Synthesis and Cytotoxic Evaluation of Some Novel Quinoxalinedione Diarylamide Sorafenib Analogues

Research in Pharmaceutical Sciences, April 2018; 13(2): 168-176 School of Pharmacy & Pharmaceutical Sciences Received:September 2017 Isfahan University of Medical Sciences Accepted: January 2018 Original Article Synthesis and cytotoxic evaluation of some novel quinoxalinedione diarylamide sorafenib analogues Mojtaba Khandan, Sedighe Sadeghian-Rizi, Ghadamali Khodarahmi, and Farshid Hassanzadeh* Department of Medicinal Chemistry, School of Pharmacy and Pharmaceutical Science, Isfahan University of Medical Sciences, Isfahan, I.R. Iran Abstract A series of novel sorafenib analogues containing a quinoxalinedione ring and amide linker were synthesized. A total of 9 novel compounds in 6 synthetic steps were synthesized. Briefly, the amino group of p-aminophenol was first protected which then followed by O-arylation with 5-chloro-2-nitroaniline to provide compound d. Reduction of the nitro group of compound d and cyclization of the diamine group of compound e with oxalic acid afforded compound f which on deacetylation yeilded compound g. Then compound g was reacted with different acyl halides to afford the target compounds 1h-1p. Chemical structures of synthesized compounds were confirmed by 1H NMR and FT-IR analysis. All compounds were evaluated at 1, 10, 50 and 100 μM concentrations for their cytotoxicity against HeLa and MCF-7 cancer cell lines. Some of the compounds showed good cytotoxic activity, especially compounds 1i and 1k-1n with the IC50 values of 19, 16, 22, 18, and 16 µM against MCF-7 cell line and 20, 18, 25, 20, and 18 µM against HeLa cell line, respectively. Keywords: Cytotoxicity; Sorafenib; Quinoxalinedione; Amide INTRODUCTION cancer types such as metastatic colorectal, brain, leukemia, breast, glioblastoma, Cancer, one of the most serious illnesses, advanced gastric, cervical, thyroid, non-small considered the second leading cause of death cell lung cancer, prostate, bladder and worldwide after cardiovascular diseases. neuroendocrine cancers. Sorafenib prevents According to the World Health Organization tumor growth by the inhibition of C-RAF and (WHO) reports, more than 13 million dying of B-RAF serine/threonine kinase activities and cancer worldwide are awaiting to happen in tumor angiogenesis by canceling vascular 2030 (1-3). endothelial growth factor receptor (VEGFR) Many research articles have lately reported and platelet derived growth factor receptors the potential antiproliferative activity of signaling. However, therapy at full dose (800 diarylureas and diarylamides against some mg/day) induces a number of side effects cancer cell lines. Thus far, with the same which emphasize on the urgent need to origin nine drugs including sorafenib, optimize the effectiveness and reduce the side sunitinib, pazopanib, axitinib, vandetanib, effects of sorafenib (9). regorafenib, lenvatinib, nintedanib, and In this research, based on previous structure cediranib have been approved for cancer activity relationships (SARs) of sorafenib and therapy. (4-7). its derivatives, our main correction focused Sorafenib (Nexavar) is an example of on replacement of N-methylpicolinamide anticancer diarylurea approved by the U.S. group of sorafenib with quinoxalindione (10) Food and Drug Administration (FDA) for (Fig. 1). therapy of advanced renal cell carcinoma and advanced hepatocellular carcinoma (8). It is also exposed to clinical experiments for other *Corresponding author: F. Hassanzadeh Tel: +98-3137922575, Fax: +98-3136680011 Email: [email protected] Synthesis and cytotoxic evaluation of some novel diarylamide sorafenib analogues Fig. 1. The design concepts of the targeted compounds. Nitrogen atoms in the N-methylpicolinamide deuterated dimethyl sulfoxide (DMSO-d6) as a moiety of sorafenib are necessary for binding solvent on Bruker 400 MHz spectrometer to ATP-binding pocket of kinase. using tetramethylsilane (TMS) as an internal Quinoxalindione is an aromatic heterocycle standard. All chemical shifts are given in containing two nitrogen atoms in its structure δ scale (ppm). Infrared (IR) analyses (KBr which is comparable with N-methylpicolinamide disks) were done using a Bruker Fourier moiety in terms of size and polarity (11,12). transform infrared spectroscopy (FT-IR) Replacing the urea linker with an amide instrument (Bruker Bioscience, Billerica, maintains hydrogen bonding with VEGFR-2 MA, USA). while reduce the flexibility of compounds (Fig. 1). Quinoxaline and its derivatives has Details of preparation procedures and broad spectrum of pharmacological activities chemistry of synthesized compounds such as anti-HIV, anti-inflammatory, and Synthesis of N-(4-hydroxyphenyl)acetamide (b) anti-cancer activity. Therefore, considerable para-amino phenol (a) (229 mmol, 25 g), biological activities of quinoxalindione water (68 mL) and acetic anhydride (29 mL, derivatives make it a good moiety in drug 263 mmol) were stirred for 2 h in 100 ºC to design (13-16). produce a uniform brown solution. The