WO2001023342A1

WO2001023342A1 - Phloroglucinol compounds

Info

Publication number: WO2001023342A1
Application number: PCT/IB2000/001168
Authority: WO
Inventors: Jacobus Johannes Marion Meyer; Abbey Danny Matome Mathegka
Original assignee: University of Pretoria
Current assignee: University of Pretoria
Priority date: 1999-09-30
Filing date: 2000-08-25
Publication date: 2001-04-05
Anticipated expiration: 2002-03-30
Also published as: AU6586900A

Abstract

Phloroglucinol compounds of Formula (1) or salts, esters or derivatives thereof are useful in the treatment and/or control of tuberculosis caused by Mycobacterium tuberculosis and infection caused by other pathogenic bacteria and fungi. R typically represents an H, OH, OCH3 or OCH2CH3 group or a similar ether and R1 typically represents a CH¿3? or CH2CH3 group or a similar hydrocarbon derivative.

Description

PHLOROGLUCINOL COMPOUNDS

BACKGROUND OF THE INVENTION

THIS invention relates to phloroglucinol compounds, their use in the treatment and control of tuberculosis caused by Mycobacteriυm tuberculosis as well as the treatment and control of other bacteria and fungi.

The World Health Organisation estimates that 80% of the people living in developing countries almost exclusively use traditional medicine. This means that in the order of 3300 million people depend on medicinal plants on a regular basis (Eloff 1998). Medicinal components from plants play an important role in conventional medicine. The number of resistant strains of microbial pathogens are growing since penicillin resistant and multi- resistant pneumococci and streptococci cause a major problem in hospitals. Berkowitz (1995) calls the emerging drug resistant bacteria a medical catastrophe.

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 Centre, 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 the invention there is provided a phloroglucinol compound of Formula 1:

wherein,

R represents an H, OH, OCH₃ or OCH₂CH₃ group or a similar ether; and

R¹ represents a CH₃ or CH₂CH₃ group or a similar hydrocarbon derivative,

or a pharmaceutically acceptable salt, ester or derivative thereof.

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

R¹ in the compound of Formula 1 is preferably a CH₃ group.

In particular the acylated phloroglucinol compound of Formula 1 is 2- methyl-4-[2',4',6'-trihydroxy-3-(2-methylpropanoyl)phenyl]but-2-enylacetate, hereinafter referred to as Caespitate.

The invention extends to a compound of Formula 1 or a salt, ester or derivative thereof for use in a method of treating and/or controlling tuberculosis in a patient caused by Mycobacterium tuberculosis or infection caused by other pathogenic bacteria and fungi.

The invention also extends to the use of a compound of Formula 1 or a salt, ester or derivative thereof in the manufacture of a medicament for use in a method of treating and/or controlling tuberculosis in a patient caused by Mycobacterium tuberculosis or infection caused by other pathogenic bacteria and fungi.

According to a further aspect of the invention there is provided a method of treating and/or controlling tuberculosis in a patient caused by Mycobacterium tuberculosis or infection caused by other pathogenic bacteria and fungi comprising administering to a pateint in need thereof a phloroglucinol compound or Formula 1 or a salt, ester or derivative thereof.

DESCRIPTION OF PREFERRED EMBODIMENTS

The present invention is directed to phloroglucinol compounds, salts, esters or derivatives thereof and their use in the treatment and/or control of tuberculosis caused by Mycobacterium tuberculosis and infection caused by other pathogenic bacteria and fungi. In particular, phloroglucinol compounds of the general Formula 1 have been found to be effective against Mycobacterium tuberculosis and infection caused by other pathogenic bacteria and fungi.

FORMULA 1

Typically, compounds of formula 1 are provided in which R represents an H, OH, OCH₃ or OCH₂CH₃ group or a similar ether and R¹ represents a CH₃ or CH₂CH₃ group or a similar hydrocarbon derivative. In particular caespitate, a phloroglucinol compound of Formula 1 in which R is OH and R¹ is CH₃, has been found to inhibit several antibiotic resistant as well as antibiotic susceptible strains of Mycobacterium tuberculosis as well as other pathogenic bacteria and fungi.

The inventors of the present application undertook an extensive research program in order to identify antituberculosis, antibacterial and antifungal agents that can readily and simply be produced from local resources.

