WO2012016266A2

WO2012016266A2 - Tetrahydro-anthracenone derivative

Info

Publication number: WO2012016266A2
Application number: PCT/AT2011/000334
Authority: WO
Inventors: Alexander Pretsch; Heinz Burgmann; Apostolos Georgopoulos; Peter Proksch; Abdessamad Debbab
Original assignee: Sealife Pharma Gmbh
Priority date: 2010-08-04
Filing date: 2011-08-03
Publication date: 2012-02-09
Also published as: AT509716B1; AT509716A4; WO2012016266A3

Abstract

The invention relates to the novel chemical compound 6,6'-dimethyl-2,2',3,3',6,6',9,9'-octahydroxy-5,5',6,6'-tetrahydro-1,1'-bianthracenyl-8,8'(7H,7'H)-dione ("talaromanin"), to the use thereof as an anti-infective agent and to a method for producing same (formula (A)).

Description

Tetrahydroanthracenon derivative

The present invention relates to a novel tetrahydroanthracenone derivative, a process for its preparation and its use as an anti-infective agent, above all against multiple drug-resistant pathogens.

STATE OF THE ART

The highly successful use of antimicrobial medicines in the late 1960s and early 1970s suggested that infectious diseases would no longer pose a threat. This should turn out to be a grave error, especially since the microbes represent a greater threat than ever before 40 years later, which is why there is an urgent need for new antimicrobial agents. Today, infectious diseases are the third leading cause of death in the US and the second most common in the world. For most of these cases, ineffective antimicrobial drugs are responsible, and the resistance of some bacteria and fungi to these drugs poses a serious problem in our society. According to US statistical data, the majority of hospital-acquired infections (the so-called). nosocomial infections) of a few bacterial species, namely Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumanii, Pseudomonas aeruginosa and Enterobacter sp., which are referred to in their initial letters as "ESKAPE" pathogens (Boucher et al., IDSA Report on Development Pipeline ", CID 2009: 48, Infectious Disease Society of America, January 1, 2009), but this term is also intended to express that these resistant pathogens escape the effects of antibacterial drugs, especially Antibiotic resistance at the molecular level nothing else means al s that a microorganism has acquired the ability to oppose the growth-inhibiting or bactericidal action of an antimicrobial substance. That is, the substance becomes clinically ineffective. For example, "methicillin-resistant Staphylococcus aureus" (also abbreviated as MRSA, although more commonly used for "multidrug-resistant Staphylococcus aureus") means that the use of β-lactams is ineffective in the therapy of S. aureus, while - For example, glycopeptides show most effect. It is therefore essential to test the effect of new anti-infective agents against multidrug-resistant strains, since efficacy against susceptible species or strains does not necessarily also confer efficacy against multi-drug resistant bacteria, as well as, for example, a Gram-positive bacterial agent does not necessarily act against Gram-negative bacteria or vice versa (see, inter alia, Boucher et al., supra).

In addition to bacteria and fungi, there are currently no effective strategies against respiratory viruses. In most cases, only the symptoms are cured without fighting the virus itself. A solution for the future could be above all drug combinations.

In the past, numerous substances have been found in endophytic fungi to control the multiplication of microorganisms. These substances show very good antibacterial, antifungal and antiviral activity in a number of cases and could be used for numerous applications (GA Strobel, Crit Rev. Biotechnol., 22, 315-333 (2002)). Due to the fact that this research was predominantly academic and did not directly target the development of new drugs, there are now very few drugs on the market. In particular, screening for resistant microbes has been neglected in the past, so that only a few substances have been tested against drug-resistant microbes. Even worse is the situation with respiratory viruses against which almost no substances have been screened. In earlier work (see Austrian Patent Applications AT AM 842/09, now Patent AT 507,298, and AT AM 843/09), the inventors of the present application have found the efficacy of both known and new age olanol and Alterporriol derivatives as anti-infective agents , These are optically active anthraquinone derivatives, which can be represented by the following general formulas and whose name was derived from the fungi from which the respective first representatives could be isolated, namely Alternaria solani, a mold which contains the so-called. Drought on the Potatoes, and Altemaria porri, causative agent of purple spot disease in onion vegetables. The first compound, already isolated in 1969, was designated as Altersolanol A:

