Synthesis, Cytotoxic and Anti-proliferative Activity of Novel Thiophene, Thieno[2,3-b]pyridine and Pyran Derivatives Derived from 4,5,6,7-tetrahydrobenzo[b]thiophene Derivative

Novel tetrahydrobenzo[b]thienopyrole derivatives are synthesized from 2-amino-3-cyano-4,5,6,7-tetrahydrobenzo[b]thiophene (1) through its reaction with α-chloroacetone to give the corresponding N-alkyl derivative 3. Compound 3 undergoes ready cyclization in sodium ethoxide solution to give the tetrahydrobenzo[b]thienopyrrole 4. The latter compound 4 is used as the key starting material for the synthesis of thiophene, thieno[2,3-b]pyridine and pyran derivatives. The cytotoxicity of the synthesized products towards the human cancer cell lines namely gastric cancer (NUGC), colon cancer (DLD-1), liver cancer (HA22T and HEPG-2), breast cancer (MCF-7), nasopharyngeal carcinoma (HONE-1) and normal fibroblast (WI-38) cell lines are measured. Compounds 4, 7a, 7b, 8a, 8b, 10c, 10d, 10f, 12a, 12b, 14b and 15b exhibit the optimal cytotoxic effect against cancer cell lines. Compounds 7b and 14b show the maximum inhibitory effect and these are much higher than the reference CHS-828 (pyridyl cyanoguanidine). On the other hand, the anti-proliferative evaluations of these compounds with high potency against the cancer cell lines L1210, Molt4/C8, CEM, K562, K562/4 and HCT116 show that compounds 7b and 8b give IC50’s against Molt4/C8 and CEM cell lines higher than that of the reference, doxorubicin.

Compound 4 showed interesting reactivity towards different reagents, thus, it reacted with either malononitrile (5a) or ethyl cyanoacetate (5b) in the presence of ammonium acetate in an oil bath at 120 °C afforded the Knoevenagel condensated products 6a and 6b, respectively.The latter products underwent ready cyclization in sodium ethoxide solution to give the annulated products 7a and 7b, respectively (Scheme 2).The structures of the latter products were established on the basis of the analytical and spectral data.Thus, the 1 H-NMR spectrum of 7a showed the presence of δ 2.89 ppm assigned to the CH 3 group, a singlet at δ 4.89 ppm indicating the NH 2 group and a singlet at δ 8.33 ppm confirming the presence of the NH group.Moreover, the 13  Compound 4 was studied to produce thiophene derivatives through the Gewald's reaction [23][24][25][26] as many thiophenes were used as anticancer drugs.Thus, the reaction of compound 4 with either of malononitrile or ethyl cyanoacetate and elemental sulphur gave the thiophene derivatives 8a and 8b, respectively.On the other hand, the one pot reaction of compound 4 with either malononitrile or ethyl cyanoacetate and any of benzaldehyde, 4-chlorobenzaldehyde or 4-methoxybenzaldehyde gave the pyran derivatives 10af, respectively.The 1 H-NMR and 13 C-NMR spectra 10a-f were consistent with their respective structures.Further confirmations for the structure of compounds 10a-f were obtained through their synthesis via another synthetic root.Moreover, the reaction of either of compound 8a or 8b with ethyl cyanoacetate in refluxing dimethylformamide afforded the 2-amido derivatives 12a and 12b, respectively.Formation of the latter products was explained on the condensation of ethyl cyanoacetate with the 2aminothiophene moiety not to the 3-aminopyrrol moiety on the basis of the 1 H-NMR spectra of such products.Thus, the 1 H-NMR spectrum of either 12a or 12b displayed the missing of the NH 2 group that attached to thiophene ring which is expected to appear within the range δ 5.10-5.24ppm while that of the 3-aminopyrrole moiety existing at δ 4.81-4.83ppm.Similar acylation of the 2aminothiophene was reported before in literature. 27The high yield of compound 12a, encouraged us to make furt-her work.Thus, the reaction of 12a with either of the aryl diazonium salts 13a-d gave the aryl hydrazo derivatives 14a-d, respectively.Moreover, compounds 12a,b underwent ready cyclization in sodium ethoxide to produce the thieno [2,3-b]pyridine derivatives 15a and 15b, respectively (Scheme 4).Louis, USA).The cell cultures was obtained from the European Collection of cell Cultures (ECACC, Salisbury, UK) and human gastric cancer (NUGC), human colon cancer (DLD-1), human liver cancer (HA22T and HEPG-2), human breast cancer (MCF-7), nasopharyngeal carcinoma (HONE-1) and normal fibroblast cells (WI-38) were kindly provided by the National Cancer Institute (NCI, Cairo, Egypt).They grow as monolayer and routinely maintained in RPMI-1640 medium supplemented with 5% heat inactivated FBS, 2 mM glutamine and antibiotics (penicillin 100 U/mL, streptomycin 100 lg/mL), at 37 °C in a humidified atmosphere containing 5% CO 2 .Exponentially growing cells were obtained by plating 1.5 × 10 5 cells/mL for the six human cancer cell lines including cells derived from 0.75 × 10 4 cells/mL followed by 24 h of incubation.The effect of the vehicle solvent (DMSO) on the growth of these cell lines was evaluated in all the experiments by exposing untreated control cells to the maximum concentration (0.5%) of DMSO used in each assay.

