Chapter I. Synthesis, modification and bioactivity of bicyclic thieno[3,2-D]pyrimidines

1.1.7 Early Synthesis of Pyrimidine-2,4-diol 9

Issue with this approach was the synthesis and hydrolysis of 8; preparation of this intermediate required two equivalents of the relatively expensive ethylisothiourea hydrobromide 13 as a raw material and the yield was a modest 60−70%. In addition, there was significant decomposition of 5 under the basic reaction conditions that resulted in the formation of foul-smelling byproducts. Furthermore, the hydrolysis of 8 for the subsequent synthesis of 9 resulted in the release of ethanethiol that also caused a strong unpleasant stench which proved difficult to contain and neutralize. To overcome the issues stated above, a new streamlined and cost-effective process for the synthesis of 9, in which inexpensive urea was used in place of the much more costly ethyl-isothiourea hydrobromide (13) was developed. 12 This new process completely eliminated the strong stench associated with both the synthesis of 8 and the liberation of ethanethiol upon its hydrolysis. In addition, the overall cost of the drug substance was significantly reduced and the new process provided a stable, easy-to-purify, solid intermediate, eliminating the complications associated with the transport or storage of crude and noncrystalline [24-26].
1.1.8 General produce for synthesis reactions to cyclisations
General procedure for synthesis of 2a-2b To a mixture of 1a-1b (0.12 mol) in acetic acid (150 mL), was slowly added potassium cyanate in water (75 mL) at 0 C and stirred for 2 h. After completed, the reaction solution was diluted with water (100 mL) and filtered to obtain white cake. The cake was dried in vacuo and then dissolved in 6% NaOH solution (250 mL), stirred at 130 C for 4 h. After completed, the mixture was cooled to room temperature and the pH value was buffered close to 6 with HCl. The solid separated out was filtered to obtain 2a-2b

General procedure for synthesis of 3a-3b To a mixture of 2a-2b (0.06 mol) in POCl3 (100 mL), was added a drop of DMF, and stirred at 110 C for 2 h. After completed, the mixture was slowly added into ice water and the solid precipitated was filtered and dried in vacuo to obtain 3a-3b[27-29].

General procedure for synthesis of 4a-4b To a mixture of 3a-3b (0.036 mol) in methyl alcohol (80 mL), was added morpholine (0.091 mol) at 0 C. The mixture was stirred at 25 C for 2 h and much white solid was precipitated. After completed, the reaction liquid was filtered and the cake was dried in vacuo to obtain 4a-4b[30, 31].

General procedure for synthesis of 5a-5b To a mixture of 4a-4b (0.037 mol) in dry THF (120 mL), was added i-PrMgCl (2 mol/L, 9.25 mL, 0.018 mol) under N2 atmosphere at 70 C. After stirred for 30 min, n-BuLi (2.5 mol/L, 17.76 mL, 0.045 mol) was added slowly and the color changed from white to light red. After 2.5 h, the reaction was poured into a mixture of acetic acid (25 g), HCl (10 g), isopropanol (100 mL) and water (100 mL). The mixture was stirred at 50 C for another 1.5 h, and THF was removed in vacuo, the residue was filtered and dried to obtain 5a-5b[32, 33].

The general synthetic routes of the target compounds are illustrated in Scheme . Commercially available methyl 3-aminothiophene-2- carboxylate (a) as the starting material reacted with molten urea to provide intermediate b, which was chlorinated using phosphorus oxychloride to afford intermediate c.

Commercially available methyl 3-aminothiophene-2-carboxylate (a) as the starting material reacted with molten urea to provide intermediateb, which was chlorinated using phosphorus oxychloride to afford intermediate c The synthesis of the title compound by five steps was described in this reaction sistem. The commercially available methyl 3-amino-2-thiophenecarboxylate was condensed with urea at 190 C for 2.0 h to give thieno[3,2-d]pyrimidine-2,4-diol. Chlorination of two with phosphorus oxychloride proceeded smoothly to 2,4-dichlorothieno[3,2-d]pyrimidine as a pale solid. 4-chloro of 2,4-dichlorothieno[3,2-d]pyrimidine was substituted by 1- methylindole to give 2-chloro-4-(1-methyl-1H-indol-3-yl)thieno[3,2-d]pyrimidine, which was then condensed with hydrazine hydrate to the key intermediate 6. Finally, intermediate six condensed with 2-fluorobenzaldehyde in EtOH at refluxing condition for 8 h to yield the title compound and its structure was established by IR, 1 H NMR, MS and elemental analyses. All data of the title compound confirmed its structural integrity. To develop novel therapeutic agents with anticancer activities, two series of novel 2,4-bismorpholinyl-thieno[3,2-d]pyrimidine and 2-morpholinothieno[3,2-d]pyrimidinone derivatives were designed, synthesized and evaluated for their biological activities. Among them, compound A12 showed the most potent antitumor activities against HCT116, PC-3, MCF-7, A549 and MDA-MB-231 cell lines with IC50 values of 3.24 μM, 14.37 μM, 7.39 μM, 7.10 μM, and 16.85 μM, respectively. Further explorations in bioactivity were conducted to clarify the anticancer mechanism of compound A12. The results showed that compound A12 obviously inhibited the proliferation of A549 cell lines and decreased mitochondrial membrane potential, which led to the apoptosis of cancer cells and suppressed the migration of tumor cells[34-37].
Histone acetylation and deacetylation, catalyzed by multisubunit complexes, play a key role in the regulation of eukaryotic gene expression. The acetylation status of histones is determined by two sets of enzymes: histone acetyltransferases (HATs) and histone deacetylases (HDACs). HDACs also interact with retinoblastoma tumor-suppressor protein and this complex is a key element in the control of cell proliferation and differentiation. Together with metastasis-associated protein-2 MTA2, HDACs deacetylates p53 and modulates its effect on cell growth and apoptosis. HDAC inhibitors demonstrated prominent antitumor efficacy on broad spectrum neoplasms in preclinical and clinical studies[38].

Reagents and conditions: a) microwave, 4-5 mins; b) H2SO4/HNO3, 0 oC-r.t;
Thienopyrimidine derivatives have attracted considerable interest in pharmaceutical discovery in cancer and antivirus research. 1-9 Therefore, the development of new efficient and mild syntheses of the thienopyrimidine framework is a useful task, particularly when one-step procedures from readily-available reagents can be employed[39].
The Gewald thiophenes 12 are used as starting materials in many routes to thieno[2,3-d]pyrimidines. We have recently reported that alkyl 2-(1H-tetrazol-1- yl)-4-R1 -5-R2 -thiophene-3-carboxylates, obtained from alkyl 2-amino-thiophene-3- carboxylates by the reaction with triethyl orthoformate and sodium azide under conditions of hydrazinolyzation of the ester group, underwent recyclization including cleavage of the tetrazole ring, elimination of the nitrogen molecule and annulation of the pyrimidinone core. This simple and convenient synthetic path opened access to 2,3-diaminothieno[2,3-d]pyrimidin-4(3H)-ones and allowed us to presume that the tetrazole ring cleavage and loss of nitrogen could proceed under mild conditions (hydrazine solution). The ready access to the cyanamide moiety makes alkyl 2-(1H-tetrazol-1-yl)-4-R1 -5-R2 -thiophene-3-carboxylates the potentially useful precursors to a range of thieno[2,3-d]pyrimidin-4(3H)-ones, a transformation elaborated here[40].