Diseño, síntesis y estudio de acoplamiento molecular de híbridos de quinazolinona-tiazolidin-4-onas como agentes anticancerígenos

Nara, Sukanya; Garlapati, Achaiah; Nara, Sukanya; Garlapati, Achaiah

doi:10.30827/ars.v59i3.7360

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Ars Pharmaceutica (Internet)

versión On-line ISSN 2340-9894

Ars Pharm vol.59 no.3 Granada jul./sep. 2018 Epub 19-Oct-2020

https://dx.doi.org/10.30827/ars.v59i3.7360

Original Articles

Design, Synthesis and molecular docking study of hybrids of quinazolin-4(3H)-one as anticancer agents

Diseño, síntesis y estudio de acoplamiento molecular de híbridos de quinazolinona-tiazolidin-4-onas como agentes anticancerígenos

Sukanya Nara¹, Achaiah Garlapati¹^*

^¹Medicinal Chemistry Research Lab., University College of Pharmaceutical Sciences (UCPSc), Kakatiya University, Warangal, Telangana, India

ABSTRACT

A series of 4-(2-(4-substituted phenyl)-4-oxoquinazolin-3(4H)-yl)-N-(2-(4-fluorophenyl)-4-oxo-5-(arylidene)thiazolidin-3-yl) benzamides (VIa-n) have been synthesized by condensation of N-(2-(4-fluorophenyl)-4-oxothiazolidin-3-yl)-4-(4-oxo-2-(4-substituted phenyl)quinazolin-3(4H)-yl)benzamides (Va-b) with various aryl/heteroaryl aldehydes using conventional methodology. All compounds were screened for their in vitro anticancer activity against the human breast cancer cell lines (MCF-7), human lung cancer cell lines (A549) using MTT assay method and doxorubicin is used as standard drug. Compound VId, VIk and VIn showed high potency against A549 cell lines with IC₅₀ values 0.035±0.002 µM, 0.031±0.002 µM and 0.030±0.002 µM respectively compared to 0.023±0.002 µM showed by the standard. However, highest activity against MCF-7 cell lines was exhibited by Va, Vb, VIk and VIn with IC₅₀ values between 0.040 - 0.050 µM. All the remaining compounds showed moderate anticancer activity against both the MCF-7 and A549 cell lines. To understand the interactions with active binding site of receptor, molecular docking study was also performed.

Keywords: Quinazolin-4(3H)-one; Thiazolidin-4-one; Anticancer activity; Molecular docking

RESUMEN

Una serie de 2- (4-sustituido fenil) -4-oxoquinazolin-3 (4H) -il) -N- (2- (4-fluorofenil) -4-oxo-5- (arilideno) tiazolidin-3-ilo) benzamidas (VIa-n) han sido sintetizadas por condensación de N- (2- (4-fluorofenil) -4-oxotiazolidin-3-il) -4- (4-oxo-2- (4-fenil sustituido) quinazolin-3 (4H) -il) benzamidas (Va-b) con diversos aldehídos de arilo / heteroarilo usando metodología convencional. Todos los compuestos se cribaron para su actividad anticancerosa in vitro contra las líneas celulares de cáncer de mama humano (MCF-7), líneas celulares de cáncer de pulmón humano (A549) usando el método de ensayo MTT y se usa doxorrubicina como fármaco estándar. El compuesto VId, VIk y VIn mostraron alta potencia contra las líneas celulares A549 con valores IC₅₀ de 0.035 ± 0.002 μM, 0.031 ± 0.002 μM y 0.030 ± 0.002 μM, respectivamente, en comparación con 0.023 ± 0.002 μM mostrada por el estándar. Sin embargo, la actividad más alta contra líneas celulares MCF-7 fue exhibida por Va, Vb, VIk y VIn con valores de CI50 entre 0.040 - 0.050 μM. Todos los compuestos restantes mostraron una actividad anticancerígena moderada contra las líneas celulares MCF-7 y A549. Para comprender las interacciones con el sitio de unión activa del receptor, también se realizó el estudio de acoplamiento molecular.

Palabras clave: Quinazolin-4 (3H) -ona; Thiazolidin-4-one; Actividad anticancerosa; Atraque molecular

INTRODUCTION

Cancer is a disease of worldwide importance. Its incidence in the developed countries is increasing, and it occupies the second rank in the order of death causes¹. Genotoxicity and cytotoxicity of anti-cancer drugs to the normal tissues is major problems in cancer therapy. However, cancer research always requires the discovery of new drugs that can kill the cancer cells or stop the growth of cancer cells more specifically and overcome the limitation of toxicity to normal tissue leading to many undesirable side effects². Quinazoline derivatives have been identified as a new class of cancer chemotherapeutic agents with significant therapeutic efficacy against solid tumors³. Hybrid anti-cancer agents, which combine quinazoline and phenylquinoxaline moieties in one were reported and found to possess enhanced cytotoxicity and specificity⁴. 4-Oxo-thiazolidine derivatives have occupied a unique place in the field of medicinal chemistry due to a wide range of biological activities⁵. 4-Thiazolidinones have been reported to inhibit tumor cell proliferation through inducing cell cycle arrest⁶. Recently 2-phenyl-(4-oxothiazolidin-3-yl)-quinazolin-4(3H)-one derivatives were found to possess potent activity against MCF-7 and HepG2 cell lines⁷. In our present study, we have designed and synthesized a new series of quinazolin-4(3H)-one and 4-thiazolidinone hybrid molecules as new anticancer agents with improved activity profile. In the process the C2 position of quinazolin-4(3H)-one was attached with substituted phenyl ring and the C5 position of 4-thiazolidinone substituted with various aryl and heteroaryl ring systems (Figure 1).

Figure 1: General structure of the synthesized compounds (VIa-n).

