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Synthesis of New Quinolone Derivatives Linked to Benzothiazole or Benzoxazole Moieties as Anticancer and Anti-Oxidant Agents | OMICS International
ISSN: 2161-0444
Medicinal Chemistry

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Synthesis of New Quinolone Derivatives Linked to Benzothiazole or Benzoxazole Moieties as Anticancer and Anti-Oxidant Agents

Mohamed A Abdelgawad1,2*, Phoebe F Lamie1* and Osama M Ahmed3

1Pharmaceutical Organic Chemistry Department, Faculty of Pharmacy, Beni-Suef University, Beni Suef, Egypt

2Pharmaceutical Chemistry Department, College of Pharmacy, Al Jouf University, Sakaka, Al Jouf, Saudi Arabia

3Zoology Department, Faculty of Science, Beni-Suef University, Beni Suef, Egypt

*Corresponding Author:
Mohamed A Abdelgawad
Pharmaceutical Chemistry Department
College of Pharmacy, Al Jouf University
Sakaka, Al Jouf-2014, Saudi Arabia
E-mail: [email protected]
 
Phoebe F Lamie
Pharmaceutical Organic Chemistry Department
Faculty of Pharmacy, Beni-Suef University
Beni-Suef, Egypt
Tel: 002012233214514
E-mail: [email protected]

Received date: October 12, 2016; Accepted date: October 24, 2016; Published date: October 27, 2016

Citation: Abdelgawad MA, Lamie PF, Ahmed OM (2016) Synthesis of New Quinolone Derivatives Linked to Benzothiazole or Benzoxazole Moieties as Anticancer and Anti-Oxidant Agents. Med Chem (Los Angeles) 6: 652-657. doi:10.4172/2161-0444.1000410

Copyright: © 2016 Abdelgawad MA, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Abstract

A new series of substituted quinolones linked to benzothiazole and/or benzoxazole moieties 5a-l was synthesized. 6-Benzoxazol-2-yl/benzothiazol-2-yl-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid ethyl esters 3a&b were reacted with hydrazine to give the hydrazide derivatives 4a&b and finally, 4a&b were reacted with different aromatic aldehydes giving the target compounds 5a-l. The benzylidene derivatives 5a-l were screened for their cytotoxic activities against breast carcinoma cell lines (MCF-7) and anti-oxidant properties. All the tested compounds 5a-l showed from high to moderate activity as anticancer and anti-oxidant agents. Compounds 5h and 5l showed the highest cytotoxicr activity against MCF-7 (IC50: 0.058 and 0.052 uM, respectively) than 4-(benzothiazol-2-yl) aniline the reference drug (IC50: 0.065 uM). Moreover, compounds 5e, 5g and 5h showed the highest anti-oxidant activity. The structure of the compounds 5a-l was confirmed using IR, 1H NMR, mass spectroscopy and elemental analysis.

Keywords

Quinolones; Benzothiazoles; Benzoxazoles; Anti-oxidant activity; Anticancer effect

Introduction

The cytotoxic activity of quinolone derivatives has become the source of new anticancer agents, which might also help addressing side-toxicity and resistance [1]. Moreover, the quinolone ring is considered an important structural unit in many anti-oxidant agents [2]. New synthesized 4-arylchalcogenyl-7-chloroquinolines were screened in vitro for antioxidant activity by previous publication which demonstrated that compound presented a potent antioxidant effect [3].

Quinolones were used especially as radicals scavenger like quercetol (A) or coumestrol (B) and the copper or iron chelating molecules such as clioquinol (C) [4,5]. Moreover, quinolone containing hydroxyl group compounds exhibited antiradical activity against DPPH radical and anion superoxide tests activity [6].

On the other hand, benzothiazoles showed potent scavenging activities against DPPH radical and 2,2'-azino-bis (3-ethylbenzthiazoline- 6-sulphonic acid) (ABTS) (D) radicals had reducing power, and strong inhibitory capacity on lipid peroxidation. Also, benzothiazoles or benzoxazoles containing compounds were found to be cytotoxic against CNS cancer cell line SNB-75 [7]. Moreover, 4-(benzothiol-2- yl) aniline (Eb) showed a promising cytotoxic activity against breast, overian, lung and renal cell lines [8-10]. Activity was partially retained in benzoxazole analogue Ea [8] (Figure 1). Compounds of thiol and aminothiol derived from benzothiazoles showed an promising antioxidant property [11].

medicinal-chemistry-Chemical-previously-synthesized

Figure 1: Chemical structures of previously synthesized anticancer and antioxidant agents containing benzoxazole, benzothiazole and quinolone moieties (A-E) and the design of the target compounds 5a-1.

