Synthesis , Characterization and Cytotoxicity of Substituted [ 1 ] Benzothieno [ 3 , 2e ] [ 1 , 2 , 4 ] triazolo [ 4 , 3-a ] pyrimidines

A new series of 4-benzyl-6,7,8,9-tetrahydro[1]benzothieno[3,2-e][1,2,4]triazolo[4,3-a]pyrimidines was synthesized motivated by the widely reported anticancer activity of thieno[2,3-d]pyrimidines and triazolothienopyrimidines. The in vitro cytotoxic activity of some selected compounds was evaluated against two human cell lines: prostate cancer (PC-3) and colon cancer (HCT-116). A preliminary study of the structure–activity relationship of the target compounds was discussed. Most of the synthesized compounds showed remarkable activity on the tested cell lines, while compound 16c had the highest potency against the PC-3 cell line with an IC50 of 5.48 μM compared to Doxorubicin (IC50 = 7.7 μM), the reference standard used in this study. On the other hand, 6c and 18c were the most active against HCT-116 (IC50 = 6.12 and 6.56 μM, respectively) relative to IC50 = 15.82 μM of the standard. Thus, some of the synthesized thienopyrimidine derivatives, specially 6c, 16c and 18c, have the potential to be developed into potent anticancer agents.


Introduction
Despite decades of research that have resulted in an enormous leap in cancer therapy, cancer remains a major cause of death worldwide thus there is a continuous need for the discovery and development of new anticancer agents. 1,2It is worth mentioning that 60% of world's total new annual cases occur in Africa, Asia and Central and South America. 3hiophenes have been reported to possess interesting biological activities particularly as anticancer agents. 4,5any research groups reported the synthesis of biologically active thiophene derivatives through the well-known Gewald reaction. 6,7As an example, Mohareb et al. 8 synthesized some thiophene derivatives and investigated their antitumor activity.The prepared compounds exhibited GI 50 ranging from 0.02 to 0.08 μM against MCF-7, NCI-H450 and SF-268 cell lines compared to Doxorubicin.
Meanwhile, thieno [2,3-d]pyrimidines represent an important class of bioactive heterocycles attracting much attention due to their wide range of biological and pharmaceutical activities. 9,10e presence of pyrimidine ring in the basic building scaffolds of DNA and RNA modules (thymine, cytosine and uracil) is probably the reason of their diverse biological activities. 11][14][15][16] On the other hand, the 1,2,4-triazole heterocycle is of great value as a building block in the structure of several anticancer drug candidates. 11,17,180][21] Among these heterocycles, the mercapto substituted 1,2,4-triazole ring systems have been well studied and so far a variety of biological activities have reported for them. 17,22,23ecently, 4-amino-1,2,4-triazol-3-thione was used as an intermediate for the synthesis of several biologically active fused heterocyclic compounds where the amino and mercapto groups are appropriate nucleophile centers for many chemical modifications. 249][30] For example, the fusion of a triazole ring to cycloalkylthieno [2,3-d]pyrimidine (VII) showed significant in vitro cytotoxic activity against human colorectal cancer cells (HCT-116) (IC 50 = 2.8 μg/mL) compared to the reference drug Doxorubicin (Fig. 2). 31n our search for new classes of potential anticancer agents, the aforementioned findings prompted us to synthesize a series of 4-benzyl [1]benzothieno [3,2e] [1,2,4]triazolo [4,3-a] pyrimidines with varying the substitution at position 1 (Target compound A) in order to investigate the effect of combining these bioactive moieties on the anticancer activity.Moreover, we aimed in this work to prepare a series of 4-benzyl [1]benzothieno [3,2e] [1,2,4]triazolo [4,3-a] pyrimidines bearing various S-(substituted amino alkyl) moieties at position 1 (Target compound B) to act as cytotoxic agents.In this series, different alkyl linkers and different aliphatic and aromatic amines were used to study the effect of these variations on the cytotoxic activity.Some selected compounds were tested for possible anti-cancer activity against two cell lines (PC-3 and HCT-116).