EXPERIMENTAL

All the chemicals and solvents were supplied by Sigma-Aldrich Pvt Ltd. The column chromatography was performed on silica gel 60-100 (Merk) for purification of the compounds using ethylacetate: hexane and chloroform: methanol in different ratios as mobile phase. Melting points were determined using MEL TEMP electrothermal apparatus. IR spectrum was acquired on Perkin Elmer FT-IR spectrometer. Both ¹H-NMR and ¹³C-NMR spectra of the compounds were taken on Bruker Avance-II 400 NMR Spectrometer operating at 400 MHz and 100 MHz respectively at IICT, Hyderabad and Andhra University, Vishakapatnam. Mass spectra were recorded at IICT, Hyderabad. The elemental analysis was carried out using Elementar Vario Micro Cube at UCPSc, Kakatiya University, Warangal.

Chemistry

Preparation of intermediate compounds 2-(4-substituted phenyl)benzo[d]1,3-oxazin-4-ones Ia-b were carried out according to the literature method⁸.

Preparation of ethyl 4-[2-(4-sustituted phenyl)-4-oxoquinazolin-3(4H)-yl] benzoate (IIa-b)

A mixture of compound (Ia-b) (0.01 mol) and ethyl 4-amino benzoate (0.012 mol) were fused at a temperature of 180-200^oC for 25 min. After cooling, the reaction mixture was treated with methanol carefully portion wise the solid was separated by filtration, dried and recrystallized from ethanol to give the corresponding derivatives IIa & IIb. [(IIa, R = CH ₃; C₂₄H₂₀N₂O₃; % Yield 63, M.P. 162-166^oC); (IIb, R = Cl; C₂₃H₁₇ClN₂O₃; % Yield 57, M.P. 186-190^oC)].

Preparation of 4-[2-(4-substituted phenyl)-4-oxoquinazolin-3(4H)-yl] benzohydrazide (IIIa-b)

A mixture of 4-[2-(4-substituted phenyl)-4-oxoquinazolin-3(4H)-yl] benzoates (IIa-b) (0.01 mol) and excess of hydrazine hydrate (99%, 0.015 mol) in methanol (20 ml) were transferred to a round bottom flask and refluxed for 3 h. After the reaction was completed, the reaction mixture was poured into crushed ice to afford dark brown precipitates. These precipitates were collected by filtration and washed with cold water. The resulting solids were recrystallized from ethanol (99%) to give the corresponding hydrazides (IIIa & IIIb). [(IIIa, R = CH ₃; C₂₂H₁₈N₄O₂; % Yield 75, M.P. 182-186^oC); (IIIb, R = Cl; C₂₁H₁₅ClN₄O₂; % Yield 68, M.P. 204-208^oC)]

Preparation of N-(4-fluorobenzylidene)-4-(4-oxo-2-(4-substituted phenyl)-quinazolin-3(4H)-yl) benzohydrazide (IVa-b)

The compounds IVa & IVb were prepared by refluxing 4-[2-(4-substituted phenyl)-4-oxoquinazolin-3(4H)-yl)] benzohydrazides (IIIa-b; 0.01 mol) and 4-fluorobenzaldehyde (0.012 mol) in methanol (30 ml) containing small amount of glacial acetic acid for 5 h. After the reaction was completed, the Schiff bases (IVa-b) were precipitated out. The precipitate was collected by filtration. The resulting solids were recrystallized from methanol. [(IVa, R = CH ₃; C₂₉H₂₁FN₄O₂; % Yield 58, M.P. 224-228^oC; IVb, R = Cl; C₂₈H₁₈ClFN₄O₂; % Yield 53, M.P. 240-244^oC)].

Preparation of N-(2-(4-fluorophenyl)-4-oxothiazolidin-3-yl)-4-(4-oxo-2-(4-substituted phenyl)-quinazolin quinazolin-3(4H)-yl)benzamide (Va-b)

The synthesis of compounds (Va-b) was achieved by refluxing N-(4-fluorobenzylidene)-4-(4-oxo-2-(4-substituted phenyl)-quinazolin-3(4H)-yl) benzo hydrazides (IVa-b; 0.01 mol) and thioglycolic acid in 1,4 dioxan (20 ml) for 6 h. After the reaction was completed, the solution was poured into ice-cold aqueous solution of sodium bicarbonate to neutralize unreacted thioglycolic acid. The yellow to brown solids separated were filtered and then washed with cold water. The resulting solids were recrystallized from ethanol. [(Va, R = CH ₃; C₃₁H₂₃FN₄O₃S; % Yield 76, M.P. 190-194^oC); (Vb, R = Cl; C₃₀H₂₀ClFN₄O₃S; % Yield 65, M.P. 214-218^oC)].

General procedure for synthesis of N-(5-(aryl)-2-(4-fluorophenyl)-4-oxo thiazolidin-3-yl)-4-(4-oxo-2-(4-substituted phenyl)-quinazolin-3(4H)-yl) benzamides (VIa-n).

A mixture of a solution of N-(2-(4-fluorophenyl)-4-oxothiazolidin-3-yl)-4-(4-oxo-2-(4-substituted phenyl)-quinazolin-3(4H)-yl)benzamides (Va-b; 0.01 mol) and appropriate aryl aldehyde (0.01 mol) in 1,4 dioxane (20 ml) were added with a few drops of glacial acetic acid and refluxed for 8 h. After the reaction was completed, the reaction mixture was poured into crushed ice to afford a precipitate. This was collected by filtration, and then washed with cold water. The above procedure was applied to synthesize the arylidene derivatives VIa-n. All the derivatives were purified by column chromatography using mixture of ethyl acetate and hexane as mobile phase. (% Yield 54-71, M.P. 190-252^oC).