Benzothiazoles and benzoxazoles containing compounds were showed anticancer activity against various cell lines [12,13]. New synthesized compounds containing benzothiazoles or benzoxazoles linked to quinolone showed anticancer and antimicrobial activities [14,15].

According to the aforementioned facts and as a continuation of our previous studies in the field of anticancer screening and anti-oxidant evaluation, [16-19] we attempt to design novel quinolone derivatives through:

• Substitution at quinolone nucleus with benzothiazole and benzoxazole rings (which have antioxidant or anticancer activity) at 6 position of quinolone.

• Maintain the main structure which responsible for receptor coupling of quinolone.

• Substitution of carboxyl group at 3-postion by substituted phenylhydrazone to increase its lipophilicity.

• Over all incorporation of benzoxazole or benzothiazole and quinolone in one scaffold structure.

All the synthesized compounds were evaluated for their anticancer activity against human breast adenocarcinoma cell line (MCF-7) and antioxidant activity.

Materials and Methods

Chemistry

Melting points were determined on a Graffin apparatus and were uncorrected. Element analyses (C, H, and N) were carried out on Perkin-Elmer 2400 analyzer (Perkin-Elmer, Norwalk, CT, USA) at the Micro analytical unit of Cairo University, Egypt. All compounds were within ± 0.4% of the theoretical values. IR spectra were determined as KBr discs on Shimadzu IR 435 Spectrophotometer and values were represented in cm-1. 1H NMR spectra were carried out on a Bruker 400 MHz NMR Spectrophotometer in Beni Suef University, Beni Suef, Egypt, using (Bruker, Munich, Germany) in DMSO-d6 as a solvent, TMS as internal standard and chemical shifts were recorded in ppm on δ scale. Mass spectra were run on Hewlett Packard 5988 Spectrometer, Micro analytical center, Cairo University, Egypt. Progress of the reactions was monitored by TLC using TLC sheets percolated with UV fluorescent silica gel MERCK 60 F 254 that were visualized by UV lamp.

General procedure for the synthesis of 6-(benzo[d]oxazole or thiazol-2-yl)-4-oxo-1,4-dihydroquinoline-3-carbohydrazide 4a&b

To a suspension of compounds 3a&b (0.01 mol) in absolute ethanol (30 mL), hydrazine hydrate 99% (5 g, 0.1 mol) was added. The mixture was heated under reflux for 20 h. The precipitated solid was filtered, dried and crystallized from DMF/ethanol.

6-(Benzo[d]oxazole-2-yl)-4-oxo-1,4-dihydroquinoline-3- carbohydrazide (4a): Yield: 53%; yellow crystals ;mp: 390-392°C;IR (cm-1): 3465-3192 (2NH and NH2), 1661,1613 (2C=O); 1H NMR (DMSO-d6) δ ppm 4.65 (s, 2H, NH2, D2O exchangeable), 7.47 (m, 2H, Ar-H), 7.86 (m, 2H, Ar-H), 7.92 (d, 1H, J= 9.2 Hz, Ar-H), 8.53 (d, 1H, J= 7.6 Hz, Ar-H), 8.82 (s, 1H, Ar-H), 9.04 (s, 1H, Ar-H), 10.63 (s, 1H, CONH, D2O exchangeable), 13.06 (s, 1H, NH, D2O exchangeable); MS m/z: 320 [(M) +, 7.98%], 288 [(C17H8N2O3)+, 100%]. Anal. Calcd. for C17H12N4O3: C, 63.75; H, 3.78; N, 17.49. Found: C, 63.70; H, 3.50; N, 17.20.

6-(Benzo[d]thiazol-2-yl)-4-oxo-1,4-dihydroquinoline-3- carbohydrazide (4b): Yield: 53%; yellow crystals; mp: 396-398°C;IR (cm-1): 3422-3176 (2NH and NH2), 1661,1624 (2C=O); 1H NMR (DMSO-d6) δ ppm 4.51 (s, 2H, NH2, D2O exchangeable), 7.49 (m, 1H, Ar-H), 7.57 (m, 1H, Ar-H), 7.84 (d, 1H, J= 8.4Hz, Ar-H), 8.11 (d, 1H, J= 7.2 Hz, Ar-H), 8.18 (d, 1H, J= 7.2 Hz, Ar-H), 8.40 (d, 1H, J= 8 Hz, Ar-H), 8.81 (s, 1H, Ar-H), 8.89 (s, 1H, Ar-H), 10.84 (s, 1H, CONH, D2O exchangeable), 13.29 (s, 1H, NH, D2O exchangeable); MS m/z: 336 [(M) +., 20.70%], 57 [(C4H9)+, 100%]. Anal. Calcd. For C17H12N4O2S: C, 60.70; H, 3.60; N, 16.66. Found: C, 60.60; H, 3.40; N, 16.50.

