Synthesis of Tetrahydro-benzothieno [ 2 , 3-d ] pyrimidine and Tetrahydrobenzothieno [ 3 , 2e ]-[ 1 , 2 , 4 ] triazolo [ 4 , 3c ] pyrimidine Derivatives as Potential Antimicrobial Agents

Five series of tetrahydrobenzothieno[2,3-d]pyrimidine and tetrahydrobenzothienotriazolopyrimidine derivatives have been synthesized namely: 4-(substituted amino)-5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidines 4a–d, 4-substituted (methylidenehydrazino)-5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidines 6a–c, 4-(3,5-disubstituted pyrazol-1-yl)-5,6,7,8-tetrahydro[1]benzothieno[2,3-d]pyrimidines 7a,b, 3-substituted-8,9,10,11-tetrahydro[1]benzothieno[3,2-e][1,2,4]triazolo[4,3-c]pyrimidines 8a,b, N-(phenyl or 4-substituted phenyl)-2-(8,9,10,11-tetrahydro[1]benzothieno[3,2-e][1,2,4]triazolo[4,3-c]pyrimidin-3-ylsulfanyl)acetamides 10a–c. Preliminary antimicrobial testing revealed that compounds 4a and 10b were the most active among the tested compounds against C. albicans showing IZ = 22 mm and MIC = MBC = 31.25 μg/ml, with no significant antibacterial activity. Compounds 6b and 6c showed the highest antibacterial activity against S. aureus (IZ = 21 mm, MIC = 62.5 μg/ml, MBC = 125 μg/ml for 6b; IZ = 21 mm, MIC = MBC = 125 μg/ml for 6c). Compounds 4c and 6c showed the highest antibacterial potency against P. aeruginosa among the tested compounds (IZ = 19–20 mm, MIC = MBC = 62.5 μg/ml). None of the tested compounds showed significant antibacterial activity against E. coli. 756 R. Soliman et al.: Sci Pharm. 2009; 77; 755–773.


Introduction
Thienopyrimidines are potential bioactive molecules as they are structural analogs of biogenic purines and can be considered as potential nucleic acid antimetabolites.They are characterized by a broad spectrum of biological activities.Thienopyrimidine derivative Ia showed significant antibacterial and antimycobacterial activity [1,2] (Fig. 1) while compound Ib was proved to possess anti-inflammatory activity [3].Moreover, compound II displayed IC 50 of 0.019 and 0.83 µg/ml against cyclin dependant kinases Cdk4 and Cdk2, respectively.Thus it might be useful for treatment of hyperproliferative diseases such as cancer [4].
From the standpoint of biological activity, fused heteroaromatic systems are often of much greater interest than the constituent monocyclic compounds.The appearance of qualitatively new properties of an annelated molecule, enlargement of the possibility of varying pharmacophore groups in different positions of the molecule and the ability of the latter to interact with a wider spectrum of receptors adopting various conformations are apparently of crucial importance [5].

Sch. 2.
The peaks due to quaternary carbon atoms of the structure disappeared on DEPT experimentation.The IR spectrum of 5 showed three NH absorption bands at 3306, 3234 and 3156 cm -1 .Heating the hydrazine derivative 5 under reflux with the appropriate aldehydes e.g.1,3-diphenylpyrazole-5-carbaldehyde, thiophene-3-carbaldehyde or pyridine-4-carbaldehyde in absolute ethanol afforded 4-substituted (methylidenehydrazino)-5,6,7,8-tetrahydro [1]benzothieno [2,3-d]pyrimidine derivatives 6a-c in good yields (Scheme 2).IR spectra of compounds 6a-c showed absorption bands for NH group at 3288-3108 cm -1 , beside the absorption bands due to C=N, C=C and C−S−C functions. 1H NMR spectra of 6a, 6b and 6c showed a singlet at 9.08, 8.51 and 7.55 ppm, respectively due to N=CH.The spectra of 6a, 6b and 6c also showed a deuteriumexchangeable signal for the NH proton at 11.68, 10.48 and 8.10 ppm, respectively in addition to the other signals at their expected chemical shifts. 13 10a-c.The IR spectra of compounds 10a-c showed absorption bands due to NH function at 3383-3114 and C=O moiety at 1676-1685 cm -1 .The 1 H NMR spectra of compounds 10a, 10b and 10c showed a singlet at 4.04, 4.03 and 4.13 ppm, respectively for SCH 2 protons.The spectra of 10a, 10b and 10c also showed a deuterium-exchangeable singlet at 10.19, 10.11 and 10.18 ppm, respectively for NH protons.In addition to the other signals at their expected chemical shifts. 13C NMR spectrum of 10b showed in addition to the signals due to the tetrahydrobenzothienotriazolopyrimidine carbons at their expected chemical shifts, two shielded signals due to CH 3 and SCH 2 at 20.98 and 39.20 ppm, respectively and two signals at 119.66 and 129.67 ppm due to four methine carbons of the 4-tolyl moiety indicating the equivalency between the two ortho and two meta carbons.The spectrum also showed two signals corresponding to the quaternary C 4 and C 1 of the phenyl group at 133.12 and 136.55 ppm respectively while the most deshielded signal at 166.10 ppm was characterized for C=O.The peaks due to quaternary carbon atoms of the structure disappeared on DEPT experimentation.