Ageolanol A:

7-Methoxy-2-methyl- (1 /,, 2S, 3 /,, 4S) -1, 2,3,4-tetrahydro-1,2,3,4,5-penta-ydroxyant racene-9, 10 dion

Altersolanols generally correspond to one (2):

where R ¹ to R ^{4 may} each be H or OH, which in the case of OH in formula (2) in each case gives a chiral carbon atom which may be present both in the R and in the S configuration. The "dimeric" aged porcines are generally of the following formula (3):

where similarly diverse substitution and hydrogenation patterns on the aromatic rings are possible as for the "monomeric" age olanols. Due to limited Because of the freedom of rotation about the axis of the chemical bond between the anthraquinone nuclei, numerous Alterporriol derivatives exist in the form of two atropisomes. The inventors have found, in addition to the few earlier reports on possible anti-infective efficacy of such compounds, that it is impossible to predict whether or not a particular age olanol or altipyriol isomer or derivative will show anti-infective activity, let alone against which genera or even species of (micro) organisms (ie viruses, bacteria, fungi).

Of course, this applies equally to other, structurally similar mono- and bis-anthracenones. Known representatives, such as the atrovirin B (for the first time isolated from Cortinarius atrovirens), icterinidine and flavomannine, which are listed below as examples of possible substitution pattern variations, have mostly also been isolated from fungi and occasionally also find use in medicine. For example, flavomannins and methyl ethers have been found to be effective anti-tumor agents because of their antiproliferative activity.

Atrovirin B Icterinoidin

Flavomannin Such compounds have at least one chiral center in the molecule and are therefore present in the form of enantiomers, with atropisomerism generally also occurring. However, reports on the antimicrobial efficacy of such compounds are largely missing. Thus, only one study is known in which the two atropisomers of atrovirin B, isolated from Talaromyces wortmannii, were tested against microorganisms. As a result, both isomers showed no activity whatsoever against Bacillus subtilis, Saccharomyces cerevisiae, Cladospohum cucumerinum and Cladosporium herbarum (ID Intriani, "Biodiversity of fungi of marine origin and identification of their secondary metabolites", Diss., Heinrich Heine University Düsseldorf (2007)) ,

Against this background, the object of the invention was the identification, isolation and preparation of new substances for use as antimicrobial agents in pharmaceutical compositions, especially compounds which show activity against multiple drug resistant (MDR) pathogens.

In the course of their research, the inventors have now been able to obtain a substance with previously unpublished structure, i. identify a new chemical compound that displays good activity against microorganisms, especially against M DR pathogens.

DISCLOSURE OF THE INVENTION

Specifically, the inventors have isolated and characterized from a Talaromyces worfman / culture solution the new chemical compound shown in the formula below: e.ö'-dimethyl ^^ 'a.S'.e.e' ^ '-octahydroxy-ö.S'.e.e'-tetrahydro-l, 1'-bianthracenyl-8,8' (7W, 77- /) -dione. This compound is most likely in the form of several optical isomers because of the two asymmetric carbon atoms at positions 6/6 'and the limited freedom of rotation around the bond between the two anthracenone moieties, their separation and structure elucidation are still not satisfactory a successful degree. The new compound was called by the inventors because of their origin as "Talaromanin":

Talaromanin

With regard to the nomenclature, it should be noted that for such anthracene dimer structures instead of bianthracenyl the terms biathracene or bisanthracene are also known, the numbering of the carbon atoms sometimes beginning at the site of the keto group, which makes the new compound a 8,8'-bi (s) - anthracene. However, based on biphenyl and other biaryls such compounds are according to IUPAC recommendations as 1, 1'-Bianthracenyle to call.

Since the new compound has demonstrated excellent antimicrobial activity in screening experiments, a second aspect of the invention is the use of this novel compound as an anti-infective agent, preferably against Gram-positive and Gram-negative bacteria, in particular as anti-infective agents against multiple drug resistant, MDR) pathogens, propionibacteria and salmonella.

