Abstract
In this study, some N-[4-(Benzothiazole-2-yl) phenyl]-2-aryloxyacetamide derivatives were prepared by reacting N-[4-(benzothiazole-2yl)phenyl]-2-chloroacetamide and different substituent phenol or thiophenol derivatives. The anticancer activities of the compounds obtained were investigated. It was observed that some of the compounds, namely 25 and 38, showed notable anticancer activity.
Introduction
Cytotoxic agents conventionally used in cancer treatment (conventional phase-specific purine, pyrimidine bases and non-phase-specific alkylating agents) are known to be toxic and have been criticized because of this, but will continue to be used until the introduction of less harmful agents. Another limitation of cancer treatment is that resistance to anticancer agents develops in tumor cells during clinical use. In addition to the aforementioned drugs, intensive investigation continues into new compounds targeting cancer-specific proteins and affecting different mechanisms, such as topoisomerase and telomerase inhibitionCitation1–3.
In previous studies, 2-(4-aminophenyl) benzothiazole derivatives I were shown to have high antitumor activity in vitro and in vivoCitation4–6, mainly against breast, renal, colon, ovarian, lung and prostate cancers, despite their simple structuresCitation7–10. Analysis of structure-activity relationships showed that benzothiazole residues were essential for high activity. A phenyl ring on the 2-position of benzothiazole and alkyl and halogen residues on the 3-position were shown to be effective in increasing activityCitation10. The binding of aminophenyl benzothiazoles to β-amyloid using for has been used for imaging and therapy in Alzheimer’s diseaseCitation11–12. The several differentially-substituted 2′-(3-aminophenyl)- and 2′-(4-aminophenyl) benzothiazoles were prepared as building blocks and their inhibitory activities were determined on kinase by initially testing in vitroCitation13. In addition, other notable structures in terms of anticancer activity are compounds of aryloxy-acetamido-thiazole II residue. These compounds were reported to be effective therapeutic agents in prostate cancerCitation14.
In this study, 2-(aminoaryl)benzothiazole and aryloxyacetamide residues were combined in a single structure in the light of the data above, N-[4-(Benzothiazole-2-yl)phenyl]-2-aryloxyacetamide compounds were obtained and their effects were evaluated against nine cancer types comprised of approximately sixty cell-lines.
Experimental
Chemistry
The synthesis of 2-(4-aminophenyl)benzothiazole molecules was done using Milestone microwawe reaction apparatus. Melting points were determined by using an Electrothermal IA9000 digital melting point apparatus. Spectroscopic data were recorded on the following instruments: a Bruker Tensor 27 IR spectrophotometer; a 1H-NMR (nuclear magnetic resonance) Bruker DPX-400 FT-NMR spectrophotometer, and an MS (mass spectroscopy) Agilent 1100 MSD spectrophotometer. Analyses for C, H and N were within 0.4% of the theoretical values. 4-(benzothiazole-2-yl)phenylamine and N-(4-benzothiazole-2-yl)phenyl-2-chloroacetamide were prepared according to the methods described in the literatüreCitation1,Citation4. Some characteristics of the compounds are given in and the processes for their synthesis are shown in .
Table 1. Some characteristic of the compounds.
Scheme 1. Synthesis of compounds 3–39. Reagents and conditions: (a) PPA/MW, 125°C, 35 minutes (b) DMF/Et3N (c) acetonitril, K2CO3, heating at reflux.
General method for the preparation of N-[4-(benzothiazole-2-yl)phenyl)-2-phenoxyacetamide and N-[4-(benzothiazole-2-yl)phenyl)-2-thiophenoxyacetamide derivatives 3–33 and 34–39
A mixture of N-[4-benzothiazole-2-yl)phenyl]-2-chloroacetamide (1.65 mmol, 0.5 g), the appropriate substitue phenol or thiophenol derivatives (1.98 mmol) and K2CO3 (1.98 mmol, 0.3 g) in acetonitril was refluxed for 6 hours. The cooled mixture was filtered and recrystallized from DMSO/alcohol.
The characterization of compounds (25 and 38) showing high activity are given below. The remaining final compounds characterization are given as supporting data.
