WO2012035305A1

WO2012035305A1 - Bioactive alkaloids

Info

Publication number: WO2012035305A1
Application number: PCT/GB2011/001359
Authority: WO
Inventors: Margey Tadesse; Morten B. STRØM; Johan Svenson; Marcel Jaspars; Klara STENSVÅG; Tor Haug
Original assignee: Lytix Biopharma As
Priority date: 2010-09-16
Filing date: 2011-09-16
Publication date: 2012-03-22
Also published as: GB201015537D0

Abstract

The present invention relates to compounds of formula (I) (Formula (I)); wherein R₁ denotes either hydrogen or chlorine, R₂ and R₃ denote hydrogen, or where C_α and N_β are covalently linked to form a fused ring, R₂ and R₃ are absent. The invention further relates to pharmaceutical and veterinary formulations containing these compounds and the use of these compounds in therapy, particularly as antimicrobial and anti-tumour agents.

Description

59.67.104096

Bioactive Alkaloids The present invention relates to novel compounds exhibiting antibacterial, antifungal and anticancer activities and to the medical and other uses thereof.

More specifically, these compounds include a 4-oxazolidinone ring.

The emergence and re-emergence of resistant bacteria is one of the major challenges facing the pharmaceutical industry today. Even though the extent and speed of emergence of bacterial resistance to antimicrobial agents vary with different types of drugs, resistance has so far developed to all known antimicrobial drugs. The prevalence of infectious diseases caused by antimicrobial resistant human pathogens is rapidly increasing. This includes the worldwide emergence of multidrug-resistant Mycobacterium tuberculosis. Other examples of microbial resistance to conventional antibiotics include vancomycin resistance in

Staphylococcus aureus (VRSA) and enterococci (VRE), resistance to beta-lactam antibiotics, such as the cephalosporins in the gram-negative bacilli Pseudomonas aeruginosa and Escherichia coli, and penicillin resistance (often multidrug-resistant) in pneumococci. Resistance has also spread to a variety of non-bacterial pathogens, such as viruses, fungi and parasites. The development of resistance to antifungal agents by opportunistic fungal pathogens such as Candida albicans and Saccharomyces cerevisiae which can cause lifethreatening systemic infections in immunocompromised individuals such as HIV and cancer patients, is on the rise.

Resistance develops by genetic mutations or by the acquisition of exogenous genetic material. Chemical modification of known antibiotics has been the most frequently employed method to address the problem of resistance. For example derivation of the basic nuclear structure of the penicillins, 6-amino- penicillinic acid, yielded compounds with activity against gram-negative bacilli (ampicillin, amoxicillin, carbenicillin, ticarcillin, mezlocillin, aziocillin, piperacillin, and a variety of other "broadspectrum" penicillins), and β-lactams with activity against β- lactamase producing S. aureus and coagulase negative staphylococci (methicillin and a variety of other antistaphylococcal penicillins). Modifications of the cephalosporin molecule have resulted in alterations in its in vitro spectrum of activity, its resistance to β-lactamases, and its pharmacokinetic properties.

Nonetheless, as each new analogue has been introduced over the years, it has ultimately been succeeded by the emergence of resistant organisms. One of the major contributing factors to this is the abuse and overuse of new antibiotics. The picture is further complicated by the fact that the speed of discovery and

development of new antimicrobial drugs active against multidrug-resistant organisms have slowed down considerably, although billions of dollars are annually invested in this research area. There are several reasons for this apparently contradictory situation, including the even greater costs of bringing a new antibiotic from discovery to the market. This is currently estimated at between $100 million and $350 million in the United States alone. Another reason is the between $100 million and $350 million in the United States alone. Another reason is the limited revenue from sales expected by pharmaceutical companies due to the short duration of treatment with antibiotics relative to other drugs, such as cholesterol and hypertension agents, which are consumed for prolonged periods and relieve symptoms rather than provide a cure. Only two of the few antibiotics introduced in the last 20 years, the oxazolidinones (which inhibit bacterial protein synthesis) and cationic peptides (which permeabilize bacterial membranes), have unconventional modes of action.