1. Chemistry
All melting points were determined with Stuart SMP10 apparatus and the values given are uncorrected.IR spectra (KBr, cm -1 ) were determined on Shimadzu IR 8400s spectrophotometer (Faculty of Pharmacy, Cairo University, Egypt). 1 H-NMR and 13 C-NMR spectra were recorded on Mercury 300-BB 300 MHz (Microanalytical Center, Faculty of Science, Cairo University, Egypt) and Bruker 400-BB 400 MHz spectrometers (Microanalytical Unit, Faculty of Pharmacy, Cairo University, Egypt) using TMS as the internal standard.Chemical shift values are given in ppm on δ scale.Mass spectra were recorded on Hewlett Packard 5988 spectrophotometer (Microanalytical Center, Faculty of Science, Cairo University, Egypt).Elemental analyses were carried out at the Regional center for Mycology and Biotechnology, Faculty of Pharmacy, Al Azhar University, Egypt; values found were within ±0.35% of the theoretical ones.Progress of the reactions was monitored by TLC using aluminum sheets precoated with UV fluorescent silica gel (Merck 60F 254) and visualized using UV lamp.The solvent system used was chloroform : benzene : methanol [9:5:2].

1. 3. 4-Benzyl
A mixture of 3 (0.32 g, 1 mmol) and acetic acid (10 mL, 70 mmol) was heated under reflux for 6 h.The reaction mixture was poured onto ice cold water (25 mL).The white precipitate formed was collected by filtration, washed with water, dried and crystallized from acetonitrile.To a solution of 3 (1 g, 3 mmol) in dry DMF (10 mL), chloroacetyl chloride (1.5 mL, 20 mmol) was added dropwise with cooling.The solution was then heated under reflux in a boiling water bath for 9 h.After cooling, the reaction mixture was poured onto ice-cold water and the suspension formed was stirred at room temperature for 2 h.The separated solid was collected by filtration, washed with cold water, dried and crystallized from methanol.

1. 7. General procedure for the preparation of compounds 8a-e
A mixture of 3 (0.32 g, 1 mmol) and the appropriate isothiocyanate (2 mmol) in absolute ethanol (30 mL) was heated under reflux for 8 h.The precipitated product was collected by filtration, dried and crystallized from ethanol/CHCl 3 (2:1).[1,2,4] ]triazolo[ [4,3-a] ]pyrimidin-5(4H)-one (8a).Yield: 43%; mp: 206-208 °C; IR (KBr, cm   A mixture of 3 (0.32 g, 1 mmol) and diethyl malonate (2 mL, 13 mmol) was refluxed for 9 h.The reaction was allowed to cool, the formed residue was triturated with ethanol, collected by filtration, dried and crystallized from isopropanol to yield the title compound 9.To an ice cold solution of ammonium thiocyanate (0.17 g, 2 mmol) in dry acetone (5 mL), a solution of benzoyl chloride (0.3 mL, 2 mmol) in acetone (5 mL) was added dropwise.An ice-cold suspension of 3 (0.34 g, 1 mmol) in acetone (15 mL) was added to the previous mixture.The reaction mixture was heated on a water-bath for 15 h.The reaction mixture was cooled and filtered.The filtrate was evaporated and the obtained product was crystallized from ethanol/CHCl 3 (  A mixture of 3 (1.4g, 4.3 mmol), KOH (0.42 g, 7.5 mmol) and CS 2 (4.5 mL, 7.5 mmol) in absolute ethanol (70 mL) was heated under reflux for 25 h.The solvent was evaporated under reduced pressure.The obtained residue was dissolved in H 2 O (20 mL) followed by acidification with dilute HCl (1 mL).The precipitated product was collected by filtration, dried and crystallized from methanol.

1. Materials and Methods
The prostate tumor cell line (PC-3) and the colon tumor cell line (HCT-116) were obtained frozen in liquid nitrogen (-180 °C) from the American Type Culture Collection (ATCC) and were maintained in the National Cancer Institute, Cairo, Egypt, by serial sub-culturing.All chemicals used in this study were of high analytical grade.They were obtained from either Sigma-Aldrich or Bio-Rad.

