Synthesis, Characterization and Biological Studies of Some Novel Thieno[2,3-d]pyrimidines

Several 2-unsubstituted thieno[2,3-d]pyrimidines have been prepared from 2-aminothiophene-3-carboxylic acid esters and their carbonitrile analogs. Some triazolo-thienopyrimidine and 2-thioxothienopyrimidine representatives have also been synthesized using thermal and microwave (MW) irradiation techniques. Structures of the prepared compounds were elucidated on the basis of IR, NMR, 2D NMR and mass spectral data. The biological activity of some selected synthesized compounds was also examined.


Introduction
Heterocycles containing the thienopyrimidine moiety ( Figure 1) are of interest because of their interesting pharmacological and biological activities [1][2][3][4][5][6]. Thus, over the last two decades many thienopyrimidines have been found to exhibit a variety of pronounced activities, for example, as antiinflammatory [3,7], antimicrobial [3,8], antiviral [9] and analgesic [7,10] agents. Some thienopyrimidine derivatives showed good antitumor activity [11], while compounds with the general structure designated by E (Figure 1) showed potent and specific cytotoxicity against several leukemia cell lines [4]. Motivated by the aforementioned biological and pharmacological importance of the title OPEN ACCESS compounds, and as continuation with our previous work on thienopyrimidines [12,13], we report herein the synthesis of some new heterocycles incorporating a thienopyrimidine moiety. Representative compounds among the synthesized thienopyrimidines were tested and evaluated as antibacterial agents and for cytotoxicity against some cancer cell lines.