N-(2-(4-fluorophenyl)-4-oxo-5-(furan-2-ylmethylene)-4-oxothiazolidin-3-yl)-4-(4-oxo-2-(p-tolyl) quinazolin-3(4H)-yl) benzamide (VIa)

¹H - NMR: (400 MHz; DMSO-d₆) ? (ppm): 2.28 (s, 3H, CH₃); 5.79 (s, 1H, -N-CH-S-); 7.37-7.48 (m, 2H, H); 7.53-7.89 (m, 9H, H); 7.98-8.10 (m, 4H, H); 8.19-8.37 (m, 4H, Ar-H); 8.84 (s, 1H, -C=CH-Ar); 9.12 (s, 1H, -CO-NH-). ¹³C - NMR: (100 MHz; DMSO-d₆) ? (ppm): 168.24, 166.46, 163.87, 162.35, 157.87, 153.21, 147.82, 144.17, 139.78, 137.89, 137.50, 136.92, 133.78, 130.06, 129.84, 127.69, 126.75, 125.47, 124.97, 120.85, 116.12, 110.97, 108.74, 68.47, 19.43. IR: 3328.20 (N-H str); 3108.50, 3086.40 (C-H Ar); 2932.70 (C-H Ali); 1709.80 (C=O str); 1674.70 (C=O str); 1567.40 (C=N str); 1241.10 (C-F str). Mass (m/z) (ESI-MS): [M⁺] 629. C, H, N: Calcd. C-68.78, H-4.01, N-8.91; Found C-69.10, H-4.33, N-9.23.

N-(2-(4-fluorophenyl)-4-oxo-5-(thiophen-2-ylmethylene)-4-oxothiazolidin-3-yl)-4-(4-oxo-2-(p-tolyl) quinazolin-3(4H)-yl) benzamide (VIb)

¹H - NMR: (400 MHz; DMSO-d₆) ? (ppm): 2.23 (s, 3H, CH₃); 5.88 (s, 1H, -N-CH-S-); 7.39-7.50 (m, 2H, Ar-H); 7.56-7.873 (m, 10H, Ar-H); 7.98-8.26 (m, 7H, Ar-H); 8.84 (s, 1H, -C=CH); 9.11 (s, 1H, -CO-NH-). ¹³C - NMR: (100 MHz; DMSO-d₆) ? (ppm): 166.27, 165.83, 162.17, 161.85, 157.74, 153.86, 149.25, 144.38, 139.78, 137.89, 137.50, 136.92, 135.47, 134.06, 131.64, 130.08, 129.65, 127.41, 126.35, 125.68, 124.16, 120.53, 116.92, 112.37, 110.74, 69.23, 19.78. IR: 3304.83 (N-H str); 309875, 3057.84 (C-H Ar); 2987.10 (C-H Ali); 1729.04 (C=O str); 1683.60 (C=O str); 1584.10 (C=N str); 1198.78 (C-F str). Mass (m/z) (ESI-MS): [M⁺] 645. C, H, N: Calcd. C-67.06, H-3.91, N-8.69; Found C-67.43, H-4.23, N-9.04.

N-(2-(4-fluorophenyl)-4-oxo-5-(pyridin-3-ylmethylene) thiazolidin-3-yl)-4-(4-oxo-2-(p-tolyl) quinazolin-3(4H)-yl) benzamide (VIc)

¹H - NMR: (400 MHz; DMSO-d₆) ? (ppm): 2.28 (s, 3H, CH₃); 5.90 (s, 1H, -N-CH-S-); 7.41-7.64 (m, 8H, Ar-H); 7.72-7.84 (m, 2H, Ar-H); 7.94-8.12 (m, 4H, Ar-H); 8.19-8.28 (m, 2H, Ar-H); 8.76-8.92 (m, 4H, -C=CH & Ar-H); 9.26 (d, 2H, J=8.528, -CO-NH- & Ar-H). ¹³C - NMR: (100 MHz; DMSO-d₆) ? (ppm): 167.63, 164.75, 163.12, 160.09, 157.78, 150.09, 148.93, 147.81, 141.07, 137.82, 136.43, 135.04, 132.65, 132.48, 130.07, 129.61, 129.13, 127.28, 126.97, 125.65, 124.51, 124.03, 121.36, 117.18, 68.31, 19.62. IR: 3312.63 (N-H str); 3112.44, 3097.32 (C-H Ar); 2962.46 (C-H Ali); 1712.64 (C=O str); 1682.09 (C=O str); 1547.25 (C=N str); 1221.17 (C-F str). Mass (m/z) (ESI-MS): [M⁺] 640. C, H, N: Calcd. C-69.47, H-4.10, N-10.95; Found C-69.12, H-3.75, N-10.62.

N-(5-(4-fluorobenzylidene)-2-(4-fluorophenyl)-4-oxothiazolidin-3-yl)-4-(4-oxo-2-(p-tolyl) quinazolin-3(4H)-yl) benzamide (VId)

¹H - NMR: (400 MHz; DMSO-d₆) ? (ppm): 2.40 (s, 3H, CH₃); 5.91 (s, 1H, -N-CH-S-); 7.22-7.32 (m, 3H, Ar-H); 7.48-7.56 (m, 2H, Ar-H); 7.68-7.76 (m, 5H, Ar-H); 7.98-8.08 (m, 4H, Ar-H); 8.30-8.38 (m, 3H, Ar-H); 8.61-8.72 (m, 3H, Ar-H); 8.87 (s, 1H, -C=CH); 9.26 (s, 1H, -CO-NH-). ¹³C - NMR: (100 MHz; DMSO-d₆) ? (ppm): 166.07, 164.82, 160.35, 157.21, 149.27, 140.18, 137.89, 134.12, 130.81, 129.84, 127.53, 126.96, 126.07, 124.85, 116.74, 72.48, 20.08. IR: 3298.90 (N-H str); 3096.73, 3042.52 (C-H Ar); 2943.89 (C-H Ali); 1720.36 (C=O str); 1671.37 (C=O str); 1586.10 (C=N str); 1198.95 (C-F str). Mass (m/z) (ESI-MS): [M⁺] 657. C, H, N: Calcd. C-69.50, H-3.99, N-8.53; Found C-69.18, H-3.67, N-8.21.