25: IR(KBr)υmax (cm−1): 3399 (N-H), 3050 (aromatic C-H), 2904 (aliphatic C-H), 1689 (C=O), 1605, 1539, 1499 (C=C, C=N), 1300-1000 (C-N), 1245 (C-O-C), 596 (C=C-S), 664 (C-S) NMR(400 MHz)(DMSO-d6) δ (ppm): 3.72 (3H, s, OCH3) 4.8 (2H, s, (-O-CH2-)), 6.89 (2H, d, J:8.78 Hz, Ar-H), 7.05 (2H, d, J:8.34 Hz, Ar-H), 7.44 (1H, dt, J:7.62 Hz, J:7.52Hz, benzothiazole, C6-H), 7.55 (1H, dt, J:7.64 Hz, J:7.49Hz, benzothiazole,C5-H), 7.78 (1H, dd, J: 1.66 Hz, J: 1.64 Hz, Ar-H), 7.82 (1H, s, Ar-H), 8.12 (1H, d, J: 7.67 Hz, benzothiazole C7-H), 8.21 (1H, d, J:7.68 Hz, Ar-H), 8.38 (1H, d, J: 8.29 Hz, benzothiazole C4-H), 9.36 (1H, s, NH) MS (ES+): 425.1 (100%) M+1, 426.1 (26%) M+2, 427.1 (40.3%) M+3, 428.1 (10%) M+4.
38: IR(KBr)υmax (cm−1): 3249 (N-H), 3079 (aromatic C-H), 2916 (aliphatic C-H), 1683 (C=O), 1600, 1531, 1477 (C=C, C=N), 1300-1000 (C-N), 734 (C=C-S) 1H-NMR(400 MHz) (DMSO-d6) δ (ppm): 3.94 (2H, s, (-S-CH2-)), 7.5 (5H, m, Ar-H, benzothiazole, C6-H), 7.59 (1H, dt, J:8.26 Hz, J: 8.26 Hz, benzothiazole C5-H) 7.64 (1H, dd, J:2.11 Hz, J: 2.05 Hz, Ar-H), 8.02 (1H, d, J:2.04 Hz, Ar-H), 8.1 (1H, d, J:8.03 Hz, benzothiazole C7-H), 8.19 (1H, d, J:7.81 Hz Ar-H), 8.27 (1H, d, J: 8.69 Hz, benzothiazole C4-H), 10.70 (1H, s, NH) MS (ES+): 445(100%) M+1, 446 (27%) M+2, 447 (74%) M+3, 448(19%) M+4, 449 (20%) M+5.
Anticancer activity
The cyctotoxic and/or growth inhibitory effects of the compounds were evaluated in vitro against approximately 60 human tumor cell lines derived from nine neoplastic diseases, namely: leukemia (L), non-small cell lung cancer (NSCLC), colon cancer (CC), central nervous system cancer (CNSC), melanoma (M), ovarian cancer (OC), renal cancer (RC), prostate cancer (PC) and breast cancer (BC). The evaluation of anticancer activity was performed at the National Cancer Institute (NCI), Bethesda, USA. The in vitro screening program was based upon the use of multiple panels of 60 human tumor cell lines, against which our compounds were tested at 10-fold dilutions of five concentrations ranging from 10−4 to 10−8 M. The percentage growth was evaluated spectrophotometrically against controls not treated with test agents. A 48-hour continuous drug exposure protocol was followed and a sulforhodamine B (SRB) protein assay was used to estimate cell growthCitation15.
Result and discussion
Chemistry
N-[4-benzothiazole-2-yl]phenyl]-2-aryloxyacetamide derivatives were synthesized using the sequence of reactions depicted in . The initial compounds, 2-(4-aminophenyl)benzothiazoles 1a-d, were prepared via polyphosphoric acid mediated oxidative condensation of 2-aminothiophenol with 4-aminobenzoic acid in microwave conditions.
N-[4-(benzothiazole-2-yl)phenyl]-2-chloroacetamide compounds 2a-d were prepared by reacting 2-(4-aminophenyl)benzothiazole and chloroacetyl chloride in triethylamine and DMF to produce the halides.