General procedure for the synthesis of (ZE)-6-(benzo[d] oxazol or thiazol-2-yl)-N'-substituted benzylidene-4-oxo-1,4- dihydroquinoline-3-carbohydrazide 5a-l

A mixture of 4a&b (0.01 mol) and the appropriate aromatic aldehyde (0.01 mol) in absolute ethanol, catalytic amount of glacial acetic acid was added (0.5 mL). The reaction mixture was heated under reflux for 8-10 h (monitored by TLC). The separated solid was filtered, dried and crystallized from DMF.

(ZE)6-(Benzo[d]oxazol-2-yl)-N'-benzylidene-4-oxo-1,4- dihydroquinoline-3-carbohydrazide (5a): Yield: 53%; yellow solid; mp: 349-351°C; IR (cm-1): 3439, 3141 (2NH), 3089 (CH aromatic), 1697, 1615 (2C=O); 1H NMR (DMSO-d6) δ ppm 7.47 (m, 6H, Ar-H), 7.86 (m, 5H, Ar-H), 8.47 (s, 1H, Ar-H), 8.72 (s, 1H, Ar-H), 8.95 (s, 1H, N=CH), 9.09 (s, 1H, CONH, D2O exchangeable), 13.26 (s, 1H, NH, D2O exchangeable); MS m/z: 409 [(M+1)+., 8.18%], 408 [(M) +., 13.48%], 288 [(C17H8N2O3)+., 100%]. Anal. Calcd. for C24H16N4O3: C, 67.91; H, 3.80; N, 13.20. Found: C, 68.00; H, 3.60; N, 12.90.

(ZE)6-(Benzo[d]oxazol-2-yl)-N'-(2-hydroxybenzylidene)-4- oxo-1,4-dihydroquinoline-3-carbohydrazide (5b): Yield: 53%; yellow solid; mp: 350-352°C; IR (cm-1): 3425-3141 (OH and 2NH), 3089 (CH aromatic), 1696, 1609 (2C=O); 1H NMR (DMSO-d6) δ ppm 6.95 (m, 2H, Ar-H), 7.47 (m, 4H, Ar-H), 7.96 (m, 4H, Ar-H), 8.69 (s, 1H, Ar- H), 8.95 (s, 1H, Ar-H), 9.08 (s, 1H, N=CH), 9.09 (s, 1H, CONH, D2O exchangeable), 11.33 (s, 1H, OH, D2O exchangeable),13.20 (s, 1H, NH, D2O exchangeable); MS m/z: 425 [(M+1)+., 3.52%], 424 [(M) +., 5.37%], 80 [(C3N2O)+., 100%]. Anal. Calcd. for C24H16N4O3: C, 67.92; H, 3.80; N, 13.20. Found: C, 67.80; H, 3.50; N, 13.00.

(ZE)6-(Benzo[d]oxazol-2-yl)-N'-(4-florobenzylidene)-4-oxo- 1,4-dihydroquinoline-3-carbohydrazide (5c): Yield: 53%; yellow solid; mp: 354-356°C; IR (cm-1): 3441, 3141 (2NH), 3088 (CH aromatic), 1696, 1623 (2C=O); 1H NMR (DMSO-d6) δ ppm 7.34 (m, 2H, Ar-H), 7.47 (m, 2H, Ar-H), 7.96 (m, 4H, Ar-H), 7.99 (m, 2H, Ar-H), 8.48 (s, 1H, Ar-H), 8.54 (s, 1H, Ar-H), 8.95 (s, 1H, N=CH), 9.09 (s, 1H, CONH, D2O exchangeable), 13.26 (s, 1H, NH, D2O exchangeable); MS m/z: 427 [(M+1)+., 21.86%], 426 [(M) +., 29.96%], 79 [(C5F)+, 100%]. Anal. Calcd. For C24H15FN4O3: C, 67.60; H, 3.55; N, 13.14. Found: C, 67.40; H, 3.30; N, 13.10.