Chemistry
The synthetic pathways depicted in Schemes 1-4 outline the chemistry of the present study. Thus, the 2-amino-3-thiophene carboxylic ester starting materials 1a-d and 32a,b are easily prepared following the well established procedure reported in the literature [14,15]. Treatment of 1a,b with ClCO(CH 2 ) 4 Cl gave the corresponding amides 2a,b, which in turn gave compounds 3a,b upon refluxing in DMF with 1-(methoxyphenyl)piperazine in the presence of potassium carbonate (Scheme 1). The IR spectra of 2a,b and 3a,b showed two peaks in the 1,675-1,678 cm -1 and 1,658-1,662 cm -1 range corresponding to ester and amide carbonyl absorptions, respectively. The structure of the unpreviously unknown 2a,b and 3a,b were unambiguously assigned on the basis of their NMR and MS data (see Experimental). Reaction of 3a,b with hydrazine in hot methanol led to cyclization to the corresponding thienopyrimidines in moderate yields. The IR spectra of 4a,b exhibited a strong peak corresponding to the carbonyl group at 1,672-1,680 cm -1 . The 1 H-NMR spectrum of 4a in CDCl 3 showed a broad singlet at δ 4.96 (NH 2 ), a methoxyl proton singlet and four aromatic proton resonances (Table 1). This spectrum also showed the presence of the cyclohexane and piperazine ring methylene protons as well as the butyl moiety, as judged from the DEPT spectrum. The mass spectrum of 4a showed a prominent molecular ion peak [M + ] as the base peak and a fragmentation pattern consistent with its structure. The 13 C-NMR, combined with the DEPT spectrum of 4a, confirmed the existence of 25 carbons, including a benzene ring, 12 methylene carbons (10 signals, taking in consideration the equivalency of methylene protons in the piperazine ring), methoxyl and thienopyrimidine carbons. HMBC spectroscopy (Table 1) were used to elucidate the structure and to establish the complete NMR assignments of 4a. Starting from H-2` the most shielded aromatic proton, showed two-bond correlations with C-1` and C-3`, and showed three-bond correlations with C-4` and C-6`. H-5` and methoxyl protons showed three-bond correlations with C-1`. Further, H-5` and C-a` of the piperazine ring showed respectively two-bond and three-bond correlations to C-6`.  25.16 (CH 2 ) 2.71 C-6, 10 C-5, 7, 11 10 22.24 (CH 2 ) 1.83 C-9, 11 C-6, 12 11 22.95 (CH 2 ) 1.83 C-10, 12 C-7, 9 12 25.39 (CH 2  33.99(CH 2 ) 2.98(t) C-2", 2 C-3"
A two bond-correlation observed from a`-CH 2 to C-a, established the assignments of the chemical shifts of the piperazine ring methylene groups. H-1`` and the amino protons showed two-bond and three-bond correlations with C-2, respectively. The three-bond correlation from a-CH 2 to C-4`` assigned the chemical shifts of the butyl protons in compound 4a. On the other hand, the 9-CH 2 showed three-bond correlations with C-5 and C-7 and two-bond correlations with C-6 and C-10, while 12-CH 2 exhibited three-bond correlation to C-6 and two-bond correlations to C-7 and C-11 carbons. These correlations unequivocally confirmed the assignments of all carbons of the cyclohexane and thiophene rings. The signal at δ C 141.34 must correspond to C-8 since this carbon has no correlation with any protons in the HMBC spectrum of 4a.The mass spectrum of 4b exhibited a molecular ion peak at m/z 481. Its NMR spectral data are similar to the corresponding data of 4a with an additional δ H at 1.82 and δ C at 23.11 for the methylene group.
In another pathway, thienopyrimidine dione 6 and the 2-unsubstituted-thienopyrimidines 7-9 have been synthesized as depicted in Scheme 2. The structures of 6-8 were elucidated on the basis of their various spectral data (see Experimental), including their mass spectral data which were consistent with the proposed structures. Compound 8 failed to give the condensation product 9. None of the thienopyrimidines in Scheme 2 were previously prepared, with the exception of 7a,b [13]. The 1 H-NMR spectrum of 8a revealed two singlets at δ 7.62 and δ 13.14, each integrating for one proton, corresponding to the protons at position 2 and for the NH, respectively. Compounds 11a,c,d were synthesized from 10 upon treatment with sodium ethoxide. The IR spectra of 11a,c,d are characterized by two bands in the 1,664-1,681 cm -1 and 3,157-3,427 cm -1 range due to the C=O and NH stretching frequencies, respectively. These spectra also showed a band in the 1,110-1,174 cm -1 range, which corresponds to the C=S absorption in 11. 13 C-NMR spectra of the latter compounds revealed two signals at around δ 173 and 157 which were attributed to the C=S and C=O, respectively, in addition to the other carbon signals at the expected values. Two singlets at δ 4.43 and 4.24 in the 1 H-NMR spectrum of 11d, each integrating for two protons, correspond to the CH 2 of the benzyl group and the CH 2 at position 2 of the piperidine moiety in the molecule, respectively. This spectrum also displayed other two multiplet signals at δ 3.15 (CH 2 ) and δ 3.39 (CH 2 ) in addition to the signals of the protons for two NH groups at δ 11.40 (br s) and δ 12.50.
Compound 11a reacted with hydrazine to give the corresponding hydrazino derivative 14 which in turn was transformed into the pyrazolothienopyrimidines 15-17 upon treatment with triethyl orthoformate, triethyl orthoacetate and carbon disulphide. A singlet at δ 9.01 in the 1 H-NMR of 15 corresponds to the proton in the triazole ring, while a singlet at δ 2.98 in the similar spectrum of 16 corresponds to the methyl substituent in the latter heterocylic ring. Other than that, the data of both spectra were almost identical. Expected 13 C-NMR chemical shifts for both compounds were observed (see Experimental).
The condensation products 12 and 13 were also obtained upon treatment of 11a with some aromatic hydrazides, as elaborated in Scheme 2. Accordingly, cyclization of 11a to the triazolo-derivatives 12 and 13 occured through the NH at position 1, as shown by the HMBC spectrum of 13 which indicated a two bond correlation of the hydrogen of NH at position 3 to the C-4, and in fact this would be the expected case where the latter NH is an amide. A literature survey revealed that these condensed thienopyrimidines 12-17 have not been previously prepared, and their NMR spectral data were in full agreement with the proposed structures. The assignment of the various carbons in the 13 C-NMR spectra of 12-17 was accomplished with the aid of DEPT-135 and HETCOR experiments. The HMBC correlations of 13, helpful in the assignment of chemical shifts, are shown in Figure 2.

