Utility of thieno[2,3-b] pyridine derivatives in the synthesis of some condensed heterocyclic compounds with expected biological activity

On the pharmaceutical account of the reported anticancer activity of thieno[2,3-b] pyridine and condensed thieno[2,3-b] pyridine, new compounds containing thieno[2,3-b] pyridine condensed with each of pyridine, cyclopentyl, tetrahydroquinoline, pyrimidine, 1,6-naphthiridin, benzofuro[2,3-b] pyridine, imidazo[1,2-c] pyrimidine, [1,2,3] triazolo[1,5-a] pyrimidine were synthesized through different chemical reactions. The obtained compounds were evaluated for their in vitro antitumor activity against Liver HepG-2 and Breast MCF-7 cell lines compared to the reference drug (doxorubicin). Compounds 5, 7, 12, 23, 24, 37 and 39 were found to be the most active against both cell lines exhibiting IC50 values ranging from 10.33-43.90 μM/L and 9.70-48.80 μM/L against HepG-2 and MCF-7 cell lines; respectively. From which compound 5 was the most active compound exerting comparable activity to the reference drug against both cell lines, showing IC50 values 10.33 and 9.70 μM/L comparable to doxorubicin that exerted IC50 values 8.55 and 8.90 μM/L against HepG-2 and MCF-7 cell lines; respectively.


Results and discussion Chemistry
The o-aminonitrile thienopyridine derivative 2 was synthesized through treatment of the pyridine thione derivative 1 with chloroacetonitrile in presence of sodium hydroxide [32]. "( Scheme 1)". Fusion of the o-aminonitrile derivative 2 with cyclic ketones namely; cyclopentanone and cyclohexanone in the presence of anhydrous zinc chloride [33] yielded the tetracyclic compounds 3 and 4; respectively. Reactions utilizing cyclohexanone resulted in higher yields of the product compared to cyclopentanone, a fact that could be attributed to the steric flexibility of cyclohexanone [34]. Reactions of compound 2 with ethyl methyl ketone furnished the tricyclic pyridothienopyridine derivative 5. The reactions of α-aminonitriles with various cyclic ketones in presence of Lewis acid were reported to be achieved through the nucleophilic attack of the lone pair of amino nitrogen on the carbonyl carbon to form Schiff's base, followed by complex formation between Lewis acid, zinc chloride and the nitrile triple bond that accelerates the intramolecular cyclization [35].
Furthermore, the o-aminonitrile derivative 2 was refluxed with malononitrile in presence of a catalytic amount of triethylamine [36] to yield pyridothienopyridine-3-carbonitrile derivative 6. acid [33] furnished pyrimidinone derivatives 14a and 14b; respectively. The 1 H NMR spectra of compounds 14a and 14b revealed two deuterium oxide exchangeable singlets at d 12.60, 10.50 ppm corresponding to pyrimidine NH protons; respectively. However, 1 H NMR spectrum of compound 14b showed an additional singlet at d 3.07 ppm integrated for two protons corresponding to CH 2 -Cl protons.
The o-aminonitrile derivative 2 was reacted with triethyl orthoformate [41] which underwent a nucleophilic substitution reaction in the presence of acetic anhydride introducing a replaceable ethoxy group furnishing the intermediate compound 15. The 1 H NMR spectrum of compound 15 displayed a triplet at δ 1.06 ppm and a quartet at δ 3.44 ppm attributed to ethyl protons of the ethoxymethylene group. In addition to a singlet due methine proton at δ 7.25 ppm.
Furthermore, compound 15 upon reaction with excess hydrazine hydrate [42] yielded novel pyridothienopyrimidine derivative 16. The reaction mechanism was reported [41] to be accomplished through addition of a hydrazine molecule on the enamine double bond followed by elimination of an ethanol molecule and intramolecular cyclization to yield the target compound 16. The 1 H NMR spectrum of compound 16 showed two deuterium oxide exchangeable singlets at δ 6.06 and δ 8.90 ppm corresponding to the vicinal amino and imino groups; respectively.
Similarly, compound 2 was refluxed with formic acid to yield pyridothienopyrimidinone derivative 17 which its synthesis was rationalized via a sequence of N-formylation followed by cyclization processes involving the interaction between nucleophilic hydroxyl group and electrophilic nitrile carbon to give the 4-iminoxazine intermediate which underwent rearrangement to yield the pyrimidinone ring [33]. The 1 H NMR spectrum of the compound 17 displayed a deuterium oxide exchangeable singlet corresponding to OH proton at δ 12.80 ppm. The interaction of the o-aminonitrile derivative 2 with phenyl isothiocyanate in presence of pyridine [36] led to the formation of pyridothienopyrimidine-2-thione 18. "(Scheme 4)". The 1 H NMR spectrum of compound 18 showed two deuterium oxide exchangeable singlets at d 7.10 and 9.75 ppm attributed to pyrimidine NH and imino protons; respectively.

