Naphthoquinone derivatives and their use in the treatment and control of tuberculosis

Abstract

Naphthoquinone derivatives of Formula (1): wherein R, represents an OH group, methyl ether, ethyl ether or a similar ether, R1 represents a methyl, ethyl or similar aliphatic hydrocarbon derivative: R2 and R3 each independently represent hydrogen or a group selected from: (A), (B), or (C) wherein R5 represents an PH group, methyl ether, ethyl ether or a similar ether and R6 represents a methyl, ethyl or similar aliphatic hydrocarbon derivative: R4 represents hydrogen or a group selected from: (D), (E) or (F) wherein R7 represents an OH group, methyl ether, ethyl ether or a similar ether and R8 represents a methyl, ethyl or similar aliphatic hydrocarbon derivative: or pharmaceutically acceptable salts thereof, are useful for the treatment and/or control of a tuberculosis in a patient caused by Mycobacterium tuberculosis.

Description

BACKGROUND OF THE INVENTION

THIS invention relates to the treatment and control of tuberculosis caused by Mycobacterium tuberculosis and in particular to the use of naphthoquinone derivatives in such treatment and control.

Tuberculosis ( TB ) remains a serious health problem in many regions of the world, especially in developing nations. It is a contagious disease and is becoming epidemic in some parts of the world. It is estimated that 30-60% of adults in developing countries are infected with Mycobacterium tuberculosis . Approximately 8-10 million individuals develop clinical TB and 3 million die of TB each year (WHO/IUATLD, 1989).

In South Africa, over 3 in every thousand people die of TB , the highest rate in the world, while one out of every 200 people suffers from active tuberculosis. Tuberculosis is the most commonly notified disease in South Africa and the fifth largest cause of death among the black population (South African Tuberculosis Association, 1998).

In the United States, the number of TB cases steadily decreased until 1986 when an increase was noted. Since then TB cases have continued to rise. Ten million individuals are infected in the U.S.A., with approximately 26000 new cases of active disease each year (National Jewish Medical and Research Center, 1994).

Individuals infected with Human Immunodeficiency Virus (HIV) are very susceptible to tuberculosis and often develop this disease before other manifestations of AIDS become apparent (Grange and Davey, 1990). Control of the TB epidemic linked with HIV infection will depend largely on the adequate treatment of TB , and possibly of effective chemoprophylaxis, not just for HIV-infected persons but for communities as well (WHO/IUATLD, 1989).

TB therapy has been revolutionized and the present treatment regimes for TB are based on multidrug therapy with usually 3 or 4 antituberculosis drugs. However, the problem of multidrug resistant tubercle bacilli is emerging for various drugs such as isoniazid, ethambutol, rifampicin and streptomycin, for example (Girling, 1989; Grange and Davey, 1990). Drug-resistant TB is very difficult to treat requiring greater numbers and varieties of medications for a longer period of treatment. The need for new antituberculosis agents is urgent due to the increasing resistance of mycobacteria to these classic antituberculosis drugs. A recent WHO report states that, globally, 2% of all cases of tuberculosis are multidrug resistant—by definition, resistance to rifampicin plus isoniazid (plus/minus other resistances). Such cases can be treated in the USA and other high resource regions but at a great cost (>US$ 250,000 per case!) and using very long courses of rather toxic drugs, thereby raising serious problems of compliance (WHO, 1997). South Africa is witnessing an explosion in the number of cases of drug-resistant tuberculosis . In some parts of South Africa, 1 in 10 cases of TB is resistant to treatment (New Scientist, March 1997). It is essential to have new antituberculosis agents, preferably those that can readily and simply be produced from some local source.

SUMMARY OF THE INVENTION

According to a first aspect of the invention there is provided a naphthoquinone derivative of Formula 1:

wherein,

R represents an OH group, methyl ether, ethyl ether or a similar ether;

R1 represents a methyl, ethyl or similar aliphatic hydrocarbon derivative;

R2 and R3 each independently represent hydrogen or a group selected from:

wherein R5 represents an OH group, methyl ether, ethyl ether or a similar ether and R6 represents a methyl, ethyl or similar aliphatic hydrocarbon derivative;

R4 represents hydrogen or a group selected from:

wherein R7 represents an OH group, methyl ether, ethyl ether or a similar ether and R8 represents a methyl, ethyl or similar aliphatic hydrocarbon derivative:

or pharmaceutically acceptable salts thereof, for use in a method of treating and/or controlling tuberculosis in a patient caused by Mycobacterium tuberculosis.