(ZE)6-(Benzo[d]oxazol-2-yl)-N'-(4-chlorobenzylidene)-4-oxo- 1,4-dihydroquinoline-3-carbohydrazide (5d): Yield: 53%; yellow solid; mp: 378-380°C; IR (cm-1): 3430, 3141 (2NH), 3088 (CH aromatic), 1696, 1623 (2C=O); 1H NMR (DMSO-d6) δ ppm 7.47 (m, 1H, Ar-H), 7.68 (m, 2H, Ar-H), 7.81 (d, 2H, J= 8.4 Hz, Ar-H), 7.87 (m, 3H, Ar-H), 7.93 (m, 2H, Ar-H), 7.97 (s, 1H, Ar-H), 8.48 (s, 1H, Ar-H), 8.95 (s, 1H, N=CH), 9.09 (s, 1H, CONH, D2O exchangeable), 13.26 (s, 1H, NH, D2O exchangeable); MS m/z: 443 [(M) +., 6.22%], 80 [(C3N2O)+., 100%]. Anal. Calcd. For C24H15ClN4O3: C, 65.09; H, 3.41; N, 12.65. Found: C, 64.80; H, 3.10; N, 12.70.

(ZE)6-(Benzo[d]oxazol-2-yl)-N'-(4(dimethylamino) benzylidene)-4-oxo-1,4-dihydroquinoline-3-carbohydrazide (5e): Yield: 53%; orange solid; mp: 354-356°C; IR (cm-1): 3436, 3141 (2NH), 3085 (CH aromatic), 1696, 1600 (2C=O); 1H NMR (DMSO-d6) δ ppm 3.6 (s, 6H, 2 NCH3), 7.55 (m, 6H, Ar-H), 8.26 (m, 6H, Ar-H), 8.39 (s, 1H, CONH, D2O exchangeable), 8.93 (s, 1H, N=CH), 14.26 (s, 1H, NH, D2O exchangeable); MS m/z: 451 [(M) +., 9.64%], 80 [(C5H6N)+., 100%]. Anal. Calcd. For C26H21N5O3: C, 69.17; H, 4.69; N, 15.51. Found: C, 68.80; H, 4.40; N, 15.50.

(ZE)6-(Benzo[d]oxazol-2-yl)-N'-(4-nitrobenzylidene)-4-oxo- 1,4-dihydroquinoline-3-carbohydrazide (5f): Yield: 53%; brown solid; mp: 347-349°C; IR (cm-1): 3434, 3141 (2NH), 3089 (CH aromatic), 1694, 1617 (2C=O); 1H NMR (DMSO-d6) δ ppm 7.54 (m, 4H, Ar- H), 7.76 (m, 2H, Ar-H), 8.13 (m, 3H, Ar-H), 8.82 (s, 2H, N=CH and CONH, D2O exchangeable), 8.87 (m, 3H, Ar-H),11.19 (s, 1H, NH, D2O exchangeable); MS m/z: 454 [(M+1)+., 23.62%], 453 [(M) +., 40.94%], 61 [(C5H)+, 100%]. Anal. Calcd. For C24H15N5O5: C, 63.58; H, 3.33; N, 15.45. Found: C, 63.20; H, 3.00; N, 15.20.

(ZE)6-(Benzo[d]thiazol-2-yl)-N'-benzylidene-4-oxo-1,4- dihydroquinoline-3-carbohydrazide (5g): Yield: 53%; yellow solid; mp: 396-398°C; IR (cm-1): 3404, 3259 (2NH), 3061 (CH aromatic), 1661, 1622 (2C=O); 1H NMR (DMSO-d6) δ ppm 7.45 (m, 4H, Ar- H), 7.56 (m, 2H, Ar-H), 7.78 (m, 2H, Ar-H), 8.16 (m, 2H, Ar-H), 8.79 (s, 2H, Ar-H), 8.80 (s, 1H, N=CH), 8.91 (d, 1H, J= 14.8 Hz, Ar- H), 10.87 (s, 1H, CONH, D2O exchangeable), 14.02 (s, 1H, NH, D2O exchangeable); MS m/z: 425 [(M+1)+., 43.94%], 424 [(M) +., 53.03%], 55 [(C4H7)+, 100%]. Anal. Calcd. For C24H16N4O2S: C, 67.91; H, 3.80; N, 13.20. Found: C, 67.70; H, 3.80; N, 13.10.