Reagents
A facile reaction occurred when o-aminonitrile derivative 2 was refluxed in excess formamide [43] to furnish the corressponding 4-aminopyrimidine derivative 19. The reaction mechanism was reported to be proceeding first through o-cyanoformamidine formation followed by intramolecular cyclization via nucleophilic attack of the lone pair of the formamide amino group on the electrophilic nitrile carbon [43].
Diazotization of compound 2 was accomplished through its reaction with cold hydrochloric acid and saturated aqueous sodium nitrite solution to yield thienopyridine-3-diazonium chloride 20 which was further coupled with compounds bearing active methylene functions namely; malononitrile and ethyl cyanoacetate [44] to afford the corresponding hydrazono derivatives 21 and 22; respectively. The 1 H NMR spectra of compounds 21 and 22 revealed deuterium oxide exchangeable singlets at d 8.50 and d 8.28 ppm attributed to NH protons; respectively.
Hydrazono derivatives 21 and 22 were cyclized upon treatment with hydrazine hydrate in boiling ethanol [44] to afford the expected 3,5-diaminopyrazole derivative 23 and 3-amino-5-oxopyrazole derivative 24. The 1 H NMR spectrum of compound 23 revealed a deuterium oxide exchangeable singlet at δ 9.85 ppm integrated for four protons attributed to two NH 2 protons. The 1 H NMR spectrum of the compound 24 displayed three deuterium oxide exchangeable singlets corresponding to pyrazole-NH, pyrazole-C 3 -NH 2 and thiophene-C 3 -NH protons at δ 7.00, 7.22 and 8.60 ppm; respectively.
Reagents: i) C 6 H 5 -NCS/ dioxane/ pyridine; ii) HCONH 2 ; iii) NaNO 2 / HCl/ 0-5°C; iv) CNCH 2  Compound 2 was also reacted with ethylene diamine in the presence of carbon carbon disulphide disulfide [45] to afford 2-imidazolylthienopyridine derivative 25 "(Scheme 5)". The reaction mechanism is proposed to proceed through addition of carbon disulphide and an ethylene diamine molecules on the nitrile function followed by the elimination of 2-thioxoimidazolidine moiety to yield thiocarboxamide derivative which further reacted with one molecule of ethylene diamine with simultaneous elimination of ammonia and hydrogen sulphide molecules to yield the imidazolidine ring. 1 H NMR spectrum of compound 25 showed two deuterium oxide exchangeable singlets at d 6.90 and 7.20 ppm attributed to NH and NH 2 protons; respectively.
Compound 25 was further subjected to cyclization into tetracyclic imidazopyridothienopyrimidine systems in different ways [45]. Treatment of compound 25 with triethyl orthoformate gave the unsubstituted imidazopyridothienopyrimidine 26, while refluxing of compound 25 with benzaldehyde yielded the corresponding 5-phenylimidazopyridothienopyrimidine 27. On the other hand, imidazopyridothienopyrimidine-5thione 28 was obtained by heating compound 25 with in boiling pyridine.
The 1 H NMR spectrum of compound 27 displayed a singlet at d 8.71 ppm corresponding to pyrimidine-C 2 proton besides to a deuterium oxide exchangeable singlet at d 9.65 ppm attributed to NH proton. Furthermore, the 1 H NMR spectrum of compound 28 revealed a deuterium oxide exchangeable singlet at d 9.85 ppm attributed to NH proton.
Reagents: i) NH 2 (CH 2 ) 2 NH 2 / CS 2 ; ii) HC(OC 2 H 5 ) 3 /gl.AcOH; iii) C 6 H 5 -CHO/absolute EtOH; iv) CS 2 /pyridine. Scheme 5. Synthetic pathways for compounds 25, 26, 27 and 28. The thienopyridine o-aminoester derivative 29 was prepared through the reaction of pyridine thione 1 with ethyl chloroacetate in presence of sodium hydroxide [46] "(Scheme 6)". The amino group in o-aminoester derivative 29 was diazotized by nitrososulfuric acid followed by the addition of sodium azide solution [47] to yield azide derivative 30. Compound 30 was further reacted with different nitriles namely; malononitrile and 2-(benzo[d]thiazol-2-yl) acetonitrile by refluxing in sodium methoxide [47] to yield pyridothienotriazolopyrimidinones 31a and 31b; respectively. Moreover, methylthiocarbonothioylamino derivative 32 was prepared in a one pot reaction by treating a vigorously stirred solution of o-amino ester derivative 29 with carbon disulphide and sodium hydroxide solution [48] to yield the sodium salt of dithiocarbamic acid which was not isolated and was further treated with dimethyl sulphate. Compound 32 was further cyclized by refluxing with hydrazine hydrate in ethanol [48] to yield 2-thioxopyridothienopyrimidin-4-one 33. The 1 H NMR spectrum of compound 32 revealed a singlet at δ 2.71 ppm attributed to S-CH 3 protons. In addition to a deuterium oxide exchangeable singlet at d 7.32 ppm attributed to NH proton. However, the 1 H NMR spectrum of compound 33 revealed two deuterium oxide exchangeable signlets at d 4.30 and d 6.61ppm attributed to NH 2 and pyrimidine NH protons; respectively.

