Abstract
A unique series oxadiazoles were synthesized by cyclization of benzophenone hydrazide, followed by the nucleophillic alkylation of heterocyclic scaffold. Synthesized title compounds were characterized by the FT-IR, LCMS and NMR spectral techniques. The newly synthesized compounds were screened for the anticancer activity. IC50 values of the 7h observed for in-vitro anti-cancer activities were 112.6 µg/ml and 126.7 µg/ml, against the MCF-7 and KB cell lines respectively. Most active compounds were found to be less toxic, which were determined by MTT assay method with normal cell line (L292). Biological screening of the synthesized series of compounds reveals that, Compound 7h was the potent molecule.
Graphic abstract
Similar content being viewed by others
1 Introduction and experimental
The recognition of prime molecular targets for cancer therapy has paved the way to a paradigm shuffle in drug discovery and thus more up thrust is now placed on molecules for drug designing. A plethora of heterocycles containing nitrogen have been the center of attention for their high therapeutic values in the past few decades. Cancer is one of the most life threatening diseases for mankind. Approximately 38.4% of men and women will be diagnosed with cancer at some point during their lifetimes and according to the World Health Organization (WHO), in 2018 globally 9.6 million deaths was due to the cancer and one in six deaths is from the cancer. When diseases become tolerant to pharmaceutical treatments this phenomenon is known as Drug resistance [1]. Multidrug resistance continues to be the principal limiting factor to achieving cures in patients with cancer. To overcome the MDR limitations many research has taken place and in that oxadiazole is one of the promising scaffold in the medicinal chemistry [2]. Oxadiazoles are physiologically active heterocyclic compounds owing to a vast sphere of biological activities, including anticancer [3,4,5,6,7,8,9,10,11], antidiabetic [12], antiviral [13, 14], anti-inflammatory [15, 16], antibacterial [17, 18], antifungal [19], antimycobacterial [20], analgesic [21], anticonvulsant [22], tyrokinase inhibitor [23] and cathepsin K inhibitor [24] antioxidant [25] properties.
The presence of morpholine and piperidine kernels in several categories of pharmaceutical agents has made these irreplaceable anchors for the development of new therapeutic agent. Piperidine and morpholine are heterocyclic organic compounds which are major class of elementary units in organic synthesis. Morpholine and piperidine derivatives are known to display various pharmacological properties such as antimicrobial, antitubercular, anticonvulsant, anti-inflammatory, analgesic [26], tachykinin receptor binding [27] and antiviral [28, 29]. An awareness of morpholine for their several factors, firstly, the participation of oxygen atom in donor–acceptor type interactions and secondly, electronegative effects of the oxygen atom is on a high fame.
As the synthesis of heterocyclic compounds comprising of multi-structured framework is gaining an incredible progress in recent years. Conjugation of the different types of the functional unit makes the hybrid molecular structure, which exhibit the different phase of the activities. Benzophenone based compounds is known to exhibit a broad range of biological activity. Primarily, it acts as target specific anti-angiogeneic pharmacophores which is in the processes of drug development progression, it encouraged us to envisage the combination of oxadiazole, benzophenone, morpholine and piperidine. In the current context and continuation of our drug discovery research [30, 31], a new series of morpholine and piperidine tagged oxadiazole compounds have been synthesized and evaluated for anticancer activity.
2 Materials and methods: chemistry
The chemicals were procured from Sigma Aldrich Chemical Co. Ltd, Mumbai. TLC was performed on aluminium-backed silica plates and visualized by UV-light. Melting points were measured by the open capillary method and were uncorrected. FT-IR spectra were determined on Perkin Elmer spectrophotometer version 10.03.09 instrument over the range of 600–4000 cm−1. 1H and 13C NMR spectra were recorded on a Bruker 400 MHz in deuterated chloroform and the chemical shifts were recorded in ppm downfield using TMS as the internal standard. Mass spectra were obtained with a VG70-70H spectrophotometer and the elemental analysis of the compounds was performed on a Perkin Elmer 2400 Elemental Analyzer. The results of elemental analyses were within ± 0.4% of the theoretical values.
2.1 Synthesis of benzophenone-mercapto oxadiazole derivatives
The protocol for the synthesis of hydroxy benzophenone analogues and benzophenone-mercapto oxadiazole derivatives are shown in Schemes 1 and 2, respectively.
Synthesis of oxadiazole conjugated benzophenone derivatives
Synthesis of benzophenone derivatives
2.1.1 General procedure for the synthesis of substituted hydroxy benzophenone analogues (3a–g)
The starting benzoates were synthesized by the reaction of substituted phenols with substituted benzoyl chlorides in 10% sodium hydroxide solution. The reaction mixture was stirred for 3–4 h at 0–5 °C and was monitored by thin layer chromatography (TLC) using 8:2 (n-hexane: ethyl acetate) solvent mixture. Further, hydroxy benzophenones 3a–g was synthesized by Fries rearrangement. Substituted benzoates and anhydrous aluminium chloride (0.002 mol) were homogenized and the mixture was heated upto 150–170 °C without solvent for 2–3 h. Then the reaction mixture was cooled to 0 °C and quenched with 6 N hydrochloric acid in ice cold water. The reaction mixture was stirred for about 2–3 h. The solid was filtered and recrystallized with ethanol. Compound 3a is taken as a representative example to explain characterization data.
2.1.2 Synthesis of hydroxy benzophenone (3a)
Yield 85%; M.P: 133–135 °C FT-IR (KBr, cm−1): 1640 (C=O), 3518 (OH); 1H NMR (CDCl3): 6.76–7.86 (m, 9H, Ar–H), 12.2 (bs, 1H, OH); 13C-NMR (CDCl3): δ 116.1, 129.08, 131.1, 132.6,132.9, 133.5, 139.1, 163.13, 195.1; LC–MS m/z 199 (M + 1). Elemental Analysis: Calcd. for C13H10O2: C, 79.22; H, 5.70%. Found: C, 78.88; H, 5.69%.
