Design and characterisation of piperazine-benzofuran integrated dinitrobenzenesulfonamide as Mycobacterium tuberculosis H37Rv strain inhibitors

Abstract Molecular hybridisation of four bioactive fragments piperazine, substituted-benzofuran, amino acids, and 2,4-dinitrobenzenesulfonamide as single molecular architecture was designed. A series of new hybrids were synthesised and subjected to evaluation for their inhibitory activity against Mycobacterium tuberculosis (Mtb) H37Rv. 4d–f and 4o found to exhibit MIC as 1.56 µg/mL, equally active as ethambutol whereas 4a, 4c, 4j displayed MIC 0.78 µg/mL were superior to ethambutol. Tested compounds demonstrated an excellent safety profile with very low toxicity, good selectivity index, and antioxidant properties. All the newly synthesised compounds were thoroughly characterised by analytical methods. The result was further supported by molecular modelling studies on the crystal structure of Mycobacterium tuberculosis enoyl reductase.


General procedure for the synthesis of 2a-2j: To solution of Ethyl 5-(piperazin-1-yl)
benzofuran-2-carboxylate (1 eq) in DMF (10 vol), Amino acid (1.1 eq), HATU (1.5 eq) and DIPEA (3 eq) were added and stirred at 25 o C for 2h. TLC showed the completion of starting material and formation of non-polar spot. The reaction mixture was quenched with water, extracted with ethyl acetate. The obtained crude product was purified by silica gel (100-200 mesh) column chromatography using ethyl acetate in hexane as eluent to give 2a-2j 1.2. General procedure for the synthesis of 3a-3j: To solution of appropriate 2a-2j derivatives (1 eq) in DCM (10 vol), TFA (4 eq) were added and stirred at 25 o C for 2h. TLC showed the completion of starting material and formation of polar spot. The reaction mixture was concentrated dry to give 3a-3j, the obtained crude was used directly in next step.

General procedure for the synthesis of 6a-6d:
To solution of acid derivative 5 (1 eq) in DMF (10 vol), Amine (1.5eq), HATU (1.5eq) and DIPEA (3 eq) were added and stirred at 25 o C for 2h. TLC showed the completion of starting material and formation of non-polar spot. The reaction mixture was quenched with water, extracted with ethyl acetate, dried over sodium sulphate, concentrated to dry and purified by silica gel (100-200 mesh) column chromatography using ethyl acetate in hexane as eluent to give 6a-6d.

General procedure for the synthesis of 7a-7d:
To solution of appropriate 6a-6d derivatives (1 eq) in DCM (10 vol), TFA (3 eq) was added and stirred at 25 o C for 2h. TLC showed the completion of starting material and formation of polar spot. The reaction mixture was concentrated to dry to give 7a-7d, used directly in next step.

In-vitro Mtb MABA assay
Briefly, the inoculum was prepared from fresh LJ medium re-suspended in 7H9-S medium (7H9 broth, 0.1% casitone, 0.5% glycerol, supplemented oleic acid, albumin, dextrose, and catalase [OADC]), adjusted to an OD590 1.0, and diluted 1:20; 100 µl was used as inoculum. Each drug stock solution was thawed and diluted in 7H9-S at four-fold the final highest concentration tested. Serial two-fold dilutions of each drug were prepared directly in a sterile 96-well microtiter plate using 100 µl 7H9-S. A growth control containing no antibiotic and a sterile control were also prepared on each plate. Sterile water was added to all perimeter wells to avoid evaporation during the incubation. The plate was covered, sealed in plastic bags and incubated at 37 o C in normal atmosphere. After 7 days incubation, 30 µl of Alamar blue solution was added to each well, and the plate was re-incubated overnight. A change in colour from blue (oxidised state) to pink (reduced) indicated the growth of bacteria, and the MIC was defined as the lowest concentration of drug that prevented this change in colour.

Cytotoxicity assay
Most active anti-TB compounds were further examined for toxicity in human cell lines A549 at the concentration of 50 mg/mL. After 72 h of exposure, viability was assessed on the basis of cellular conversion of MTT into a formazan product using the Promega Cell Titer 96 nonradioactive cell proliferation assay.

DPPH radical scavenging activity
The hydrogen atom or electron donation ability of some compounds were measured from the bleaching of the purple colored methanol solution of 1,1-diphenyl-1-picrylhydrazyl (DPPH). The spectrophotometric assay uses the stable radical DPPH as a reagent. 1 mL of various concentrations of the test compounds (5, 10, 25, 50 and 100 μg/mL) in methanol was added to 4 mL of 0.004% (w/v) methanol solution of DPPH. The reaction mixture was incubated at 37 °C.
The scavenging activity on DPPH was determined by measuring the absorbance at 517 nm after