The two groups of final compounds are N-[4-(benzothiazole-2-yl)phenyl)-2-phenoxyacetamide derivatives and N-[4-(benzothiazole-2-yl)phenyl)-2-(phenylthio) acetamide derivatives, derivative numbers 3–33 and 34–39 respectively. N-[4-(benzothiazole-2-yl)phenyl)-2-phenoxyacetamide derivatives 3–33, were synthesized by reacting N-[4-(benzothiazole-2-yl)phenyl]-2-chloro acetamide and appropriate substituent phenols in acetonitrile solvent. The other derivatives, 34–39, were synthesized by reacting N-[4-benzothiazole-2-yl)phenyl]-2-chloro acetamide and appropriate substituent thiophenols in acetonitrile solvent. The structures of the compounds obtained were elucidated using spectral data. In the IR spectra, characteristic amide carbonyl functions were observed in the 1709-1670 cm−1 region, both separately and as a single band. The NMR spectra of compounds 3–33 exhibited singlets resulting from resonances of the N-[4-(benzothiazole-2-yl)phenyl)-2-phenoxyacetamide residue assigned to −O-CH2- protons at 4.7-4.8 ppm, and N-H protons at 9.3–10.92 ppm. N-[4-(benzothiazole-2-yl)phenyl]-2-phenylthio) acetamide derivative residue was assigned to −S-CH2- protons at 3.85-3.94 ppm and N-H protons at 9.61-10.70 ppm. For the other compounds, the same protons were taking part in multiplets, because they were overlapping with aromatic and benzothiazole protons.
Anticancer activity
In the first step, compounds 3, 7, 13, 17, 20, 25, 26, 35 and 38 were selected by NCI for the anticancer tests. The selected compounds were evaluated in vitro against 60 human tumor cell lines derived from nine neoplastic diseases and the test results were determined as growth percentage values for 10−5 M concentration. The obtained growth percent values are shown in .
Table 2. Anticancer activity of some compounds as % growth.
Compounds 25 and 38 showed notably low growth of 29.64% and 30.93%, respectively, against CNSC and PC cell lines. With respect to mean values, the same two compounds exhibited the lowest growth percent values of 50.94 and 59.73%, respectively. As required by the test methods, the compounds with a growth percentage lower than 75% were accepted for the further screening test. Thus, compounds 25 and 38 were used in the second stage of the study, in which the selected compounds were tested at 10-fold dilutions of five concentrations ranging from 10−4 to 10−8 M. The results are given as logGI50 (GI50: growth inhibition of 50%). The detailed test results are given in .
Table 3. Mean logGI50 values of compounds 25 and 38 and control anticancer agents.
The test method stated that compounds having logGI50 values greater than −4 are considered as inactive. It can be seen that the logGI50 values for the compounds are less than −4. Therefore, we may conclude that the two compounds under investigation provide a notable activity level. Melphalan and cisplatin (cis-diaminodichloroplatinum) are two commonly used chemotherapeutic agents and are used as control anticancer agents. When the mean graph midpoint (MG-MID) values of the compounds melphalan and cisplatin (i.e. −5.09 and −6.20, respectively) are considered, it is observed that the two compounds provide high activity levels. The MG-MID values of the compounds are lower than that of the control compound, melphalan.
The present study analyzed the effect levels of 2-(aminoaryl)benzothiazole and aryloxyacetamide, two groups of compounds known to have an anticancer effect, after their residues were combined in a single structure. However, although the obtained compounds showed relatively high activity values, they did not achieve the same activity level as compounds containing both of the residues separately within their structures.
Compound 25 contained R: 2-OCH3 and R’: 4-Cl as a substituent and compound 38 contained R: 3-Cl and R’: 4-Cl as substituents. Moreover, while compound 25 has an aryl ether structure, Compound 38 is an aryl thioether. Therefore, it is remarkable that the common feature of these most effective two compounds is the R’ group being 4-CI. Except for this feature, it does not seem possible to establish a structure-activity relationship according to the structure of the compounds and substituents.
Acknowledgements
This work was supported by the Commission of Scientific Research Projects of Eskisehir Osmangazi University (ESOGU/200819010). The authors gratefully acknowledge the financial support by Eskişehir Osmangazi University. Authors also would like to thank to National Cancer Institue (NCI), Bethesda, MD, USA for in vitro screening of our compounds in human cancer cell lines.
Declaration of interest
The authors report no conflicts of interest.
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