Heterocyclic compounds containing five-membered or six-membered heterocyclic units have a diversity of valuable biological effects. In the present work, some novel tri-heterocyclic benzamides, 8a-g, were synthesized in multi-steps. The benzamide containing electrophile, {4-[4-(chloromethyl)benzoyl]-1-piperazinyl}(2-furyl) methanone (3), was synthesized by the reaction of 4-(chloromethyl)benzoyl chloride (2) and 2-furoyl-(1-piperazinyl) methanone (1) in a basic aqueous medium. In parallel series of steps, substituted-benzoic acids (4a-g) were refluxed with ethanol and conc. sulfuric acid to form respective ethyl substituted-benzoates (5a-g). These esters were further refluxed with N₂H₄.H₂O in methanol solution to acquire substituted-benzohydrazides (6a-g). These hydrazides were cyclized into heterocyclic core by refluxing with CS₂ in the presence of KOH and ethanol solvent, whereby yielding various 5-(substituted-phenyl)-1,3,4-oxadiazol-2-thiols (7a-g). In the final step, the electrophile 3, was refluxed with synthesized 1,3,4-oxadiazoles, 7a-g, in acetonitrile and potassium carbonate to acquire the targeted novel tri-heterocyclic benzamides, 8a-g. The structural characterization of these newly synthesized molecules was done by IR, ¹H-NMR, ¹³C-NMR, and EI-MS spectral data. All these compounds were evaluated for their hemolytic activity to ascertain their cytotoxicity profile.

Keywords: novel tri-heterocycles, ¹H-NMR, ¹³C-NMR, EI-MS, hemolytic activity, heterocyclic compounds

OLED, organic light-emitting diodes; KOH , potassium hydroxide; NMR, nuclear magnetic resonance; TLC, thin layer chromatography; EDTA, ethylene diamine tetra acetic acid; PBS, Phosphate-buffered saline; HEC, higher education commission; EI-MS, electron ionization mass spectrometry

Oxadiazoles are the heterocyclic compounds containing one oxygen and two nitrogen atoms in a five membered ring,¹ possessing a diversity of useful biological effects.² Oxadiazole is considered to be resultant from furan by replacement of two methane (–CH=) groups by two pyridine type nitrogen atoms (–N=) at position 3 and 4.³ Oxadiazole is a very weak base due to the inductive effect of the extra heteroatom.⁴ The replacement of (–CH=) groups in furan by two pyridine type nitrogen (–N=) reduces aromaticity of the resulting oxadiazole ring to such an extent that the oxadiazole ring exhibits the character of conjugated diene.⁵ Due to relatively low electron density on the carbon atom, the oxadiazole ring is extremely resistant towards electrophillic substitutions at carbon atom; however the attack of electrophile occurs at nitrogen, if oxadiazole ring is substituted with electron releasing groups. Nucleo-philic attack is quite difficult in oxadiazole ring; however, halogen substituted oxadiazoles can undergo nucleophilic substitution with replacement of halogen atom by nucleophiles.⁶ These derivative compounds have been found to exhibit diverse biological activities such as analgesic,⁷ anti-inflammatory,⁸ antimicrobial,⁹ anti-HIV,¹⁰ antimalarial,¹¹ antifungicidal,¹² and other biological properties. Some 1,3,4-oxadiazole derivatives have also been applied in the fields of photosensitizers,¹³ liquid crystals,¹⁴ and organic light-emitting diodes (OLED).¹⁵ Consequently, the synthesis of compounds containing this heterocyclic core has attracted considerable attention, and a wide variety of methods has been used for their assembly. The most common synthetic protocol toward the preparation of these compounds involves the dehydrative cyclization of diacylhydrazides using usually strong acidic reagents such as thionyl chloride,¹⁶ phosphorus pentoxide,¹⁷ phosphorus oxychloride,¹⁸ and sulfuric acid.¹⁹

Literature survey showed that slight modifications in the structure of 1,3,4-oxadiazole can result in quantitative as well as qualitative variations in the biological activity.²⁰ So, in the present study we have synthesized various tri-heterocyclic benzamides through a multi-step process to incorporate multi-functionalities in their skeleton. Then, cytotoxicity of these molecules was profiled through hemolytic study on the membrane of red blood cells.

Chemistry

All the chemicals, along with analytical grade solvents, were purchased from Sigma Aldrich, Alfa Aesar (Germany), or Merck through local suppliers. Pre-coated silica gel Al-plates were used for TLC with ethyl acetate and n-hexane as solvent system. Spots were detected by UV₂₅₄. Gallonkamp apparatus was used to detect melting points in capillary tubes. IR spectra (ν_max, cm^–1) were recorded by KBr pellet method in the Jasco-320-A spectrophotometer. ¹H-NMR spectra (δ, ppm) were recorded at 600 MHz (¹³C-NMR spectra, at 150 MHz) in CDCl₃ using the Bruker Advance III 600 As- cend spectrometer using BBO probe. EI-MS spectra were measured on a JEOL JMS-600H instrument with data processing system.