N-(5-(2-chlorobenzylidene)-2-(4-fluorophenyl)-4-oxothiazolidin-3-yl)-4-(4-oxo-2-(p-tolyl) quinazolin-3(4H)-yl) benzamide (VIe)

¹H - NMR: (400 MHz; DMSO-d₆) ? (ppm): 2.39 (s, 3H, CH₃); 5.81 (s, 1H, -N-CH-S-); 7.27-7.33 (m, 2H, Ar-H); 7.38-7.47 (m, 6H, Ar-H); 7.63-7.72 (m, 3H, Ar-H); 7.78-7.85 (m, 3H, Ar-H); 7.92-7.99 (m, 4H, Ar-H); 8.42-8.49 (m, 2H, Ar-H); 8.78 (s, 1H, -C=CH); 9.21 (s, 1H, -CO-NH-). ¹³C - NMR: (100 MHz; DMSO-d₆) ? (ppm): 165.13, 164.28, 161.39, 156.84, 149.27, 140.13, 137.46, 134.92, 133.27, 131.48, 130.12, 129.85, 129.36, 127.83, 126.75, 125.87, 125.26, 120.96, 115.82, 69.24, 19.82. IR: 3294.82 (N-H str); 3100.12, 3053.64 (C-H Ar); 2964.17 (C-H Ali); 1707.68 (C=O str); 1644.83 (C=O str); 1589.75 (C=N str); 1209.78 (C-F str). Mass (m/z) (ESI-MS): [M⁺] 673. C, H, N: Calcd. C-67.80, H-3.89, N-8.32; Found C-68.10, H-4.22, N-8.63.

N-(5-(4-chlorobenzylidene)-2-(4-fluorophenyl)-4-oxothiazolidin-3-yl)-4-(4-oxo-2-(p-tolyl) quinazolin-3(4H)-yl) benzamide (VIf)

¹H - NMR: (400 MHz; DMSO-d₆) ? (ppm): 2.26 (s, 3H, CH₃); 5.87 (s, 1H, -N-CH-S-); 7.38-7.62 (m, 5H, Ar-H); 7.79-8.10 (m, 8H, Ar-H); 8.17-8.33 (m, 4H, Ar- H); 8.43-8.60 (m, 3H, Ar-H); 8.90 (s, 1H, -C=CH); 9.26 (s, 1H, -CO-NH-). ¹³C - NMR: (100 MHz; DMSO-d₆) ? (ppm): 165.13, 164.28, 161.39, 156.84, 149.27, 140.13, 137.46, 134.92, 133.67, 130.97, 130.04, 129.85, 129.36, 127.83, 126.75, 125.87, 125.26, 120.96, 115.82, 69.24, 19.82. IR: 3289.12 (N-H str); 3111.45, 3097.24 (C-H Ar); 2978.10 (C-H Ali); 1718.05 (C=O str); 1665.85 (C=O str); 1578.42 (C=N str); 1208.63 (C-F str). Mass (m/z) (ESI-MS): [M⁺] 673. C, H, N: Calcd. C-67.80, H-3.89, N-8.32; Found C-68.14, H-4.23, N-8.64.

N-(2-(4-fluorophenyl)-5-(4-methoxybenzylidene)-4-oxothiazolidin-3-yl)-4-(4-oxo-2-(p-tolyl) quinazolin-3(4H)-yl) benzamide (VIg)

¹H - NMR: (400 MHz; DMSO-d₆) ? (ppm): 2.18 (s, 3H, CH₃); 3.90 (s, 3H, OCH₃); 5.88 (s, 1H, -N-CH-S-); 7.07-7.22 (m, 4H, Ar-H); 7.25-7.36 (m, 2H, Ar-H); 7.43-7.68 (m, 6H, Ar-H); 7.73-7.89 (m, 3H, Ar-H); 7.92-8.07 (m, 3H, Ar-H); 8.26-8.40 (m, 2H, Ar-H); 8.82 (s, 1H, -C=CH); 9.24 (s, 1H, -CO-NH-). ¹³C - NMR: (100 MHz; DMSO-d₆) ? (ppm): 165.13, 164.28, 161.39, 156.84, 149.27, 140.13, 137.46, 134.92, 133.27, 131.48, 130.12, 129.85, 129.36, 127.83, 126.75, 125.87, 125.26, 120.96, 115.82, 69.24, 19.82. IR: 3278.20 (N-H str); 3104.50, 3082.40 (C-H Ar); 2923.70 (C-H Ali); 1711.45 (C=O str); 1680.35 (C=O str); 1574.56 (C=N str); 1217.24 (C-F str). Mass (m/z) (ESI-MS): [M⁺] 669. C, H, N: Calcd. C-70.04, H-4.37, N-8.38; Found C-69.65, H-4.06, N-8.02.

4-(2-(4-chlorophenyl)-4-oxo quinazolin-3(4H)-yl)-N-(2-(4-fluorophenyl)-4-oxo-5-(furan-2-ylmethylene) thiazolidin-3-yl) benzamide (VIh)

¹H - NMR: (400 MHz; DMSO-d₆) ? (ppm): 5.79 (s, 1H, -N-CH-S-); 7.56-7.83 (m, 11H, Ar-H); 7.98-8.14 (m, 3H, Ar-H); 8.20-8.49 (m, 5H, Ar-H); 8.83 (s, 1H, -C=CH); 9.11 (s, 1H, -CO-NH-). ¹³C - NMR: (100 MHz; DMSO-d₆) ? (ppm): 165.37, 164.10, 161.28, 157.06, 152.48, 149.24, 144.60, 138.18, 136.83, 135.46, 133.81, 129.56, 128.83, 127.63, 126.42, 125.06, 121.75, 116.54, 113.68, 110.35, 72.17. IR: 3386.75 (N-H str); 3189.60, 3046.32 (C-H Ar); 2934.61 (C-H Ali); 1708.13 (C=O str); 1676.02 (C=O str); 1591.52 (C=N str); 1175.18 (C-F str); 723.92 (C-Cl str). Mass (m/z) (ESI-MS): [M⁺] 649. C, H, N: Calcd. C-64.76, H-3.42, N-8.63; Found C-64.42, H-3.10, N-8.31.