In this work, the structure of novel sorafenib container was placed in an ice bath and when analogues containing a quinoxalindione ring the crystals were formed, filtered and washed with amide linker were synthesized and with cold water. confirmed by proton nuclear magnetic resonance (1H NMR) and Fourier transform Synthesis of N-(4-(3-amino-4-nitrophenoxy) infrared (FT-IR) (Fig. 1). Cytotoxic effects of phenyl)acetamide (d) compounds on MCF-7 (17) and HeLa (18) N-(4-hydroxyphenyl)acetamide (b) (32.98 cell lines were evaluated by 3-(4,5- mmol, 4.98 g), was added to NaOH (33 mmol, dimethylthiazol-2-yl)-2,5-diphenyltetrazolium 1.32 g), water (3 mL) and DMSO (11 mL) the bromide (MTT) assay method. mixture was stirred for 30 min. 5-chloro-2- nitro-aniline (c) (29.82 mmol, 5.13 g) was MATERIALS AND METHODS added to the mixture and stirred overnight at 100 °C in oil bath. The reaction mixture was Instrumentation poured onto aqueous NaOH (2 M) to form a All starting materials, reagents, and solid, which was collected by filtration, solvents were purchased from commercial washed with water and dried to give suppliers of Merck (Germany) and Aldrich compound d. (USA) companies. The purity of the prepared compounds was proved by thin layer Synthesis of N-(4-(3,4-diaminophenoxy) chromatography (TLC) using various solvents phenyl)acetamide (e) of different polarities. Proton nuclear magnetic N-(4-(3-amino-4-nitrophenoxy) phenyl) resonance (1H NMR) spectra were taken in acetamide (d) (19.51 mmol, 5.6 g) was 169 Khandan et al. / RPS 2018; 13(2): 168-176 dissolved in ethanol (140 mL) and water solvent was removed in vacuo. To the obtained (55 mL). Then sodium dithionite (14 g, oily residue was added chloroform (2 mL) and 80 mmol) was added and the mixture of the stirred for 5 min to form a solid. The reaction was refluxed with intense stirring precipitate was filtered with dichloromethane, until the yellow color of mixture converted to ethyl acetate, and diethyl ether to give the brown. Then the reaction mixture was cooled target compounds 1h-1p. down to room temperature. The reaction mixture was filtered and evaporated to Cell culture condense. The mixture was extracted with Two cell lines were used in this study water and ethyl acetate. The organic layer was including Michigan Cancer Foundation-7 dried by magnesium sulfate and the solvent (MCF-7) and Henrietta Lacks (HeLa). Cell was evaporated to afford the desired product. lines were obtained from the National Cell Bank of Iran (Pasteur Institute, Tehran, Iran). Synthesis of N-(4-((2,3-dioxo-1,2,3,4-tetra- The cells were cultured in RPMI 1640 (Gibco, hydroquinoxalin-6-yl)oxy)phenyl)acetamide Scotland ) supplemented with 10% fetal (f) bovine serum (FBS) (Gibco, Scotland ) and A mixture of oxalic acid (1 mmol), water (1 100 Units/mL penicillin-G and 100 µg/mL mL) and HCl (1.5 mL, 0.75 M) was heated to streptomycin at 37 °C in humidified air 100 °C. Then N-(4-(3,4-diaminophenoxy)-2- containing 5 % CO2. 4, 5-dimethylthiazole-2- methylphenyl)acetamide (e) (1 mmol, 0.257 g) yl, 2, 5-diphenyl tetrazolium (MTT) was from was added and stirred for 2 h. The progress of Merck (Germany). RPMI1640, Dulbecco's the reaction was monitored by TLC until the phosphate buffered saline (DPBS), and reaction was complete. The reaction mixture penicillin-G/streptomycin were products of was put in ice bath and the solid Biosera (Ringmer, UK). precipitate (compound f) was filtered and washed with water. MTT assay MCF-7 and HeLa cells were seeded in 96- Synthesis of 6-(4-aminophenoxy)-1,4-dihydro- well plate at a density of 5 × 104 cell/mL, quinoxaline-2,3-dione (g) respectively and incubated for 24 h. A mixture of N-(4(2,3-dioxo-1,2,3,3- Doxorubicin was used as the positive control tetrahydroquinoxalin-6-yloxy)-2-methylphenyl) and the wells containing DMSO (1%) and cell acetamide (compound f) (2800 mg, 8.99 suspension was regarded as the negative mmol), 125 mL ethanol/HCl (3 M) was controls. Then 20 µL of various concentrations refluxed for 2 days. The progress of the of target compounds (1, 10, 50, 100 µM) were reaction was monitored by TLC until the added and plates were incubated for 48 h. reaction was completed. The reaction mixture After the treatment period, 20 µL of MTT was put in ice bath and the pH was adjusted to 0.5% (Merck, Germany) was added and 8 by ammonia solution. The solid precipitate further incubated for 3 h. Then supernatant of (compound g) was filtered and washed with each well was replaced with 150 µL of DMSO water. (Merck, Germany) to dissolve the formed formazan crystals. The absorbances were General procedure for synthesis of target determined at 570 nm with an enzyme-linked compounds (1h-1p) immunosorbent assay (ELISA) plate reader 6-(4-aminophenoxy)-1,4- (BioTek, USA). Each assay was carried out dihydroquinoxaline-2,3-dione(45 mg, 0.169 three times at three different days, and the mmol) and diisopropylethylamine (35 µL, results of the experiment were summarized in 0.203 mmol) were mixed in dry Fig.

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