Procedure for the preparation of {4-[4-(chloromethyl)benzoyl]-1-piperazinyl}(2-furyl)methanone (3)

2-Furyl(1-piperazinyl)methanone (12.8mmol; 1) was taken in an iodine flask (250mL) containing 15mL of distilled water and 10% Na₂CO₃ (sodium carbonate) solution to adjust pH at 9-10. Then equimolar 4-(chloromethyl)benzoyl chloride (2) was added dropwise to the reaction medium in 2-5 min. After complete addition, the iodine flask was vigorously shaken (manually) and then set to stir at room temperature for 4 h till the formation of solid precipitates. The progress of reaction was monitored by thin layer chromatography (TLC) till single spot. The obtained precipitates were filtered, washed with distilled water and dried to yield the titled electrophiles.^20,21

Procedure for the preparation of ethyl substituted-benzoates (5a-g)

Substituted-benzoic acids (50mmol; (4a-g, one in each reaction) were taken into a 250mL round bottom flasks with a reflux condenser, then absolute ethanol (40mL) and conc. sulphuric acid (1/2mL) were added into the flask and the reaction mixture was refluxed for 3-4h. After maximal completion by thin layer chromatography (TLC), excess water was added and pH was adjusted to 8-10 by aqueous solution of sodium carbonate (Na₂CO₃; 10%). The title compounds were extracted by chloroform.

Procedure for the preparation of substituted-benzohydrazides (6a-g)

Esters, 5a-g, (40mmol) and N₂H₄.H₂O (hydrazine monohydrate; 40mmol) were taken in a 100mL round bottom flask with a reflux condenser and were refluxed for 4-6h with 25mL methanol. After final thin layer chromatography (TLC), excess water was added to acquire precipitates which were separated by filtration.

Procedure for the preparation of 5-(substituted-phenyl)-1,3,4-oxadiazol-2-thiols (7a-g)

Acid hydrazides, 6a-g, (30mmol) were refluxed for 1/2 h with solid KOH (potassium hydroxide; 30mmol) in 45mL ethanol in a 100mL round bottom flask. Then CS₂ (carbon disulfide; 60mmol) was added and further refluxed for 3-6 h. After final thin layer chromatography (TLC), excess water was added followed by conc. HCl to adjust pH of 2. The mixture was left for 3h for precipitation. Precipitates were collected through filtration and washed with water.

General procedure for the preparation of {4-[4-({[5-(substituted-phenyl)-1,3,4-oxadiazol-2-yl]sulfanyl}methyl)benzoyl]-1-piperazinyl}(2-furyl)methanone (8a-g)

5-(Substituted-phenyl)-1,3,4-oxadiazol-2-thiols (24mmol; 7a-g, one in each reaction) were dissolved in acetonitrile (20-30mL) in 100mL round bottom flask. Then solid K₂CO₃ (potassium carbonate; 12mmol) was added. The mixture was refluxed for 1/2 h and then the equimolar (24mmol) desired electrophile, 3 were added. The mixture was further refluxed for 4-5 hours. Thin layer chromatography (TLC) was carried out to check the reaction completion. Distilled water was added to the reaction mixture to acquire the precipitates. Precipitates were filtered, washed and dried to get the titled compounds.^8,17

{4-[4-({[5-(2-Chlorophenyl)-1,3,4-oxadiazol-2-yl]sulfanyl}methyl)benzoyl]-1-piperazinyl}(2-furyl)methanone (8a)