Twenty-eight South African medicinal plants used to treat pulmonary diseases were screened by the inventors 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 caespititium, 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 caespititium, 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 for the isolation of caespitate from H. caespititium and other species in this genus, as well as any other plants that may synthesise caespitate or other compounds of formula 1 , their salts, esters or derivatives thereof.

1. Identification of plant species

The aerial plant parts of H. caespititium were collected near Harrismith 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 aerial plant parts of H. caespititium were shaken in acetone for 5 minutes, 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 antimicrobial compound. 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-ethylacetate (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 2mg/ml p-iodonitrotetrazolium violet to visualise the bacterial cells. The plates were then reincubated at 37°C for 2-3 hours.

Four zones of bacterial growth inhibition could be seen on TLC plates sprayed with S. aureus. Activity was more pronounced in the R_f 0.57 zone (chloroform-ethylacetate (1 :1)) than in the other 3 zones. 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 tested for antibacterial activity on TLC to detect the fraction containing the active compound of R_f 0.57.

High performance liquid chromatography

The compound was further purified by HPLC utilising an analytical Phenomenex reverse phase 250x4.60 mm column, at a flow rate of 1.0 ml/min, oven temp. 40°C and a wavelength of 206nm. An ethanol-water (50:50) solution was employed as mobile phase. The pure compound was collected from the eluent. The chemical structure was confirmed by ¹H-, ¹³C-, COSY-, DEPT- and HETCOR- nmr and ms to be:

Caespitate (2-methyl-4-[2',4',6'-trihydroxy-3-(2-methylpropanoyI) phenyl] but-2-enylacetate. Molecular weight: 322.14363

ANTIBACTERIAL ACTIVITY

The activity of caespitate was examined against ten bacteria by the agar dilution method (Turnbull & Kramer, 1991). Referring to Table 1 , it is evident that caespitate significantly inhibited the growth of all the Gram- positive bacteria tested at a concentration of between 0.5 and 5μg/ml.

TABLE 1. Antibacterial activity (MIC)^a of the crude acetone extract of the aerial parts of Helichrysum caespititium and caespitate isolated from the extract.

a Minimum inhibitory concentration b Not active

Caespitate had no activity against all the Gram-negative bacteria tested. These results are in accordance with previous reports (Tomas-Barberan, Iniesta-Sanmarin, Tomas-Lorente, & Rumbero, 1990; Dekker, Fourie, Snyckers & Van der Schyf, 1983) of similar antimicrobial activity of related compounds against Gram-negative bacteria. Most bacillus species are regarded as having little or no pathogenic potential, however, both Bacillus cereus and B. subtilis have been known to act as primary invaders or secondary infectious agents in a number of cases and have been implicated in some cases of food poisoning (Turnbull & Kramer, 1991). Staphylococcus aureus is a human pathogen whose infections are amongst the most difficult to combat with conventional antibiotics (Tomas-Barberan, Msonthi & Hostettmann, 1988; Tomas-Barberan, Iniesta-Sanmarin, Tomas- Lorente, & Rumbero, 1990). This study provided a probable scientific explanation for the therapeutic potency attributed to H. caespititium.

ANTIFUNGAL ACTIVITY

The growth of six fungi, Aspergillus niger, A. flavus, Cladosporium cladosporioides, C. cucumerium, C. sphaerospermum and Phythophthera capsici, were significantly inhibited at very low MIC's by caespitate (Table 2). A. flavus and A. niger are some of the most important fungi responsible for human systemic infections. These organisms were inhibited at 1.0 μg/ml. It is generally agreed that at least one acidic hydroxyl group and a certain degree of lipophilicity are required for biological activity compound (Tomas-Barberan, Iniesta-Sanmarin, Tomas-Lorente, & Rumbero, 1990). Lipophilicity is important because many antifungal metabolites exert their toxicity by some membrane associated phenomenon, and it is known that acidic hydroxyl groups may act by uncoupling oxidative phosphorylation. In this case the antifungal caespitate isolated from H. caespititium bears three acidic hydroxyls (phenolic hydroxyls) and lipophilicity (3'-isobutyrylphenyl and but-2-enyl acetate residues). On the other hand, antibacterial activity, against Gram-positive bacteria seems to be related to the presence of phenolic hydroxyls (phenol itself is a well known antibacterial compound (Tomas-Barberan, Iniesta-Sanmarin, Tomas- Lorente, & Rumbero, 1990).