Specifically, the compound is preferably against multiple drug-resistant strains of methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli, Klebsiella sp., Pseudomonas aeruginosa, Enterococcus faecalis or faecium, Streptococcus pneumoniae, Staphylococcus epidermis, Acinetobacter baumannii, Enterobacter, Propionibacterium acnes, Clostridium difficile and Enterobacter sp. and against Salmonella enteritidis and Salmonella typhimurium, as evidenced by the later embodiments in detail. As noted in various tests, the new compound is virtually nontoxic. Therefore, it should also be taken in relatively high doses, such as several hundred milligrams per day, or administered topically, without causing unwanted side effects. The exact dosages, routes of administration, and formulations are the subject of current research by the inventors, but may be determined by the skilled artisan for the particular purpose without undue experimentation. Because of this and their excellent activity against Salmonella and Propionibakte- rien also a use of the new compound as a feed additive for farm animals, especially pigs, and as a component for medical or cosmetic preparations, especially against acne, for example as a cream, tincture, lotion or the like, taken into consideration. Other impurities of such feed additives and cosmetic preparations are not particularly limited as long as the effect of the novel compound of the present invention is not impaired, and may include, for example, other pharmaceutical or cosmetic agents, pharmaceutically acceptable carriers and excipients, vitamins, etc.

Finally, the present invention also relates to a process for the preparation of the novel compound which consists in fermenting a microorganism producing the compound or a precursor thereof under growth conditions, and the compound, optionally after destruction of the cells of the microorganism to increase the yield to win the culture. In such a fermentation process, a pure strain of Talaromyces wortmannii is preferably used as microorganism since the inventors were able to obtain the best yields with this species. Alternatively, however, any other microorganisms capable of producing the new compound or precursors thereof can also be used for the fermentation. If precursors are obtained by the fermentation, these can be converted into the desired new compound by any method well known to those skilled in the art of organic synthesis, wherein optionally also enzyme catalysis process steps can be used to ensure higher stereoselectivity. A possible synthetic route is to isolate a compound from the fermentation broth having a methyl group at C6 and one or more groups (eg, OH groups) at C5, C6 and C7, respectively, which can be conveniently eliminated to form a double bond between C5 and C6 or C6 and C7, and thus to obtain a prochiral center at C6. Subsequently, this double bond can be enzymatically (re) hydrated, which results in a suitable choice of the stereospecificity of the enzyme (eg hydratase, peroxygenase) the desired compound of the present invention. The same procedure can, of course, also be carried out on the corresponding atoms of the second anthracenone main body in the molecule.

Suitable enzymes for the synthesis steps for the reaction of precursors of the desired compound can in some cases also be isolated from that used for fermentation or else from another microorganism. The latter case may be useful if, for example, a microorganism produces the desired product, but e.g. in so small amounts or so heavily contaminated that it is more economical to obtain a precursor of the product by culturing another strain (or even another species or genus) and to convert it to the target compound by enzymatic synthesis.

The extraction of the compound is carried out according to the present invention, however, preferably by extraction and subsequent isolation from a crude extract of a culture, for example by fractional crystallization or chromatography, more preferably preparative HPLC, which has so far again yielded the best yields with the highest purity. Of course, especially in approaches on a larger scale, other isolation methods, such as direct fractional crystallization of the crude extract or absorption methods, conceivable. However, the person skilled in the art can determine the isolation method suitable for the particular culture (or synthesis or partial synthesis) step without undue experimentation. EXAMPLES

The invention will be described in more detail below with reference to specific embodiments, which are not intended to limit the scope of protection. The novel compound of the invention was obtained by cultivating Talaromyces worfmannii and subsequent extraction.

Isolation of the microorganism

The leaves of Aloe vera (true aloe) minced into small pieces were used to insulate the endophytic fungus. The surface of the pieces was sterilized twice with 70% ethanol for 2 minutes and then rinsed with sterile water to remove the alcohol. To distinguish any remaining epiphytic fungi from endophytic fungi, an imprint of the leaf surface on biomalt agar was made. Small tissue samples from the interior were aseptically sliced and pressed into petri dishes on malt agar medium containing antibiotic to suppress bacterial growth. The composition of the insulating medium was as follows: 15 g / l malt extract, 15 g / l agar and 0.2 g / l chloramphenicol in distilled water, pH 7.4-7.8). From the cultures, a pure mushroom strain was obtained by repeated inoculation on malt agar plates.