KOH, EtOH,reflux 1h
The thioureido derivatives 20-23 were obtained in moderate yields upon reaction of 1a,d with some aromatic isothiocyanates using either the classical heating method [12] or microwave irradiation [13], although the reaction was cleaner in the case of the latter method (Scheme 3). Thioureido compounds 21 and 22 were transformed to thienopyrimidine 18 through the corresponding pyridinium salts 24 and 25, respectively. Compounds 18a,d were obtained directly, although in low yields, on refluxing 1a,d with the aromatic isothiocyanates for longer times. Condensation of 18d with hydrazine hydrate gave the novel 2-hydrazino derivative 19. Compounds 20-23 were also condensed with hydrazine hydrate to give the corresponding 3-aminothienopyrimidines 26-29. The latter amines reacted smoothly with aromatic aldehydes, as exemplified by the reaction of 26 with two different aldehydes. Thienopyrimidines 28-31 have not been previously synthesized and their structures were firmly established on the basis of their NMR (δ H,C ) and mass spectral data (see Experimental).
The starting 2-amino-3-cyanothiophene derivatives 32a,b reacted with formamide to give the corresponding thienopyrimidine derivatives 33a,b (Scheme 4). Structural elucidation of the latter was based on the various spectroscopic methods and comparison of the NMR data of 33b with those given in the literature [13] for the same compound.  Further, condensation of 32a,b with TEOF and TEOA followed by hydrazine hydrate led to the formation of the novel thienopyrimidines 36-39, whose structures were elucidated from their IR, NMR and MS spectral data (see Experimental). Compounds 40-43 were obtained in moderate yields upon the reaction of 37-39 with TEOF and TEOA. Reaction of 36, 38 and 39 with carbon disulphide in pyridine gave the condensed thienopyrimidines 44-46. The structures of 42-46 were confirmed from their 1 H-NMR, 13 C-NMR and mass spectral data. The assignment of the NMR chemical shifts of the thienopyrimidines were made on the basis of COSY 1 H-NMR and DEPT-135 experiments. HETCOR and HMBC spectra were further used in the structural elucidation of 38, 43, 45 and 46. Thus, the 1 H-NMR spectrum of 43 displayed four multiplets and two singlets in the aliphatic region attributable to the protons in five methylenes and two methyl groups. The signals of these aliphatic carbons in the 13 C-NMR spectrum were verified through the corresponding DEPT-135 technique. The remaining signals in the latter spectrum correspond to the aromatic carbons in 43, which were unambiguously assigned on the basis of the analysis of HETCOR and HMBC spectral data. Figure 3 shows the HMBC spectrum correlations in 43. were used as test organisms. The filter paper disc method [16,17] was used for the antimicrobial screening of selected compounds (4a, 8c, 12, 15, 17, 41 and 46). The tested compounds were dissolved in a suitable solvent (the concentration of the compounds was 10%). Standard blank paper discs (5 mm in diameter) were separately soaked in the solutions of each compound and after 3-5 min transferred onto the surface of growth media seeded with the test organism. After an incubation period under suitable conditions for the test organism (35 ºC) and after 24 h, the diameter of the inhibition zones around the discs were measured in millimeters. The effects were compared with the reference antibiotics vancomycin (VA) in the case of S. aureus and cefatzidine (CAZ) in the case of other tested organisms. The antibiotics VA and CAZ were used at a concentration of 20 mg/mL as references. The obtained results are summarized in Table 2. Most of the compounds (except 17) showed very good activity against Staphylococcus aureus comparing with the used reference standards. Some of the tested compounds showed very weak or moderate activities aginst Klepsela monas, Pseudomonas aeruginosa and Escherichia coli, as can be noticed from Table 2. Table 2. Antimicrobial activity of the select synthesized compounds.

