Antimycobacterial activity of diospyrin derivatives and a structural analogue of diospyrin against Mycobacterium tuberculosis in vitro

N. Lall1, M. Das Sarma2, B. Hazra2 and J. J. M. Meyer1,*

1 Department of Botany, University of Pretoria, Pretoria 0002, South Africa; 2 Department of Pharmaceutical Technology, Jadavpur University, Calcutta-700032, India

Received 16 April 2002; returned 15 August 2002; revised 16 October 2002. accepted 24 October 2002


    Abstract
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 Abstract
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 Materials and methods
 Results and discussion
 References
 
Three derivatives and one structural analogue of diospyrin were synthesized and investigated for their inhibitory activity against Mycobacterium tuberculosis employing the rapid radiometric method in vitro. A novel aminoacetate derivative was found to be more active than the parent compound, the MICs being 50 and 100 mg/L, respectively, for a drug-susceptible strain, H37Rv, of M. tuberculosis. This derivative also exhibited an MIC of 50 mg/L for a few multidrug-resistant strains of M. tuberculosis. The other two derivatives and the analogue did not show any significant antimycobacterial activity at the highest concentration (100 mg/L) tested.


    Introduction
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Tuberculosis (TB), an airborne lung infection, is becoming epidemic in some parts of the world and kills ~1 million children each year. It is estimated that between now and 2020, nearly 1 billion more people will be newly infected, 200 million people will become sick and 70 million will die from TB if control is not strengthened.1 Moreover, TB has also been recognized as one of the most frequent opportunistic infections in persons suffering from human immunodeficiency virus (HIV) in developing countries, particularly in Africa.

The presently available drug regimes comprise four to five drugs administered in combination for 6–9 months, and the patients often stop taking drugs as soon as the symptoms are ameliorated. As a consequence, there is a rise in multidrug-resistant TB (MDR-TB) cases, which are much more complicated to treat than those infected with a susceptible strain of Mycobacterium tuberculosis.2 Hence, a specific drug is needed that can cure TB in a much shorter time.

During our investigation of South African medicinal plant species traditionally used in the treatment of TB, a significant activity of the crude extracts of Euclea natalensis A. DC. (Ebenaceae) and that of a bisnaphthoquinonoid, diospyrin, isolated from this plant was observed against drug-susceptible and -resistant strains of M. tuberculosis.3 Incidentally, diospyrin had also been isolated from Diospyros montana Roxb., an Indian species belonging to the same family, and was found to possess significant anti-tumour and anti-leishmanial activities.4,5

Subsequently, a series of derivatives was synthesized from diospyrin that had vastly improved activity against not only Leishmania donovani but also other Trypanosomatidae parasites, e.g. Trypanosoma b. brucei and Trypanosoma cruzi.5 Thus, it was envisaged that a few derivatives/analogues of diospyrin could be studied to find out whether these would show enhanced antimycobacterial activity compared with diospyrin. Thus, an aminoacetate substituent was introduced into diospyrin to get a new aminoacetate derivative (of diospyrin dimethyl ether), which along with three other analogues were tested against M. tuberculosis and are reported here.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Plant material

The stem bark of D. montana Roxb. was collected from Hooghly district, a suburb of West Bengal, India, and the voucher specimen was authenticated at the Botanical Survey of India, Calcutta.

Compounds

Diospyrin was isolated from the stem-bark of D. montana Roxb. as described previously.4 Diospyrin dimethyl ether hydroquinone was synthesized from the dimethyl ether derivative of diospyrin by reduction with sodium dithionite. Diospyrin diethyl ether was prepared from diospyrin by using ethyl iodide and silver oxide. For synthesis of the aminoacetate derivative, the dimethyl ether of diospyrin was used as the starting material, which was then converted into an amino-substituted derivative by using hydroxylamine hydrochloride, followed by acetylation with acetyl chloride and pyridine (M. Das Sarma, R. Ghosh, A. Patra and B. Hazra, unpublished results). The structural analogue was prepared through dimerization of lawsone, a naphthoquinonoid plant product (Sigma Chemical Co., St Louis, MO, USA), as described previously.6 After synthesizing these compounds (Figure 1) they were purified meticulously through column, thin layer and preparative chromatography, followed by crystallization. The structures were authenticated through determination of melting point and standard instrumental techniques involving ultra-violet, infra-red, NMR and mass spectroscopy.