Measurement of Potential Cytotoxicity
The cytotoxic activity of some selected compounds was measured in vitro against human prostate cancer cell line (PC-3) and colon cancer cell line (HCT-116) at five different doses (0, 5.0, 12.5, 25.0 and 50.0 μg/mL).The screening was carried out at the Pharmacology Unit, Cancer Biology Department, National Cancer Institute, Cairo University using Sulforhodamine-B (SRB) assay, applying the method of Skehan et al. 41 as follows.
Cells were plated in 96 multi-well plate (104 cells/well) for 24 h before treatment with the tested compound to allow attachment to the wall of the plate.Different concentrations of the compounds (0, 5.0, 12.5, 25.0 and 50.0 μg/mL) were added to the cell monolayer in tri-Botros et al.: Synthesis, Characterization and Cytotoxicity ... plicate and wells were prepared for each individual dose.Monolayer cells were incubated with the compounds for 48 h at 37 °C in atmosphere of 5% CO 2 .After 48 h, cells were fixed, washed and stained with Sulforhodamine-B stain.Excess stain was washed with acetic acid and the attached stain was recovered with Tris EDTA buffer.Color intensity was measured in an ELISA reader.The relation between surviving fraction and drug concentration was plotted to get the survival curve of each tumor cell line.IC 50 values (the concentration required for 50% inhibition of cell viability) were calculated using sigmodial dose response curve-fitting models (GraphPad, Prizm software incorporated), each concentration was repeated three times.The results are given in Table 1 and represented graphically in Fig. 3.