In vitro Cytotoxicity Assay
The heterocyclic compounds, prepared in this study, were evaluated according to standard protocols 28,29 for their in vitro cytotoxicity against the six human cancer cell lines including cells derived from human gastric cancer (NUGC), human colon cancer (DLD-1), human liver cancer (HA22T and HEPG-2), human breast cancer (MCF-7), nasopharyngeal carcinoma (HONE-1) and a normal fibroblast cells (WI-38).All of IC 50 values were listed in Table 1.Some heterocyclic compounds were observed with significant cytotoxicity against most of the cancer cell lines tested (IC 50 =10-1000 nM).Normal fibroblasts cells (WI-38) were affected to a much lesser extent (IC 50 >10,000 nM).The reference compound used was the CHS-828 which is the pyridyl cyanoguanidine anti-tumor agent. 30It is a new chemotherapeutic drug in addition it has low toxicity and lacks known patterns of multidrug resistance. 31

3. Structure-activity Relationship
From Table 1 it is clear that the thiophene moiety was found to be crucial for the cytotoxic effect of the cyclic compounds 3 -15a,b.Compounds 4, 7a, 7b, 8a, 8b, 10c, 10d, 10f, 12a, 12b, 14b and 15b exhibited optimal cytotoxic effect against cancer cell lines, with IC 50 's in the nM range.Comparing the cytotoxicity of the tetrahydrobenzothiophene 3 and the cyclized product 4, it is obvious that the cytotoxicity of compound 4 is higher than that of compound 3.The presence of the pyrrol ring through the tetrahydrobenzo[b]thiophene in compound 4 is responsible for its high potency.The condensation reac- The thiophene derivatives 12a and 12b showed high cytotoxicity similarl to that of compounds 8a,b.Moreover, compound 12b with the COOE-t showed high potency than that of compound 12a.The coupling of the diazonium salts 13a-d with compound 12a afforded the arylhydrazone derivatives 14a-d.Compound 14b with the Cl group showed the maximum cytotoxicity among the arylhydrazone derivatives 14a-d.Finally, considering the thieno[2,3-b]pyridine derivatives 15a,b where the presence of the OH in compound 15b conserved an interesting cytotoxicity against the cancer cell lines HA22T, HEPG-2 and HONE-1 with the IC 50 's 377, 740, 253 nM, respectively.It is of great value to notice that compounds 7b, 8b and 12b showed the maximum cytotoxicity among the tested compounds.

4. Anti-proliferative Cell Activity Against Cancer Cell Lines
We used a panel of tumor cell lines to test the cytotoxicity of the new compounds, especially those showed high potency against the six cancer cell lines through Table 2. Importantly, this panel included the cell lines and their isogenic sub-lines with the determinants of drug resistance: murine leukemia L1210, T-lymphocyte cell lines Molt4/C8 and CEM, human leukemia R562 and its MDR subline K562/4 that over expressed P-glycoprotein, and the colon carcinoma HCT116.The above determinants alter the response of cells to many anticancer drugs including doxorubicin.Data on cytotoxic (anti-proliferative) activity are presented in Table 2 in which IC 50 values represent the concentrations that inhibit cell proliferation by 50%.It is clear from Table 2 that tested compounds 4, 7a, 7b, 8a, 8b, 10c, 10d, 10f, 12a, 12b, 14b and 15b showed high potency against the cell lines.The benzo[4',5']thieno[3',2':4,5]pyrrolo[3,2-b]pyridine derivative 7b and the benzo [4,5]thieno-[2,3-b]pyrrol-2-yl)-thiophene derivative 8b showed high potency against Molt4/C8 and CEM cell lines and their IC 50 's are higher than that of the reference doxorubicin.It is clear from Table 2 that the twelve tested compounds showed high IC 50 against K562/4 cell line than doxorubicin.

