Synthesis and antimicrobial activity of some new thienopyrimidine derivatives

Due to the biological activities of pyrimidine and thienopyrimidine as antimicrobial agents, so in the present work, a series of new heterocyclic compounds containing thienopyrimidine moiety were synthesized such as, tetrazolo[1",5":1',6']pyrimido[4',5':4,5]thieno[2,3-d]pyrimidine derivatives (8-12b), pyrimido[3",2":1',6']pyrimido[4',5':4,5]thieno[2,3-d]pyrimidine derivatives (14,16) and pyrimido[5',4':4,5]thieno[2,3-e]pyrimido[1,2-c] [1,2,3]triazine derivative (15) by using 5-amino-4-phenyl-2-(p-tolylamino)thieno[2,3-d]pyrimidine-6carbonitrile (7) as starting material. All synthesized compounds were evaluated for their antimicrobial activity. And Compounds (8-13) exhibited high antibacterial activity. Also, compounds (12b-18) exhibited high antifungal activity.

ARKAT USA, Inc  (8).Aminotetrazolothienopyrimidine 8 was considered as precursor to the synthesis of other heterocyclic compounds, Thus, compound 8 was reacted with triethyl orthoformate in the presence of glacial acetic acid to afford the corresponding 9-p-tolylamino-7phenyltetrazolo[1",5":1',6']pyrimido [4',5':4,5]thieno [2,3-d]pyrimidine (9).Compound 8 reacted with benzaldehyde in refluxing ethanol in the presence of a few drops of piperidine to afford 5,7-diphenyl-9-(p-tolylamino)-5,6-dihydrotetrazolo[1",5":1',6']pyrimido [4',5':4,5]thieno [2,3-d]pyrimidine 10.Also, when 8 was allowed to react with carbon disulfide in the presence of pyridine on a steam bath for 3h. it gave the corresponding tetrazolopyrimidothienopyrimidinethione derivative 11.Reaction of compound 11 with alkylating agents such as ethyl chloroacetate and p-methoxychloroacetanilid in refluxing ethanol in the presence of fused sodium acetate yielded the S-alkylated tetrazolopyrimidothienopyrimidine derivatives 12a,b.The structures of the produced alkylthiotetrazolopyrimidothienopyrimidine derivatives were established by IR and 1 H NMR spectra.The IR spectrum of compound 12a showed a peak at 1736 cm -1 for C=O ester.The 1 H NMR spectrum showed  (15).Also, compound 13 condensed with benzaldehyde in the presence of few drops of piperidine as a basic catalyst yielding compound 16.Chloroacetylation of compound 13 with chloroacetyl chloride in dioxane on steam bath for 3h., gave the chloroacetylamino compound 17 which reacted with aniline to afford the corresponding phenylaminoacetamide derivative 18.The IR spectrum of compound 17 revealed the disappearance of bands at 3468, 3363 cm -1 characteristic for the NH2 group and the appearance of an absorption band at 3291 cm -1 for the NH and another at 1686 cm -1 for the CO of the amide. 1 H NMR spectrum of compound 17 revealed the disappearance of a signal characteristic for NH2 groups and the appearance of signals at δ: 4.02 characteristic for the CH2 and at 11.60 for the NH.Also compound 17 was confirmed by the mass spectrum which showed a molecular ion peak at m/z 491.21 (M + , 66 %). in agreement with the proposed structure (Scheme 3).Antimicrobial activities (i) Antibacterial evaluation.Using the agar well-diffusion method 32 , all of the synthesized compounds in this paper were screened in vitro for their antimicrobial activity against two pathogenic gram positive bacteria.Staphylococcus aureus, Bacillus cereus and two gram negative strains, Escherichia coli and Pseudomonas aeruginosa.The inhibition zone (mm) was compared with a series of antibiotics according to the sensitivity of each bacteria type to the most effective antibiotic for it.(Table S1, fig.2).The minimum inhibition concentrations (MICs) were recorded.All compounds exhibit significant antibacterial activities.Compounds 8,  9, 10, 11 , 12b, 13, 14, 15, 16, 17 and 18 revealed the existence of a remarkable activity against bacteria.Compound 13 showed the highest antibacterial activity against all strains of bacteria, with values almost similar to the corresponding reference antibiotics (Ofloxacin, Levofoxacin, Clindamycin and Nitrofuration, respectively).This highest activity due to the formation of 3,4,5,6-tetrahydropyrimidinyl ring.However, the presence of sulfanyl acetate group in ethyl 9-p-tolylamino-7-phenyltetrazolo[1",5":1',6']pyrimido [4',5':4,5]  thieno [2,3-d] pyrimidine-5-ylsulfanyl] acetate 12a decrease the antibacterial activity compared with other tested compounds especially compound 12b which has higher antibacterial activity than compound 12a due to the introduce the aromatic ring into the structure by sulfanylmethoxyacetanilide group.
(ii) Antifungal evaluation.The tested compounds were also screened for their antifungal activities against four antifungal species, Candida albicans, Geotrichum candidum, Aspergillus flavus and Trichophyton rubrum.As shown in (Table S2, fig. 3) most of the tested compounds are active.Compounds 13, 14, 15, 16, 17 and 18 showed high antifungal activity against Trichophyton rubrum, Aspergillus flavus and Geotrichum candidum, while compounds 12b, 13, 15, 18 showed the highest antifungal activity against Geotrichum candidum.Compounds 13, 17 showed the highest antifungal activity against Candida albicans.However, compounds 7, 8, 9, and 12b showed moderate fungal activity, while compound 12a showed low activity against all strain of fungi.From this results we showed that compounds 13, 15 and 18 showed highest antifungal activity against most fungi species that due to the presence of 3,4,5,6-tetrahydroprimidinyl ring.And when the triazino ring formed in compound 15 this increase the activity of compound.On the hand , the introduce the phenylaminoacetamide group to compound 13 the antifungal activity increasing.

Figure 1 .
Figure 1.Structures of the three fundamental thienopyrimidines
Numbers out parentheses represent the diamer of inhibition zone in (mm) of compounds 8-18; (b) Numbers in parentheses represent the MIC (minimum inhibition concentration) in (µg mL -1 ) of tested compounds; (c) (-), no activity.

Figure 2 .
Figure 2. Comparison of (inhibition zone, mm) for anti-bacterial activity of the synthesized compounds 8-18 with standard Microorganisms.

Figure 3 .
Figure 3.Comparison of (inhibition zone, mm) for anti-fungal activity of the synthesized compounds 8-18 with standard Microorganisms.