Antitumor studies
The synthesized thienopyrimidines 4a, 7d, 8a, 12, 15, 29 and 42 were tested against the following human tumor cell lines: colorectal carcinoma (HCT116), hepatocellular carcinoma (HEPG2), cervix adenocarcinoma (HELA), larynx carcinoma (HEP2), human breast adenocarcinoma (MCF7). The drug doxorubicin [18] has been used as a reference in the present study to compare the inhibition effect for tested compounds on the growing cancer cells. Measurement of Potential Cytotoxicity by the SRB Assay was used [19]. Compounds 8, 12 and 29 showed very good anticancer activity against HEPG2, while most of the compounds were very active against HELA, except 4a and 7d. Compounds 7d and 15 were active toward MCF7. For cell line HCT116 only 7d and 42 were very active, on the other hand 7d, 8a and 15 were very active against HEP2. Results are summarized in Table 3.

General
Melting points were determined on an Electrothermal IA9000 series digital capillary melting point apparatus. IR spectra were run (KBr discs) on a Shimadzu FT spectrophotometer 1000. 1 H-and 13 C-NMR spectra were recorded in DMSO-d 6 (or in CDCl 3 ) on a JEOL ECP 400 NMR spectrometer operating at 400/100 MHz, with TMS as internal standard. DEPT and HETCOR experiments were recorded on a 500 MHz instrument (Bruker, J.F.B. 288) at King Saud University (Pharmacy Research Centre). Chemical shifts are given in δ (ppm) and coupling constants (J) are given in Hz. The assignments of all carbons are made by comparison to 13 C-NMR spectra of structurally related compounds [12,13] and theoretical grounds [20,21], and with the aid various modern NMR techniques in many cases. Electron impact (EI) MS spectra were recorded on a Shimadzu GCMSQP5050A spectrometer (DB-1 glass column 30 m × 0.25 mm, ionization energy 70 eV), at the Chemistry Department, College of Science, King Saud University. Antimicrobial and anti-cancer tests of some of the synthesized compounds were run in King Abdul Aziz Hospital for the National Guard, Al-hasa, Saudi Arabia and the Pharmacology Unit, National Cancer Institute, Cairo University, Egypt, respectively. The reactions were monitored by TLC, and the purity of the compounds were routinely checked by TLC silica gel plates while the spots were visualised by UV (Uvitec). The starting materials, ethyl 2-amino-4,5-disubstituted thiophene-3-carboxylates 1a-d and the 2-amino-4,5disubstituted thiophene-3-carbonitrile 32, were prepared by condensation of ketones, elemental sulfur, ethyl cyanoacetate or malononitrile as described [14,15].

General procedure for synthesis of 2a,b
Compounds 2a,b were both prepared according to a method reported in the literature [22] for similar compounds. 5-Chlorovaleryl chloride (1.3 mL, 10 mmol) was added to a solution of amino ester 1a,b (10 mmol) in chloroform (20 mL) and the solution was refluxed for 4 h. After cooling, the solution was concentrated under reduced pressure to give a dark oil. Addition of a small amount of water and ethanol yielded a solid that was collected, dried and recrystallized from ethanol.

General procedure for synthesis of 4,5-disubstituted-3H-thieno[2,3-d] pyrimidin-4-thiones 8a,c,d
A mixture of compound 7a,c,d (10 mmol), phosphorous pentasulphide (4.02 g, 30 mmol) and dry pyridine (50 mL) was refluxed with stirring for 2 h. The mixture was evaporated to dryness under reduced pressure and the residue was boiled with water (100 mL) for one hour. After cooling overnight in refrigerator, the formed solid was recrystallized from a suitable solvent.