1. Chemistry
The synthetic strategies adopted for the synthesis of the intermediate and final compounds are illustrated in Schemes 1 and 2. In Scheme 1, the starting compound ethyl 2-amino-4,5,6,7-tetrahydro [1]benzothiophene-3carboxylate (1) was prepared according to the well-known Gewald procedure. 32Reacting 1 with benzyl isothiocyanate in acetonitrile afforded the corresponding 3-benzyl-2-sulfanylthienopyrimidine derivative 2. The 2 formed was treated with 99% hydrazine hydrate in dry pyridine to give the 2-hydrazino derivative 3. Structural elucidation of 3 was based on IR and 1 H-NMR spectroscopy.Reacting the key intermediate 3 with formic acid or acetic acid induced cyclization to the corresponding triazolo derivatives 4a and 4b.IR and 1 H-NMR spectra confirmed the cyclization through the disappearance of NH and NH 2 signals.The presence of a signal at δ 12.07 ppm in 13 C-NMR verified the presence of the CH 3 group in 4b.Compound 5 was obtained upon treatment of 3 with chloroacetyl chloride in dry DMF.The notable feature in the 1 H-NMR spectrum was the appearance of a singlet peak at δ 5.18 ppm indicating CH 2 Cl group.The successful formation of the intermediate 5 prompted us to investigate the nucleophilic replacement of the active chlorine atom with different amines.Compound 5 underwent nucleophilic substitution with various substituted piperazines to afford 6a-d.The 1 H-NMR spectra of the products 6a-d showed the appearance of the protons of the piperazine moiety in the range of δ 2.43-3.19ppm.Moreover, a singlet signal at δ 3.94-3.96ppm characteristic to the CH 2 linking the triazole ring and the piperazine ring confirmed the successful incorporation of piperazine moieties.
Compound 7 was obtained in good yield by heating the key intermediate 3 with N,N-carbonyldiimidazole in dry benzene.IR spectrum of 7 showed absorption bands at ν 1720 and 1632 cm -1 indicating the presence of two C=O groups of the triazole ring and the pyrimidinone ring, res-pectively.Furthermore, the 1 H-NMR spectrum displayed an exchangeable singlet signal at δ 12.0 ppm corresponding to the NH proton of the triazole ring. 13C-NMR spectrum of 7 showed two signals at δ 149.17 and 156.63 ppm confirming the presence of two carbonyl moieties.Furthermore, the reaction of 3 with various isothiocyanates yielded the corresponding 1-substituted aminotriazolo derivatives 8a-e. 1 H-NMR spectra of 8a-e showed D 2 O exchangeable signals in the range of δ 9.29-9.39ppm assignable to the NH.
On the other hand, reacting 3 with diethyl malonate in acetic acid afforded the unexpected product 1-methyltriazolo derivative 4b.The formation of 4b may be explained by the hydrolysis and decarboxylation of the ester group in the intermediate compound 9 in acidic medium.However, the direct interaction of 3 with excess diethyl malonate in the absence of solvent at the refluxing temperature afforded the expected product 9.The IR spectrum showed the presence of two C=O moieties at ν 1740 and 1666 cm -1 while the 1 H-NMR spectrum confirmed the presence of the ethyl ester group.Further evidence was obtained from the 13 C-NMR spectrum of 9 which confirmed the presence of ethyl ester group through signals at δ 14.45 and 62.0 ppm in addition to a signal at δ 32.31 ppm corresponding to the -CH 2 -flanked between the thienopyrimidin-2-ylsulphanyl group and carbonyl function.Furthermore, the reaction of 3 with benzoyl chloride and ammonium thiocyanate in dry acetone afforded the benzoyl thiourea derivative 10. 1 H-NMR spectrum of compound 10 showed the presence of three D 2 O exchangeable signals assignable to three NH moieties at δ 9.79, 11.71 and 12.49 ppm.
In Scheme 2, the reaction of 3 with carbon disulfide in ethanolic potassium hydroxide followed by acidification with hydrochloric acid yielded the thiol (11) / thione (11a) tautomers.One of the objectives of this work was to prepare a series of S-alkylated triazolopyrimidine derivatives with varying the linker skeleton as well as varying the bioactive amine to test their cytotoxicity.
Herein, a series of alkylated mercapto 1,2,4-triazoles was synthesized via the reaction of the key intermediate 11 with various alkyl halides or αand β-chloroamides ( 13a-d, 14a-d, 15a-d) in absolute ethanol in the presence of anhydrous sodium acetate to afford the corresponding S-alkyl derivatives ( 12a,b, 16a-d, 17a-d, 18a-d).The success of alkylation was confirmed by the absence of SH or NH signals in 1 H-NMR spectra of 12a and 12b together with the appearance of peaks characteristic to methyl and ethyl moieties in each compound, respectively.Moreover, the mass spectrum of 12a,b showed their corresponding molecular ion peaks at m/z 382 and 396, respectively.
The structure of the mercapto alkylated derivatives 16a-d, 17a-d and 18a-d linked to different secondary amines with different linkages was supported by elemental analyses and spectral data.IR spectra of all target compounds indicated the appearance of new amide C=O absorption band at ν 1629-1643 cm -1 .Moreover, 1 H-NMR spectra of compounds 16a-d, 17a-d and 18a-d showed the disappearance of the SH signal at δ 14.06 ppm.Besides, the alkyl protons in the linker between the triazolopyrimidine ring and the amine appeared as follows;  CH 2 -CO moieties, respectively.In addition, 1 H-NMR spectra of all the target products 16a-d, 17a-d and 18a-d displayed the expected signals of the morpholino, 4-chloroanilino and substituted piperazine moieties.

