Synthesis, Characterization and Biological Evaluation of some New Thieno[2,3-d]Pyrimidine Derivatives

10-Oxo-4,6,7,8,9,10-hexahydroprazolo[1,5-a][1]benzothieno[2,3-d]pyrimidine-3-carbaldehyde (2) was prepared by Vilsmeier-Haack reaction of 3-amino-2-methyl-5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidin-4(3H)-one (1). Reaction of carbaldehyde derivative 2 with malononitrile afforded arylidene malononitrile 3. Cyclization of the latter compound with thiourea yielded pyrimidinethione 4. Interaction of carbaldehyde derivative 2 in presence of thiourea with ketocompounds such as ethyl acetoacetate, or acetylacetone, or dimedone or ethyl cyanoacetate gave pyrimidine derivatives 5-8. Hydrazinolysis of carbaldehyde derivative 2 gave the hydrazone 9. Reaction of the latter with phenyl isothiocyanate afforded thiosemicarbazone 10, which underwent cyclization with oxalyl chloride to give thioxoimidazolidinedione 11. Condensation of compound 2 with thiosemicarbazide furnished thiosemicarbazone derivative 12. Reaction of compound 2 with aminopyrazolone in the presence of an acid and/or a base afforded pyrazolones 13 and 14. Treatment of carbaldehyde derivative 2 with cyanoacetohydrazide gave acrylohydrazide 15. Interaction of the latter with carbon disulfide yielded mercaptooxadiazole 16. Condensation of compound 2 with acetylpyridazinone 17 produced chalcone 18. Reaction of compound 18 with malononitrile in pyridine gave cyanopyran 19, while its reaction with malononitrile in presence of ammonium acetate in ethanol yielded cyanopyridine 20. Structures of the newly synthesized products have been deduced on the basis of elemental analysis and spectral data. The synthesized compounds were screened for their antimicrobial activity.


INTRODUCTION
A literature survey has revealed the diversified biological and pharmacological significance of several nitrogen and sulphur heterocycles. This aspect has been drawing the attention of many researchers towards exploiting the biological importance of various heterocyclic compounds and to establish the relationship between their biological, pharmacological potency and structural features [1]. Heterocycles containing the thienopyrimidine moiety are of interest because of their interesting pharmacological and biological activities [2][3][4][5][6][7]. Thus, over the last two decades many thienopyrimidines have been found to exhibit a variety of pronounced activities, for example, as anti-inflammatory [4,8], antimicrobial [4,9], antiviral [10], and analgesic agents [8,11]. Some thienopyrimidine derivatives showed good antitumor activity [12][13][14]. Consequently, thienopyrimidines have become a well-sought privileged class of compounds in drug discovery programs. In view of these reports and in continuation of our work on biologically active nitrogen and sulfur heterocycles [15][16][17][18], we have synthesized some novel thienopyrimidines and evaluated them for their antimicrobial properties.
The structures of compounds 3 and 4 were characterized from their spectroscopic as well as elemental analytical data. Thus, the IR spectrum of compound 3 revealed absorption bands at 1685, 2210 and 3447 cm −1 corresponding to C=O, C≡N and NH functions, respectively. Its 1 H NMR spectrum showed the presence of the cyclohexane ring methylene protons at δ 1.72-1.74, 2.70 and 2.83 and a D2O-exchangeable signal at δ 9.71 due to NH proton. The IR spectrum of compound 4 showed absorption bands at 1198, 1653, 2209 and 3420-3200 cm −1 corresponding to C=S, C=O, C≡N, NH and NH2 functions, respectively. Its 1 H NMR spectrum revealed a D2O-exchangeable signal at δ 8.79 ppm corresponding to three NH protons in addition to a D2O-exchangeable signal at δ 3.57 due to NH2 protons.
The structures of compounds 5-8 were confirmed on the basis of spectroscopic data and elemental analyses. The 1 H NMR spectrum of compound 5 showed a triplet signal at δ 1.27 (J = 6.9 Hz) corresponding to CH3 protons, a quartet signal at δ 4.20 (J = 6.9 Hz) due to CH2 protons, a doublet signal at δ 5.30 corresponding to CH-4 proton, and two D2O-exchangeable signals at δ 6.90 and 7.07 due two NH protons. The mass spectrum of compound 7 revealed the molecular ion peak at m/z 453 corresponding to the molecular formula C22H23N5O2S2, which agree well with the molecular weight (453.58) and supports the identity of the structure. The IR spectrum of compound 8 showed absorption bands at 1701, 1678, 2220, 2380 and 3424 cm -1 corresponding to two C=O groups, C≡N, SH and two NH functions, respectively. The 1 H NMR spectrum of the same compound revealed a D2O-exchangeable signal at δ 1.23 due to SH proton and two D2O-exchangeable signals at δ 8.39 and 8.58 due to two NH protons. O c t o b e r 2 9 , 2 0 1 3 Treatment of 2 with hydrazine hydrate in presence of triethylamine as a catalyst in DMF gave the corresponding hydrazone 9, which was allowed to react with phenyl isothiocyanate in DMF containing few drops of piperidine to give the thiosemicarbazone derivative 10. Heterocyclization of compound 10 with oxalyl chloride in boiling DMF containing few drops of triethylamine produced the novel thioxoimidazolidinedione derivative 11 (Scheme 3). Also, condensation of 2 with thiosemicarbazide in refluxing glacial acetic acid afforded the corresponding thiosemicarbazone 12 (Scheme 3). The structures of the products were determined from spectroscopic as well as elemental analytical data. Thus, IR spectrum of compound 9 exhibited characteristic absorption bands at 3340-3197 (NH2, NH), 1670 (C=Opyrimidinone) and 1607 cm -1 (C=N). Also, its 1 H NMR spectrum showed D2O-exchangeable signals at δ 3.36 and 11.51 ppm assigned to the NH2 and NH protons, respectively. The 1 H NMR spectra of compound 10 showed characteristic D2O-exchangeable signals at δ 8.66, 8.88 and 12.12 ppm assigned to three N-hydrogens. The IR spectrum of compound 12 showed absorption bands at 3445-3300 cm -1 due to NH2 and NH functions. Its 1 H NMR spectrum showed two D2O-exchangeable signals at δ 11.22, 8.36 ppm corresponding to two NH protons, D2O-exchangeable signal at δ 3.47 ppm due to NH2 protons and a singlet signal at δ 9. . The structures of the products were characterized from their spectroscopic as well as elemental analytical data. Thus, the IR spectrum of compound 13 revealed absorption bands at 1660 and 3373 cm -1 corresponding to two C=O groups and two NH functions, respectively. Its 1 H NMR spectrum showed two D2Oexchangeable signals at δ 8.14 and 9.52 ppm corresponding to two NH protons, in addition to a singlet signal at δ 4.83 ppm assigned to the methylene protons. The 1 H NMR spectrum of compound 14 showed an exchangeable signal at δ 5.22 ppm attributed to the NH2 protons, while the 1 H NMR spectrum of compound 15 showed characteristic D2Oexchangeable signals at δ 9.97 and 11.24 ppm assigned to two N-hydrogens and D2O-exchangeable signal at δ 5.63 ppm due to NH2 protons, in addition to singlet signal at δ 7.21 ppm assigned to the methine proton. The synthesis of cyanopyran 19 and cyanopyridine 20 from chalcone 18 was performed as shown in Scheme 5. In the initial step, chalcone 18 was synthesized by condensing formyl derivative 2 with 4-acetyl-5,6-diphenylpyridazin-3(2H)-one (17) [20], in presence of a catalytic amount of a base. Finally, cyanopyran 19 was synthesized by reacting chalcone 18 with malononitrile in pyridine, while cyanopyridine 20 was synthesized by reacting chalcone 18 with malononitrile in presence of ammonium acetate in ethanol (Scheme 5). The 1 H NMR spectra of compound 18 showed characteristic signals at δ 7.10 and 7.66 ppm assigned to ethylienic protons. Also, the mass spectra of compound 18 revealed the molecular ion peaks at m/z 545 which agree well with the molecular weight for compound 18. The 1 H NMR spectrum of compound 19 showed a characteristic doublet at δ 7.94 and 8.41 ppm attributed to pyran-H4 and -H5, respectively, in addition to an exchangeable signal at δ 4.45 ppm assigned to the NH2 protons, while the 1 H NMR spectrum of compound 20 showed an exchangeable signal at δ 5.40 ppm attributed to the NH2 protons.