According to a second aspect of the invention there is provided the use of a naphthoquinone derivative having the Formula 1 as set out above in the manufacture of a medicament for use in a method of treating and/or controlling tuberculosis in a patient caused by Mycobacterium tuberculosis.

According to a third aspect of the invention there is provided a method of treating and/or controlling tuberculosis caused by Mycobacterium tuberculosis comprising administering to a patient in need thereof an effective amount of a naphthoquinone derivative having the Formula 1 as set out above.

The naphthoquinone derivative of Formula 1 is typically a compound of Formula 1a or Formula 1b:

wherein R and R1 are as defined for Formula 1 above.

R in the compound of Formula 1a or 1b is preferably an OH group.

R1 in the compound of Formula 1a or 1b is preferably a CH 3 group.

In particular, the naphthoquinone derivative of Formula 1 is 5,5′ dihydroxy 7,7′ binaphthoquinone (diospyrin) or 5-hydroxy-7-methyl-1,4-naphtoquinone (methyljuglone).

DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention is directed at the use of naphthoquinone derivatives in the treatment and/or control of tuberculosis caused by Mycobacterium tuberculosis . In particular, naphthoquinone derivatives of the general Formula 1

wherein,

R represents an OH group, methyl ether, ethyl ether or a similar ether;

R1 represents a methyl, ethyl or similar aliphatic hydrocarbon derivative;

R2 and R3 each independently represent hydrogen or a group selected from:

wherein R5 represents an OH group, methyl ether, ethyl ether or a similar ether and R6 represents a methyl, ethyl or similar aliphatic hydrocarbon derivative;

R4 represents hydrogen or a group selected from:

wherein R7 represents an OH group, methyl ether, ethyl ether or a similar ether and R8 represents a methyl, ethyl or similar aliphatic hydrocarbon derivative,

have been found to be effective against Mycobacterium tuberculosis.

Particular naphthoquinone derivatives of Formula 1a and 1b have been found to be particularly effective:

In particular diospyrin and methyljuglone, naphthoquinone derivatives of Formula 1a and Formula 1b, respectively, in which R is OH and R1 is a methyl group, have been found to inhibit several antibiotic resistant as well as antibiotic susceptible strains of Mycobacterium tuberculosis . Although diospyrin and methyljuglone are particularly preferred, naphthoquinone derivatives of Formula 1a and 1b in which R is a methyl ether, ethyl ether or similar ether and R1 is an ethyl or similar aliphatic hydrocarbon derivative are also provided.

An extensive research program was undertaken in order to identify antituberculosis agents that can readily and simply be produced from local resources.

Twenty South African medicinal plants used to treat pulmonary diseases were screened for activity against drug-resistant and sensitive strains of M. tuberculosis . A preliminary screening of acetone and water plant extracts, against a drug-sensitive strain of M. tuberculosis ; H37Rv, was carried out by the agar plate method. Fourteen of the 20 acetone extracts showed inhibitory activity at a concentration of 0.5 mg/ml against this strain. Acetone as well as water extracts of Cryptocarya latifolia, Euclea natalensis, Helichrysum melanacme, Nidorella anomala and Thymus vulgaris inhibited the growth of M. tuberculosis . Given the activity of 14 acetone extracts at 0.5 mg/ml against the drug-sensitive strain by the agar plate method a further study was carried out employing the rapid radiometric method to confirm the inhibitory activity. These active acetone extracts were screened against the H37Rv strain as well as a strain resistant to the drugs, isoniazid and rifampicin. The minimal inhibitory concentration of Croton pseudopulchellus, Ekebergia capensis, Euclea natalensis, Nidorella anomala and Polygala myrtifolia was 0.1 mg/ml against the H37Rv strain by the radiometric method. Extracts of Chenopodium ambrosioides, Ekebergia capensis, Euclea natalensis, Helichrysum melanacme, Nidorella anomala and Polygala myrtifolia were active against the resistant strain at 0.1 mg/ml. Eight plants showed activity against both the strains at a concentration of 1.0 mg/ml.

The following procedure was developed by the applicant for the isolation of diospyrin and methyljuglone from E. natalensis and other species in this genus, as well as any other plants that may synthesise diospyrin or methyljuglone or other quinone derivatives.