4-(2-(4-chlorophenyl)-4-oxoquinazolin-3(4H)-yl)-N-(2-(4-fluorophenyl)-4-oxo-5-(thiophen -2-ylmethylene) thiazolidin-3-yl) benzamide (VIi)

¹H - NMR: (400 MHz; DMSO-d₆) ? (ppm): 5.79 (s, 1H, -N-CH-S-); 7.48-7.61 (m, 3H, Ar-H); 7.67-7.86 (m, 8H, Ar-H); 7.98-8.24 (m, 3H, Ar-H); 8.20-8.49 (m, 5H, Ar-H); 8.43-8.56 (m, 2H, Ar-H); 8.83 (s, 1H, -C=CH); 9.11 (s, 1H, -CO-NH-). ¹³C - NMR: (100 MHz; DMSO-d₆) ? (ppm): 165.42, 163.92, 161.15, 157.23, 148.94, 138.05, 136.70, 135.68, 133.76, 131.28, 130.65, 129.86, 128.73, 127.52, 126.75, 125.16, 121.28, 116.42, 71.85. IR: 3316.54 (N-H str); 3127.43, 3098.12 (C-H Ar); 2914.16 (C-H Ali); 1718.23 (C=O str); 1686.42 (C=O str); 1545.12 (C=N str); 1185.34 (C-F str); 726.21 (C-Cl str). Mass (m/z) (ESI-MS): [M⁺] 665. C, H, N: Calcd. C-63.20, H-3.33, N-8.42; Found C-63.57, H-3.70, N-8.79.

4-(2-(4-chlorophenyl) -4-oxo quinazolin-3(4H)-yl)-N-(2-(4-fluorophenyl)-4-oxo-5-(pyridin-3-ylmethylene) thiazolidin-3-yl) benzamide (VIj)

¹H - NMR: (400 MHz; DMSO-d₆) ? (ppm): 5.95 (s, 1H, -N-CH-S-); 7.42-7.59 (m, 3H, Ar-H); 7.69-7.83 (m, 3H, Ar-H); 7.92-8.11 (m, 4H, Ar-H); 8.18-8.30 (m, 3H, Ar-H); 8.43-8.62 (m, 3H, Ar-H); 8.78-8.90 (m, 3H, Ar-H); 9.04 (s, 1H, -C=CH); 9.24 (s, 1H, -CO-NH-). ¹³C - NMR: (100 MHz; DMSO-d₆) ? (ppm): 164.96, 163.84, 161.67, 160.43, 157.18, 151.60, 148.45, 139.12, 137.42, 136.74, 134.45, 132.87, 130.67, 129.52, 128.90, 127.45, 126.76, 125.38, 124.12, 121.60, 116.85, 72.10. IR: 3296.72 (N-H str); 3142.35, 3042.12 (C-H Ar); 2914.67 (C-H Ali); 1711.43 (C=O str); 1684.32 (C=O str); 1568.72 (C=N str); 1186.23 (C-F str); 734.02 (C-Cl str). Mass (m/z) (ESI-MS): [M⁺] 660. C, H, N: Calcd. C-65.50, H-3.51, N-10.61; Found C-65.15, H-3.16, N-10.26.

4-(2-(4-chlorophenyl) - 4-oxoquinazolin-3(4H)-yl)-N-(5-(4-fluoro benzylidene)-2-(4-fluoro phenyl)-4-oxothiazolidin-3-yl) benzamide (VIk)

¹H - NMR: (400 MHz; DMSO-d₆) ? (ppm): 5.87 (s, 1H, -N-CH-S-); 7.22-7.30 (m, 3H, Ar-H); 7.48-7.56 (m, 2H, Ar-H); 7.67-7.86 (m, 5H, Ar-H); 7.98-8.06 (m, 4H, Ar-H); 8.28-8.36 (m, 3H, Ar-H); 8.49-8.68 (m, 3H, Ar-H); 8.95 (s, 1H, -C=CH); 9.27 (s, 1H, -CO-NH-). ¹³C - NMR: (100 MHz; DMSO-d₆) ? (ppm): 165.57, 164.42, 161.82, 160.35, 157.21, 151.65, 149.27, 140.18, 137.89, 134.12, 130.81, 129.84, 127.53, 126.96, 126.07, 124.85, 116.74, 72.48. IR: 3316.05 (N-H str); 3118.40, 3074.32 (C-H Ar); 2925.46 (C-H Ali); 1712.18 (C=O str, amide); 1684.62 (C=O str); 1597.12 (C=N str); 1167.28 (C-F str); 728.14 (C-Cl str). Mass (m/z) (ESI-MS): [M⁺] 677. C, H, N: Calcd. C-65.63, H-3.42, N-8.27; Found C-66.01, H-3.78, N-8.64.

N-(5-(2-chlorobenzylidene)-2 - (4-fluorophenyl)-4-oxothiazolidin-3-yl)-4-(2-(4-chloro phenyl)-4-oxo quinazolin-3(4H)-yl) benzamide (VIl)

¹H - NMR: (400 MHz; DMSO-d₆) ? (ppm): 5.82 (s, 1H, -N-CH-S-); 7.37-7.46 (m, 6H, Ar-H); 7.63-7.74 (m, 3H, Ar-H); 7.80-7.85 (m, 3H, Ar-H); 7.92-8.00 (m, 4H, Ar-H); 8.23-8.30 (m, 2H, Ar-H); 8.48-8.56 (m, 2H, Ar-H); 8.76 (s, 1H, -C=CH); 9.21 (s, 1H, -CO-NH-). ¹³C - NMR: (100 MHz; DMSO-d₆) ? (ppm): 165.74, 164.12, 162.32, 161.08, 157.25, 149.37, 139.18, 137.89, 135.72, 133.56, 130.48, 130.48, 129.84, 128.90, 128.53, 126.76, 125.20, 121.38, 116.85, 72.15. IR: 3307.62 (N-H str); 3118.96, 3078.43 (C-H Ar); 2914.98 (C-H Ali); 1711.43 (C=O str); 1656.62 (C=O str); 1584.62 (C=N str); 1168.94 (C-F str); 727.49 (C-Cl str). Mass (m/z) (ESI-MS): [M⁺] 694. C, H, N: Calcd. C-64.07, H-3.34, N-8.08; Found C-64.39, H-3.66, N-8.40.