General procedure for the synthesis of 10a,c,d
A mixture of 1a,c,d (10 mmol) and ethoxycarbonyl isothiocyanate (10 mmol) in ethanol (5 mL) was placed in a 50 mL conical flask covered with a funnel glass and then irradiated with microwaves (950 W) for 35-40 seconds. The cold reaction mixture was treated with ethanol and the solid product was filtered and recrystallized from a suitable solvent. -4,5,6,7-tetrahydrobenzo [4,5] [4,5]

General procedure for the synthesis of 11a,c,d
These susbstances were prepared according to a method reported in the literature [23]. Compound 10 (1 mmol) was dissolved in solution of sodium ethoxide (230 mg sodium and 15 mL of absolute ethanol) and the solution was heated under reflux for 30 min. The solvent was then evaporated under vacuum, some water was added to the residue, and the pH of the mixture was adjusted to 4 with hydrochloric acid. The product that separated was collected and crystallized from a suitable solvent. [4,5]thieno [2,3-d] -2,3,5,6,7,8-hexahydro-1H-pyrido [4`,3`:4,5]thieno [2,3-d]

Synthesis of 12 and 13
To a solution of 11a (1.19 g, 5 mmol) was added aroyl hydrazine (5 mmol) in n-butanol (15 mL) and the mixture was heated under reflux for 20 h. The solid obtained was cooled, collected and recrystallized from butanol.

Synthesis of disubstituted thienyl-2-thioureides 20-23
3.18.1. Method A 2-Amino-3-ethoxycarbonyl thiophene 1a,d (100 mmol) was dissolved in hot ethanol (100 mL) and phenyl (or p-clorophenyl) isothiocyanate (110 mmol) was added dropwise with stirring. The reaction mixture was heated under reflux on water bath for 2 h, then left to cool overnight and the separated crude solid was filtered, washed with ethanol, and recrystallized from ethanol [12].

Method-B
A mixture of 1a,d (20 mmol) and phenyl (or p-clorophenyl) isothiocyanate (20 mmol) placed in a 50 mL conical flask covered with a funnel glass and then irradiated with microwaves (950 W) for 35-40 seconds. The cold reaction mixture was then treated with ethanol and the solid product was filtered off and recrystallized from ethanol [13].

General procedure for the preparation of the monopotassium salts of 24 and 25
A mixture of the compounds 21, 22 (13.5 mmol) and potassium hydroxide (760 mg, 13.5 mmol) in absolute ethanol (55 mL) was heated under reflux with stirring for 1h. The suspension was filtered while hot, and the solid was washed with hot absolute ethanol to give 24) [13] and 25 which were both used without any further purification.
3.20. General procedure for the preparation of 18a,d

Method A
A suspension of potassium salts of 24, 25 in water (50 mL) was acidified with concentrated hydrochloric acid and stirred at room temperature for 30 min. The solid was collected by filtration, washed with water and recrystallized from the suitable solvent to give 18a,d.

Method B: Synthesis of 18d
A mixture of 1d (3.16 g, 10 mmol) and phenyl isothiocyanate (1350 mg, 10 mmol) in acetonitrile (30 mL) was heated under reflux for 15 h in the presence of anhydrous potassium carbonate (1.4 g). The reaction mixture was then cooled, filtered, diluted with water (10 mL) and neutralized with 2M hydrochloric acid. The product obtained was filtered, washed with water, dried and recrystallized from acetic acid.

General procedure for the preparation of 26-29
A mixture of thiouredo derivatives 20-23 (10 mmol) and hydrazine hydrate (20 mmol) in ethanol (100 mL) was heated under reflux for 3-4 h. The solid that separated upon cooling was filtered, washed with water, dried and recrystallized from ethanol.