in addition to the stretching vibration of three carbonyl groups at 1701 and 1649 cm −1 . Its 1 H-NMR spectrum revealed a triplet signal at 3.00 ppm assignable to CH 3 protons, a quartet signal at 3.71 ppm assignable to CH 2 , two singlet signals at 3.01 and 3.09 ppm assignable to two N-CH 3 , a singlet signal at 4.45 ppm assignable to NH 2 , a singlet signal at 5.63 ppm assignable to NH and a singlet signal at 8.49 ppm assignable to N=CH proton. The mass spectrum showed the molecular ion peak at m/z = 325 (M + ), corresponding to the molecular formula C 12 H 15 N 5 O 4 S.
Finally, cyclization of compound 27 in DMF and TEA to afford the corresponding 8-amino-1,3-dimethylthieno[2,3-d:4,5-d']dipyrimidine-2,4,9(1H,3H, 8H)tri o ne (28) succeeded. The assignment of structure 28 was supported by elemental analysis and spectral data. The IR spectrum showed absorption bands at 3443, 3385 cm −1 due to NH 2 groups, in addition to the stretching vibration of three carbonyl groups at 1649 cm −1 . Its 1 H-NMR spectrum revealed two singlet signals at 3.01 and 3.09 ppm assignable to two N-CH 3 , a singlet signal at 5.45 ppm assignable to NH 2 and a singlet signal at 8.43 ppm assignable to pyrimidine-H 7 . Also, 13 C-NMR spectra showed a signal at 148.8 ppm due to C7 of pyrimidine ring. The mass spectrum showed the molecular ion peak at m/z = 279 (M + ), corresponding to the molecular formula C 10 H 9 N 5 O 3 S.

1. Cytotoxic Activity
The newly synthesized target compounds were evaluated for their in vitro anticancer effects via the standard MTT method, 30-32 against a panel of four human tumor cell lines namely; Hepatocellular carcinoma (HePG-2), Colorectal carcinoma (HCT-116), Mammary gland (MCF-7) and Epitheliod Carcinoma (Hela). The cell lines were obtained from ATCC via the Holding company for biological products and vaccines (VACSERA), Cairo, Egypt. 5-Fluorouracil (5-FU) was used as the standard anticancer drug for comparison. The data of cytotoxic activity are reported in Table 1.
The results are expressed as IC 50 (inhibitory concentration 50%), the concentration of compounds which inhibits the tumor cell growth by 50%. The obtained results revealed that eight of the tested compounds namely; 2, 3, 5, 7, 9, 10, 26 and 27 exhibited variable degrees of inhibitory activity towards the four tested human tumor cell lines. The compounds 2, 3 and 26 showed the highest cytotoxic activity against all four cell lines. In addition, compounds 5, 9, 10 and 27 exhibited a strong cytotoxic activity against the four cell lines, whereas, compound 7 had a moderate activity against HePG-2 cell line and a strong cytotoxic activity against remaining three cell lines. Finally, the rest of compounds showed the lowest activity against all four cell lines. On the other hand, compound 2 showed activity better than that of the standard drug against HePG-2 (IC 50 = 6.9 ± 0.69), and the most potent activity against Hela (IC 50 = 8.7 ± 0.73), whereas compound 3 had the most potent activity against HCT-116 and MCF-7 (IC 50 = 5.6 ± 0.47) and (IC 50 = 5.6 ± 0.59), respectively.
Regarding the structure-activity relationship we can reveal that compounds, such as 2, containing a pyridine ring besides a thienopyrimidine ring and as the number of heterocyclic rings increases, especially when a six membered ring which contains one heteroatom is present, possess a high potency and their activity increases in comparison with the other compounds. In addition, compound 3 showed a more potent activity due to the presence of a sulfonamide moiety that was shown to be able to selectively concentrate in tumor tissues in addition to playing a unique role in carbonic anhydrase inhibition. 33,34 Also, compound 26 has a strong activity due to the presence of phenylhydrazinyl moiety and compound 27 has a very good activity due to the hydrazenyl group. Moreover, compounds 5, 9 and 10 showed very good activities due to the presence of sulfathiazole, thiophene and hydroxyquinoline rings, respectively; as a heterocyclic ring increases the activity otherwise, not very strong like compound 2 due to steric hindrance. Finally, compound 7 has a moderate activity due to the introduction of dihydroimidazol-4-one ring beside the thienopyrimidine reducing the activity towards the four cell lines.

Conclusion
The present study describes the synthesis of a series of novel tricyclic compounds bearing thienopyrimidine scaffolds having been the focus of great interest because of their promising pharmacological activities. This work has advantages of cheap starting materials, excellent yields, mild reaction conditions and simple experimental procedures. The compounds 2, 3 and 26 showed the most potent antitumor activity against HepG2, HCT-116, MCF-7 and Hela cell lines.