N-(5-(4-chlorobenzylidene)-2-(4-fluorophenyl)-4-oxothiazolidin-3-yl)-4-(2-(4-chloro phenyl)-4-oxo quinazolin-3(4H)-yl) benzamide (VIm)

¹H - NMR: (400 MHz; DMSO-d₆) ? (ppm): 5.86 (s, 1H, -N-CH-S-); 7.38-7.61 (m, 5H, Ar-H); 7.80-8.08 (m, 8H, Ar-H); 8.16-8.32 (m, 4H, Ar-H); 8.40-8.62 (m, 3H, Ar-H); 8.90 (s, 1H, -C=CH); 9.28 (s, 1H, -CO-NH-). ¹³C - NMR: (100 MHz; DMSO-d₆) ? (ppm):165.75, 164.12, 161.08, 157.20, 149.62, 139.86, 136.94, 135.78, 133.45, 131.40, 130.52, 129.76, 128.74, 128.62, 126.47, 125.26, 121.30, 116.74, 71.83. IR: 3365.16 (N-H str); 3123.46, 3074.13 (C-H Ar); 2945.08 (C-H Ali); 1712.78 (C=O str); 1672.61 (C=O str); 1584.52 (C=N str); 1178.07 (C-F str); 728.19 (C-Cl str). Mass (m/z) (ESI-MS): [M⁺] 694. C, H, N: Calcd. C-64.07, H-3.34, N-8.08; Found C-64.39, H-3.66, N-8.40.

4-(2-(4-chlorophenyl) - 4-oxoquinazolin-3(4H)-yl)-N-(2-(4-fluorophenyl)-5(4-methoxy benzylidene)-4-oxothiazolidin-3-yl) benzamide (VIn)

¹H - NMR: (400 MHz; DMSO-d₆) ? (ppm): 3.92 (s, 3H, Ar-OCH₃); 5.88 (s, 1H, -N-CH-S-); 7.16-7.32 (m, 4H, Ar-H); 7.42-7.63 (m, 6H, Ar-H); 7.74-7.89 (m, 3H, Ar-H); 7.91-8.10 (m, 3H, Ar-H); 8.22-8.40 (m, 4H, Ar-H); 8.82 (s, 1H, -C=CH); 9.23 (s, 1H, -CO-NH-). ¹³C - NMR: (100 MHz; DMSO-d₆) ? (ppm):165.80, 164.63, 162.17, 161.25, 160.10, 157.20, 149.62, 138.56, 136.42, 135.78, 134.23, 131.84, 130.52, 129.26, 128.94, 127.48, 126.67, 125.26, 124.53, 121.30, 116.74, 114.75, 71.90, 56.47. IR: 3345.17 (N-H str); 3139.60, 3086.32 (C-H Ar); 2932.61 (C-H Ali); 1712.84 (C=O str); 1694.02 (C=O str); 1572.32 (C=N str); 1164.18 (C-F str); 724.92 (C-Cl str). Mass (m/z) (ESI-MS): [M⁺] 689. C, H, N: Calcd. C-66.23, H-3.80, N-8.13; Found. C-65.87, H-3.44, N-7.87.

Cell culture

The MCF-7 and A549 cell lines were maintained in culture with MEM medium, supplemented with 10% FBS and the antibiotics penicillin/streptomycin (0.5 mg/ml); in atmosphere of 5% CO₂ and 95% air at 37ºC. Stock solutions of synthesized compounds were made in DMSO and kept in aliquots at -20ºC. For MTT assay, each test compound was weighed separately and dissolved in DMSO, made up the final concentration with media to 1 mg/ml and the cells were treated with series of concentrations from 10 to 100 μm.

Molecular Docking

Quinazolinone derivatives were reported to possess anticancer activity by inhibiting target enzyme phosphoinositide-3-OH kinase PI(3)K⁹^{), (}¹⁰. Hence, in the present study, the titled compounds were docked on the PI(3)K to understand the underlying mechanism. Docking studies were performed by Molegro Virtual Docker (MVD) programmer¹¹. The 3D coordinates of crystal structure of phosphoinositide-3-OH kinase (PI(3)K) was obtained from protein data bank (PDB ID: 2WXQ) with resolution of 2.7 Å. The ligands were developed using Chem Draw Ultra 12.0. Ligands were further 3D optimized and saved in mol2 file format. MVD performs flexible ligand docking, so the optimal geometry of the ligand will be determined during the docking.

RESULTS

Synthesis of Compounds

The titled compounds (VIa-n) were synthesized by the reported multi-step reaction protocol¹²^{), (}¹³. The synthetic procedures were carried out as shown in Scheme 1. The starting materials (Ia-b) were prepared by the reaction of 5-chloroanthranilic acid with substituted benzoyl chlorides¹⁴, which were treated with ethyl-p-amino benzoate to give (IIa-b). The compounds (IIIa-b) were obtained by the reaction of (IIa-b) with hydrazine hydrate. Upon fusion of (IIIa-b) with 4-fluoro phenyl afforded the (IVa-b). The reaction of compounds (IVa-b) with thioglycolic acid gave (Va-b). The desired compounds (VIa-n) were obtained by refluxing the (Va-b) with various heteroaryl/ substituted aldehydes in 1,4 dioxane by adding few drops of glacial acetic acid. The target compounds were obtained in 54-71% yields (Table 1).

Scheme 1: Synthetic route of the final compounds (Via-n). (a) Pyridine; stirring at 15-20 ºC, 1-2 hrs; (b) Ethyl 4-amino benzoate, heated on an oil bath at 180-200 ºC for 25 min; (c) NH₂-NH₂.H₂O, methanol, 3 hrs reflux; (d) Methanol, glacial acetic acid, 5 hrs reflux; (e) HSCH₂COOH, 1,4-dioxane, 6 hrs reflux; (f) Ar-CHO, 1,4-dioxane, glacial acetic acid, 8 hrs reflux.