Light brown liquid; yield: 85%; Molecular formula: C₂₅H₂₁ClN₄O₄S; molecular mass: 508g/mol; IR (KBr, υ_max cm^–1): 3415 (N-H), 3063 (Ar C-H), 2872 (C-H), 1654 (C=O), 1576 (C=C), 1200 (C-O-C), 1112 (C-N-C), 644 (C-S); ¹H-NMR (600 MHz, CDCl₃, δ in ppm): δ 7.93 (d, J = 6.0 Hz, 2H, H-2'' & H-6''), 7.55 (d, J = 6.6 Hz, 2H, H-3'' & H-5'') 7.52 (br s, 1H, H-5), 7.42-7.39 (m, 4H, H-3'''', H-4'''', H-5'''' & H-6''''), 7.07 (d, J = 3.4 Hz, 1H, H-3), 6.50 (dd, J = 1.7, 3.5 Hz, 1H, H-4), 4.54 (s, 2H, H-8''), 3.82 (br s, 4H, H-3' & H-5'), 3.46 (br s, 4H, H-2' & H-6'); ¹³C-NMR (150 MHz, CDCl3, δ in ppm): 170.09 (C-7''), 164.33 (C-5'''), 164.15 (C-2'''), 159.2 (C-6), 147.65 (C-2), 143.96 (C-5), 137.96 (C-4''), 134.95 (C-1''''), 133.03 (C-1''), 132.45 (C-6''''), 131.3 (C-4''''), 130.92 (C-2''''), 129.48 (C-5''''), 127.66 (C-3'' & C-5''), 127.11 (C-3''''), 122.81 (C-2'' & C-6''), 117.26 (C-3), 111.54 (C-4), 52.00 (C-2', C-3', C-5' & C-6'), 36.27 (C-8''); EI-MS (m/z): 383 [M]+, 259 [C₁₆H₂₅N₂O]+, 261 [C₁₅H₂₁N₂O₂]+, 204 [C₁₃H₁₈NO]+, 193 [C₁₀H₁₃N₂O₂]+, 150 [C₉H₁₂NO]+, 134 [C₈H₈NO]+, 107 [C₇H₉N]•+, 92 [C₇H₈]•+, 95 [C₅H₃O₂]+.

{4-[4-({[5-(3-aminophenyl)-1,3,4-oxadiazol-2-yl]sulfanyl}methyl)benzoyl]-1-piperazinyl}(2-furyl)methanone (8b)

Light brown liquid; yield: 81%; Molecular formula: C₂₅H₂₃N₅O₄S; molecular mass: 489g/mol; IR (KBr, υmax cm^–1): 3413 (N-H), 3071 (Ar C-H), 2886 (R C-H), 1658 (C=O), 1580 (Ar C=C), 1203 (C-O-C), 1106 (C-N-C), 653 (C-S); ¹H-NMR (600 MHz, CDCl₃, δ in ppm): δ 7.96 (d, J = 7.9 Hz, 1H, H-4''''), 7.59 (s, 1H, H-2''''), 7.53-7.49 (m, 2H, H-2'' & H-6''), 7.50 (d, J = 8.1 Hz, 1H, H-6''''), 7.49 (br s, 1H, H-5), 7.42 (t, J = 7.6 Hz, 1H, H-5''''), 7.30 (d, J = 7.8 Hz, 2H, H-3'' & H-5''), 7.07 (d, J = 2.0 Hz, 1H, H-3), 6.50 (dd, J = 1.6, 3.4 Hz, 1H, H-4), 4.64 (s, 2H, H-8''), 3.85 (br s, 4H, H-3' & H-5'), 3.52 (br s, 4H, H-2' & H-6'); ¹³C-NMR (150 MHz, CDCl₃, δ in ppm): 170.09 (C-7''), 164.97 (C-2'''), 164.49 (C-5'''), 159.23 (C-6), 147.63 (C-2), 143.81 (C-5), 137.28 (C-4''), 133.56 (C-1''), 132.06 (C-3''''), 131.7 (C-1''''), 130.38 (C-2''''), 129.29 (C-4''''), 128.15 (C-5''''), 127.88 (C-3'' & C-5''), 126.92 (C-6''''), 122.8 (C-2'' & C-6''), 117.56 (C-3), 111.87 (C-4), 45.50 (C-2', C-3', C-5' & C-6'), 36.76 (C-8''); EI-MS (m/z): 489 [M]+, 421 [C₂₁H₁₉N₅O₃S]•+, 392 [C₂₀H₁₈N₅O₂S]+, 310 [C₁₆H₁₂N₃O₂S]+, 246 [C₁₃H₁₄N₂O₃]•+, 243 [C₁₂H₉N₃OS]•+, 179 [C₉H₁₁N₂O₂]+, 161 [C₈H₇N₃O]+, 151 [C₈H₉NO₂]+, 95 [C₅H₃O₂]+.

6{4-[4-({[5-(3-nitrophenyl)-1,3,4-oxadiazol-2-yl]sulfanyl}methyl)benzoyl]-1-piperazinyl}(2-furyl)methanone (8c)