(ZE)6-(Benzo[d]thiazol-2-yl)-N'-(2-hydroxybenzylidene)-4- oxo-1,4-dihydroquinoline-3-carbohydrazide (5h): Yield: 48%; yellow solid; mp: 362-364°C; IR (cm-1): 3400-3166 (OH and 2NH), 3057 (CH aromatic), 1658, 1617 (2C=O); 1H NMR (DMSO-d6) δ ppm 6.96 (m, 2H, Ar-H), 7.54 (m, 4H, Ar-H), 7.84 (m, 1H, Ar-H), 8.09 (d, 2H, J= 1.2 Hz, Ar-H), 8.40 (m, 1H, A-H), 8.62 (s, 1H, N=CH), 8.90 (s, 2H, Ar- H), 10.69 (s, 1H, CONH, D2O exchangeable), 11.46 (s, 1H, OH, D2O exchangeable), 13.78 (s, 1H, NH, D2O exchangeable) ; MS m/z: 441 [(M+1)+., 61.22%], 440 [(M) +., 56.12%], 135 [(C7H7N2O)+, 100%]. Anal. Calcd. For C24H16N4O3S: C, 65.44; H, 3.66; N, 12.72. Found: C, 65.60; H, 3.40; N, 12.50.

(ZE)6-(Benzo[d]thiazol-2-yl)-N'-(4-flurobenzylidene)-4- oxo-1,4-dihydroquinoline-3-carbohydrazide (5i): Yield: 48%; yellow solid; mp: 395-397°C; IR (cm-1): 3426, 3262 (2NH), 3063 (CH aromatic), 1661, 1623 (2C=O); 1H NMR (DMSO-d6) δ ppm 7.54 (m, 4H, Ar-H), 8.08 (m, 2H, Ar-H), 8.13 (m, 3H, Ar-H), 8.84 (m, 2H, Ar- H), 9.60 (s, 1H, N=CH), 9.61 (s, 1H, Ar-H), 11.00 (s, 1H, CONH, D2O exchangeable), 13.26 (s, 1H, NH, D2O exchangeable); MS m/z: 443 [(M+1)+., 3.21%], 442 [(M) +., 21.57%], 69 [(C5H9)+, 100%]. Anal. Calcd. For C24H15FN4O2S: C, 65.15; H, 3.42; N, 12.66. Found: C, 64.90; H, 3.20; N, 12.70.

(ZE)6-(Benzo[d]thiazol-2-yl)-N'-(4-chlorobenzylidene)-4- oxo-1,4-dihydroquinoline-3-carbohydrazide (5j): Yield: 48%; yellow solid; mp: 398-400°C; IR (cm-1): 3432, 3209 (2NH), 3047 (CH aromatic), 1660, 1621 (2C=O); 1H NMR (DMSO-d6) δ ppm 7.53 (m, 4H, Ar-H), 7.77 (m, 3H, Ar-H), 7.79 (m, 4H, Ar-H), 8.38 (s, 1H, Ar-H), 8.91 (s, 1H, N=CH), 10.08 (s, 1H, CONH, D2O exchangeable), 14.41 (s, 1H, NH, D2O exchangeable) ; MS m/z: 459 [(M) +., 19.07%], 458 [(MH)+, 17.53%], 54 [(C4H6)+., 100%]. Anal. Calcd. For C24H15ClN4O2S: C, 62.81; H, 3.29; N, 12.21. Found: C, 62.60; H, 3.40; N, 12.10.

(ZE)6-(Benzo[d]thiazol-2-yl)-N'-(4-(dimethylamino) benzylidene)-4-oxo-1,4-dihydroquinoline-3-carbohydrazide (5k): Yield: 48%; orange solid; mp: 378-380°C; IR (cm-1): 3420, 3263 (2NH), 3065 (CH aromatic), 1661, 1622 (2C=O);1H NMR (DMSO-d6) δ ppm 3.45 (s, 6H, 2CH3), 7.00 (d, 2H, J= 4.4 Hz, Ar-H), 7.49 (m, 4H, Ar-H), 7.54 (m, 2H, Ar-H), 8.16 (d, 1H, J= 7.6 HZ, Ar-H), 8.34 (d, 1H, J= 8.4 Hz, Ar-H), 8.81 (s, 1H, Ar-H), 8.90 (s, 1H, N=CH), 8.92 (s, 1H, Ar- H) 10.96 (s, 1H, CONH, D2O exchangeable), 14.22 (s, 1H, NH, D2O exchangeable) ; MS m/z: 468 [(M+1)+., 25%], 467 [(M) +., 7.41%], 217 [(C12H15N3O)+., 100%]. Anal. Calcd. For C26H21N5O2S: C, 66.79; H, 4.53; N, 14.98. Found: C, 66.90; H, 4.40; N, 14.90.