Table 1: Physical data of the synthesized compounds

Sl. No.	Compound code	R	Aryl	Mol. Formula	Mol. Wt.	% Yield	R_f	M. P. (_oC)
1	VIa	CH₃	2-Furan	C₃₆H₂₅FN₄O₄S	628.67	58	0.51	226-230
2	VIb	CH₃	2-Thiophene	C₃₆H₂₅FN₄O₃S₂	644.74	67	0.67	208-212
3	VIc	CH₃	3-Pyridine	C₃₇H₂₆FN₅O₃S	639.70	54	0.35	222-228
4	VId	CH₃	4-Fluoro phenyl	C₃₈H₂₆F₂N₄O₃S	656.70	71	0.74	230-234
5	VIe	CH₃	2-Chloro phenyl	C₃₈H₂₆ClFN₄O₃S	673.15	69	0.61	202-206
6	VIf	CH₃	4-Chloro phenyl	C₃₈H₂₆ClFN₄O₃S	673.15	62	0.53	194-198
7	VIg	CH₃	4-Methoxy phenyl	C₃₉H₂₉FN₄O₄S	668.74	60	0.47	190-194
8	VIh	Cl	2-Furan	C₃₅H₂₂ClFN₄O₄S	649.09	56	0.29	244-248
9	VIi	Cl	2-Thiophene	C₃₅H₂₂ClFN₄O₃S₂	665.16	62	0.41	232-236
10	VIj	Cl	3-Pyridine	C₃₆H₂₃ClFN₅O₃S	660.12	56	0.52	240-244
11	VIk	Cl	4-Fluoro phenyl	C₃₇H₂₃ClF₂N₄O₃S	677.12	69	0.36	248-252
12	VIl	Cl	2-Chloro phenyl	C₃₇H₂₃Cl₂FN₄O₃S	693.60	63	0.68	226-230
13	VIm	Cl	4-Chloro phenyl	C₃₇H₂₃Cl₂FN₄O₃S	693.60	61	0.46	218-222
14	VIn	Cl	4-Methoxy phenyl	C₃₈H₂₆ClFN₄O₄S	689.15	57	0.53	212-216

MTT assay

Cell viability was evaluated by the MTT Assay with three independent experiments with five concentrations of compounds. MCF-7 and A549 cells were seeded in 96 well plates at a density of 5X10³ cells/well. The cultures were treated with a series of concentration of 10-100 µg/ml of test and standard compounds and incubated at 37^oC for 48 hrs., followed by addition of MTT solution (5 mg/ml) to each plate and were incubated at 37^oC for 3 hrs. At the end of incubation time, precipitates are formed as a result of the reduction of the MTT salt to chromophore formazan crystals by the cells with metabolically active mitochondria. The optical density of solubilized crystals in DMSO was measured at 570 nm on a microplate reader¹⁵^{), (}¹⁶. Percentage growth inhibition was calculated using the following formula (Formula 1). Results were indicated with mean and standard deviation (M±S.D.) of three independent experiments and were documented in Table 2.

Formula 1: Percentage growth inhibition.

Table 2: Anticancer activity of synthesized compounds (VIa-n)

Sl. No.	Compound	R	Aryl	IC₅₀ (µM)
Sl. No.	Compound	R	Aryl	MCF-7	A549
1	Va	CH₃	----	0.053±0.003	0.065±0.002
2	Vb	Cl	----	0.048±0.004	0.055±0.002
3	VIa	CH₃	2-Furanyl	0.074±0.003	0.064±0.001
4	VIb	CH₃	2-Thiophenyl	0.116±0.002	0.140±0.002
5	VIc	CH₃	3-Pyridinyl	0.075±0.002	0.125±0.002
6	VId	CH₃	4-Fluorophenyl	0.066±0.003	0.035±0.002
7	VIe	CH₃	2-Chlorophenyl	0.093±0.002	0.135±0.002
8	VIf	CH₃	4-Chlorophenyl	0.079±0.003	0.123±0.002
9	VIg	CH₃	4-Methoxyphenyl	0.066±0.002	0.042±0.003
10	VIh	Cl	2-Furanyl	0.063±0.002	0.040±0.003
11	VIi	Cl	2-Thiophenyl	0.099±0.003	0.109±0.002
12	VIj	Cl	3-Pyridinyl	0.065±0.003	0.092±0.002
13	VIk	Cl	4-Fluorophenyl	0.054±0.003	0.031±0.003
14	VIl	Cl	2-Chlorophenyl	0.075±0.002	0.106±0.002
15	VIm	Cl	4-Chlorophenyl	0.066±0.002	0.093±0.002
16	VIn	Cl	4-Methoxyphenyl	0.055±0.002	0.030±0.002
17	Standard	---	---	0.028±0.002	0.023±0.002
Standard: Doxorubicin

DISCUSSION

Structures of synthesized compounds were ascertained by IR, NMR and Mass spectral studies. Elemental analysis results were in good agreement (±0.4%) with the calculated values. The mass spectra ESI-MS showed molecular ion peak at M⁺ corresponding to exact molecular weight of the respective compound. The IR spectral data of quinazolin-4(3H)-one-thiazolidinone derivatives (VIa-n) showed characteristic absorption bands. The vibration frequencies of N-H stretching were found in the range of 3386-3278 cm^-1; C=O stretching bands were observed in the range of 1729-1707 cm^-1 and 1686-1644 cm^-1 whereas C-F stretching at 1241-1164 cm^-1. The ¹H-NMR spectra of all compounds showed a singlet in the range of ? 9.28 - 9.09 ppm corresponding to amide (-CO-NH-) proton. The proton of thiazolidinone-5-arylidene substitution (-C=CH-Ar-) appeared as singlet between ? 8.95- 8.76 ppm; whereas thiazolidinone ring (-N-CH-S-) proton appeared as singlet between ? 5.91-5.79 ppm. The substituents of phenyl rings -CH₃ (VIa-g) and -OCH₃ (VIg & VIn) appeared as singlet signals between ? 2.38-2.18 ppm and ? 3.90-3.92 ppm respectively. ¹³C-NMR spectra of VIa-n showed signals at frequencies in the range of ? 167-158 ppm associated with C=O of quinazolinone, thiazolidinone and amide. Further, the aliphatic carbons of CH₃ and OCH₃ appeared at ? ~20 ppm and ? ~57 ppm respectively. All the other carbons of aromatic and hetero aromatic rings were observed in the region at ? 110-165 ppm.