2.1.3 General protocol for the synthesis of (4-benzoyl phenoxy)-acetic acid ester analogues (4a–g)
To a mixture of substituted hydroxy benzophenones 3a–g (0.05 mol) and chloro ethyl acetate (0.075 mol) in dry acetone (40 ml), anhydrous potassium carbonate (0.075 mol) was added and were refluxed for 8–10 h. The reaction mixture was cooled and solvent removed by distillation. The residual mass was triturated with cold water to remove potassium carbonate, and extracted with ether (3 × 30 ml). The ether layer was washed with 10% sodium hydroxide solution (3 × 30 ml) followed by water (3 × 30 ml) and then dried over anhydrous sodium sulphate and evaporated to afford compounds 4a–g. Compound 4a is taken as a representative example to explain characterization data.
2.1.4 Synthesis of (4-benzoyl phenoxy)-acetic acid ester (4a)
Yield: 93%. M.P: 52–54 °C. IR (KBr) νmax (cm−1): 1672 (C=O), 1719 (ester, C=O). 1H NMR (400 MHz, CDCl3) δ (ppm): 1.35 (t, 3H, CH3 of ester), 4.21 (q, 2H, CH2 of ester), 5.01 (s, 2H, OCH2), 6.81–7.85 (m, 9H, Ar–H). 13C-NMR (CDCl3): δ 14.5, 61.15, 65.8, 115.3, 129.08, 131.8, 132.1,133.3, 133.9, 139.4, 161.7, 169.8, 194.6; LC–MS m/z 285 (M + 1). Elemental Analysis: Cal. for C17H16O4 (212): C, 72.22; H, 5.70. Found: C, 72.20; H, 6.16%.
2.1.5 General protocol for the synthesis of (4-benzoyl phenoxy)-acetic acid hydrazide derivatives (5a–g)
To the solution of ester analogs 4a–g (0.05 mol) in ethanol (20 ml), hydrazine hydrate (0.06 mol) was added and the reaction mixture was stirred at room temperature for 5–6 h. Reaction completion was monitored by thin layer chromatography using hexane:ethylacetate (3:1) solvent mixture and the reaction mixture was allowed to stand overnight. The white crystals 5a–g formed were filtered, washed and after drying recrystallized from ethanol. Compound 5a is taken as a representative example to explain characterization data [32].
2.1.6 Synthesis of (4-benzoyl phenoxy)-acetic acid hydrazide (5a)
Yield 92%; M.P: 115–117 °C; FT-IR (KBr, cm−1): 3318 (NH2), 3230 (NH), 1675 (amide, C=O), 3240–3350 (NH-NH); 1642 (C=O); 1H NMR (CDCl3): δ 3.86 (d, 2H, NH2), 5.09 (s, 2H, OCH2), 8.46 (t, 1H, NH), 6.81–7.85 (m, 9H, Ar–H). 13C-NMR (CDCl3): δ 65.9, 116.3, 129.78, 131.6, 132.3, 133.6, 133.9, 132.4, 161.7, 169.8, 194.9; LC–MS m/z 271 (M + 1). Elemental Analysis: Calcd. for C15H14N2O3: C, 66.89; H, 5.52; N, 10.96. Found: C, 66.78; H, 5.56; N, 10.08%.
2.1.7 General procedure for the synthesis of (4-benzoyl phenoxy)-[1,3,4]oxadiazole-2-thiol derivatives (6a–g)
A mixture of hydrazide analogs 5a–g (3 mmol), carbon disulfide (3 ml) and potassium hydroxide (6 mmol) in ethanol (60 ml) was refluxed till evolution of hydrogen sulfide was ceased. Afterwards the reaction mixture was cooled to room temperature. The solvent was removed at reduced pressure, poured to cold water and acidified with dil HCl solution to precipitate [1,3,4]oxadiazole-2-thiol derivatives by bringing the pH between 3 and 4. The precipitate thus separated out was allowed to stand overnight, filtered, washed, dried and recrystallized from acetone. Compound 6a is taken as a representative example to explain characterization data [33].
2.1.8 Synthesis of (4-benzoyl phenoxy)-[1,3,4]oxadiazole-2-thiol (6a)
Yield 84%; M.P: 140–142 °C; FT-IR (KBr, cm−1): 1612 (C = N), 2557 (SH), 1164 (C–O–C); 1H NMR (CDCl3): δ 5.05 (s, 2H, OCH2), 6.95 (d, J = 8.80 Hz, 2H, Ar–H), 7.32 (d, J = 8.85 Hz, 2H, Ar–H), 7.41 (d, J = 8.8 Hz, 2H), 7.54 (d, J = 7.6 Hz, 2H), 10.72 (s, 1H, SH); LC–MS m/z 243 (M + 1). Elemental Analysis: Calcd. for C9H7N2O2S: C, 44.54; H, 2.91; N, 11.54. Found: C, 44.70; H, 2.82; N, 11.65.
2.1.9 General synthetic procedure for [(4-benzoyl phenoxy)-[1,3,4]oxadiazol-2-ylsulfanyl)-ethyl]-morpholine/piperidine derivatives (7a–n)
A mixture of 1,3,4 oxadiazole-2-thiol derivatives 6a–g (1 mmol), 4-(2-chloroethyl) morpholine/piperidine hydrochloride (3 mmol) and anhydrous potassium carbonate (3 mmol) in DMF (30 ml) was refluxed for 10–15 h. After completion of the reaction, the reaction mixture was cooled down, excess of solvent was evaporated under reduced pressure. The residual crude was poured into crushed ice to precipitate the desired target compound as potassium carbonate is highly soluble in water.
2.1.10 Synthesis of Phenyl(4-((5-((2-(piperidin-1-yl)ethyl)thio)-1,3,4-oxadiazol-2-yl)methoxy) phenyl) methanone (7a)
Yield 88%; M.P: 58–60 °C; FT-IR (KBr, cm−1): 1654 (C=O), 1333 (C = N), 1272 (C–O–C); 1H NMR (CDCl3): δ 2.52 (t, 4H, CH2NCH2 of piperidine ring), 2.74 (t, 2H, N CH2), 3.44 (t, 2H, CH2S), 1.54 (m, 4H, CH2CH2 of piperidine ring), 1.44 (t, 2H, CH2 of piperidine ring), 5.12 (s, 2H, OCH2), 6.81–7.85 (m, 9H, Ar–H); 13C-NMR (CDCl3): δ 24.66, 26.2, 30.7, 56.8, 57.80, 71.99, 114.2, 128.8, 130.6, 130.8, 131.6, 132.6, 138.9, 160.1, 162.9, 163.5, 194.7; LC–MS m/z 424 (M + 1)+. Elemental Analysis: Calcd. for C23H25N3O3S: C, 65.23; H, 5.95; N, 9.92; S, 7.57%. Found: C, 65.29; H, 5.90; N, 9.98; S, 7.67%.
