Synthesis and in vitro Anticancer Activity of Novel Heterocycles Utilizing Thiophene Incorporated Thioureido Substituent as Precursors

Abstract: Ethyl 2-(3-allylthioureido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (1) was used as a building block for synthesis of new heterocycles. Pyrimidine and thiazole moieties were achieved upon condensation of compound 1 with various reagents such as chloroacetic acid, dietyl malonate, ninhydrin, 2,3-epoxy-2,3-dihydro-1,4-naphthoquinone, and hydrazine hydrate. The structures of the newly synthesized compounds were confirmed using spectral measurements. The prepared products were evaluated for their anticancer activity against colon HCT-116 human cancer cell line. Compounds 6, 9, 10a, 11, 12, 15 have displayed potent activity. Ethyl 2-(3-allylthioureido)-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate (1) was used as a building block for synthesis of new heterocycles. Pyrimidine and thiazole moieties were achieved upon condensation of compound 1 with various reagents such as chloroacetic acid, dietyl malonate, ninhydrin, 2,3-epoxy-2,3-dihydro-1,4-naphthoquinone, and hydrazine hydrate. The structures of the newly synthesized compounds were confirmed using spectral measurements. The prepared products were evaluated for their anticancer activity against colon HCT-116 human cancer cell line. Compounds 6, 9, 10a, 11, 12, 15 have displayed potent activity.


Introduction
Heterocyclic compounds containing thiophene ring have been widely reported to have numerous pharmaceutical importance. For example, such compounds exhibited anticancer, 1-4 antibacterial, 5 antifungal, 6 anti-inflammatory, 7 anti-ulcer, 8 anti-diabetic, 9 antileishmanial, 10 antimicrobial, 11,12 antitubercular, 13 COX-2 selective inhibition, 14 antiproliferative 15 activities. Thiophene derivatives were utilized as inhibitors for hepatitis C virus polymerase, 16 novel BACE1, 17 alkaline phosphatases, 18 and kinesin spindle protein 19 and they are more effective in the treatment of Alzheimer's disease. 20 Moreover, thiophene derivatives have acquired great importance in drug discovery studies [21][22][23] which are available in markets, such as methaphenilene that acts as antiallergic agent; tiagabine acts as anticonvulsant agent; Sertaconazole acts as anti-fungal drug and is available as a cream for treatment of skin infections, such as athlete's foot, and the important drug biotin which is used for treating the deficiency of biotin related to pregnancy. The interest in these heterocycles has been attributed to their promising utilization as dye-sensitized solar cells, 24 organic semiconductors, 25 potential light-emitting materials for OLEDs 26 and in textile dyeing. 27,28 Accordingly, several methods for the synthesis of thiophene and related heterocycles earn great attention. Normally, the most important method for their preparation is Gewald method involving the reaction of an equimolar amount of elemental sulfur with α-methylene ketones and acetonitrile in a basic medium. 29,30 Polyfunctionalized thiophenes were synthesized via the multicomponent reactions from starting materials, such as β-ketodithioesters with cyclohexylisocyanide and α-haloketones. 31 Two-step synthesis of benzothiophene derivatives using iodocyclization followed by etherification reaction sequence has been reported. 32 On the other hand, incorporation of thiourea chain residues was used to obtain key intermediates necessary to produce important heterocyclic compounds. [33][34][35] It has been reported that compounds containing pyrimidine [36][37] and thiazole [38][39] moieties have potent anticancer activity. According to these facts, we decided to develop the synthesis of new heterocycles utilizing thiophene moiety bearing thioureido chain substituent as a building block and to characterize the prepared products by spectral and analytical techniques. The activity of the newly synthesized heterocyclic compounds against HCT-116 cell lines was tested.

1. General
Most reagents and chemicals were purchased, utilized without additional purification, and bought from Sigma-Aldrich. Melting points are uncorrected and an electrothermal apparatus was used for their measurements. The purity of reagents and reaction validation were checked by thin-layer chromatography (TLC) technique on silica gel plates and the spots were imagined under UV light (254 nm) using mobile phase (pethroleum etherethyl acetate). Infrared spectra were detected using KBr discs on Shimadzu FT-IR 8201 PC spectrophotometer. 1 H NMR and 13 C NMR spectra were recorded on a Bruker instrument at 850 MHz spectrometer using tetramethylsilane (TMS) as an internal standard in CDCl 3 or DMSO-d 6 as solvents; chemical shifts are expressed in δ (ppm); spectroscopic measurements were carried out in the Microanalysis unit at the Universities of Qassim and Abdul-Aziz (KSA). Mass spectra were recorded on a GCMS-QP1000 EX spectrometer at 70 eV. Anticancer analysis was performed at the National cancer institute (NCl), Cairo, Egypt. Starting materials were already prepared according to the reported method. 39 "