TABLE 2. Antifungal activity of the crude acetone extract of the aerial parts of Helichrysum caespititium and caespitate isolated from the extract.

MIC ^a

Fungal species Crude Extract Caespitate mg/ml μg/ml

Aspergillus flavus 1.0 1.0

A. niger 0.01 1.0

Cladosporium cladosporioides 0.01 5.0

C. cucumerium 0.01 0.5

C. sphaerospermum 0.01 0.5

Phytophthora capsici 1.0 1.0

a Minimum inhibition concentration.

ANTITUBERCULOSIS ACTIVITY

The effect of caespitate 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 3.

The results show that caespitate controls the Mycobacterium tuberculosis bacterium effectively. Oral administration of caespitate 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 caespitate is readily absorbed intestinally.

However, it is believed that caespitate 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. TABLE 3. Inhibition of Mycobacterium tuberculosis strains by caespitate

'ΔGI values are means ± s.d.

SYNTHESIS OF CAESPITATE

It is believed that it may be possible to increase the concentration of caespitate and other acylated phloroglucinols in H. caespititium or similar species by phytoalexic stimulation or by the biotechnological manipulation of tissue cultures and/or intact plants. Acylation ofglucinol derivatives

Since glucinols have phenolic hydroxyl groups and are very acidic, a mild base such as pyridine is enough to obstruct the H⁺ of the phenolic OH^". Acylated phloroglucinols would, therefore, generally be synthesised by dissolving glucinol in pyridine in equimolar quantities of acetic anhydride, under reflux for a period of 30 minutes.

The reaction is quenched by neutralising the reaction mixture by either adding water or NaOH. The product can then be extracted into an organic phase such as chloroform or ethyl acetate (Yuste, F. et al., 1978; Yoneyama, K. et al., 1989)

As far as the applicant has been able to establish, caespitate has never previously been synthesised in a laboratory.

It is believed that caespitate and related acylated phloroglucinol derivatives are viable alternatives to conventional drugs in the treatment and control of tuberculosis and infections caused by other pathogenic bacteria and fungi in humans.

Claims

A phloroglucinol compound of Formula 1 :

wherein,

R represents an H, OH, OCH₃ or OCH₂CH₃ group or a similar ether; and

R¹ represents a CH₃ or CH₂CH₃ group or a similar hydrocarbon derivative,

or a pharmaceutically acceptable salt, ester or derivative thereof.

A compound according to claim 1 , wherein R each occurrence is an OH group.

A compound according to claim 1 or claim 2, wherein R¹ is a CH₃ group.

A compound according to claim 1 which is 2-methyl-4-[2',4',6'- trihydroxy-3-(2-methylpropanoyl)phenyl]but-2-enylacetate.

5. A compound of Formula 1 as defined in claim 1 or a salt, ester or derivative thereof for use in a method of treating and/or controlling tuberculosis in a patient caused by Mycobacterium tuberculosis or infection caused by other pathogenic bacteria and fungi.

6. A method according to claim 5, wherein the compound of Formula 1 is 2-methyl-4-[2',4',6'-trihydroxy-3-(2-methylpropanoyl)phenyl]but-2- enylacetate.

7. The use of a compound of Formula 1 as defined in claim 1 or a salt, ester or derivative thereof in the manufacture of a medicament for use in a method of treating and/or controlling tuberculosis in a patient caused by Mycobacterium tuberculosis or infection caused by other pathogenic bacteria and fungi.

8. The use of claim 7, wherein the compound of Formula 1 is 2-methyl- 4-[2',4',6'-trihydroxy-3-(2-methylpropanoyl)phenyl]but-2-enylacetate.

9. A method of treating and/or controlling tuberculosis in a patient caused by Mycobacterium tuberculosis or infection caused by other pathogenic bacteria and fungi comprising administering to a patient in need thereof a therapeutically effective amount of a compound of Formula 1 as defined in claim 1 or a pharmaceutically acceptable salt, ester or derivative thereof.

10. A method according to claim 9, wherein the compound of Formula 1 is 2-methyl-4-[2',4',6'-trihydroxy-3-(2-methylpropanoyl)phenyl]but-2- enylacetate.