2.1.11 Synthesis of (4-((5-((2-morpholinoethyl)thio)-1,3,4-oxadiazol-2-yl)methoxy) phenyl) methanone (7b)
Yield 85%; M.P: 63–66 °C; FT-IR (KBr, cm−1): 1650 (C=O), 1338 (C = N), 1278 (C–O–C); 1H NMR (CDCl3): δ 2.46 (t, 4H, CH2NCH2 of morpholine ring), 2.65 (t, 2H, NCH2), 3.41 (t, 2H, CH2S), 3.59 (t, 4H, CH2OCH2 of morpholine ring), 5.22 (s, 2H, OCH2), 6.71–7.95 (m, 9H, Ar–H); 13C-NMR (CDCl3): δ 30.9, 55.9, 57.9, 56.8, 57.80, 71.99, 114.2, 128.8, 130.6, 130.8, 131.6, 132.6, 138.9, 160.1, 162.9, 163.5, 194.7; LC–MS m/z 426 (M + 1)+. Anal. Calcd. for C22H23N3O4S: C, 62.10; H, 5.45; N, 9.88; S, 7.54%. Found: C, 62.29; H, 5.50; N, 9.99; S, 7.65%.
2.1.12 Synthesis of (4-fluorophenyl){3-methyl-4-[(5-((2-(piperidin-1-yl)ethyl)thio)-1,3,4-oxadiazole-2-yl)methoxy] phenyl}methanone (7c)
Yield 84%; M.P: 54–58 °C; FT-IR (KBr, cm−1): 1652 (C=O), 1330 (C=N), 1270 (C–O–C); 1H NMR (CDCl3): δ 2.59 (t, 4H, CH2NCH2 of piperidine ring), 2.79 (t, 2H, N CH2), 3.47 (t, 2H, CH2S), 1.59 (m, 4H, CH2CH2 of piperidine ring), 1.48 (t, 2H, CH2 of piperidine ring), 5.18 (s, 2H, OCH2), 6.90–7.95 (m, 7H, Ar–H); 13C-NMR (CDCl3): δ 15.6, 24.66, 25.9, 30.4, 56.9, 57.6, 71.9, 113.6, 115.6, 124.5, 128.28, 131.7, 131.9, 133.8, 134.4, 160.2, 162.9, 163.5, 166.54, 194.3; LC–MS m/z 456 (M + 1)+. Elemental Analysis: Calcd. for C24H26FN3O3S: C, 63.28; H, 5.75; F, 4.17; N, 9.22; S, 7.04% Found: C, 64.29; H, 5.80; F, 4.19 N, 9.28; S, 7.07%.
2.1.13 Synthesis of 4-(fluorophenyl){3-methyl-4-[(5-((2-morpholinoethyl)thio)-1,3,4oxadiazol-2-yl) methoxy] phenyl} methanone (7d)
Yield 83%; M.P: 49–52 °C; FT-IR (KBr, cm−1): 1651 (C=O), 1340 (C=N), 1280 (C–O–C); 1H NMR (CDCl3): δ 2.48 (t, 4H, CH2NCH2 of morpholine ring), 2.69 (t, 2H, NCH2), 3.48 (t, 2H, CH2S), 3.64 (t, 4H, CH2OCH2 of morpholine ring), 5.36 (s, 2H, OCH2), 6.88–7.97 (m, 7H, Ar–H); 13C-NMR (CDCl3): δ 15.7, 30.9, 55.9, 57.9, 66.9, 72.4, 113.9, 115.8, 124.9, 128.3, 132.4, 132.9, 134.4, 135.1, 160.2, 163.2, 164.5, 166.64, 195.3; LC–MS m/z 458 (M + 1)+. Elemental Analysis: Calcd. for C23H24FN3O4S: C, 60.38; H, 5.29; F, 4.15; N, 9.18; S, 7.01%. Found: C, 60.29; H, 5.40; F, 4.45; N, 9.19; S, 7.05%.
2.1.14 Synthesis of (2-chlorophenyl)(3-methyl-4-((5-((2-(piperidin-1-yl)ethyl)thio)-1,3,4 oxadiazol-2-yl)methoxy) phenyl) methanone (7e)
Yield 81%; M.P: 58–60 °C; FT-IR (KBr, cm−1): 1658 (C=O), 1337 (C=N), 1275 (C–O–C); 1H NMR (CDCl3): δ 2.53 (t, 4H, CH2NCH2 of piperidine ring), 2.74 (t, 2H, N CH2), 3.43 (t, 2H, CH2S), 1.75 (m, 4H, CH2CH2 of piperidine ring), 1.52 (t, 2H, CH2 of piperidine ring), 5.3 (s, 2H, OCH2), 7.29–8.69 (m, 7H, Ar–H); 13C-NMR (CDCl3): δ 15.8, 24.7, 25.7, 30.7, 56.8, 57.5, 71.7, 113.8, 115.7, 124.8, 128.4, 131.9, 132.3, 134.8, 139.4, 160.2, 162.9, 163.5, 166.54, 198.3; LC–MS m/z 472 (M + 1)+. Elemental Analysis: Calcd. for C24H26ClN3O3S: C, 61.28; H, 5.55; Cl, 7.57; N, 8.92; S, 7.04% Found: C, 62.29; H, 5.70; Cl, 7.67; N, 8.28; S, 7.07%.