Brick red liquid; yield: 82%; Molecular formula: C₂₅H₂₁N₅O₆S; molecular mass: 519g/mol; IR (KBr, υ_max cm^–1): 3414 (N-H), 3087 (Ar C-H), 2870 (R C-H), 1665 (C=O), 1579 (Ar C=C), 1190 (C-O-C), 1114 (C-N-C). 645 (C-S); ¹H-NMR (600 MHz, CDCl₃, δ in ppm): δ 7.53-7.49 (m, 2H, H-2'' & H-6''), 7.49 (br s, 1H, H-5), 7.44 (s, 1H, H-2''''), 7.41-7.39 (m, 3H, H-4'''', H-5'''' & H-6''''), 7.30 (d, J = 7.8 Hz, 2H, H-3'' & H-5'') 7.07 (d, J = 2.0 Hz, 1H, H-3), 6.50 (dd, J = 1.6, 3.4 Hz, 1H, H-4), 4.64 (s, 2H, H-8''), 3.85 (br s, 4H, H-3' & H-5'), 3.52 (br s, 4H, H-2' & H-6'); ¹³C-NMR (150 MHz, CDCl₃, δ in ppm): 170.05 (C-7''), 164.34 (C-5'''), 164.17 (C-2'''), 159.23 (C-6), 149.53 (C-3''''), 147.6 (C-2), 143.91 (C-5), 137.99 (C-4''), 133.09 (C-1''), 130.78 (C-6''''), 129.23 (C-5''''), 128.48 (C-1''''), 127.66 (C-3'' & C-5''), 126.04 (C-4''''), 125.4 (C-2''''), 122.8 (C-2'' & C-6''), 117.26 (C-3), 111.55 (C-4), 48.05 (C-2', C-3', C-5' & C-6'), 36.23 (C-8''); EI-MS (m/z): 397 [M]+, 273 [C₁₇H₂₅N₂O]+, 261 [C₁₅H₂₁N₂O₂] +, 218 [C₁₄H₂₀NO]+, 193 [C₁₀H₁₃N₂O₂]+, 164 [C₁₀H₁₄NO]+, 148 [C₉H₁₀NO]+, 121 [C₈H₁₁N]•+, 106 [C₈H₁₀]•+, 95 [C₅H₃O₂]+.

{4-[4-({[5-(4-methylphenyl)-1,3,4-oxadiazol-2-yl]sulfanyl}methyl)benzoyl]-1-piperazinyl}(2-furyl)methanone (8d)

Light brown liquid; yield: 88%; Molecular formula: C₂₆H₂₄N₄O₄S; molecular mass: 488g/mol; IR (KBr, υmax cm^–1): 3410 (N-H), 3088 (Ar C-H), 2879 (C-H), 1661 (C=O), 1578 (C=C), 1199 (C-O-C), 1111 (C-N-C), 657 (C-S); ¹H-NMR (600 MHz, CDCl₃, δ in ppm): δ 7.87 (d, J = 6.3 Hz, 2H, H-2'' & H-6''), 7.55 (d, J = 6.4 Hz, 2H, H-3'' & H-5'') 7.46 (br s, 1H, H-5), 7.40 (d, J = 6.4 Hz, 2H, H-2'''' & H-6''''), 7.29 (d, J = 6.3 Hz, 2H, H-3'''' & H-5''''), 7.07-7.05 (m, 1H, H-3), 6.51-6.49 (m, 1H, H-4), 4.52 (s, 2H, H-8''), 3.9 (br s, 4H, H-3' & H-5'), 3.5 (br s, 4H, H-2' & H-6'), 2.42 (s, 3H, H-7''''); ¹³C-NMR (150 MHz, CDCl3, δ in ppm): 170.09 (C-7''), 166.2 (C-5'''), 163.04 (C-2'''), 159.2 (C-6), 147.64 (C-2), 143.96 (C-5), 142.39 (C-4''), 138.13 (C-1''), 134.89 (C-4''''), 129.79 (C-3'' & C-5''), 128.83 (C-2'' & C-6''), 127.64 (C-2'''' & C-6''''), 126.64 (C-3'''' & C-5''''), 120.74 (C-1''''), 117.28 (C-3), 111.55 (C-4), 45.45 (C-2', C-3', C-5' & C-6'), 36.28 (C-8''), 21.64 (C-7''''); EI-MS (m/z): 488 [M]+, 273 [C₁₇H₂₅N₂O]+, 261 [C₁₅H₂₁N₂O₂] +, 218 [C₁₄H₂₀NO]+, 193 [C₁₀H₁₃N₂O₂]+, 164 [C₁₀H₁₄NO]+, 148 [C₉H₁₀NO]+, 121 [C₈H₁₁N]•+, 106 [C₈H₁₀]•+, 95 [C₅H₃O₂]+.