(ZE)6-(Benzo[d]thiazol-2-yl)-N'-(4-nitrobenzylidene)-4-oxo- 1,4-dihydroquinoline-3-carbohydrazide (5l): Yield: 48%;brown solid; mp: 356-358°C; IR (cm-1): 3419, 3212 (2NH), 3160, 3053 (CH aromatic), 1664, 1623 (2C=O); 1H NMR (DMSO-d6) δ ppm 7.48 (m, 3H, Ar-H), 8.16 (m, 7H, Ar-H), 8.41 (s, 1H, Ar-H), 8.92 (s, 1H, N=CH), 8.93 (s, 2H, Ar-H and CONH, D2O exchangeable), 14.62 (s, 1H, NH, D2O exchangeable) ; MS m/z: 470 [(M+1)+., 23.77%], 469 [(M) +., 60.66%], 428 [(C23H14N3O3S)+, 100%]. Anal. Calcd. For C24H15N5O3S: C, 61.40; H, 3.22; N, 14.92. Found: C, 61.20; H, 3.50; N, 14.70.

Biological Evaluation

Anticancer screening

Human tumor cell lines: Breast carcinoma cell lines (MCF-7) used in this study were obtained from the American Type Culture Collection (ATCC, Minisota, USA) through the Tissue Culture Unit, the Egyptian Organization for Biological Products and Vaccines, Vacsera, 51 Wezaret EI Zeraa St., Agouza, Giza, Egypt. The tumor cell lines were maintained at Center for Genetic Engineering, Al-Azhar University, Cairo, Egypt by serial sub-culturing.

Chemicals

Dimethylsulphoxide (DMSO), Dulbecco's Modified Eagle Medium (DMEM), trypan blue, Fetal Bovine Serum, Penicillin/ Streptomycin antibiotic and Trypsin- EDTA Sigma Aldrich Chemical Co., St. Louis, Mo, USA. Tris buffer was obtained from Applichem, Germany. All chemicals and reagents used in this study are of highest analytical grade.

Methods

Preparation of test compounds

The tested derivatives 5a-l were prepared by dissolving in dimethylsulfoxide (DMSO) and the prepared stock was stored at -20°C. Different concentrations of the compounds 0, 6.25, 12.5, 25, 50 and 100 μg/ml in culture medium were used.

Preparatory steps prior to cytotoxicity investigation

Maintenance of the breast carcinoma cell lines (MCF-7) in the laboratory, cryopreservation of cells, collection of cells by trypsinization and determination and counting of viable cells are performed according to the reported methods [20,21].

Determination of potential cytotoxicity of drug on human cancer cell line

The cytotoxicity was carried out using Sulphorhodamine-B (SRB) assay following the reported method [22] SRB is a bright pink aminoxanthrene dye with two sulphonic groups. It is a protein stain that binds to the amino groups of intracellular proteins under mildly acidic conditions to provide a sensitive index of cellular protein content.

Cells of MCF-7 cell lines are seeded in 96 well microliter plates at a concentration of 1000-2000 cells/well, 100 μl/well. After 24 h, cells will be incubated for 72 h with various concentrations of drugs (0, 6.25, 12.5, 25, 50 and 100 μg/ml). Dulbecco's Modified Eagle Medium (DMEM) with 10% foetal calf serum, sodium pyruvate, 100 U/ml penicillin and 100 mg/ml streptomycin at 37°C and 5% CO2, was used as culture medium. At the end of the incubation, the medium is discarded. The cells are fixed with 150 μl cold trichloroacetic acid 10% final concentration for 1 hour at 4°C. The plates were washed with distilled water using (automatic washer Tecan, Germany) and stained with 50 μl 0.4% SRB dissolved in 1% acetic acid for 30 minutes at room temperature in dark. The plates were washed with 1% acetic acid to remove unbound dye and air-dried (24 h). The dye was solubilized with 150 μl/well of 10 mMtris base (PH 7.4) for 5 min on a shaker at 1600 rpm. The optical density (OD) of each well will be measured spectrophotometrically at 490 nm with an ELISA microplate reader. The mean background absorbance was automatically subtracted and mean values of each derivative and 4-(benzothiazol-2-yl) aniline (reference drug) concentration was calculated. The experiment was repeated three times. The percentage of cell survival was calculated by using formula, surviving percent=[OD (treated cells)/OD (control cells)] × 100. The IC50 values (the concentrations of derivatives required to produce 50% inhibition of cell growth) were also calculated using linear trend linear equation.