2.1.15 Synthesis of (2-chlorophenyl){3-methyl-4-[(5-((2-morpholinoethyl)thio)-1,3,4-oxadiazol-2-yl) methoxy]phenyl}methanone (7f)
Yield 89%; M.P: 51–55 °C; FT-IR (KBr, cm−1): 1656 (C=O), 1346 (C=N), 1286 (C–O–C); 1H NMR (CDCl3): δ 2.49 (t, 4H, CH2NCH2 of morpholine ring), 2.66 (t, 2H, NCH2), 3.44 (t, 2H, CH2S), 3.66 (t, 4H, CH2OCH2 of morpholine ring), 5.34 (s, 2H, OCH2), 6.99–7.97 (m, 7H, Ar–H); 13C-NMR (CDCl3): δ 15.7, 30.9, 55.9, 57.9, 66.9, 72.4, 113.9, 115.8, 124.9, 128.3, 132.4, 132.9, 134.8, 139.1, 160.2, 163.2, 164.5, 166.64, 195.3; LC–MS m/z 474 (M + 1)+. Elemental Analysis: Calcd. for C23H24ClN3O4S: C, 58.38; H, 5.29; Cl, 7.45; N, 9.18; S, 6.71%. Found: C, 58.29; H, 5.40; Cl, 7.49; N, 9.19; S, 6.85%
2.1.16 Synthesis of (2-chlorophenyl){3-methyl-4-[(5-((2-(piperidin-1-yl)ethyl)thio)-1,3,4-oxadiazol-2-yl)methoxy] phenyl} methanone (7g)
Yield 80%; M.P: 54–56 °C; FT-IR (KBr, cm−1): 1654 (C=O), 1334 (C=N), 1279 (C–O–C); 1H NMR (CDCl3): δ 2.53 (t, 4H, CH2NCH2 of piperidine ring), 2.77 (t, 2H, N CH2), 3.48 (t, 2H, CH2S), 1.76 (m, 4H, CH2CH2 of piperidine ring), 1.54 (t, 2H, CH2 of piperidine ring), 5.33 (s, 2H, OCH2), 7.55–8.65 (m, 7H, Ar–H); 13C-NMR (CDCl3): δ 15.7, 24.9, 25.8, 30.9, 56.4, 57.9, 71.6, 113.8, 124.8,126.8, 128.4, 128.7, 129.9, 131.9, 131.3, 133.8, 136.5, 139.4, 160.2, 162.9, 163.5, 198.3; LC–MS m/z 472 (M + 1)+. Elemental Analysis: Calcd. for C24H26ClN3O3S: C, 61.28; H, 5.55; N, 8.92; S, 7.04% Found: C, 62.29; H, 5.70; N, 8.28; S, 7.07%.
2.1.17 Synthesis of (2-chlorophenyl){3-methyl-4-[(5-((2-morpholinoethyl)thio)-1,3,4-oxadiazol-2-yl) methoxy]phenyl}methanone (7h)
Yield 89%; M.P: 49–52 °C; FT-IR (KBr, cm−1): 1656 (C=O), 1346 (C=N), 1286 (C–O–C); 1H NMR (CDCl3): δ 2.42 (t, 4H, CH2NCH2 of morpholine ring), 2.63 (t, 2H, NCH2), 3.44 (t, 2H, CH2S), 3.66 (t, 4H, CH2OCH2 of morpholine ring), 5.34 (s, 2H, OCH2), 6.99–7.97 (m, 7H, Ar–H); 13C-NMR (CDCl3): δ 15.5, 30.6, 55.5, 57.6, 66.7, 72.1, 113.4, 124.95, 126.7, 128.7, 128.5,129.9, 131.4, 131.9, 133.8, 136.4, 139.1, 160.2, 162.2, 163.5, 195.5; LC–MS m/z 474 (M + 1)+. Elemental Analysis: Calcd. for C23H24ClN3O4S: C, 58.38; H, 5.29; N, 9.18; S, 6.71%. Found: C, 58.29; H, 5.40; N, 9.19; S, 6.85%.
2.1.18 Synthesis of phenyl{3-chloro-5-fluoro-4-[(5-((2-(piperidin-1-yl)ethyl)thio)-1,3,4-oxadiazol-2-yl) methoxy]phenyl}methanone (7i)
Yield 80%; M.P: 52–56 °C; FT-IR (KBr, cm−1): 1654 (C=O), 1334 (C=N), 1279 (C–O–C); 1H NMR (CDCl3): δ 2.53 (t, 4H, CH2NCH2 of piperidine ring), 2.79 (t, 2H, N CH2), 3.52 (t, 2H, CH2S), 1.98 (m, 4H, CH2CH2 of piperidine ring), 1.65 (t, 2H, CH2 of piperidine ring), 5.67 (s, 2H, OCH2), 7.67–8.75 (m, 7H, Ar–H); 13C-NMR (CDCl3): δ 24.8, 25.4, 30.6, 56.4, 57.9, 71.2, 114.4, 123.8,126.4, 128.9, 130.4, 132.9, 135.5, 138.4, 153.4, 153.6, 160.2, 163.5, 195.3; LC–MS m/z 476 (M + 1)+. Anal. Calcd. for C23H23ClFN3O3S: C, 58.28; H, 4.85; N, 8.82; S, 6.74% Found: C, 58.29; H, 4.70; N, 8.88; S, 6.77%.
2.1.19 Synthesis of phenyl {3-chloro-5-fluoro-4-[(5-((2-morpholinoethyl)thio)-1,3,4-oxadiazol-2-yl) methoxy]phenyl}methanone (7j)
Yield 84%; M.P: 59–62 °C; FT-IR (KBr, cm−1): 1660 (C=O), 1344 (C=N), 1283 (C–O–C); 1H NMR (CDCl3):δ 2.47 (t, 4H, CH2NCH2 of morpholine ring), 2.73 (t, 2H, NCH2), 3.54 (t, 2H, CH2S), 3.76 (t, 4H, CH2OCH2 of morpholine ring), 5.69 (s, 2H, OCH2), 7.97–8.95 (m, 7H, Ar–H); 13C-NMR (CDCl3): δ 30.6, 55.5, 57.6, 66.7, 71.5, 114.6, 123.6,126.8, 128.4, 130.9, 132.7, 135.6, 138.8, 153.1, 153.3, 160.2, 163.6, 195.6; LC–MS m/z 478 (M + 1)+. Elemental Analysis: Calcd. for C22H21ClFN3O4S: C, 55.38; H, 4.66; N, 8.72; S, 6.74% Found: C, 55.29; H, 4.70; N, 8.88; S, 6.77%.