32,33, 34 and 35.
The 3-carbohydrazide derivative 36 was prepared by treatment of o-aminoester 29 with hydrazine hydrate [46]. "(Scheme 7)". Compound 36 was further refluxed with triethyl orthoformate in presence acetic anhydride and with acetic anhydride only [50] to yield the formimidate derivative 37 and the acetamide derivative 38; respectively. The 1 H NMR spectrum of compound 37 displayed two singlets at d 7.87 ppm and d 8.36 ppm due to N=CH proton and pyrimidine C 2 proton; respectively.
Furthermore, the reaction of acid hydrazide derivatives with ethyl acetoacetate were reported to yield either pyrazolone derivatives [50,51] or the open chain imine derivative [50]. However, compound 36 upon reaction with ethyl acetoacetate in presence of sodium ethoxide [50] yielded the pyrazolone derivative 39 which its 1 H NMR spectrum revealed a singlet at d 2.26 ppm integrated for five protons corresponding to pyrazole-C 3 -CH 3 and pyrazole-CH 2 protons. It also showed a deuterium oxide exchangeable singlet at d 16.53 ppm attributed to H-bonded NH 2 protons.

Biological evaluation
The synthesized compounds were screened for their in vitro cytotoxic activity against human hepatocellular liver carcinoma (HepG2). (Table 1) and human breast cancer cell line (MCF-7) ( Table 2). Doxorubicin was used as the reference drug. It is to be noted that, introduction of a pyrazole ring to the thienopyridine backbone either through an azo junction as in 3,5-diaminopyrazole derivative 23 or a hydrazo function in 3-aminopyrazol-5-one derivative 24 or a carbonyl group as in3-methyl pyrazol-5-one derivative 39 afforded significant activity against both cell lines. Also, the introduction of ethoxymethyleneamino moiety at the N 3 of pyrimidine-4one ring fused to the thieno[2,3-b]pyridine back bone as in compound 37 also resulted in marked increase in activity against both HepG2 and MCF-7 cell lines.

General procedure for the synthesis of compounds 3, 4 and 5
An equimolar mixture of compound 2 (2.65 g, 10 mmol) and cyclic/alicyclic ketones (10 mmol), in presence of anhydrous ZnCl 2 (0.68g, 5 mmol) was fused at 120-130° C for 5h. The reaction mixture was allowed to cool, triturated with H 2 O and neutralized with sodium hydroxide 10%. The separated solid was filtered off, washed with water, left to dry then recrystallized from ethanol.

-b]pyridine-3carbonitrile; 11
A mixture of compound 2 (2.65 g, 10 mmol) and cyanothioacetamide (1 g, 10 mmol) was refluxed for 8 h in absolute ethanol (30 mL) containing 3 drops of piperidine. The reaction mixture was allowed to cool, then poured onto ice-cold water, and neutralized with hydrochloric acid (10%). The separated solid was filtered and recrystallized from ethanol.

General procedure for the synthesis of compounds 14a,b
A mixture of compound 13 (2.65 g, 10 mmol) and the appropriate acid chloride (10 mmol) namely; benzoyl chloride and chloroacetyl chloride was refluxed in acetic acid (10 mL) for 10 h. The reaction mixture was allowed to cool, and then poured onto ice-cold water. The separated solid was filtered, washed with water and recrystallized from ethanol.