Anti-oxidant assay

DPPH radical scavenging activity: The effect of the synthesized organic compounds on DPPH radicalwas estimated using the reported methods [23,24] with some modifications. A solution of 200 μmol DPPH in ethanol was prepared and 100 μl of this solution was mixed with 0.9 ml of varying concentrations of the synthesized derivatives (dissolved in ethanol) to reach a final concentration of 0.25, 0.5 and 1 mg/ml. The reaction mixture wasvortexed and left in the dark for 30 min (room temperature). The color became light yellow from deep violet and the absorbanceof the mixture was determined at 570 nm. The control was prepared by using adding 100 μl DPPH to 0.9 ml ethanol solution.

DPPH radical scavenging activity (%)=½[(Acontrol-Asample)/Acontrol] × 100

Where Acontrol is the absorbance of DPPH radical+ethanol and Asample is the absorbance of DPPH radical+sample of derivative compound dissolved in ethanol.

Results and Discussion

Chemistry

In this work, the synthesis of different Schiff bases at C-3 of quinolone moiety was described. Biologically important benzoxazoles and benzothiazoles were merged with quinolone nucleus. 6-Benzoxazole/ benzothiazol-2-yl-4-oxo-1,4-dihydro-quinoline-3-carboxylic acid ethyl esters 3a&b were employed as the starting materials. They were synthesized following the precautions of their reported method [14]. Direct conversion of 3a&b into hydrazide derivatives 4a&b was achieved in good yield by the treatment of 3a&b with hydrazine hydrate 99%. Several solvents were carried out to prepare these intermediates 4a&b such as absolute ethanol, glacial acetic acid and dioxane. Absolute ethanol was the solvent of choice since it gave more pure products (using: TLC) as well as higher yields (Scheme 1). The structure of compounds 4a&b was established on the basis of IR, 1H NMR, mass spectral data and elemental analysis. IR spectra of 4a&b revealed the appearance of new absorption bands at 3465-3176 cm-1 due to 2NH and NH2 groups.

medicinal-chemistry-Reagents-ethanol-diphenyl

Scheme 1: Reagents and conditions: i) EMME, ethanol, reflux for 4 h; ii) diphenyl ether, reflux for 1 h.

The structure of the compounds 4a&b was confirmed by 1H NMR which was performed in DMSO as a solvent, appearance of D2O exchangeable singlet signal at δ 4.51 and 4.65 ppm indicated NH2 protons, beside the appearance of another exchangeable singlet signal corresponding to CONH proton at δ 10.63 and 10.84 ppm, respectively. Moreover, mass spectra of 4a&b showed molecular ion peaks at m/z 320 and 336, sequentially. Heating compounds 4a&b for 8-10 hours with different aromatic aldehydes in absolute ethanol containing catalytic amount of glacial acetic acid led to the formation of 5a-l. The formation of the target Schiff bases 5a-l was substantiated on basis of spectral data and elemental analysis (see Experimental Section).

1H NMR spectra of compounds 5a-l showed disappearance of D2O exchangeable singlet signal of NH2 protons of the parent hydrazide derivatives 4a&b and the appearance of new singlet signal due to azomethine proton (N=CH) at δ 8.82-9.08 ppm in benzoxazole derivatives 5a-f or at δ 8.80-9.60 ppm for benzothiazole derivatives 5g-l.

Additionally, the mass spectrum of compound 5d reveled molecular ion peaks at m/z 443 and 445 corresponding to (M) +and (M+2)+, respectively in ratio of 3:1 (Cl pattern). The reactivity of the applied aromatic aldehydes was appeared in parallel manner with the yield of the resulting targets 5a-l, which ranged between 45% up to 79% starting from benzaldehyde to 4-nitrobenzaldehyde in both benzoxazole and benzothiazole derivatives.

Anticancer activity

The data showing the anti-proliferative effects of the tested derivatives on breast carcinoma cell lines (MCF-7) are illustrated in Figures 1 and 2. All derivatives from 5a to 5l produced a marked gradual decrease in the survival percent of MCF-7 as the dose of derivatives increased from 0 to 100 μg/ml. Based on the values of IC50, the derivatives are arranged according to their tumor cytotoxic potencies in the following order: derivatives 5l, 5h, 5g, 5k, 5e, 5f, 5a, 5i, 5d, 5j, 5c and 5a recording IC50 of 40.783, 45.461, 48.953, 50.627, 52.138, 53.665, 54.163, 55.344, 57.462, 58.452, 69.214 and 82.300 μg/ml, respectively Scheme 2. Thus, derivative 5l produced the most potent tumor cytotoxic efficacy, while derivative 5a followed by derivative 5c are the least potent (Figure 2 and Table 1).

medicinal-chemistry-Chemical-structure-compounds

Figure 2: Chemical structure of the target compounds 5a-l.

medicinal-chemistry-Reagents-absolute-ethanol

Scheme 2: Reagents and conditions: i) N2H4, absolute ethanol; ii) ArCHO, absolute ethanol, drops of glacial acetic acid.