2.1.20 Synthesis of (4-iodophenyl){3-chloro-5-fluoro-4-[(5-((2-(piperidin-1-yl)ethyl)thio)-1,3,4-oxadia-zol-2-yl) methoxy]phenyl}methanone (7k)
Yield 79%; M.P: 54–57 °C; FT-IR (KBr, cm−1): 1654 (C=O), 1334 (C=N), 1279 (C–O–C); 1H NMR (CDCl3): δ 2.63 (t, 4H, CH2NCH2 of piperidine ring), 2.70 (t, 2H, N CH2), 3.55 (t, 2H, CH2S), 1.99 (m, 4H, CH2CH2 of piperidine ring), 1.75 (t, 2H, CH2 of piperidine ring), 5.77 (s, 2H, OCH2), 7.69–8.89 (m, 6H, Ar–H); 13C-NMR (CDCl3): δ 24.8, 25.4, 30.6, 56.6, 57.6, 71.2,98.3, 114.8, 123.9,126.8, 131.9, 135.9, 137.5, 137.9, 153.4, 153.6, 160.2, 163.6, 194.3; LC–MS m/z 602 (M + 1)+. Elemental Analysis: Calcd. for C23H22ClFIN3O3S: C, 45.98; H, 3.68; N, 6.98; S, 5.33% Found: C, 45.99; H, 3.70; N, 6.95; S, 5.44%.
2.1.21 Synthesis of (4-iodophenyl){3-chloro-5-fluoro-4-[(5-((2-morpholinoethyl)thio)-1,3,4-oxadiazol-2-yl)meth-oxy] phenyl}methanone (7l)
Yield 84%; M.P: 56–59 °C; FT-IR (KBr, cm−1): 1659 (C=O), 1344 (C=N), 1289 (C–O–C); 1H NMR (CDCl3):δ 2.47 (t, 4H, CH2NCH2 of morpholine ring), 2.73 (t, 2H, NCH2), 3.54 (t, 2H, CH2S), 3.76 (t, 4H, CH2OCH2 of morpholine ring), 5.69 (s, 2H, OCH2), 7.97–8.95 (m, 6H, Ar–H); 13C-NMR (CDCl3): δ 30.6, 55.6, 57.7, 66.9, 71.2,98.7, 114.3, 123.3,126.3, 131.6, 135.3, 137.6, 137.8, 153.3, 153.8, 160.2, 163.8, 194.7; LC–MS m/z 604 (M + 1)+. Elemental Analysis: Calcd. for C22H20ClFIN3O4S: C, 55.38; H, 4.66; N, 8.72; S, 6.74% Found: C, 55.29; H, 4.70; N, 8.88; S, 6.77%.
2.1.22 Synthesis of (4-fluorophenyl){4-[(5-((2-(piperidin-1-yl)ethyl)thio)-1,3,4-oxadiazole-2-yl) methoxy] phenyl}methanone (7m)
Yield 83%; M.P: 54–58 °C; FT-IR (KBr, cm−1): 1652 (C=O), 1330 (C=N), 1270 (C–O–C); 1H NMR (CDCl3): δ 2.59 (t, 4H, CH2NCH2 of piperidine ring), 2.79 (t, 2H, N CH2), 3.47 (t, 2H, CH2S), 1.59 (m, 4H, CH2CH2 of piperidine ring), 1.48 (t, 2H, CH2 of piperidine ring), 5.18 (s, 2H, OCH2), 6.90–7.95 (m, 8H, Ar–H); 13C-NMR (CDCl3): δ 24.66, 25.9, 30.4, 56.9, 57.6, 71.9, 113.6, 114.6, 115.6, 130.4, 131.4, 133.7, 134.4, 160.2, 162.4, 163.5, 166.54, 194.3; LC–MS m/z 442 (M + 1)+. Elemental Analysis: Calcd. for C23H24FN3O3S: C, 62.28; H, 5.85; N, 9.82; S, 7.24%. Found: C, 62.29; H, 5.88; N, 9.98; S, 7.37%.
2.1.23 Synthesis of 4-(fluorophenyl){4-[(5-((2-morpholinoethyl)thio)-1,3,4-oxadiazol-2-yl) methoxy] phen yl} methanone (7n)
Yield 82%; M.P: 55–59 °C; FT-IR (KBr, cm−1): 1677 (C=O), 1334 (C=N), 1267 (C–O–C); 1H NMR (CDCl3): δ 2.48 (t, 4H, CH2NCH2 of morpholine ring), 2.69 (t, 2H, NCH2), 3.48 (t, 2H, CH2S), 3.64 (t, 4H, CH2OCH2 of morpholine ring), 5.36 (s, 2H, OCH2), 6.88–7.97 (m, 8H, Ar–H); 13C-NMR (CDCl3): δ 30.9, 55.9, 57.9, 66.9, 72.4,113.7, 114.8, 115.7, 130.5, 131.3, 133.8, 134.4, 160.2, 162.4, 163.3, 166.5, 194.5;LC–MS m/z 444 (M + 1)+. Elemental Analysis: Calcd. for C22H22FN3O4S: C, 59.68; H, 5.09; N, 9.28; S, 7.21%. Found: C, 59.79; H, 5.10; N, 9.29; S, 7.25%.
3 Biology
3.1 Anticancer activity studies using MTT Assay
The cell lines used for the study were MCF-7 (Breast cancer), KB (Oral cancer), L929 (normal cell line for cytotoxicity) procured from NCCS, Pune. The cell lines were maintained in 96 wells micro titer plate containing DMEM media with low glucose supplemented with 10% heat inactivated fetal bovine serum (FBS), and 1% antibiotic–antimycotic 100X solution and incubated in CO2 incubator at 37ºC in 5% CO2 and 95% humidity until the completion of reaction.