Tab. 1.
The inhibition zones (IZ) in mm diameter.Most of the prepared compounds were in vitro evaluated for their antimicrobial activity using the cup diffusion technique [35] against Staphylococcus aureus as Gram-positive bacteria, Escherichia coli and Pseudomonas aeruginosa as Gram-negative bacteria in addition to Candida albicans as fungi.The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for the active compounds, having inhibition zones (IZ) ≥ 18mm, were studied and compared with Ampicillin and Clotrimazole as reference antibiotics.The compounds displayed variable activities against the four tested microorganisms (Table 1,   Compounds 2 [29,30], 3 [31], and 9 [34] were prepared according to the published procedures.Compound 8a has been previously synthesized in a two step procedure [32,33] through condensation of the 4-hydrazino derivative 5 [32] with 3-chlorobenzaldehyde and further ring closure of the produced Schiff 's base using bromine in acetic acid at 45°C.

Inhibition zone measurement
Using the cup diffusion technique [35], the products as 1 mg/ml solution in DMF were in vitro evaluated for antibacterial activity against Staphylococcus aureus (ATCC 6538), Escherichia coli (ATCC 8735) and Pseudomonas aeruginosa (ATCC 9027) and for antifungal activity against Candida albicans (ATCC 10231).
The activities were estimated as zones of inhibition in mm diameter (Table 1).A 5 μg/ml solution of Ampicillin and a solution containing 0.01% of Clotrimazole in DMF were used as reference standards.Each cup (8 mm in diameter) received 0.1 ml of the test compound or reference standard solution.DMF did not show any inhibition zones.

Minimal inhibitory concentration (MIC) measurement
The bacteriostatic activity of the active compounds (having inhibition zones (IZ) ≥ 18 mm) was then evaluated using the two fold serial dilution technique [36].Two fold serial dilutions of the test compounds and reference drugs solutions were prepared using the proper nutrient broth.The final concentration of the solutions varied between 500 and 7.81 μg/ml with the concentration of DMF not exceeding 2.5%.The tubes were then inoculated with the test organisms, grown in their suitable broth at 37 °C for 24 hours for bacteria and 48 hours for fungi (about 1×10 6 cells/ml), each 5 ml received 0.1 ml of the above inoculum and were incubated at 37 °C for 48 hours.The lowest concentration showing no growth was taken as the minimum inhibitory concentration (MIC) (Table 2).

Minimal bacteriostatic concentration (MBC) measurement
To determine the minimum bactericidal concentration (MBC), a loopful from the tube not showing visible growth in the MIC experiment was spread over a quarter of Muller-Hinton agar plate.After incubation for 18 hours, the plates were examined for growth.The tube containing the lowest concentration of the test compound that prevented growth on subculture plates were judged to contain the MBC of that compound for the respective test organism (Table 2).

Conclusions
The overall results indicated that, most of the tested compounds (4a, 4b, 6b, 6c, 8b, 10b and 10c) showed antifungal activity against C. albicans, few compounds exhibited antibacterial activity against S. aureus (4b, 6b and 6c) and P. aeruginosa (4c and 6c) while none of the tested compounds showed significant antibacterial activity against E. coli.
13 C NMR spectrum of 4c showed the signals corresponding to the tetrahydrobenzothienopyrimidine carbons present almost at the same chemical shift as in compound 4a.In addition, the spectrum showed three highly shielded signals due to the equivalent piperazine C 2 & C 6 and piperazine C 3 & C 5 at 50.12 and 52.60 ppm, respectively and the benzyl-CH 2 at 62.98 ppm.It also showed three signals due to the five methine carbons in the aromatic region indicating the equivalency between the two ortho and the two meta carbons of the phenyl moiety and a deshielded signal for the phenyl C 1 quaternary carbon.