Compound No. IC50 (uM)
5a 0.2
5b 0.117
5c 0.15
5d 0.106
5e 0.078
5f 0.085
5g 0.072
5h 0.058
5i 0.099
5j 0.104
5k 0.074
5l 0.052
4-(benzothiazol-2-yl)aniline (standard) 0.065

Table 1: IC50 of the test compounds (5a-l) against breast cancer (MCF-7).

Anti-oxidant activity

The antioxidant capacity was evaluated by detection of DPPH radical scavenging activity. Different concentrations of derivatives were tested. All tested derivatives had marked antioxidant activity. At lowest dose, 0.25 mg/ml, derivative 5a followed by 5e seemed to have the most potent DPPH radical scavenging activity percent recording 26.33 and 21.58%, respectively. At medium dose (0.5 mg/ml) and high dose (1 mg/ml), derivatives 5e, 5g and 5h were the most efficient in having antioxidant activity. Derivative 5c had no antioxidant activity at high dose although it had marked efficacy at low and medium doses. Moreover, while the antioxidant activity of 5a, 5b and 5l was more or less unchanged as the dose increased from 0.5 to 1 mg/ml, it was more potentiated for derivatives 5d, 5f, 5g, 5h, 5i, 5j and 5k (Table 2).

Derivatives 0.25 mg/ml 0.5 mg/ml 1 mg/ml
5a 26.33 12.16 13.50
5b 15.58 10.75 11.50
5c 10.25 20.00 0.00
5d 15.75 13.75 18.33
5e 21.58 31.84 39.75
5f 3.58 2.5 9.83
5g 12.97 25.52 40.83
5h 12.97 25.52 40.82
5i 13.64 19.39 31.65
5j 8.92 6.58 11.75
5k 11.59 20.63 27.28
5l 12.33 14.83 15.92

Table 2: DPPH radical scavenging activity (%) of derivative compounds (5a-l) at various concentrations.

Conclusion

The synthesized derivatives exhibited various degrees of cytotoxic effects on breast carcinoma cell line (MCF-7) in vitro. Comparing the new compounds with 4-(benzothiazol-2-yl)aniline – known with its cytotoxic activity- which has IC50 0.065μM, we observed that: (ZE)-6-(bnzo[d]thiazol-2-yl)-N'-(4-nitrobenzylidene)-4-oxo-1,4- dihydroquinoline-3-carbohydrazide (5l) and (ZE)-6-(Benzo[d]thiazol-2-yl)-N'-(2-hydroxybenzylidene)-4-oxo-1,4-dihydroquinoline-3- carbohydrazide (5h) seemed to have the most potent antitumor action (IC50: 0.052 μM and 0.058 μM, respectively), in addition to five new compounds 5e, 5f, 5g, 5i and 5k showed good activity with IC50 between 0.072 μM and 0.099 μM. Only five compounds (5a, 5b, 5c, 5d, 5j) exhibit moderate activity. with IC50 in the range of 0.10 μM and 0.20w μM. On the other hand, (ZE)-6-(benzo[d] oxazol-2-yl)-N'-(4-(dimethylamino)benzylidene)-4-oxo-1,4- dihydroquinoline-3-carbohydrazide (5e), (ZE)-6-(benzo[d]thiazol- 2-yl)-N'-benzylidene-4-oxo-1,4-dihydroquinoline-3-carbohydrazide (5g) and (ZE)-6-(benzo[d]thiazol-2-yl)-N'-(2-hydroxybenzylidene)- 4-oxo-1,4-dihydroquinoline-3-carbohydrazide (5h) have the most efficient antioxidant activity as indicated by the results of DPPH radical scavenging capacity. Finally, it was found that compound 5h bearing 4-hydroxyphenyl moiety and benzothiazole nucleus has dual anticancer and anti-oxidant activity and need further investigations.

Conflict of Interest

The authors declared that there is no conflict of interest.

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