{4-[4-({[5-(4-hydroxyphenyl)-1,3,4-oxadiazol-2-yl]sulfanyl}methyl)benzoyl]-1-piperazinyl}(2-furyl)methanone (8e)

Black brown liquid; yield: 84%; Molecular formula: C₂₅H₂₂N₄O₅S; molecular mass: 490g/mol; IR (KBr, υ_max cm^–1): 3416 (N-H), 3089 (Ar C-H), 2870 (C-H), 1661 (C=O), 1574 (C=C), 1191 (C-O-C), 1116 (C-N-C), 649 (C-S); ¹H-NMR (600 MHz, CDCl₃, δ in ppm): δ 7.93 (d, J = 9.0 Hz, 2H, H-2'''' & H-6''''), 7.80 (d, J = 6.9 Hz, 2H, H-2'' & H-6''), 7.56 (d, J = 7.5 Hz, 2H, H-3'' & H-5'') 7.51 (d, J = 1.9 Hz, 1H, H-5), 7.09 (d, J = 3.7 Hz, 1H, H-3), 6.92-6.90 (d, J = 6.3 Hz, 2H, H-3'''' & H-5''''), 6.51 (dd, J = 1.9, 4.3 Hz, 1H, H-4), 4.52 (s, 2H, H-8''), 3.9 (br s, 4H, H-3' & H-5'), 3.56 (br s, 4H, H-2' & H-6'); ¹³C-NMR (150 MHz, CDCl₃, δ in ppm): 170.48 (C-7''), 166.22 (C-5'''), 162.40 (C-2'''), 160.09 (C-4''''), 159.27 (C-6), 147.53 (C-2), 144.07 (C-5), 140.29 (C-4''), 138.30 (C-1''), 130.03 (C-3'' & C-5''), 129.12 (C-1''''), 127.31 (C-2'' & C-6''), 117.45 (C-3), 116.31 (C-2'''' & C-6''''), 115.40 (C-3'''' & C-5''''), 111.59 (C-4), 52.24 (C-2', C-3', C-5' & C-6'), 36.35 (C-8''); EI-MS (m/z): 397 [M]+, 273 [C₁₇H₂₅N₂O]+, 261 [C₁₅H₂₁N₂O₂] +, 218 [C₁₄H₂₀NO]+, 193 [C₁₀H₁₃N₂O₂]+, 164 [C₁₀H₁₄NO]+, 148 [C₉H₁₀NO]+, 121 [C₈H₁₁N]•+, 106 [C₈H₁₀]•+, 95 [C₅H₃O₂]+.

{4-[4-({[5-(2,4-dichlorophenyl)-1,3,4-oxadiazol-2-yl]sulfanyl}methyl)benzoyl]-1-piperazinyl}(2-furyl)methanone (8f)

Light brown liquid; yield: 80%; Molecular formula: C₂₅H₂₀ClN₄O₄S; molecular mass: 542g/mol; IR (KBr, υ_max cm^–1): 3405 (N-H), 3082 (Ar C-H), 2880 (R C-H), 1655 (C=O), 1583 (Ar C=C), 1199 (C-O-C), 1110 (C-N-C), 644 (C-S); ¹H-NMR (600 MHz, CDCl₃, δ in ppm): δ 7.89 (d, J = 8.5 Hz, 2H, H-2'' & H-6''), 7.56-7.55 (m, 2H, H-5'''' & H-6''''), 7.54 (s, 1H, H-3''''), 7.41 (d, J = 6.3 Hz, 2H, H-3'' & H-5'') 7.49 (br s, 1H, H-5), 7.06 (d, J = 2.5 Hz, 1H, H-3), 6.50 (dd, J = 1.7, 3.4 Hz, 1H, H-4), 4.56 (s, 2H, H-8''), 3.85 (br s, 4H, H-3' & H-5'), 3.54 (br s, 4H, H-2' & H-6'); ¹³C-NMR (150 MHz, CDCl₃, δ in ppm): 170.04 (C-7''), 164.38 (C-5'''), 163.58 (C-2'''), 159.18 (C-6), 147.62 (C-2), 143.96 (C-5), 138.18 (C-4''), 137.85 (C-4''''), 134.99 (C-1''''), 133.75 (C-6''''), 131.55 (C-1''), 131.24 (C-5''''), 129.58 (C-3'' & C-5''), 129.48 (C-2''''), 127.66 (C-3''''), 127.45 (C-2'' & C-6''), 117.26 (C-3), 111.54 (C-4), 48.50 (C-2', C-3', C-5' & C-6'), 36.24 (C-8''); EI-MS (m/z): 369 [M]+, 245 [C₁₅H₂₁N₂O]+, 261 [C₁₅H₂₁N₂O₂]+, 193 [C₁₀H₁₃N₂O₂]+, 190 [C₁₂H₁₆NO]+, 136 [C₈H₁₀NO]+, 120 [C₇H₆NO]+, 93 [C₆H₆N]•+, 78 [C₆H₆]•+, 95 [C₅H₃O₂]+.