1. Identification of Plant Species

Roots and the aerial plant parts of E. natalensis were collected near Durban and identified at the HGWJ Schweickerdt Herbarium of the University of Pretoria and also at the herbarium of the National Botanical Institute, Pretoria.

2. Extraction

Dried roots of E. natalensis were ground to a powdery form with a dry mill and extracted over 48 hours with acetone. The extract was filtered and concentrated to dryness at reduced pressure on a rotary evaporator.

3. Thin Layer Chromatography

A direct antibacterial bioassay (Dilika & Meyer 1996) on TLC-plates was employed to speedup the activity guided isolation of the antituberculosis compounds. M. tuberculosis cannot be tested in this way because of its very slow growth rate. The direct antibacterial bioassays of the acetone extract were done on TLC plates (Merck) developed with chloroform-hexane (1:1). After development, the TLC plates were dried and sprayed with a 24 hr old Staphylococcus aureus culture in nutrient broth. After 24 hr incubation, the plates were sprayed with an aqueous solution of 2 mg/ml p-iodonitrotetrazolium violet to visualise the bacterial cells. The plates were then reincubated at 37° C. for 2-3 hours.

Two zones of bacterial growth inhibition could be seen on TLC plates sprayed with S. aureus . Activity was more pronounced in the R f 10.30 zone (chloroform-hexane (1:1)) than in the R f 0.54 zone.

4. Column Chromatography

The crude extract of the plant was dried, its mass determined and resuspended in chloroform. Column chromatography was performed on silica gel 60 using chloroform as eluent. The antibacterial fractions collected were then subjected to a Sephadex LH-20 column chromatography using ethanol as eluent. The fractions collected were again tested for antibacterial activity on TLC to detect the fractions containing the active compounds of R f 0.30 and R f 10.54.

5. High Performance Liquid Chromatography

The compounds were further purified by HPLC utilising an analytical Phenomenex reverse phase 250×4.60 mm column, at a flow rate of 1.0 ml/min, oven temp. 40° C. and a wavelength of 206 nm. An ethanol-water (50:50) solution was employed as mobile phase. The pure compounds were once again subjected to a Sephadex LH-20 column chromatography and proved to be pure. The chemical structures were confirmed by 1 H and 13 C nmr and ms to be:

Diospyrin (5,5′ dihydroxy 7,7′ binaphthoquinone); C 22 H 14 O 6 . Molecular weight: 374.35

7-methyljuglone (5-hydroxy-7-methyl-1,4-naphtoquinone); C 11 H 8 O 3 Molecular weight: 188.19

The effect of diospyrin and methyljuglone on the growth of the sensitive strain (H37Rv) and resistant strains of Mycobacterium tuberculosis as determined by the radiometric method are set out in Table 1 and Table 2.

TABLE 1
Effect of diospyrin on the growth of the sensitive strain (H37Rv) and
resistant strains of Mycobacterium tuberculosis as determined by the
radiometric method.
		ΔGI a
		values	ΔGI
		of	values
		plant	of the
	MIC	extracts	control vial
Mycobacterium tuberculosis strains	(mg/ml)	(mg/ml)	(mg/ml)
H37 sensitive strain	0.1	−1 ± 1.41	20 ± 4.24
2 drug resistant strain (res. to	0.1	3.5 ±	25 ± 7.07
Isoniazid and rifampicin).		0.70
3 drug resistant strain (res. to	0.1	4 ± 2.12	29 ± 1.41
streptomycin, isoniazid and
ethambutol).
4 drug resistant strain (res. to	0.1	5 ± 2.82	25 ± 2.82
streptomycin, isoniazid, rifampicin
and ethambutol).
5 drug resistant strain (res to	0.1	10 ± 1.41	22.5 ± 3.53
isoniazid, streptomycin, rifampicin,
thiacetazone and cyclocerine).
6 drug resistant strain (res. to	0.1	9 ± 2.82	30 ± 1.0
isoniazid, rifampicin, ethionamide,
terizidone, thiacetazone and
ofloxacin).
7 drug resistant strain (res to	0.1	13.5 ± 3.2	28 ± 3.1
isoniazid, streptomycin,
ethambutol, kanamycin, rifampicin,
and ethionamide)
a ΔGI values are means ± s.d.