1. General
All melting points were determined on an electrothermal apparatus (Büchi 535, Switzerland) in an open capillary tube and are uncorrected. 13C-NMR and 1 H-NMR spectra were recorded on Bruker DPX200 instrument in DMSO with TMS as internal standard for protons and solvent signals as internal standard for carbon spectra.Chemical shift values are mentioned in ä (ppm).Mass spectra

1. 2. General Procedure for the Synthesis of Thieno[ [2,3-b] ]pyrrol Derivatives 6a and 6b
To the dry solid of compound 4 (2.34 g, 0.01 mol) either malononitrile (0.66 g, 0.01mol) or ethyl cyanoacetate (1.13 g, 0.01 mol) was added followed by ammonium acetate (0.50 g, 0.01 mol).The whole reaction mixture was heated in an oil bath at 120 °C for 1h then left to cool.The solidified product was boiled with ethanol then left to cool.The formed solid product was collected by filtration and crystallized from acetic acid.
Method (B): To a solution of compound 4 (2.34 g, 0.01 mol) in 1,4-dioxane (40 mL) containing triethylamine (0.50 mL) either malononitrile (0.66 g, 0.01 mol) or ethyl cyanoacetate (1.13 g, 0.01 mol) was added.The whole reaction mixture, in each case, was heated under reflux for 4 h then poured onto ice/water containing few drops of hydrochloric acid.The formed solid product was collected by filtration and crystallized from acetic acid.

Derivatives 8a and 8b
To a solution of compound 4 (2.34 g, 0.01 mol) in 1,4-dioxane (40 mL) containing triethylamine (0.50 mL) and elemental sulfur (0.32 g,0.01 mol) either malononitrile (0.66 g, 0.01 mol) or ethyl cyanoacetate (1.13 g, 0.01 mol) was added.The reaction mixture, in each case was heated under reflux for 2 h then was left to cool and the formed solid product, in each case, was collected by filtration and crystallized from ethanol.

1. 5. General Procedure for the Synthesis of Pyran Derivatives 10a-f
Method (A): General procedure: To a solution of compound 4 (2.34 g, 0.01 mol) in 1,4-dioxane (40 mL) containing triethylamine (0.5 mL), either of malononitrile (0.66 g, 0.01 mol) or ethyl cyanoacetate (1.13 g, 0.01 mol) and either of benzaldehyde (1.06 g, 0.1 mol), 4-chlorobenzaldehyde (1.40 g, 0.01 mol) or 4-methoxybenzaldehyde (1.36 g, 0.01 mol) were added.The reaction mixture was heated under reflux for 1 h and the formed solid product produced from the hot solution was collected by filtration and crystallized from ethanol.Thin layer chromatography revealed just a single spot which proved the presence of a single product.
Method (B): To a solution of compound 4 (2.34 g, 0.01 mol) in 1,4-dioxane (40 mL) containing triethylamine (0.5 mL), either of the cinnamonitrile derivatives 11a-f (0.01 mol) were added.The reaction mixture was heated under reflux for 2 h and the formed solid product produced from the hot solution was collected by filtration and crystallized from ethanol.Thin layer chromatography revealed just a single spot which proved the presence of a single product.

Cyanoacetamido)thiophene Derivatives 12a and 12b
To a solution of either compound 8a (3.14 g, 0.01 mol) or 8b (3.61 g, 0.01 mol) in dimethylformamide (40 mL) ethyl cyanoacetate was added.The reaction mixture was heated under reflux for 2 h then poured onto ice/water.The formed solid product was collected by filtration and crystallized from ethanol.[4,5]
Thus, the reaction of compound 4 with the cinnamonitrile derivatives 11a-f in the presence of a catalytic amount of Mohareb et al.: Synthesis, Cytotoxic and Anti-proliferative Activity of Novel ... triethylamine gave the same products 10a-f, respectively (m.p., mixed m.p. and fingerprint IR) (Scheme 3).

Table 1 .
Cytotoxicity of the newly synthesized products against a variety of cancer cell lines [IC 50 a (nM)] a Drug concentration required to inhibit tumor cell proliferation by 50% after continuous exposure of 48 h.b NUGC, gastric cancer; DLD-1, colon cancer; HA22T, liver cancer; HEPG-2, liver cancer; HONE-1, nasopharyngeal carcinoma; MCF-7, breast cancer; WI-38, normal fibroblast cells.NA:Not Active.Mohareb et al.: Synthesis, Cytotoxic and Anti-proliferative Activity of Novel ...tion of compound 4 with either malononitrile or ethyl cyanoacetate to produce compounds 5a and 5b, respectively showed a decrease of cytotoxicity.On the other hand, the cyclization of compounds 6a and 6b to the but it is of great value to notice that compound 10d showed high cytotoxicity against five cancer cell lines and such cytotoxicity is higher than that of compound 10c.The high cytotoxicity of compound 10d is attributed to the presence of the OH and the Cl group as well.