Biological Activities
The standardized disc agar diffusion method [21] was followed to determine the activity of the synthesized compounds against the sensitive organisms Staphylococcus aureus and Bacillus subtilis as Gram-positive bacteria, Salmonella typhimurium and Escherichia coli as Gram-negative bacteria and Candida albicans as fungus strain. The compounds were dissolved in DMSO which has no inhibition activity to get concentration of 100 µg mL -1 . The test was performed on medium potato dextrose agars (PDA) which contain infusion of 200 g potatoes, 6 g dextrose and 15 g agar [22]. Uniform size filter paper disks (3 disks per compound) were impregnated by equal volume (10 µL) from the specific concentration of dissolved tested compounds and carefully placed on inoculated agar surface. After incubation for 36 h at 27 °C in the case of bacteria and for 48 h at 24 °C in the case of fungi, inhibition of the organisms was measured and used to calculate mean of inhibition zones.
The synthesized polyfused systems exhibited lower to mild antimicrobial activity. Thus, the synthesized compounds may be considered promising for the development of new antimicrobial agents as depicted in Table 1. O c t o b e r 2 9 , 2 0 1 3

EXPERIMENTAL PROCEDURE
Melting points are uncorrected and were recorded in open capillary tubes on a Stuart SMP3 melting point apparatus. IR spectra were recorded on a FT-IR Bruker Vector 22 spectrophotometer using a KBr wafer technique. The 1 HNMR spectra was recorded in DMSO-d6 on a Gemini spectrometer (300MHz) and the chemical shift in δ downfield from TMS as an internal standard. Elemental microanalyses were performed at the Main Laboratories of the War Chemical. Mass spectra were obtained using gas chromatography GCMS qp-2010 and on a Shimadzu instrument mass spectrometer (70 eV) at the Cairo University Microanalytical Center. 3-Amino-2-methyl-5,6,7,8-tetrahydro [1]benzothieno [2,3-d]pyrimidin-4(3H)-one (1) was prepared according to the reported method [19].