There is a need to target screening more broadly to ensure that rare activities of unanticipated mode-of action are not missed and to concentrate on the discovery of novel structural scaffolds to minimize the problem of resistance.

Humans have always relied on nature for medicines. Plants have long been a source of new therapeutic agents as have microorganisms. More recently the potential of marine life as a source of new drugs has been appreciated, indeed the greatest biodiversity of life is found in the oceans. Out of the 36 phyla, 34 are represented in the marine environment and 21 of these are exclusively marine.

In terms of identification of new chemical entities (NCEs), the richest source so far has been the sponges. Sponges were the first examples of multicellular organisms and all chemical classes are widely represented. A further class of widely studied marine organisms are the sea squirts (ascidians). The majority of compounds isolated from ascidians are alkaloids and nitrogen-containing cyclic peptides. The nitrogen-containing derivatives are often associated with aromatic nuclei among the alkaloids (indole, carbazole, pyridoacridine, isoquinoline) and with heteroaromatic nuclei among the cyclic peptides (thiazole, thiazoline, oxazole, oxazoline). Most of these compounds possess antibiotic, anti-tumour, antiviral and immunosuppressive activities. Some of the ascidian alkaloids have been found in other phyla of invertebrates suggesting that they might be of symbiotic bacterial origin. It is believed that most of the cytotoxic ascidian metabolites are involved in defense against predators and antifouling.

Synoicum sp. are colonial ascidians that have afforded a wide range of compounds with diverse biological activities. Among these are the cytotoxic palmerolide A, ecdysteroids, a tetrahydrocannabinol derivative, pronolides A, B, and C, a number of rubrolides, the anti-diabetic tiruchanduramine and £ Z-rubrolide O, which is an anti-inflammatory halogenated furanone. S. pulmonaria is commonly found in the arctic-boreal waters of the North-Sea, and the deep cold waters of West-Scotland and Northern-Ireland. It has been shown that S. pulmonaria, amongst other marine ascidians, contain compounds with antibacterial and antifungal activity on the basis of the activity of active fractions prepared thereform (Tadesse, M. et al., Journal of Invertebrate Pathology 99 (2008) 286-293).

The present inventors have isolated a series of novel brominate guanidines from S. pulmonaria, that have been named synoxazolidinones due to their unique 4-oxazolidinone core structure. The S. pulmonaria species was collected off the coast of Troms in Northern-Norway. These molecules have been shown to have a broad range of activity, including antibacterial, antifungal and antitumour activity.

Thus, in one aspect, the present invention provides a compound of formula

wherein:

Ri denotes either hydrogen or chlorine;

R₂ and R₃ denote hydrogen, or where C_a and N_p are covalently linked to form a fused ring, R₂ and R₃ are absent; wherein the compound is selected from the group consisting of:

Synoxazolidinone A (1 )

Synoxazolidinone B (2)

Synoxazolidinone

Synoxazolidinone D (4)

; and stereoisomers thereof.

Stereoisomers include enantiomers and diastereomers e.g. geometric isomers.

Compounds 1 and 2 are particularly preferred.

Compounds 1 to 3 may be isolated from S. pulmonaria as described in the Examples hereto. Other compounds according to the present invention may be synthesised by synthetic routes known in the art; compound 4, for example, may be obtained by dechlorination of compound 2 using tributyltin hydride. The

stereochemistry of the double bond can be altered by methods familiar to the skilled man, e.g. utilising irradiation with UV-light.

Synoxazolidinones A-C (1-3) were isolated from a crude acetonitrile extract of the lyophilized S. pulmonaria specimen after separation on a preparative RP-HPLC Ci8-column using a gradient of acetonitrile/water. HPLC analysis revealed three major components with almost similar retention. Synoxazolidinone A (1 ) was isolated as a colorless semi-crystalline oil with the molecular formula C₁₅H₁₇N₄0₃CIBr₂ (HRESIMS m/z 494.9437, Δ+0.3 mmu for [M + H]⁺)¹².

Mass spectrometric analysis of 2 afforded a molecular formula of

C₁₅H₁₅Br₂CIN₄0₃ (HRESIMS m/z 492.9280, Δ+0.2 mmu for [M + H]⁺)¹⁸.