The cells were seeded at a density of approximately 5 × 103 cells/well in a 96-well flat-bottom micro plate and maintained at 37 °C in 95% humidity and 5% CO2 for overnight. Different concentrations (500, 250, 125, 62.5, 31.25, 15.625 µg/mL) of samples were treated. The cells were incubated for another 48 h. The cells in well were washed twice with phosphate buffer solution, and 20 µL of the MTT staining solution (5 mg/ml in phosphate buffer solution) was added to each well and plate was incubated at 37 °C. After 4 h, 100 µL of di-methyl sulfoxide (DMSO) was added to each well to dissolve the formazan crystals, and absorbance was recorded with a 570 nm using micro plate reader [34]. Statistical analysis of the data was performed on GraphPad Prism. All the experiments were conducted in triplicate. Surviving cells (%) = mean OD of test compound/mean OD of Negative control × 100. Using graph Pad Prism Version 5.1, the IC50 of compounds were calculated.
3.1.1 In-vitro cytotoxicity assay
Toxicity of the most active compounds (7f, 7g and 7h) was determined on the basis of measurement of in vitro growth of the normal cell line (L292) using MTT assay [35]. The IC50 values (50% inhibitory concentration) were calculated. In-vitro growth inhibition effect of test compounds was assessed by MTT assay by the normal cell line L929 (procured from NCCS, Pune).
4 Results and discussion
4.1 Chemistry
The synthetic protocol of the title compounds 7a–n is outlined in the Scheme 1. Initially, the benzoylated products 2a–g were prepared by the benzoylation of substituted phenols 1a–g with substituted benzoyl chlorides. In the presence of anhydrous aluminium chloride, the compounds 2a–g have undergone Fries rearrangement, to afford hydroxy benzophenones 3a–g, which were established by the disappearance of the carbonyl stretching band of the ester group and appearance of the –OH stretching band in IR spectra and also, the appearance of broad singlet for –OH proton and decrease in one aromatic proton in 1H NMR spectra. The compounds, benzoyl phenoxy acetic acid ethyl esters, 4a–g were obtained by refluxing substituted 3a–g with ethyl chloro acetate and were evidenced by the loss of –OH stretching and emergence of carbonyl stretching band for the ester group in the IR absorption spectra. The 1H NMR confirmed the disappearance of broad singlet for –OH group and an appearance of triplet and quartet for –CH3 and –CH2 protons respectively. Upon treating the compounds 4a–g with hydrazine hydrate, phenoxy-acetic acid hydrazide analogues 5a–g were obtained, which were confirmed by the appearance of –NH2 stretching band of amide in the IR spectrum. The appearance of the peaks for –NH2 and –NH protons and concealing of triplet and quartet peaks for –CH3 and –CH2 protons respectively in 1H NMR, confirmed the formation of the product. Upon cyclization of acid hydrazide analogues 5a–g with carbon disulfide in the presence of KOH, claimed the corresponding 1,3,4-oxadiazol-2-thiol 6a–g, which clearly evident with the absence of carbonyl and –NH2 stretching bands in the IR spectra. The presence of –SH proton and the absence of –NH2 and –NH protons of amide in the NMR spectrum also confirmed the product. Finally, the title compounds 7a–n were obtained by alkylation of benzophenone tagged 1,3,4-oxadiazol-2-thiol derivatives 6a–g with 4-(2-chloroethyl) morpholine/piperidine hydrochloride in boiling DMF with fused potassium carbonate. This was validated by the disappearance of SH stretching in the IR spectrum and also the disappearance of –SH proton and appearance of methylene protons of morpholine/piperidine in the 1H NMR spectra. Further, the mass spectral data of these compounds displayed molecular ion peaks which confirmed their molecular weights.
4.1.1 Biological activity
The synthesized unique molecules play a prime role in the development of multi target drugs with target precise action. Novel heterocyclic compounds were synthesized with benzophenone and oxadiazole cores as backbones with nucleophillic alkylation of heterocyclic analogs.
4.1.2 Structure activity relationship (SAR)
4.1.2.1 Anti-cancer study
Anti-cancer activity was studied by MTT assay. The cytotoxicity of the synthesized title compounds in two different cancer cell lines MCF-7 (Breast cancer) and KB (Oral cancer) had been executed in comparison with standard paclitaxel by MTT assay and images of MCF-7 cell line and KB cell line photographed by Phase Contrast Microscope are shown in Fig. 1. The title compounds induced a cytotoxic effect in a concentration-dependent manner. The IC50 values of each compound were calculated and are summarized in Table 1.
Anticancer and cytotoxicity activity assay was performed by MTT assay. a Photographs of KB cell line at various concentrations. Compound 7h at 125 µg/ml, compound 7g at 62.5 µg/ml, control. b Photographs of MCF 7 cell line at various concentrations. Compound 7g at 125 µg/ml, compound 7h at 125 µg/ml, control. c Photographs of L292 cell line at various concentrations. Control, compound 7f at 250 µg/ml, compound 7h at 250 µg/ml
In the series, compound 7h, with one methyl group on the phenoxy ring and one chlorine group on the ortho position of the benzoyl ring, conjugated to morpholine, executed potent activity by inducing minimum viability at inhibitory concentrations of 112.6 µg/ml and 126.7 µg/ml, against the MCF-7 cell line and KB cell line respectively under identical experimental conditions. Followed by Compound 7f with one methyl group on the phenoxy ring and one 3-position chlorine group on the benzoyl ring, conjugated to morpholine, exhibited IC50 values 113.8 µg/ml and 127.0 µg/ml against the MCF-7 cell line and KB cell line respectively by inducing approximately minimum viability, which was nearly potent to compound 7h.