The fungal culture was determined according to a molecular biological protocol by DNA amplification and sequencing of the ITS region as Talaromyces sp. identified. To clarify the species, a sample was sent to the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ) in Braunschweig, Germany, where the fungal strain was identified as Talaromyces wortmannii. Sequence data were deposited with GenBank under accession number HM 807532.

cultivation

For cultivating Talaromyces wortmannii, two 1-liter Erlenmeyer flasks each containing 100 g of rice and 100 ml of distilled water were autoclaved to obtain a swollen solid rice medium. A small portion of the above isolation medium containing the pure fungal strain became sterile was applied to the solid rice medium and the fungus was cultured at room temperature (22 ° C) for 40 d.

Extraction and isolation

After 40 days, the culture was extracted twice with 300 ml of ethyl acetate (EtOAc). The EtOAc extract was evaporated to dryness and the residue was partitioned between n-hexane and 90% methanol. Evaporation of the methanol phase gave a residue, which was column chromatographed on a Diaion HP20 column with decreasing polarity eluants, namely water / methanol, acetone / methanol, and pure acetone, using thin layer chromatography (TLC) on silica gel F245 (Merck, Darmstadt, Germany ). The fractions containing the desired compound were pooled and subjected to semipreparation HPLC (Merck, Hitachi L-7100) using a Eurosphere 100-10 C18 column (300 x 8 mm, L x id) and a linear water-methanol gradient subjected to increasing polarity as eluent. In this way the novel compound of the invention was obtained in pure form.

analytics

This compound was obtained as a brown, amorphous powder with good solubility in methanol. The UV spectrum shows maxima at 234.7, 279.4 and 410.3 nm. In the above HPLC, a retention time of 23.6 minutes was found. From the ESI-MS spectrum in positive mode, the molecular ion at m / z [M + H] ⁺ 547.4 results as the base peak, while the measurement in the negative mode yields a molecular ion peak at m / z [MH] 545.4 shows, which follows a molecular weight of about 546 g / mol. From the value m / z [M + H] ⁺ 547.2074 as Pseudomolekülion peak in HRESI-MS results in the molecular formula to C30H27O10 (calculated: 547.53), ie a molecular formula of C30H26O10, resulting in a molecular weight of 546, 52 follows. The data of the ¹ H and ¹³ C NMR spectra are given in Table 1 below. The ¹ H-NMR spectrum shows signals of a tertiary methyl group at δ 1, 43 (3H, s, H-1 1), two methylene groups at δ 2.83 (2H, m, H-7) and δ 3.05 ( 2H, m, H-5) and two aromatic protons at δ 6.72 (1H, s, H-4) and δ 6.87 (1H, s, H-10). The ¹³ C spectrum comprises 15 carbon signals which are measured by DEPT measurements of a methyl group, two methylene groups, two methine groups and ten quaternary carbon atoms can be assigned. The HMBC data confirm this and show proton-carbon distance correlations within the compound. The aromatic proton at δ 6.72 (1 H, s, H-4) correlates with three oxygen-bearing aromatic carbon atoms at δ 159.0, 161, 5, and 167.8, corresponding to C2, C3, and C9. Furthermore, H-4 correlates via ω-correlations also with the carbon atoms C10 and C10a. In addition, the COZY spectrum shows far-distance correlations between protons H-5 in an aliphatic ring and proton H-10 in an aromatic ring, which was confirmed by ROESY. Confirmation of the spin system in the aliphatic ring was made by the observed COZY crosspeaks and HMBC measurement.

Table 1: H-NMR and ¹³ C-NMR data

* ¹³ C data was taken from the HMBC spectrum.

The elucidation of the stereochemistry at the chiral centers and the R _a and S _a and plus (P) and minus (M) configurations of the most probable atropisomers is expected shortly.