Accurate mass measurement of 3 indicated a molecular formula of

C₁₅H₁₈Br₂N₄0₃ (HRESIMS m/z 460.9833, Δ+0.9 mmu for [M + H]⁺)¹⁹.

In a further aspect the present invention provides a method of preparing any one of compounds 1 to 3 which comprises taking a specimen or sample of Synoicum pulmonaria and extracting one or more of compounds 1 to 3 therefrom. Extraction will typically comprise a lyophilization step and/or the addition of an organic solvent. The organic phase may be fractionated using HPLC.

In a further aspect the present invention provides a method of preparing compound 4 which comprises the step of dechlorination of compound 2 or a precursor thereof.

In a further aspect is provided the compounds of the present invention for use in therapy, particularly for use as an antimicrobial (e.g. antibacterial) or antifungal agent but also as an anti tumour agent.

Antimicrobial and antifungal molecules also have non-therapeutic uses, for example in agriculture or in domestic or industrial situations as sterilising agents for materials susceptible to microbial contamination. Thus, in a further aspect, the present invention provides the use of the compounds of the invention as

antimicrobial or antifungal agents.

Alternatively viewed, the present invention provides an in vitro method of killing or inhibiting the growth of a microbial or fungal population which comprises contacting said population with a compound of the present invention.

Formulations comprising one or more compounds of the invention in admixture with a suitable diluent, carrier or excipient constitute a further aspect of the present invention. Such formulations may be for, inter alia, pharmaceutical (including veterinary) or agricultural purposes or for use as sterilising agents for materials susceptible to microbial contamination, e.g. in the food industry. Suitable diluents, excipients and carriers are known to the skilled man.

Methods of treating or preventing microbial (e.g. bacterial), viral or fungal infections or of treating tumours which comprises administration to a human or animal patient one or more of the compounds as defined herein constitute further aspects of the present invention. The patient will typically have been identified as in need of such treatment. Treatments may be prophylactic but generally will not be.

Preferred bacterial targets are Gram-positive bacteria and particularly preferred species are disclosed in the Examples. Preferred tumour targets are melanomas, adenocarcinomas and carcinomas with particularly preferred tumour targets being disclosed in the Examples.

Animals which may be treated include domestic animals, in particular cats and dogs and livestock animals such as pigs, cows, sheep or goats as well as horses. Marine animals and other marine organisms of economic importance may also be treated. The compositions according to the invention may be presented, for example, in a form suitable for oral, nasal, parenteral, intravenal, intratumoral, topical or rectal administration.

As used herein, the term "pharmaceutical" includes veterinary applications of the invention.

The active compounds defined herein may be presented in the conventional pharmacological forms of administration, such as tablets, coated tablets, nasal sprays, solutions, emulsions, liposomes, powders, capsules or sustained release forms.

Conventional pharmaceutical excipients as well as the usual methods of production may be employed for the preparation of these forms. Tablets may be produced, for example, by mixing the active ingredient or ingredients with known excipients, such as for example with diluents, such as calcium carbonate, calcium phosphate or lactose, disintegrants such as corn starch or alginic acid, binders such as starch or gelatin, lubricants such as magnesium stearate or talcum, and/or agents for obtaining sustained release, such as carboxypolymethylene,

carboxymethyl cellulose, cellulose acetate phthalate, or polyvinylacetate.

The tablets may if desired consist of several layers. Coated tablets may be produced by coating cores, obtained in a similar manner to the tablets, with agents commonly used for tablet coatings, for example, polyvinyl pyrrolidone or shellac, gum arabic, talcum, titanium dioxide or sugar. In order to obtain sustained release or to avoid incompatibilities, the core may consist of several layers too. The tablet coat may also consist of several layers in order to obtain sustained release, in which case the excipients mentioned above for tablets may be used.

Organ specific carrier systems may also be used.

Injection solutions may, for example, be produced in the conventional manner, such as by the addition of preservation agents, such as p

hydroxybenzoates, or stabilizers, such as EDTA. The solutions are then filled into injection vials or ampoules.