TABLE 2
Effect of 7-methyljuglone as a single agent and in combination with
diospyrin on the growth of the sensitive strain (H37Rv) and resistant
strains of Mycobacterium tuberculosis as determined by the
radiometric method.
					ΔGI
				ΔGI b	values
Mycobacterium			MIC a	values	of the
tuberculosis	Lab reference	Com-	(μg/	of plant	control
strains	no.	pound(s)	ml)	extracts	vial
H37Rv sensitive	ATCC27294	7-methyl-	50	0 ± 1	15 ±
strain		juglone			3.78
Two drug (isonia-	CCKO28469V	7-methyl-	50	0 ± 0	30 ±
zid and rifampi-		juglone			4.94
cin) resistant
strain
H37Rv sensitive	ATCC27294	Diospy-	10	3 ± 1	15 ±
strain		rin + 7-			3.78
		methyl-
		juglone
Two drug	CCKO28469V	Diospy-	10	3.33 ±	30 ±
(Isoniazid and		rin + 7-		3.05	4.94
rifampicin resis-		methyl-
tant strain)		juglone
a Minimal inhibitory concentration
b ΔGI values are means ± s.d.

The results show that diospyrin and methyljuglone control the Mycobacterium tuberculosis bacterium effectively. Oral administration of diospyrin or methyljuglone in an appropriate pharmaceutical composition with suitable diluents and carriers will typically be used to treat or control tuberculosis . This will be by way of tablet, liquid or similar oral dosage form, as diospyrin and methyljuglone are readily absorbed intestinally.

However, it is believed that diospyrin or methyljuglone administered intravenously or intramuscularly will also be absorbed effectively through blood vessels and the blood stream of a patient. Transdermal administration, via a plaster or similar transdermal administration vehicle, is also a possibility.

A combination treatment of diospyrin and methyljuglone, which may be more effective than singular treatments of the two naphthoquinones, is also envisaged.

The applicant believes that it may be possible to increase the concentration of diospyrin, methyljuglone and other quinones in E. natalensis or similar species by phytoalexic stimulation or by the biotechnological manipulation of tissue cultures and/or intact plants.

Quinones are generally synthesised from catechol (1,2-quinones) or hydroquinone (1,4-quinones) by mild oxidation.

As far as the applicant has been able to establish, diospyrin has been synthesised once in a laboratory (Yoshida, M and Mori. K. 2000. European Journal of Organic Chemistry pages 1313-1317). However, similar binapthoquinones can also be synthesised by the reaction of plumbagin (94 mg in methanol, 10 ml) and its hydroquinone (190 mg in methanol, 14 ml), buffered in phosphate to pH 6.8 at 30° C. (Sankaram et al. 1975; Kumari et al. 1982).

It is believed that diospyrin, methyljuglone and related naphthoquinone derivatives are viable alternatives to conventional drugs in the treatment and control of tuberculosis in humans.

Claims

1. A method of treating and/or controlling tuberculosis caused by Mycobacterium tuberculosis comprising:administering to a patient in need thereof a therapeutically effective amount of a naphthoquinone derivative having the Formula 1: wherein,R represents an OH group; R1 represents a methyl group; R2 and R3 each independently represent hydrogen or a group selected from: wherein R and R1 are as defined above; andR4 represents hydrogen or a group selected from: wherein R and R1 are as defined above,or pharmaceutically acceptable salts thereof.

2. A method of treating and/or controlling tuberculosis caused by Mycobacterium tuberculosis comprising:administering to a patient in need thereof a therapeutically effective amount of a naphthoquinone derivative having the Formula 1: wherein,R represents an OH group: R1 represents a methyl group, R2 and R3 each independently represent hydrogen or a group selected from: wherein R and R1 are as defined above; andR4 represents hydrogen or a group selected from: wherein R and R1 are as defined above, andwherein the naphthoquinone derivative of Formula 1 is a compound of Formula 1a or Formula 1b: or pharmaceutically acceptable salts thereof.

3. A method according to claim 1 wherein the naphthoquinone derivative of Formula 1 is 5,5′ dihydroxy 7,7′ binaphthoquinone (diospyrin) or 5-hydroxy-7-methyl-1,4-naphtoquinone (7-methyljuglone), or a mixture thereof.

4. A method according to claim 1 wherein the naphthoquinone derivative of Formula 1 is administered orally, intravenously, intramuscularly or transdermally.