Anticancer evaluation

The results revealed that all the tested compounds exhibited moderate to excellent activity against MCF-7and A549 cell lines. The compounds Va and Vb with methyl and chloro substitution exhibited the highest potency against both the cell lines greater than that of their corresponding arylidene derivatives (VIa-n). Compound Vb showed IC₅₀ of 0.048±0.004 μM and 0.055±0.002 μM against MCF-7 and A549 cell lines respectively compared to the IC₅₀ of 0.027±0.002 μM and 0.023±0.002 μM exhibited by the standard doxorubicin. Among the arylidene derivatives compound VIk (R = Cl; Ar = 4-F-C₆H₄), VIn (R = Cl; Ar = 4-OCH₃-C₆H₄) and VId (R = CH₃; Ar = 4-F-C₆H₄) showed significant activity with IC₅₀ values 0.031±0.003 μM, 0.030±0.002 μM and 0.035±0.002 μM respectively against A549 cell lines, as compared to 0.023±0.002 μM showed by the standard doxorubicin. In addition, the arylidene derivatives VIk and VIn also showed high potency against MCF-7 cell lines with IC₅₀ values 0.054±0.003 μM and 0.055±0.002 μM respectively. Among the arylidene derivatives, the compounds with 4-fluorophenyl substitution on C5 position of 4-thiazolidinone (VId & VIk) showed greater potency against both cell lines than the other with aryl and hetero aryl substitutions. The anticancer activity of synthesized derivatives and inhibition of growth of cell by VIk were depicted in Figure 2.

Molecular docking study

The lowest energy docked conformation of the ligands VIb, VIl and VIn revealed that the compounds bind with an overall binding energy of -62.964 kcal/mol, -81.968 kcal/mol, -65.086 kcal/mol making intimate contacts with the residues of active site of the target enzyme phosphoinositide-3-OH kinase (PI(3)K) through significant bonded and non-bonded interactions with higher docking scores -74.602, -99.037 and -76.787 respectively (Table 3). The ligand VIl showed three hydrogen bonding interaction with His-716, Arg-720, Lys-712 with high docking score (Figure 3), whereas the ligand VIb with least anticancer activity showed three H-bond interactions with Arg-720, His-716. The ligand VIn which showed high potency also showed one hydrogen bond interaction with Arg-720. The compounds VIk and Vd, which exhibited highest activity against A549 cell lines and MCF-7 cell lines showed least docking scores, indicating that the cytotoxicity of the most active compound may due to other mechanism than the inhibition of PI(3)K enzyme.

Figure 2: (A) Comparing the IC₅₀ (µM) of all synthesized compounds and (STD) on the MCF-7 and A549 cells. The data represented as mean±S.D. (B) Dose dependant inhibition of MCF-7 and A549 cells by compound VIk, representing the images of one out of three experiments

Table 3: Molecular docking results: molecular docking scores, docking energies and H- bond interactions & distances of compounds with target PI(3)K (PDB ID: 2WXQ)

Sl. No.	Compound	Docking score	Re-rank score	Binding energy	H-Bond & Distance
1	Va	-25.527	-21.056	-14.382	-----
2	Vb	-19.88	-13.748	-5.943	-----
3	VIa	-47.131	-35.503	-41.105	-----
4	VIb	-74.602	-57.884	-62.964	Arg 720; N(7)-6=3.44Å; His 716; N(7)-6=3.48Å & N(7)-9=3.58Å
5	VIc	-16.32	0.068	-14.902	-----
6	VId	-25.525	-19.874	-13.936	-----
7	VIe	-38.777	-32.843	-22.84	-----
8	VIf	-25.533	-20.745	-14.051	-----
9	VIg	-73.171	-61.666	-59.036	Glu 713; N(7)-24=2.60Å Gln 710; N(7)-6=3.45Å
10	VIh	-51.067	-43.871	-36.627	-----
11	VIi	-25.807	-18.079	-15.17	-----
12	VIj	-56.474	-47.974	-41.384	-----
13	VIk	-20.918	-15.237	-9.425	-----
14	VIl	-99.037	-60.212	-81.968	His 716; O(8)-10=2.60Å Arg 720; O(8)-10=3.30Å Lys 712; N(7)-27=2.90Å
15	VIm	-20.918	-17.458	-13.87	-----
16	VIn	-76.787	-62.39	-65.086	Arg 720; N(7)-6=3.44Å

Figure 3: (A) Docking orientation of compound VIl with the target enzyme (B) Compound VIl forming H-Bond interactions with amino acid residues of the target enzyme PI(3)K

CONCLUSIONS

The present study revealed a new series of N-(2-(4-fluoro phenyl)-4-oxothiazolidin-3-yl)-4-(4-oxo-2-[(4-substituted phenyl)-quinazolin-3(4H)-yl)] benzamide derivatives were successfully synthesized and analyzed by various spectral methods. All the purified compounds were screened for their in vitro anti-cancer activity against MCF-7 and A549 cell lines. Compounds Va, Vb, VId, VIh, VIk and VIn exhibited potent anticancer activity with IC₅₀ range of 0.030-0.070 µM against MCF-7 and A549 cell lines. Docking results indicate that anticancer activity of the title compounds is not due to inhibition of PI(3)K enzyme.

Agradecimientos/Acknowledgments

The authors are grateful to thank IICT, Hyderabad, Telangana, India and Andhra University, Vishakhapatnam, Andhra Pradesh, India for providing analytical instrumental facilities and Synteny life sciences Pvt. Ltd. , Hyderabad, Telangana, India for biological activity studies.

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Received: April 24, 2018; Accepted: June 22, 2018

Correspondencia/Correspondence Garlapati Achaiah, Professor, Medicinal Chemistry division, University College of Pharmaceutical Sciences, Kakatiya University, Warangal 506009, Telangana, India. Tel.: Ph: (91)-870-2446259 & 2432699 (Office), Mob: 91 9440601054 achaiah_g@yahoo.co.in

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