2. In vitro Cytotoxicity
The in vitro cytotoxic activity of 24 selected compounds was evaluated against two human cancer cell lines including cells derived from human prostate cancer (PC-3) and human colon cancer (HCT-116) according to the standard protocol for IC 50 determination.Doxorubicin (DOX), being one of the most effective anticancer agents, was chosen as the reference standard anticancer drug. 42he IC 50 values in μM are listed in Table 1 and the results are represented graphically in Fig. 3.
From the results in Table 1 it is evident that most of the tested compounds displayed moderate to potent cancer cell growth inhibition.Generally, all the tested compounds tended to be more active against HCT-116 than against PC-3.Examining the IC 50 of the tested compounds against PC-3 cell line revealed that compounds 10, 12b, 17b and 18c exhibited significant anticancer activities with lower IC 50 values compared to DOX, with compound 16c being the most potent with an IC 50 of 5.48 μM.Meanwhile, compound 12a showed equipotent activity to DOX, while compounds 6a, 6c, 8a, 17c and 18a exhibited IC 50 values (ranging from 8.25-8.97μM) very close to DOX (IC 50 = 7.7 μM) against PC-3.As for the HCT-116 cell line, compounds 6c and 18c were the most active (IC 50 = 6.56 and 6.12 μM, respectively) in contrast to 15.82 μM for the standard on the same cell line.In addition, compounds 4a, 5, 6a, 8a, 8b, 8d, 10, 12b, 17b, 17c, and 18a displayed more potent cytotoxic activity compared to the standard with IC 50 values ranging from 7.4 to 14.77 μM.Referring to the IC 50 values listed in Table 1, the following SAR can be deduced: among the triazolo derivatives 4a and 4b, the unsubstituted derivative 4a showed Botros et al.: Synthesis, Characterization and Cytotoxicity ... good activity against HCT-116.Concerning the piperazine derivatives 6a-d, compounds 6a and 6c displayed good activity against both cell lines whereas the 4-chlorophenyl piperazine derivative 6c showed 2.4 fold higher activity than DOX against HCT-116 cell line in agreement with the reported anticancer activity of derivatives incorporating piperazine scaffolds and halogen atoms. 43,44Upon analyzing the results of the substituted amino triazoles 8a-e, compounds 8a, 8b and 8d exhibited higher activity than DOX against HCT-116 but it was difficult to reach conclusions regarding the effect of varying the substituent since the cytotoxicity of 8a-e was almost the same.The N-methyl derivative 8a was the only potent analogue against PC-3 cell line.Interestingly, compound 10 displayed potent cytotoxic activity against both cell lines in accordance with the reported antitumor activity of thiosemicarbazide derivatives. 456][27] Based on the above findings, we investigated herein in Scheme 2, the structure-activity relationship of S-alkylated series of compounds 12a,b, 16a-d, 17a-d and 18a-d, focusing in particular on the effect of the linker skeleton as well as varying the bioactive amine on the cytotoxic activity, the following was observed: -The incorporation of ethyl substituent in 12b resulted in a more potent derivative than 12a against both cell lines.-Among compounds 16a-d with CH 2 linker, the 4-phenyl piperazine analogue 16c showed selective high activity against PC-3.-The cytotoxic activity of compounds 17a-d with branched alkyl linker (-CHCH 3 ) showed that the incorporation of morpholine ring (17b) and phenyl piperazine moiety (17c) resulted in compounds with potent activity against both cell lines.-The phenyl piperazine derivatives (16c and 18c) afforded better cytotoxic activity compared to other amines against PC-3 and HCT-116, respectively.-Extending the side chain caused pronounced change in the activity of the 4-chloroaniline derivative 18a against both cell lines compared to 16a with acetamide linkage and 17a with branched linker which were devoid of activity.

Conclusion
A series of substituted 4-benzyl [1]benzothieno[3,2e] [1,2,4]triazolo [4,3-a]pyrimidines was designed, synthesized, and screened for their anticancer activity against PC-3 and HCT-116 cell lines.Many of the newly synthesized compounds showed remarkable activity on the tested cell lines with higher sensitivity towards the HCT-116 cell line.Compounds 10, 12b, 17b and 18c sho-wed higher cytotoxic activity against both PC-3 and HCT-116 cell lines compared to DOX.Incorporation of a 4-phenylpiperazine moiety resulted in higher activity against both cell lines where compound 16c was the most active against PC-3 with 1.4 fold higher activity than DOX, while 18c showed 2.5 fold higher anticancer activity against HCT-116.The obtained results suggest that thienopyrimidines containing 1,2,4-triazole scaffold might be suitable candidates for further chemical modifications in order to obtain more potent and selective anticancer agents.

Figure 1 .
Figure 1.Chemical structures of anticancer drugs containing triazole moiety available on the market.

Table 1 .
Results of in vitro cytotoxic activity of some selected compounds against human prostate cancer cell line (PC-3) and colon cancer cell line (HCT-116).(Results in bold represent compounds with better activity than DOX.) * The values given are means of three experiments.