Nasal sprays administration may be formulated similarly in aqueous solution and packed into spray containers either with an aerosol propellant or provided with means for manual compression. Capsules containing one or several active ingredients may be produced, for example, by mixing the active ingredients with inert carriers, such as lactose or sorbitol, and filling the mixture into gelatin capsules. Suitable suppositories may, for example, be produced by mixing the active ingredient or active ingredient combinations with the conventional carriers envisaged for this purpose, such as natural fats or polyethyleneglycol or derivatives thereof.

Dosage units containing the active molecules preferably contain 0.1-10mg, for example 1-5mg of the active agent. The pharmaceutical compositions may additionally comprise further active ingredients, including other cytotoxic agents. Other active ingredients may include different types of antibiotics, cytokines e.g. IFN-γ, TNF, CSF and growth factors, immunomodulators, chemotherapeutics e.g. cisplatin or antibodies.

In employing such compositions systemically (intra-muscular, intravenous, intraperitoneal), the active molecule is generally present in an amount to achieve a serum level of the bioactive molecule of at least about 5 pg/ml. In general, the serum level need not exceed 500 pg/ml. A preferred serum level is about

20-100 g/ml. Such serum levels may be achieved by incorporating the bioactive molecule in a composition to be administered systemically at a dose of from 1 to about 10 mg/kg. In general, the molecule(s) need not be administered at a dose exceeding 100 mg/kg.

Methods of treating environmental or agricultural sites or products, as well as foodstuffs and sites of food production, or surfaces or tools, e.g. in a hospital environment, with one or more of the compounds of the invention to reduce the numbers of viable bacteria present or limit bacterial growth or reproduction constitute further aspects of the present invention.

The invention will now be further described with reference to the following non-limiting Examples.

Examples

Example 1 Extraction and purification of synoxazolidinone A (1), synoxazolidinone B (2) and synoxazolidinone C (3)

Specimens of S. pulmonaria were collected off the coast of Tromse in northern Norway and identified by Professor Bjarn Gulliksen (Norwegian College of Fisheries Science, University of Tromso, Tromso, Norway). Specimens (80 g, wet weight) of the organism were pooled, lyophilized and extracted with 10 volumes (v/w) of 60/40 MeCN/H₂0 containing 0.1% TFA, at 4 °C. The supernatant was removed after 24 h and the procedure repeated for another 24 h. The combined supernatants were then placed in a -20 °C freezer for 2 h, resulting in phase separation between a eCN rich organic phase and an aqueous phase. Activity was detected in the MeCN phase which was subsequently loaded on a semi-prep HPLC (column;

Waters, Sunfire C18, 250 x 10 mm). Compounds (3) (12 mg), (1) (40 mg) and (2) (20 mg) were eluted (in that order) with a linear gradient peaking at 70/30

MeCN/H₂0 (flow rate; 4.0 mlJmin). Example 2

Antibacterial assay

Test strains used were Staphylococcus aureus (ATCC 25923), Escherichia coli (ATCC 25922), Enterococcus faecalis (ATCC 29212), Psuedomonas aeruginosa (ATTC 27853) and Methicillin-resistant Staphylococcus aureus ( RSA) (ATCC

33591 ). Mueller-Hinton (MH) broth functioned both as a growth and assay medium for all strains except E. faecaelis, which was cultivated and assayed in Brain Heart Infusion (BHI) broth. Briefly, overnight cultures of bacteria were grown to mid- logarithmic growth phase and diluted in assay medium to give a final inoculum of approximately 1 ^χ 10⁵ CFU/ml. Test compounds (50 μΙ) and bacteria (50 μΙ) were added to a 96-well microtiter plate and incubated overnight at 37 °C. MIC was defined as the minimum concentration resulting in no visible bacterial growth. The absorbance at 600 nm was also measured as a control. The compounds were tested at concentrations from 5-60 pg/ml. Anticancer assay

Cell viability assays were performed on four human cell lines, MRC-5 (normal lung fibroblast), A-2058 (melanoma), MCF-7 (breast adenocarcinoma) and HT-29 (colon carcinoma). Briefly, exponentially growing cells were seeded into 96-well microtiter plates. After 24 hours of incubation at 37° C, the cells were exposed to the test compounds for 72 hours. Then the living cells were assayed by the addition of 0 μΙ of CellTiter96™ reagent (Promega). The plates were incubated for 1 hour for the color development and the absorbance at 485nm was measured in a DTX 880 multimode detector. The compounds were tested at concentrations ranging from 5- 60 pg/ml.