Compounds 7c, 7g and 7k showed moderate activity, whereas compounds 7a, 7e and 7m showed less sensitivity. Compound 7n with one fluorine group on the benzoyl ring, exhibited highest IC50 values 425.5 µg/ml and 289.4 µg/ml against the MCF-7 cell line and KB cell line respectively by inducing maximum viability, which was least potent under identical experimental conditions. Importantly, this compound does not contain a chlorine group. The graphs of percentage of cell viability versus different concentrations of each compound against KB and MCF-7 cell lines are shown in Fig. 2.
a Graph of MTT Assay of the synthesized compounds of anti-cancer activity on KB cell line. b Graph of MTT Assay of the synthesized compounds of Anti-cancer activity on MCF 7 cell line
4.1.3 In-vitro cytotoxicity assay
Cytotoxicity assay of the selected compounds on the viability of normal cell lines was satisfactory. The selected compounds are screened based on their better activities on the above discussed in vitro MTT assay against MCF-7 cell line and KB cell line. The synthesized compounds 7f, 7g and 7h were most active and hence toxicity towards the normal cell line (L292) was evaluated by MTT assay. From the experimental data (Table 2), it is revealed that, all the selected compounds were non-toxic to the normal cells at higher concentration with IC50 value > 193 μg/ml. These compounds showed the anti-cancer activity at IC50 values 113.8–119.8 µg/ml and 126.7–139 µg/ml against the MCF-7 cell line and KB cell line respectively. Minimum toxicity is the major advantage of the synthesized benzophenone derivatives.
Compound 7h acts as an eminent compound by exhibiting an excellent efficacy in vitro anti-cancer experiment. Structurally, compound 7h has morpholine group (six membered ring) attached to oxadiazole through two CH2 groups, methyl substituent at ortho-position of the phenoxy ring and chlorine group at ortho-position of benzoyl ring which showed a significant cytotoxic effect, with enhancement in the IC50 values, as verified by an MTT assay.
5 Conclusion
In the present study, we synthesized analogs of oxadiazolyl benzophenone (7a–n) which vary in the number and position of the methyl and halogen groups on the benzophenone moiety, which were then evaluated for their anti cancer activity. IC50 values for anticancer activity were 112.6 and 126.7 µg/ml, against the MCF-7 cell line and KB cell line respectively by MTT assay method. In the series of the synthesized compounds, compound containing the electron withdrawing group, along with sufficient hetero atom rich compound showed more activity. In general, the activity of the compound is influenced by the chemical structure, size & shape, molecular arrangements, and electron donating/withdrawing groups, etc.
The structure–activity relationship study reveals that, the compounds with morpholine moiety, exhibited better activity than piperidine scaffolds. All the compounds having morpholine group with chlorine substitution at 2-position on benzoyl ring showed superior potency. Incorporation of morpholine and piperidine nuclei to the benzophenone skeleton has enhanced the bioactivity of the synthesized compounds to be recognized as a drug. Structure–activity relation interrelates the biological potency and chemical scaffold of the molecule.
5.1 Statistical analysis
The values of antioxidant activity are expressed as mean ± standard deviation. The inhibition zone was measured from the antimicrobial activity of compound and analyzed using one way analysis of variance (ANOVA) followed by Tukey’s test at p < 0.05. The software Origin Pro 9.0 was employed for the statistical analysis.
References
Vasan N, Baselga J, Hyman DM (2019) A view on drug resistance in cancer. Nature 575:299–309
Salahuddin MA, Shahar YM, Mazumder R, Chakraborthy GS, Ahsan MJ, Rahman MU (2017) Updates on synthesis and biological activities of 1,3,4-oxadiazole: a review. Synth Commun 47(20):1805–1847
Aboraia AS, Abdel-Rahman HM, Mahfou NM, El-Gendy MA (2006) Novel 5-(2-hydroxyphenyl)-3-substituted-2,3-dihydro-1,3,4-oxadiazole-2-thione derivatives: promising anticancer agents. Bioorg Med Chem 14:1236–1246
Bhat JJ, Shah BR, Shah HP, Trivedi PB, Undavia NK, Desai NC (1994) Synthesis of anti-HIV, anti-cancer and anti-tubercular 4-oxo-thiazolidines, 2-imino-4-oxo-thiazolidines and their 5-arylidine derivatives. Indian J Chem 33B:189–192
Ahsan MJ, Hassan MZ, Jadav SS, Geesi MH (2020) Synthesis and Biological Potentials of 5-aryl-N-[4-(trifluoromethyl) phenyl]-1,3,4-oxadiazol-2-amines. Lett Org Chem 17:133–140
Ahsan MJ, Choupra A, Sharma RK, Jadav SS, Padmaja P (2018) Rationale design, synthesis, cytotoxicity evaluation, and molecular docking studies of 1,3,4-oxadiazole analogues. Anti-Cancer Agents Med Chem 18:121–138
Ahsan MJ, Meena R, Dubey S (2018) Synthesis and biological potentials of some new 1, 3, 4-oxadiazole analogues. Med Chem Res 27:864–883
Ahsan MJ, Yadav RP, Saini S, Hassan MZ et al (2018) Synthesis, cytotoxic evaluation, and molecular docking studies of new oxadiazole analogues. Lett Org Chem 15:49–56
Ahsan MJ (2016) Rationale design, synthesis and in vitro anticancer activity of new 2,5-disubstituted-1,3,4-oxadiazole analogs. Chem Select 15(1):4713–4720
Agarwal M, Singh V, Sharma SK et al (2016) Design and synthesis of new 2,5-disubstituted-1,3,4-oxadiazole analogs as anticancer agents. Med Chem Res 25:2289–2303
Hatti I, Sreenivasulu R, Jadav SS et al (2015) Synthesis and biological evaluation of 1,3,4-oxadiazole linked bis indole derivatives as anticancer agents. Monatsh Chem 146:1699–1705