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Figure 1. Structures of the compounds.

 
M. tuberculosis strains

Bacterial cultures used in this study were grown from specimens received from the Medical Research Council (MRC) in Pretoria. A susceptible strain of M. tuberculosis, H37Rv reference strain obtained from American type culture collection, MD, USA, was used to investigate the activity of diospyrin, its synthetic derivatives and the structural analogue. Having obtained the best activity with the aminoacetate derivative (of diospyrin dimethyl ether) against the susceptible strain its activity was further evaluated against three multidrug-resistant strains of M. tuberculosis (Table 1). Drug-resistant strains were recovered in our laboratory from clinical specimens and identified by standard biochemical tests.


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Table 1.  Effect of the derivatives of diospyrin and its structural analogue on the growth of the drug-susceptible strain H37Rv and drug-resistant strains of M. tuberculosis by the radiometric method
 
Bioassays in vitro

The radiometric respiratory technique using the BACTEC system was used for susceptibility testing of M. tuberculosis as described previously.3 Solutions of all the compounds were prepared by maceration of a requisite amount of the compounds in a known volume of dimethyl sulphoxide (DMSO) to obtain a concentration of 10 g/L and stored at 4°C until used. Subsequent dilutions were made in DMSO and added to 4 mL of BACTEC 12B (7H12 medium) broth to achieve the desired final concentrations of 100, 50 and 10 mg/L together with PANTA (Becton Dickinson & Company, Ferndale, South Africa), an antimicrobial supplement. Control experiments showed that the final amount of DMSO (1%) in the medium had no effect on the growth of M. tuberculosis. BACTEC drug susceptibility testing was also performed for the two standard anti-TB drugs streptomycin and ethambutol (Sigma Chemical Co., South Africa), at concentrations of 6 and 7.5 mg/L, respectively, for H37Rv.

A homogenized culture (0.1 mL) of all the strains of M. tuberculosis, yielding 1 x 104 to 1 x 105 cfu/mL, was inoculated in the vials containing the compound/drug as well as in the control vials.7 Two compound-free vials were used as controls (medium + 1% DMSO): one vial (V1) was inoculated in the same way as the vials containing the compounds, and the other (V2) was inoculated with a 1:100 dilution of the inoculum (1:100 control) to produce an initial concentration representing 1% of the bacterial population (1 x 102 to 1 x 103 cfu/mL). The MIC was defined as the lowest concentration of the compound that inhibited >99% of the bacterial population.

When mycobacteria grow in 7H12 medium containing 14C-labelled substrate (palmitic acid), they use the substrate and 14CO2 is produced. The amount of 14CO2 detected reflects the rate and amount of growth occurring in the sealed vial, and is expressed in terms of the growth index (GI). Inoculated bottles were incubated at 38°C and each bottle was assayed every day to measure GI, at about the same hour until cumulative results were interpretable. The difference in the GI values of the last two days is designated as {Delta}GI. The GI reading of the vials containing the test compound was compared with the control vial (V2). Readings were taken until the control vials, containing a 100 times lower dilution of the inoculum than the test vials, reached a GI of 30 or more. If the {Delta}GI value of the vial containing the test compound was less than the control, the population was reported to be susceptible to the compound. Each test was replicated three times.


    Results and discussion
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
Results were interpreted on day 6 or 7 when the V2 control vials reached a GI value of 30 or more. The MICs of diospyrin and the aminoacetate derivative (of diospyrin dimethyl ether) were found to be 100 and >10–<=50 mg/L, respectively, for the drug-susceptible H37Rv strain. A similar MIC was obtained when the aminoacetate derivative (of diospyrin dimethyl ether) was tested for its activity against drug-resistant strains of M. tuberculosis. The activity of diospyrin against drug-susceptible and -resistant strains of M. tuberculosis has been reported by us recently.3 The other three compounds did not show any activity against any strain at the highest concentration tested (Table 1).