Antifungal assay

S. cerevisiae was a gift from Dr Arne Tronsmo (The Norwegian University of Life Sciences, As, Norway) and was cultivated on potato dextrose agar with 2% glucose at room temperature. Fungal spores were dissolved in potato dextrose broth

(Difco) and the cell concentration was determined and adjusted after counting in a Biirker chamber. An aliquot (50 μΙ) of fungal spores (final concentration 2 * 10⁵ spores/ml) was inoculated in 96-well nunc™ microtitre plates along with the test compounds (50 μΙ) which were dissolved in Milli-Q water. Synthetic cecropin B was used as a positive control (6.25 μ ). MIC was defined as the minimum

concentration resulting in no visible growth after inoculation for 48 h at room temperature. Growth was determined microscopically. The compounds were tested at concentrations ranging from 5-60 pg/ml. Results

Synoxazolidinone A (1 ) and B (2), displayed MIC-values against the Gram-positive bacteria Staphylococcus aureus and methicillin-resistant Staphylococcus aureus (MRSA) at a concentration of 10 μg/ml. Synoxazolidinone A (1 ) showed an MIC- value of 6.25 g/ml against the Gram-positive bacterium Corynebacterium giutamicum and an MIC of 12.5 μg ml against the fungi Saccharomyces cerevisiae. In addition, 2 exhibited antitumor activity against cell lines A2058 (melanoma), MCF-7 (breast-adenocarcinoma) and HT-29 (colon carcinoma) at an IC₅₀ value of 15 μg/ml. Synoxazolidinone C (3) generally displayed lower activities against MRSA (MIC of 30 μg/ml) and the tumour cell lines with IC₅₀ values above 30 pg/ml in all our tumour assays.

Claims

1. A compound of formula (I),

(I) wherein:

Ri denotes either hydrogen or chlorine;

Synoxazolidinone A

Synoxazolidinone B

Synoxazolidinone C

Synoxazolidinone D

; and stereoisomers thereof.

2. A compound as claimed in claim 1 for use in therapy.

A compound as claimed in claim 1 for use in treating or preventing a microbial or fungal infection.

A compound for use as claimed in claim 3 wherein said microbial infection is a bacterial or viral infection.

A compound for use as claimed in claim 4 wherein said bacterial infection is caused by a Gram-positive bacteria.

6. A compound as claimed in claim 1 for use in treating a tumour.

A compound for use as claimed in claim 6, wherein said tumour is a melanoma, adenocarcinoma or carcinoma.

Use of a compound as claimed in claim 1 in the manufacture of a medicament for treating or preventing a microbial or fungal infection or treating a tumour.

A method of treating or preventing a microbial or fungal infection, comprising administering a compound as claimed in claim 1 to a human or animal patient.

A method of treating a tumour comprising administering a compound as claimed in claim 1 to a human or animal patient.

11. A pharmaceutical or veterinary formulation comprising one or more

compounds as claimed in claim 1 in admixture with a suitable diluent, carrier or excipient.

12. An in vitro method of killing or inhibiting the growth of a microbial or fungal population which comprises contacting said population with a compound as claimed in claim 1.

13 Use of a compound as claimed in claim 1 for treating environmental or agricultural sites or products.

14. Use of a compound as claimed in claim 1 as a sterilising agent for materials susceptible to microbial contamination.

15. A method of preparing any one of Synoxazolidinone A to C as defined in claim 1 , which comprises taking a sample of Synoicum pulmonaria and extracting one or more of Synoxazolidinone A to C therefrom.

16. A method of preparing Synoxazolidinone D as defined in claim 1 , which

comprises the step of dechlorination of Synoxazolidinone B or a precursor thereof.