General Procedure for the Synthesis of Compounds 10a and 10b
A diazonium solution was prepared by dissolving (0.02 mol) of para-toluidine or 4-aminobenzophenone in 30 mL water and 6 mL concentrated HCl; this solution was cooled to 0 °C. The solution was then treated with 0.02 mol sodium nitrite in 20 mL of water, were added gradually with stirring for 30 min with cooling in an ice bath to complete the diazotization. The formed diazonium chloride was slowly added to compound 6 in pyridine with stirring at 0-5 °C for 2 h. The mixture was added with stirring to ice-cold water. The resulting solid was filtered off, dried and recrystallized from methanol. "

General Method for Synthesis of 17 and 18
Compound 1 (0.01 mol) was mixed with hydrazine hydrate (0.015 mol) in ethanol (30 mL). The resulting solution was refluxed for 8 h. After cooling, the precipitate that formed was filtered off and crystallized to give 18 as bright white needles; yield 90%; mp 280-283 °C. The filtrate was evaporated till 10 mL and then was left overnight to cool. The formed solid was filtered off, dried and crystallized from EtOH to give 17 as white crystals; yield 82%; mp 198-199 °C (EtOH). "

3. Cytotoxic Activity
Cytotoxicity of the synthesized compounds was evaluated against HCT-116 (colon adenocarcinoma) using MTT assay. 44 The prepared compounds were dissolved in DMSO (dimethylsulfoxide) and diluted 1000-times during the test. It is essential to enable cells to be attached to the wall of the plate. Prior to the treatment with the tested compounds, these cell lines were plated in 96-multi well plate (10 4 cells/well) for 24 h. Each well was supplemented with 100 μg/mL of the tested compounds. Under a 5% CO 2 atmosphere, the monolayer cells were incubated with the samples at 37 °C for 72 h. Then, 20 μL of MTT solution at 5 mg/mL has been added and incubated for 4 h after 24 h of drug treatment. The colorimetric assay is evaluated and registered using a plate reader at 570 nm absorbance. Determination of IC 50 (the half-maximal inhibitory concentration) for three samples of the most potent inhibitions. Calculation of IC 50 values along with the respective 95% confidence intervals by plotting the relationship between the surviving fraction and the sample concentration to obtain the cancer cell line survival curve.