Mamatha SV, Mahesh B, Kumara HK, Gowda DC, Meenakshi SK (2020) Design, synthesis and SAR evaluation of mercaptooxadiazole analogs as anti-tubercular, anti-diabetic and anti-bacterial agents. Chem Data Collect 20:100343
Tan TM, Chen Y, Kong KH, Bai J, Li Y, Lim SG, Ang TH (2006) Lam Y Synthesis and the biological evaluation of 2-benzenesulfonylalkyl-5-substituted-sulfanyl-[1,3,4]-oxadiazoles as potential anti-hepatitis B virus agents. Antivir Res 71:7–14
Hajimahdi Z, Zarghi A, Zabihollahi A, Aghasadeghi MR (2013) Synthesis, biological evaluation, and molecular modeling studies of new 1,3,4-oxadiazole-and 1,3,4-thiadiazole-substituted 4-oxo-4H-pyrido[1,2-a]pyrimidines as anti-HIV-1 agents. Med Chem Res 22:2467–2475
Gadegoni H, Manda S (2013) Synthesis and screening of some novel substituted indoles contained 1,3,4-oxadiazole and 1,2,4-triazole moiety. Chin Chem Lett 24:127–130
Rathore A, Sudhakar R, Ahsan MJ, Ali A, Subbarao N, Jadav SS, Umar S, Yar MS (2017) In vivo anti-inflammatory activity and docking study of newly synthesized benzimidazole derivatives bearing oxadiazole and morpholine rings. Bioorg Chem 70:107–117
Palkar MB, Singhai A (2014) Synthesis, pharmacological screening and in silico studies of new class of Diclofenac analogues as a promising anti-inflammatory agents. Bioorg Med Chem 22:2855–2866
Malladi S, Isloor AM, Peethambar SK, Fun HK (2014) Synthesis and biological evaluation of newer analogue of 2,5-disubstituted 1,3,4-oxadiazole containing pyrazole moiety as antimicrobial agents. Arab J Chem 7:1185–1191
Zou XJ, Lai LH, Jin GY (2002) Zhang ZX Synthesis, fungicidal activity, and 3D-QSAR of pyridazinone-substituted 1,3,4-oxadiazoles and 1,3,4-thiadiazoles. J Agric Food Chem 50:3757–3760
Macaev F, Rusu G, Pogrebnoi S, Gudima A, Stingaci E, Vlad L, Shvets N, Kandemirli F, Dimoglo A, Reynolds R (2005) Synthesis of novel 5-aryl-2-thio-1,3,4-oxadiazoles and the study of their structure-anti-mycobacterial activities. Bioorg Med Chem 13:4842–4850
Amir M, Shikha K (2004) Synthesis and anti-inflammatory, analgesic, ulcerogenic and lipid peroxidation activities of some new 2-[(2,6-dichloroanilino) phenyl]acetic acid derivatives. Eur J Med Chem 39:535–545
Zarghi A, Tabatabai TA, Faizi A, Ahadian P, Navabi V, Zanganeh A (2005) Shafiee A Synthesis and anticonvulsant activity of new 2-substituted-5-(2-benzyloxyphenyl)-1,3,4-oxadiazoles. Bioorg Med Chem Lett 15:1863–1865
Khan MTH, Choudhary MI, Khan KM, Rani M, Atta-ur-Rahman S (2005) Structure–activity relationships of tyrosinase inhibitory combinatorial library of 2, 5-disubstituted-1, 3, 4-oxadiazole analogues. Bioorg Med Chem 13:3385–3395
Palmer JT, Hirschbein BL, Cheung H, McCarter J, Janc JW, Yu WJ, Wesolowski G (2006) Keto-1,3,4-oxadiazoles as cathepsin K inhibitors. Bioorg Med Chem Lett 16:2909–2914
Mamatha SV, Belagali SL, Bhat M (2019) Synthesis and SAR evaluation of mercapto triazolobenzothiazole derivatives as anti-tuberculosis agents. Anti-Infect Agents 18:1–13
Chaudhary A, Sharma PK, Verma P, Kumar N, Dudhe R (2012) Microwave assisted synthesis of novel pyrimidine derivatives and investigation of their analgesic and ulcerogenic activity. Med Chem Res 21:3629
Nishi T, Ishibashi K, Takemoto T, Nakajima K, Fukazawa T, Iio L, Itoh K, Mukaiyama O, Yamaguchi T (2000) Combined tachykinin receptor antagonist: synthesis and stereochemical structure-activity relationships of novel morpholine analogues. Bioorg Med Chem Lett 10:1665
Tafti AF, Foroumadi A, Tiwari R, Shirazi AN, Hangauer DG, Bu Y, Akbarzadeh T, Parang K, Shafiee A (2011) Thiazolyl N-benzyl-substituted acetamide derivatives: synthesis, Src kinase inhibitory and anticancer activities. Eur J Med Chem 46:4853–4858
Yurttas L, Demirayak S, Çiftçi GA, Yıldırım SU, Kaplancıkl ZA (2013) Synthesis and biological evaluation of some pyrazoline derivatives bearing a dithiocarbamate moiety as new cholinesterase inhibitors. Archiv Der Pharm 346:364–403
Mamatha SV, Bhat M, Sagar BK, Meenakshi SK (2019) Synthesis, characterization, crystal structure biological activity of 4-{2-[5-(4-fluoro-phenyl)-[1, 3, 4] oxadiazol-2-ylsulfanyl]-ethyl}-morpholine. J Mol Struct 1196:186–193
Mamatha SV, Belagali SL, Mahesh B (2019) Design, synthesis and SAR bioevaluation of benzophenone-mercaptooxadiazole analogs. Med Drug Dis 3:100017
Mahesh B, Belagali SL (2017) Synthesis, characterization and biological screening of pyrazoline conjugated benzothiazole analogues. Future Med Chem 10(1):71–87
Bala S, Kamboj S, Kajal A, Saini V, Prasad DN (2014) 1,3,4-Oxadiazole derivatives: synthesis, characterization, antimicrobial potential, and computational studies. Biomed Res Int 172791:1–18
Samundeeswari S, Chougala B, Holiyachi M, Shastri L, Kulkarni M, Dodamani S (2017) Design and synthesis of novel phenyl-1, 4-beta-carboline-hybrid molecules as potential anticancer agents. Eur J Med Chem 128:123–139
Mosmann T (1983) Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65:55–63
Acknowledgements
The authors are thankful to Institute of Excellence, University of Mysore for providing sophisticated Instruments for characterization.
Author information
Authors and Affiliations
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no competing interests.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Rights and permissions
About this article
Cite this article
Mamatha, S.V., Belagali, S.L. & Bhat, M. Synthesis, characterisation and evaluation of oxadiazole as promising anticancer agent. SN Appl. Sci. 2, 882 (2020). https://doi.org/10.1007/s42452-020-2511-z
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s42452-020-2511-z