Hemolytic profile of novel tri-heterocyclic benzamides

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


Introduction
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.

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% Na 2 CO 3 (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 substitutedbenzoates (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 (Na 2 CO 3 ; 10%). The title compounds were extracted by chloroform.

Procedure for the preparation of substitutedbenzohydrazides (6a-g)
Esters, 5a-g, (40mmol) and N 2 H 4 .H 2 O (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.

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. Phosphatebuffered 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:

Figure 1
Outline for the synthesis of novel tri-heterocyclic benzamides. e. Acetonitrile/K 2 CO 3 /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. Table 1 List of -R1 and -R2 substituents in novel tri-heterocyclic benzamides (8a-g) 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 C 26 H 24 N 4 O 4 S (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 1 H-NMR and 13 C-NMR spectra. The structure was also thorough supported by its 13

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.

Conclusion
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. 5a-g 6a-g 7a-g