Activity determination u ng

The activity of the new compound was tested in two different screening systems. The antibacterial activity was examined in an MIC test. MIC stands for the "minimal inhibitory concentration" (MIC for "minimal inhibitory concentration ") and refers to the lowest concentration of a substance that can not be observed by the naked eye to increase the number of microorganisms.The MIC is determined by a so-called titer method, in which the substance is diluted out and then the pathogen is added determines the concentration of an antibiotic that just barely inhibits the growth of a bacterial strain.

MIC is expressed in micrograms per milliliter (pg / ml) and dilutions are usually in log2 increments. Here, a starting concentration of 250 g / ml was in each case doubled, which consequently resulted in test concentrations of 250 μg / ml, 125 μg / ml, 62.5 μg / ml, 31, 25 μg / ml, 15.6 μg / ml, 7.8 pg / ml and so on. Lower values therefore reflect better activity as an anti-infective agent.

The tests were performed according to the standards required by the EUCAST (European Committee for Antimicrobial Susceptibility Testing). Table 2, overleaf, gives the test results regarding the anti-infective effect against some multidrug-resistant bacteria for the new compound as well as some known reference substances. All data are averages of multiple determinations. It is clearly evident that the novel compound of the invention exhibits - sometimes excellent - activity against all bacterial species tested, having a very broad spectrum of activity in this series of tests, at least as effective as or even better than the comparisons and thus against all bacteria tested a highly suitable candidate for active ingredients in broad spectrum antibiotics, as a feed additive, especially against Salmonella, and as an active ingredient in medical or cosmetic preparations against acne. Especially the latter application is recommended, especially since the new use in the tests against Propionibacterium acnes has shown the best activity, which was even higher than those of the known antibacterial reference substances Altenusin and Macrosporin. Table 2: MIC values for bacteria

EC E. coli

KL Klebsiella sp.

PS Pseudomonas aeruginosa

EK Enterococcus faecalis or faecium

MRSA methicillin-resistant Staphylococcus aureus

STR Streptococcus pneumoniae

SE Staphylococcus epidermis

AB Acinetobacter baumannii

EB Enterobacter sp.

PB Propionibacterium acnes

CL Clostridium difficile

SA Salmonella enteritidis

ST Salmonella typhimurium

Thus, it is clearly demonstrated that according to the present invention, a new compound is provided which shows very good activity against multi-drug-resistant bacterial pathogens as well as Salmonella and Propionibakterien and therefore broad application as anti-infective agent, as well as feed additive or as an antibacterial agent in medical or cosmetic preparations, eg against acne, can find.

Claims

1. e.e'-dimethyl ^^ 'S.S'.e.e'.Q.g'-octahydroxy-ö.S'.e.e'-tetrahydro-l, 1' -bianthracenyl 8,8 '(7H, 7'H) -dion ("Talaromanin"):

Talaromanin

2. Use of the compound according to claim 1 as Antiinfektivum.

3. Use according to claim 2, characterized in that the compound is used against Gram-positive and / or Gram-negative bacteria.

4. Use according to claim 3, characterized in that the compound is used as an anti-infective agent against multiple drug resistant ("MDR") pathogens.

5. Use according to claim 4, characterized in that the compound against multidrug-resistant strains selected from strains of Staphylococcus aureus (MRSA), Escherichia coli, Klebsiella sp., Pseudomonas aeruginosa, Enterococcus faecalis or faecium, Streptococcus pneumoniae, Staphylococcus epidermis, Acinetobacter baumannii, Clostridium difficile and Enterobacter sp., Is used.

Use according to claim 3, characterized in that the compound is used as an active ingredient against Salmonella, e.g. Salmonella enteritidis and Salmonella typhimurium.

7. Use according to claim 6, characterized in that the compound is used as a feed additive.

Use according to claim 3, characterized in that the compound is resistant to priopionibacteria, e.g. Propionibacterium acnes, is used.

9. Use according to claim 8, characterized in that the compound is used as an active ingredient in a medical or cosmetic preparation for acne.

10. A process for the preparation of the compound according to claim 1, characterized in that a compound producing microorganism, in particular a strain of Talaromyces wortmannii, is fermented under growth conditions and the compound, optionally after destruction of the cells of the microorganism, is recovered from the culture.

1 1. A method according to claim 10, characterized in that the compound is obtained by extraction and subsequent isolation from the crude extract.