Although the activities of streptomycin and ethambutol, used as positive controls, were much stronger than those of diospyrin and the aminoacetate derivative (of diospyrin dimethyl ether), it would be interesting to study the synergic effects, if any, of these novel inhibitory agents if used in combination with the established anti-tubercular drugs.

Quinonoid compounds, by virtue of their facile redox cycling capacity, are known to possess wide-ranging antimicrobial as well as anti-cancer activities. However, the prospective antimycobacterial activity of quinonoid compounds, particularly those isolated from natural sources, has remained unexplored by and large. The reported tuberculostatic activity of Lawsonia inermis Linn.8 would indicate the probable involvement of lawsone, i.e. 2-hydroxynaphthoquinone, which is known to be the major bioactive constituent of this herb. However, in the present study the dimeric form of lawsone has been found to be rather inactive. This observation is consistent with the enormous influence of structural modification on the biological activity of the bisnaphthoquinonoid, namely diospyrin, which has been amply illustrated in our earlier reports.5,9 Likewise, the present study shows the positive contribution of an aminoacetate substituent being introduced into the allylic double bond of diospyrin. Hence, newer analogues of the aminoacetate derivative (of diospyrin dimethyl ether) have to be designed in order to resolve the structure–activity relationship in this series leading to the development of more effective antimycobacterial agents.


    Acknowledgements
 
We offer our sincere thanks to Dr Karin Weyer and Jeannette Brand of the Medical Research Council (Pretoria) for their assistance. Support from the University Grants Commission, New Delhi, and the International Foundation for Science, Stockholm (RGA No. F/1836-3) is gratefully acknowledged (B.H.). M.D.S. is supported by a fellowship from the Council of Scientific and Industrial Research, New Delhi.


    Footnotes
 
* Corresponding author. Tel: +27-12-4202224; Fax: +27-12-3625099; E-mail: marion{at}scientia.up.ac.za Back


    References
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 Abstract
 Introduction
 Materials and methods
 Results and discussion
 References
 
1 . World Health Organization. (1997). Anti-Tuberculosis Drug Resistance in the World. The WHO/IUATLD Project on Anti-Tuberculosis Drug Resistance Surveillance. World Health Organization, Geneva, Switzerland.

2 . National Jewish Medical and Research Centre. (1994). Medfacts from the National Jewish Centre for Immunology and Respiratory Medicine, Colorado, USA.

3 . Lall, N. & Meyer, J. J. M. (2001). Inhibition of drug-sensitive and drug-resistant strains of Mycobacterium tuberculosis by diospyrin, isolated from Euclea natalensis. Journal of Ethnopharmacology 78, 213–6.[CrossRef][ISI][Medline]

4 . Hazra, B., Sur, P., Roy, D. K., Sur, B. & Banerjee, A. (1984). Biological activity of diospyrin towards Ehrlich ascites carcinoma in Swiss A mice. Planta Medica 51, 295–7.

5 . Yardley, V., Snowdon, D., Croft, S. & Hazra, B. (1996). Antitrypanosomal activity of an indigenous plant product, diospyrin, and its synthetic derivatives. Phytotherapy Research 10, 559–62.

6 . Hazra, B., Acharya, S., Ghosh, R., Patra, A. & Banerjee, A. (1999). Ammonium metavanadate and perchloric acid: a novel reagent for dimerisation of naphthyl derivatives. Synthetic Communications 29, 1571–6.[ISI]

7 . Heifets, L. B., Iseman, M. D., Cook, J. L., Lindholm-Levy, P. J. & Drupa, I. (1985). Determination of in vitro susceptibility of M. tuberculosis to cephalosporins by radiometric and conventional methods. Antimicrobial Agents and Chemotherapy 27, 11–5.[ISI][Medline]

8 . Sharma, V. K. (1990). Tuberculostatic activity of henna (Lawsonia inermis Linn.). Tubercle 71, 293–5.[CrossRef][ISI][Medline]

9 . Hazra, B., Ghosh, R., Banerjee, A., Kirby, G. C., Warhurst, D. C. & Phillipson, J. D. (1995). In vitro antiplasmodial effects of diospyrin, a plant-derived naphthoquinonoid, and a novel series of derivatives. Phytotherapy Research 9, 72–4.[ISI]