1. Chemistry
Our target was to convert the open-chain thiourea substituent 1 into heterocycles to enhance their biological activity. The building block thiourea derivative 1 was synthesized through multicomponent synthetic route starting by Gewald reaction of ethyl cyanoacetate with cyclohexanone in the presence of elemental sulfur and then subsequent treatment of 1 with allyl thiocyanate. 40 Spectral and analytical data of the synthesized thiourea intermediate were compatible with reported results. Refluxing of 1 with chloroacetic acid in the presence of potassium hydroxide has unexpectedly afforded the corresponding thiazole derivative 4 rather than the thiazolidinone derivative 2 40 while it was expected that the formation of 2 through the reaction as the intermediate would be possible.
The mechanism suggested for the creation of 4 begins with the electronegative sulfur attack on the active methylene group which is an electron-deficient carbon, followed by cyclization to form thiazolidinone intermediate 2. Keto-enol tautomerism leads to enol form and then O-alkylation with another molecule of chloroacetic acid takes place. The spectral data of 4 supported our explanations (Scheme 1). IR spectrum of 4 revealed the disappearance of NH absorption band. 1 H NMR spectrum confirmed the structure while it showed the presence of a singlet signal at 11.9 ppm due to -COOH group beside the other signals of the compound. Also, its 13 C NMR spectral data revealed the presence of 19 signals. Mass spectrum showed molecular ion peak at m/z M + -CO 2 381 (20%) with a base peak at m/z 365 due to M + -CH 2 COOH fragment. One-pot reaction of 1 by cyclocondensation with chloroacetic acid and benzaldehyde in boiling acetic acid and acetic anhydride in the presence of sodium acetate produced the benzylidene-thiazolidinone 5 in good yield. Spectral data have confirmed its structure. 1 H NMR spectrum revealed the appearance of multiplet signals at 7.29-7.51 ppm due to the phenyl ring protons.
Cyclocondesation of the thiourea derivative 1 with diethyl malonate in the presence of sodium ethoxide in ethanol formed the pyrimidinone derivative 6. Structure of 6 was confirmed using IR, NMR and mass spectra. IR spectrum showed significant stretching bands at 1639 cm -1 due to the amidic carbonyls beside the lack of NH absorption bands. Its 1 H NMR spectrum accentuated the presence of singlet signal at 3.88 ppm due to the methylene protons in addition to the other expected signals.
The resulting pyrimidinone 6 underwent subsequent condensation with corresponding aromatic aldehydes, namely furfural and benzaldehyde and isatin in refluxing ethanol in the presence of piperidine yielding the arylidene derivatives 7a,b and 8, respectively. Mannich base 9 was formed in good yield via Mannich reaction of 6 by amino alkylation of its acidic protons which are placed between the two carbonyl groups with para-anisidine and formaldehyde. Further treatment of 6 with aryl diazonium chloride obtained from the suitable aromatic amines (para-toluidine and 4-aminobenzophenone) in pyridine furnished the corresponding hydrazones 10a,b (Scheme 2). The spectral and analytical data for compounds 7a,b-10a,b were matching with their expected structures. Their IR and NMR spectra revealed the absence of methylene bands and new bands appeared in accordance with the proposed structures. As well, mass spectra of these synthesized compounds showed molecular ion peaks compatible with their molecular mass.
Further reaction of thiourea intermediate 1 with benzoyl hydrazide in sodium ethoxide in boiling ethanol afforded the unexpected triazolopyrimidinone 14 in very good yield. The general mechanism suggested that this reaction proceeds via the formation of the thienopyrimidine 15 followed by condensation and cyclization after the removal of H 2 S gas. Isolation of 14 was confirmed; its synthesis utilizing an alternative route by cyclization of compound 1 to give 15 in DMF and in the presence of KOH with stirring 15 was synthesized previously 42 with a different method. All spectral and analytical data were compatible with the reported results. Finally, the starting compound 1 contains many active sites that led to expect various products. Accordingly, unexpected products were synthesized by treatment of 1 with hydrazine hydrate. However, it was observed that when compound 1 was treated with hydrazine hydrate in ethanol this resulted in the formation of the thienopyrimidine derivatives 17 and 18 which were separated easily from ethanol (Scheme 4). Compound 17 was prepared previously from the reaction of 15 with hydrazine hydrate in EtOH. The spectral and analytical data of the synthesized thienopyrimidine 17 were compatible with the reported ones. Evidence of thienopyrimidine 18 was gained from its spectral measurements. Its IR spectrum revealed the presence of NH stretching band 3239 cm -1 and demised of the ester band. 1 H NMR spectrum lacked the signals of -COOEt and allyl protons, in addition, it showed signals at 1.02, 4.17 and 4.37 ppm due to 3 CH 2 protons.

2. Anticancer Activity
The newly synthesized compounds 1-18 were preliminarily screened via in vitro anticancer screening in a single high dose (100 μg/mL) concentration against colorectal carcinoma (HCT-116) human cancer cell lines. The efficacy of anticancer activity in comparison to the standard drug 5-fluorouracil and the results of cytotoxic activity are in Table 1. As shown in the table, the majority of the synthesized compounds have low to good anticancer activity versus HCT116 cell line. The obtained results revealed that compounds 6, 9, 11, 12, and 15 showed the highest activity against the cell line; so these compounds were chosen to test at inhibitory concentration 50% (IC 50 ). The structure-activity relationship according to the results obtained, indicates that incorporation of pyrimidine moieties enhances the anticancer activity of the tested compounds. Compound 6 showed the most potent activity, indicating that the presence of the pyrimidinone ring increases the anticancer activity in comparison with pyrimidine moiety. While results for compounds 9 and 10a indicate that the introduction of para-tolyl substituent with the pyrimidine moiety will increase their anticancer activity. The highest activity of compounds 11 and 12 is expected to be due to the presence of 1,2-naphthoquinone moieties and two OH groups, respectively. It is known that introducing amidic group with pyrimidine moiety enhances the anticancer activity in which its structure is similar to the most important anticancer drugs, such as Imatinib; based on that, compounds 12 and 15 showed higher activity. "

Conclusion
In conclusion, a new series of pyrimidines and thiazoles bearing thiophene ring systems were synthesized. Their structures were characterized by spectral data (IR, NMR and mass spectra). Our products were assessed for their anticancer activity against colon HCT-116 human cancer cell line. Among all the synthesized compounds, compounds 6, 9, 10a, 12, 13, 16 displayed potent anticancer activity. The rest of compounds showed a moderate to weak activity against the tested tumor cell lines.