{4-[4-({[5-(3,5-dinitrophenyl)-1,3,4-oxadiazol-2-yl]sulfanyl}methyl)benzoyl]-1-piperazinyl}(2-furyl)methanone (8g)

Light brown liquid; yield: 84%; Molecular formula: C₂₅H₂₀N₆O₈S; molecular mass: 564g/mol; IR (KBr, υ_max cm^–1): 3415 (N-H), 3062 (Ar C-H), 2880 (R C-H), 1653 (C=O), 1582 (Ar C=C), 1205 (C-O-C), 1107 (C-N-C), 658 (C-S); ¹H-NMR (600 MHz, CDCl₃, δ in ppm): δ 8.05 (s, 1H, H-4''''), 7.58 (d, J = 6.3 Hz, 2H, H-5'''' & H-6''''), 7.50-7.44 (m, 2H, H-2'' & H-6''), 7.46 (br s, 1H, H-5), 7.27 (d, J = 7.8 Hz, 2H, H-3'' & H-5'') 7.00 (d, J = 2.0 Hz, 1H, H-3), 6.54 (dd, J = 1.6, 3.4 Hz, 1H, H-4), 4.68 (s, 2H, H-8''), 3.80 (br s, 4H, H-3' & H-5'), 3.57 (br s, 4H, H-2' & H-6'); ¹³C-NMR (150 MHz, CDCl₃, δ in ppm): 170.05 (C-7''), 164.31 (C-5'''), 164.12 (C-2'''), 159.20 (C-6), 150.08 (C-3'''' & C-5''''), 147.66 (C-2), 143.94 (C-5), 137.90 (C-4''), 134.24 (C-4''''), 133.04 (C-1''), 131.46 (C-1''''), 130.79 (C-2'''' & C-6''''), 127.69 (C-3'' & C-5''), 122.86 (C-2'' & C-6''), 117.27 (C-3), 111.52 (C-4), 45.48 (C-2', C-3', C-5' & C-6'), 36.24 (C-8''); EI-MS (m/z): 564 [M]+, 496 [C₂₁H₁₆N₆O₇S]•+, 467 [C₂₀H₁₅N₆O₆S]+, 385 [C₁₆H₉N₄O₆S]+, 318 [C₁₂H₆N₄O₅S]•+, 246 [C₁₃H₁₄N₂O₃]•+, 236 [C₈H₄N₄O₅]+, 179 [C₉H₁₁N₂O₂]+, 151 [C₈H₉NO₂]+, 118 [C₆H₂N₂O]+, 95 [C₅H₃O₂]+.

Hemolytic activity assay

Bovine blood sample was collected in Ethylene Diamine Tetra Acetic acid (EDTA) that was diluted with saline (0.9% NaCl), and centrifuge at 1000xg for 10 min. The erythrocytes separated diluted in phosphate buffer saline of pH 7.4 and a suspension was made. Add 20µL of synthetic compounds solution (10mg/mL) in 180µL of RBCs suspension and incubate for 30 min at room temperature. Phosphate-buffered saline (PBS) was used as negative control and Triton 100-X was taken as positive control.^21,22 The % age of hemolysis was taken as by using formula:

Chemistry

In the present investigation, various novel tri-heterocyclic benzamides were synthesized by the nucleophilic substitution reaction of 2-furoyl-(1-piperazinyl)methanone (1) with 4-(chloromethyl)benzoyl chloride (2) in a basic aqueous medium to get an electrophile {4-[4-(chloromethyl)benzoyl]-1-piperazinyl}(2-furyl) methanone (3). In parallel set of reactions, various substituted-benzoic acids (4a-g) were refluxed with ethanol and conc. sulphuric acid to form respective ethyl substituted-benzoates (5a-g). These esters were further refluxed with N₂H₄.H₂O in methanol solution to acquire substituted-benzohydrazides (6a-g). These hydrazides were cyclized into heterocyclic core by refluxing with CS₂ in the presence of KOH and ethanol solvent, giving rise to the formation of various 5-(substituted-phenyl)-1,3,4-oxadiazol-2-thiols (7a-g). The final step in the synthesis was the coupling of electrophile, 3, with nucelophiles, 7a-g, in acetonitrile and potassium carbonate to yield the targeted tri-heterocyclic molecules, 8a-g. Structures of these novel compounds were characterized and confirmed by IR, ¹H-NMR, ¹³C-NMR and EI-MS techniques. (Figure 1) (Table 1).

Figure 1 Outline for the synthesis of novel tri-heterocyclic benzamides. Reagents & Conditions:
a. Aq. Na₂CO₃ soln./pH 9-10/stirring at RT for 4 hrs.
b. EtOH/H₂SO₄/refluxing for 3-4 hrs.
c. MeOH/N₂H₄•H₂O/refluxing for 4-6 hrs.
d. EtOH/CS₂/KOH/refluxing for 3-6 hrs.
e. Acetonitrile/K₂CO₃/refluxing for 0.5 hrs for activation of 7a-g (one in each reaction), followed by addition of 3 and finally refluxing for 4-5 hrs.