Tab. 2 .
2).The data indicate that, compounds 4a and 10b were the most active among the tested compounds against C. albicans showing IZ = 22 mm and MIC = MBC = 31.25 μg/ml, or about one-sixth the activity of Clotrimazole with no significant antibacterial activity.In addition, compounds 4b, 6b, 6c, 8b and 10c showed MIC = MBC = 31.25 μg/ml against C. albicans but their IZ = 20 mm.Replacement of the morpholino moiety at 4-position of the tetrahydrobenzothienopyrimidine nucleus in 4a by a piperidine moiety gave compound 4b that showed nearly the same activity of 4a against C. albicans (IZ = 20 mm, MIC = MBC = 31.25 μg/ ml).In addition, compound 4b showed activity against S. aureus (IZ = 18 mm, MIC = MBC = 62.5 μg/ml).On the other hand, replacement of the morpholino moiety in 4a by 4-benzylpiperazine in 4c led to increase the antibacterial potency of 4c against P. aeruginosa showing IZ = 19 mm, MIC = MBC = 62.5 μg/ml and reduced the antifungal activity.MIC and MBC in μg/ml of the most active compounds.methylidenehydrazino linkage at the 4-position of the tetrahydrobenzothienopyrimidine nucleus as in 6b enhanced the antibacterial activity against S. aureus (IZ = 21 mm, MIC = 62.5 μg/ml and MBC = 125 μg/ml) and retained the same antifungal potency as compound 4b (IZ = 20 mm, MIC = MBC = 31.25 μg/ml).Replacement of the pyridinyl function present in 6b by a thiophene moiety led to compound 6c which was equipotent with 6b against C. albicans (IZ = 20 mm, MIC = MBC = 31.25 μg/ml), while the potency of 6c against S. aureus as compaired with 6b was diminished (IZ = 21 mm, MIC = MBC = 125 μg/ml).However, 4c and 6c showed the highest antibacterial potency against P. aeruginosa among the tested compounds with IZ = 19 and 20 mm, respectively and MIC = MBC = 62.5 μg/ml or nearly one-third the activity of Ampicillin.
C NMR spectrum of 6b showed three signals for five methine carbons at 124.54, 135.42 and 148.84 ppm due to pyridine C 3,5 , N=CH and pyridine C 2,6 , respectively and one signal at 145.03 ppm corresponding to the pyridine C 4 quaternary carbon.In addition, the spectrum showed the signals due to tetrahydrobenzothienopyrimidine carbons at their expected chemical shifts.in the present study, was achieved via one pot reaction of the 4-hydrazino derivative 5 with the appropriate carboxylic acid in presence of phosphorous oxychloride.The IR spectra of compounds 8a,b lacked the absorption bands due to NH functions present in the precursor.The 1 H NMR spectra of compound 8a,b showed signals due to aromatic protons in addition to the other signals for the tetrahydrobenzothienotriazolopyrimidine-C 5 , 8-11 -protons at their expected chemical shifts. 13C NMR spectrum of 8a showed four signals for the highly shielded methylene carbons C 10 , C 9 , C 11 and C 8 at 22.30, 23.10, 25.41 and 25.67 ppm, respectively.The five methine carbons of the 3-chlorophenyl C 6 , C 2 , C 4 , C 5 and the tetrahydrobenzothienotriazolopyrimidine C [8,12]aks due to quaternary carbon atoms of the structure disappeared on DEPT experimentation.Condensation of 5 with 1,3-dicarbonyl compounds such as benzoylacetone and ethyl benzoylpyruvate in accordance with the reported procedures[8,12]afforded 4-(3,5-disubstituted pyrazol-1-yl)-5,6,7,8-tetrahydro[1]benzothieno[2,3d]pyrimidine derivatives 7a,b.The IR spectra of compounds 7a,b lacked the stretching NH bands present in the precursor.The 1 H NMR spectra of 7a and 7b showed a singlet at 6.40 and 7.35 ppm, respectively due to pyrazolyl-C 4 -H in addition to the other signals due to aromatic protons and tetrahydrobenzothienopyrimidine nucleus at their expected chemical shifts.The 13 C NMR spectrum of 7b showed two high field signals for the methyl and methylene carbons of the O-ethyl group at 14.35 and 44.17 ppm, respectively.It also showed six signals due to the methine carbons of the pyrazolyl C 4 and phenyl C 4 , C 2 , C 5 , C 3 and C 6 at 98.35, 128.29, 128.64, 128.77, 128.88 and 133.81 ppm, respectively and the signals characteristic for the four deshielded quaternary carbons, phenyl C 1 , pyrazolyl C 5 , pyrazolyl C 3 and C=O at 136.07, 142.42, 154.56 and 196.52 ppm, respectively.In addition, the spectrum showed the signals due to tetrahydrobenzothienopyrimidine carbons at their expected chemical shifts.The peaks due to quaternary carbon atoms of the structure disappeared on DEPT experimentation.The preparation of 3-substituted-8,9,10,11tetrahydro-[1]benzothieno[3,2-e][1,2,4]triazolo[4,3-c]pyrimidine derivatives 8a,b, 5 resonated as five signals at 125.78, 127.85, 130.14, 130.62 and 135.39 ppm, respectively.In addition to eight signals due to the eight quaternary carbons C 11b , C 11a , phenyl C 1 , phenyl C 3 , C 7a , C 6a , C 3 and C 11c at 120.56, 129.40, 132.16, 134.87, 139.29, 149.70, 153.65 and 163.91 ppm, respectively.Sci Pharm.2009; 77; 755-773.
Faculty of Science, Cairo University.Microanalysis were performed at the Microanalytical Unit, Faculty of Science, Cairo University and The Microanalytical Unit, Faculty of Science, Assuit University, A. R. Egypt.The results of the microanalysis were within ±0.4% of the calculated values.Follow up the reactions and checking the homogeneity of the compounds were made by ascending TLC run on silica gel G (Merck 60) coated glass plates.The spots were visualized, by exposure to iodine vapour or UV-Lamp at λ 254 nm for few seconds.