Synthesis of 2-cyano-4-methyl-6-phenylthieno[2,3-b] pyridine-3-diazonium chloride; 20
A suspension of compound 2 (2.65 g, 10 mmol) in concentrated hydrochloric acid (3 mL) was cooled to 0-5°C in an ice bath to which an ice cold solution of sodium nitrite (1.5 g, 20 mmol in 10 mL water) was added dropwise while cooling over a period of 15 minutes. The reaction mixture was then stirred for 30 minutes to yield crystals of the diazonium product 20, which was filtered and used as such in next step.

General procedure for the synthesis of compounds 21 & 22
To an ice-cold mixture of active methylene compounds namely; malononitrile and ethyl cyanoacetate (10 mmol) and anhydrous sodium acetate (4 g, 50 mmol) in absolute ethanol (50 mL), an ice cold solution of compound 20 (3.12 g, 10 mmol) in absolute ethanol (10 mL), was added dropwise over a period 15 minutes while stirring and cooling in an ice bath. Stirring was then continued for 24 h at room temperature. The reaction mixture was then filtered and the obtained product was washed with ethanol.

Synthesis of ethyl 3-azido-4-methyl-6-phenylthieno[2,3-b] pyridine carboxylate; 30
Compound 29 (3.12 g, 10 mmol) was dissolved in a mixture of concentrated sulphuric acid (2.5 mL) and water (7 mL) and cooled to 0-5°C in an ice bath. An aqueous solution of sodium nitrite (0.83 g, 120 mmol in 1 mL water) was added dropwise while stirring and maintaining the temperature below 5°C. Then, a solution of sodium azide (0.65 g, 10 mmol) in water (5 mL) was added dropwise to the reaction mixture while cooling. The reaction was stirred for 24 h at room temperature and the precipitated solid was filtered off, washed with water, left to dry and recrystallized from ethanol.

General procedure for the synthesis of compounds 31a&b
Equimolar amounts of compound 30 (3.38 g, 10 mmol) and appropriate nitrile (10 mmol) namely; malononitrile and 2-(benzo[d]thiazol-2-yl)acetonitrile were added with vigorous stirring to a solution of sodium methoxide [prepared from 0.3 g sodium and 20 mL methanol]. The reaction mixture was stirred at room temperature for 24 h. Then, poured onto crushed ice. The obtained precipitate was filtered off, washed with water, left to dry and recrystalized from ethanol.

Cell line Propagation
The cells were propagated in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, 1% L-glutamine, HEPES buffer and 50 µg/mL gentamycin. All cells were maintained at 37°C in a humidified atmosphere with 5% CO 2 and were subcultured two times a week.

Cytotoxicity evaluation using viability assay
For cytotoxicity assay, the cells were seeded in 96-well plate at a cell concentration of 1x10 4 cells per well in 100 µL of growth medium. Fresh medium containing different concentrations of the test sample was added after 24 hof seeding. Serial two-fold dilutions of the tested chemical compounds were added to confluent cell monolayers dispensed into 96-well, flat-bottomed microtiter plates (Falcon, NJ, USA) using a multichannel pipette. The microtiter plates were incubated at 37°C in a humidified incubator with 5% CO 2 for a period of 48 h. Three wells were used for each concentration of the test sample. Control cells were incubated without test sample and with or without DMSO. The little percentage of DMSO present in the wells (maximal 0.1%) was found not to affect the experiment.
After incubation of the cells for 24 h at 37°C, various concentrations of sample (50, 25, 12.5, 6.25, 3.125 & 1.56 µg) were added, and the incubation was continued for 48 h and viable cells yield was determined by a colorimetric method. After the end of the incubation period, media were aspirated and the crystal violet solution (1%) was added to each well for at least 30 minutes. The stain was removed and the plates were rinsed using tap water until all excess stain is removed. Glacial acetic acid (30%) was then added to all wells and mixed thoroughly, and then the absorbance of the plates were measured after gentle shaking on Microplate reader (TECAN, Inc.), using a test wavelength of 490 nm. All results were corrected for background absorbance detected in wells without added stain. All experiments were carried out in triplicate. The cell cytotoxic effect of each tested compound was calculated [52,53]. The cytotoxicity of the tested compounds was estimated in terms of percent growth inhibition compared to untreated control cells and their IC 50 in µM/L which is the concentration of the compound that inhibits the tumor cell growth by 50%.

Conclusion
Most of the compounds showed better activity against liver cancer HepG2 cell line than breast cancer MCF-7 cell line.