One of the compounds is discussed hereby in detail for the expediency of the readers. For example, compound 8d, IR absorption band of aromatic C-H str. appeared at 3088, aliphatic C-H str. at 2879, 1661 (C=O), 1578 (Ar C=C), 1199 (C-O-C), 1111 (C-N-C), 657 (C-S). Its molecular formula was confirmed through EI-MS showing molecular ion peak at m/z 192, 119, 95 corresponding to C₂₆H₂₄N₄O₄S (Calcd. for 488). The EI-MS spectral data was also in complete agreement for the proposed structure of 8d which was finally confirmed through its ¹H-NMR and ¹³C-NMR spectra.

In ¹H-NMR spectrum, signals of methylbenzamide moiety appeared at δ 7.87 (d, J = 6.3 Hz, 2H, H-2'', H-6''), 7.55 (d, J = 6.4 Hz, 2H, H-3'', H-5'') and 4.52 (s, 2H, H-8''). The signals of protons for 4-methylphenyl ring appeared at 7.40 (d, J = 6.4 Hz, 2H, H-2'''', H-6''''), 7.29 (d, J = 6.3 Hz, 2H, H-3'''', H-5'''') and 2.42 (s, 3H, CH3-7''''). Furan ring showed three peaks in aromatic region at δ 7.46 (br.s, 1H, H-5), 7.07-7.05 (m, 1H, H-3) and 6.51-6.49 (m, 1H, H-4). The eight protons of piperazine ring appeared at δ 3.90 (br.s, 4H, CH2-3', CH2-5') and 3.50 (br.s, 4H, CH2-2', CH2-6').

The structure was also thorough supported by its ¹³C-NMR spectrum. Six signals of 170.09 (C-7''), 142.39 (C-4''), 138.13 (C-1''), 129.79 (C-3'', C-5''), 128.83 (C-2'', C-6'') and 36.28 (C-8'') supported the 4-methylenebenzamide. Two signals of 166.20 (C-5''') and 163.04 (C-2''') corroborated the 1,3,4-oxadiazole ring present in the molecule. Five signals of 159.20 (C-6), 147.64 (C-2), 143.96 (C-5), 117.28 (C-3) and 111.55 (C-4) confirmed the furoyl. Five signals of 134.89 (C-6''''), 127.64 (C-2'''', C-6''''), 126.64 (C-3'''', C-5''''), 120.74 (C-1'''') and 21.64 (C-7'''') confirmed the 4-methylphenyl ring. Piperazine was corroborated by single signal of 45.45 (C-2', C-3', C-5', C-6'). So, on the basis of above discussed cumulative evidences, the structure of 8d was named as {4-[4-({[5-(4-Methylphenyl)-1,3,4-oxadiazol-2-yl]sulfanyl}methyl)benzoyl]-1-piperazinyl}(2-furyl)methanone. Similarly all other synthesized derivatives were characterized by aforesaid spectral techniques.

The %age hemolytic activity and structure-activity relationship (8a-g)

All the synthesized compounds were subjected to hemolytic assay to find out their cytotoxicity profile. Results of percentage hemolysis are shown in Table 2 indicate that all the compounds are nearly nontoxic for membrane of red blood cells. Maximum membrane toxicity was seen by the compound 8e (10.90 %) due to hydroxyl group substitution at para position while minimum was noted in compounds 8a (0.76 %) in which at the o-position occupied by a chloryl group. Overall very mild toxicity was observed for molecules 8d (2.98%), 8c (4.21%), 8g (4.25%), 8b (7.94%) and 8f (10.77%) relative to PBS and Triton-X having % hemolysis of 0.09% and 100% respectively.

The anticipated structures of the synthesized tri-heterocyclic molecules, 8a-g, were thoroughly supported by spectroscopic analysis. The hemolytic activity data of these molecules revealed that these have low cytotoxicity and hence might be considered as safe therapeutic agents in drug discovery program.

The Higher Education Commission (HEC) of Pakistan is highly acknowledged by the authors for financial support regarding purchasing of chemicals and spectral study.

There is no conflict of interest.

