Synthetic utility of sydnones to couple pharmacologically important heterocycles for antitubercular activity

2.1 General

Melting points were determined in open capillaries. The IR spectra were recorded on a Nicolet Impact 5200 USA FT IR using KBr pellets. ¹H NMR spectra were recorded on a Bruker Varian 300-MHz FT NMR spectrometer with TMS as an internal standard. EI mass spectral analyses were recorded on a Shimadzu Japan QP2010 S model spectrometer and elemental analyses were carried out using a Heraus CHN rapid analyzer. The purity of the compounds was checked by thin layer chromatography (TLC) on silica gel plate using benzene and ethyl acetate. The pharmacological evaluation was carried out at the Department of Microbiology and Immunology, NGH College of Dental sciences, Belgaum, Karnataka, India. The log P values have been calculated using the OSIRIS molecular property explorer software for the structural analogues of the synthesized compounds and are uncorrected. 4-Bromo-3-arylsydnone (2a–c) required for the present work and the title compounds were prepared according to reported methods (Kamble and Badami, 2002; Puranik and Suschitzky, 1967).

2.2 General procedure for the preparation of amide derivatives (7–10)

2.3 Anti-tubercular assay

The anti-tubercular activity of the test compounds was evaluated against the standard strain of Mycobacterium tuberculae H37Rv (Sahm and Washington, 1991). Antibiotic standards used were streptomycin and pyrazinamide. The procedure followed for anti-tubercular activity involves the use of Middlebrook 7H-9 broth and standard strain of M. tuberculosis H37Rv. The basal medium was prepared according to manufacturer’s instructions (Hi-Media) and sterilized by autoclaving. Then broth (4.5 ml) was poured into each one of the sterile bottles. To this, ADC (0.5 ml) supplement was added. The supplement consists of catalase, dextrose and BSA fraction v. Then a stock solution was prepared (10 mg/ml). From this appropriate amount of solution was transferred to media bottles to achieve final concentrations of 25, 50, 100 μg/ml. Finally 10 μl suspension of M. tuberculosis H37Rv strain (1 lakh organisms/ml adjusted by McFarmland’s turbidity standard) was transferred to each of the tubes and incubated at 37 °C. Along with this, one growth control without compound and drug controls were also set up. The bottles were inspected for growth twice a week for a period of three weeks. The appearance of turbidity indicated the growth and infers the resistance to the compound. The growth was confirmed by making a smear from each bottle and performing a ZN strain Scheme 1.

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Scheme 1

Formation of amide derivatives 7–10.

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3.1 Biological activity

To qualify as a drug candidate, a new molecule has to be analysed for the parameters set by Lipinski’s rule (Lipinski et al., 1997) of five using Osiris property explorer. Lipinski’s rule of five is a rule of thumb to evaluate drug likeliness or to determine if a compound with a certain pharmacological or biological activity has properties that would make it a likely orally active drug in humans. The molecular properties are important for drug pharmacokinetics in human body, including their absorption, distribution, metabolism and excretion (ADME). The rule is important for drug development where pharmacologically active lead structure is optimized step wise for increased activity and selectivity, as well as drug like properties as described by Lipinski rule. The modification of the molecular structure often leads to drugs with higher molecular weight, more rings, more rotatable bonds and higher lipophilicity. The rule states that in general an orally active drug has not more than 5 hydrogen bond donors, and not more than 10 hydrogen acceptors, a molecular weight under 500 and the partition coefficient c log P less than 5 did not title compounds do not violate the Lipinski rule and they fall well in the range as mentioned when evaluated by the Osiris property explorer (Table 1) which lead us to evaluate the compounds experimentally. Promising results were shown for all compounds as the compounds were shown as safe drugs without any effects as mentioned in Osiris property explorer. None of the compounds were found to be tumourigenic, irritant or reproductive effect drug. Compounds (7b, 8b, 9c, and 9d) with a methyl group on the phenyl ring have shown mutagenic effect. Rest of all the compounds has not shown any mutagenic effect. Almost all the title compounds showed c log P well within the range and also certain compounds have molecular weights less than 500. The drug likeliness ranged from −1.71 to 2.28 where as the drug score is in the range 0.1–0.82. The compounds 7a, 7b, 10a which have electron donating groups, such as phenyl, halogen atom and morpholine show good drug scores as depicted in Table 2. The compounds 8a–b, 9d, 10a–b and d which have benzotriazole and diphenyl amine groups showed negative drug likeliness and as a result the drug score was less, and the presence of a halogen atom did not magnify the effect, where as the compounds 9a and 10c which have benzothaizoline-2-thione moiety have also shown diversified effects based on drug likeliness and drug score but c log P values are well within the range as mentioned by the rule.

In case of anti-tubercular activity studies, depicted in Table 2 the compounds with electron withdrawing atom such as bromine, heterocycles viz., morpholine and benzotriazole, (7a–b, 8a, 8c and 10a), have exhibited excellent inhibition (MIC) at less than or equal to 5 μg/ml concentration. The compounds with benzotriazole and diphenylamine 9d, 10a–c showed moderate inhibition in the range of 10–12 μg/ml. The compounds 9a–c with benzothiazoline-2-thione showed activity only at more than 20 μg/ml concentrations as compared to standards used viz., streptomycin (7.5 μg/ml) and pyrazinamide (10 μg/ml), respectively. Encouraging activity is attributed to the presence of cycloalkylating chains (e.g., morpholine ring) and with electron withdrawing substituent such as bromo appended to phenyl ring.