1. Sengupta P, Mal M, Mandal S, et al. Evaluation of Antibacterial and Antifungal Activity of some 1, 3, 4 Oxadiazoles. Iranian J Pharm Therap. 2008;7(2):165–167.
2. Kadi AA, El-Brollosy NR, Al-Deeb OA, et al. Synthesis, antimicrobial, and anti-inflammatory activities of novel 2-(1-adamantyl)-5-substituted-1,3,4-oxadiazoles and 2-(1-adamantylamino)-5-substituted-1,3,4-thiadiazoles. Eur J Med Chem. 2007;42(2):235–242.
3. N Bhardwaj, SK Saraf, P Sharma, et al. Syntheses, Evaluation and Characterization of Some 1, 3, 4-Oxadiazoles as Antimicrobial Agents. E J Chem. 2009;6(4):1133–1138.
4. Sahu VKR, Singh AK, Yadav D. Review article on 1, 3, 4-Oxadiazole derivaties and it’s Pharmacological activities. Int J Chem Tech Res. 2011;3(3):1362–1372.
5. Nagaraj, Chaluvaraju KC, Niranjan MS, et al. 1, 3, 4-Oxadiazole: a potent drug candidate with various pharmacological activities. Int J Pharmacy and Pharm Sci. 2011;3(3):9–16.
6. Bhatia S, Gupta M. 1, 3, 4-Oxadiazole as antimicrobial agents: An overview. J Chem Pharm Res. 2011;3(3):137–147.
7. Jayashankar B, Rai KML, Baskaran N, et al. Synthesis and pharmacological evaluation of 1,3,4-oxadiazole bearing bis(heterocycle) derivatives as anti-inflammatory and analgesic agents. Eur J Med Chem. 2009;44(10):3898–3902.
8. Alam MM, Shaharyar M, Hamid H, et al. Synthesis of novel 8-hydroxy quinolin based 1,3,4-oxadiazoles and S-substituted 1,2,4-triazole derivatives and evaluation of their anti-inflammatory, analgesic, ulcerogenic and anti-microbial activities. Med Chem. 2011;7(6):663–673.
9. Gupta V, Kashaw SK, Jatav V, et al. Synthesis and antimicrobial activity of some new 3–[5-(4-substituted) phenyl-1,3,4-oxadiazole-2yl]-2- styrylquinazoline-4(3H)-ones. Med Chem Res. 2008;17(2-7):205–211.
10. El-Emam AA, Al-Deeb OA, Al-Omar M, et al. Synthesis, antimicrobial, and anti-HIV-1 activity of certain 5-(1-adamantyl)-2-substituted thio-1,3,4-oxadiazoles and 5-(1-adamantyl)-3-substituted aminomethyl-1,3,4-oxadiazoline-2-thiones. Bioorg Med Chem. 2004;12(19):5107–5113.
11. Zareef M, Rashid I, De Dominguez NG, et al. Synthesis and antimalarial activity of novel chiral and achiral benzenesulfonamides bearing 1, 3, 4-oxadiazole moieties. J Enzym Inhib Med Chem. 2007;22(3):301–308.
12. Xu W, He J, He M, et al. Synthesis and antifungal activity of novel sulfone derivatives containing 1,3,4-oxadiazole moieties. Molecules. 2011;16(11):9129–9141.
13. Wang JF, Jabbour GE, Mash EA, et al. Oxadiazole Metal Complex for Organic Light‐Emitting Diodes. Adv Mater. 1999;11(15):1266–1269.
14. Hernández-Ainsa S, Barberá J, Marcos M, et al. Liquid Crystalline Ionic Dendrimers Containing Luminescent Oxadiazole Moieties. Macromolecules. 2012;45(2):1006–1015.
15. Linton KE, Fisher AL, Pearson C, et al. Colour tuning of blue electroluminescence using bipolar carbazole–oxadiazole molecules in single-active-layer organic light emitting devices (OLEDs). J Mater Chem. 2012;22(23):11816–11825.
16. Wagner E, Al-Kadasi K, Zimecki M, et al. Synthesis and pharmacological screening of derivatives of isoxazolo[4,5-d]pyrimidine. Eur J Med Chem. 2008;43(11):2498–2504.
17. Sharba AHK, Al-Bayati RH, Aouad M, et al. Synthesis of Oxadiazoles, Thiadiazoles and Triazoles Derived from Benzo[b]thiophene. Molecules. 2005;10(9):1161–1168.
18. Husain A, Rashid M, Mishra R, et al. Benzimidazole bearing oxadiazole and triazolo-thiadiazoles nucleus: design and synthesis as anticancer agents. Bioorg Med Chem Lett. 2012;22(17):5438–5444.
19. Sharma S, Srivastava VK, Kumar A. Newer N-substituted anthranilic acid derivatives as potent anti-inflammatory agents. Eur J Med Chem. 2002;37(8):689–697.
20. Abbasi MA, Akhtar A, Aziz-ur-Rehman, et al. Synthesis, structural characterization and enzyme inhibition studies on 5-(2-nitrostyryl)-1,3,4-oxadiazole-2-thiol derivatives. J Chil Chem Soc. 2013;58(4):2186–2190.
21. Sharma P, Sharma JD. In vitro hemolysis of human erythrocytes -- by plant extracts with antiplasmodial activity. J Ethnopharmacol. 2001;74(3):239–243.
22. Stepanovic S, Vukovic D, Dakic I, et al. A modified microtiter-plate test for quantification of staphylococcal biofilm formation. J Microbiol Methods. 2000;40(2):175–179.