RU2795449C1 - New depsipeptide compounds with antibacterial activity - Google Patents

Abstract

FIELD: pharmaceuticals.

SUBSTANCE: invention relates to new antibacterial depsipeptide compounds and can be used to create agents for the treatment of microbial infections in humans and animals. Also proposed is the use of the said compound for the preparation of an agent having antibacterial activity.

EFFECT: claimed invention expands the range of agents for combating bacterial infection, which can be used in the creation of antibacterial agents.

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Description

The present invention relates to new antibacterial depsipeptide compounds and can be used to create agents for the treatment of microbial infections in humans and animals.

State of the art

Humans and animals are often exposed to microbial agents, including Streptococcus spp.; Arthrobacter spp.; Mycobacterium spp., Micrococcus spp.

The main groups of antibiotics used in the treatment of streptococcal infections (with Streptococcus spp. bacteria) are penicillins, cephalosporins, macrolides and glycosamides [https://medvestnik.ru/content/articles/Opublikovan-Kokreinovskii-obzor-sravneniya-antibiotikov-pri- streptokokkovoi-infekcii.html]. Penicillin may be the drug of choice for both mild and severe disease. For patients allergic to penicillin, erythromycin can be used.

The most widely used chemotherapeutic agents used against Mycobacterium spp. are the antibiotics clarithromycin, azithromycin, rifampicin, rifabutin, ethambutol, streptomycin and amikacin.

One of the groups of compounds active against these microorganisms are depsipeptides (DP) - peptides in which one or more amide groups are replaced by ester groups. DPs are nonribosomal peptides that are synthesized by enzymatic complexes of peptide synthetases consisting of repeating domains with a modular organization for activation and binding of amino acids. The main producers of natural DPs are soil bacteria, marine organisms, and fungi.

These compounds may have antibacterial, immunomodulatory, antitumor activity, and are also effective against various microorganisms and helminths.

Thus, compounds related to depsipeptides with anthelmintic activity are known from the prior art (patent RU 2715556 C2 publ. 02.03.2020). The use of depsipeptides in the treatment of fungal infections is known (patent RU 2745671 C2, publ. 30.03.2021).

Cyclic depsipeptide - daptomycin - having passed all the stages of study, registered as an antibacterial drug [Tedesco KL, Rybak MJ. Daptomycin. pharmaceutical therapy. 2004 Jan;24(l):41-57. Review.]. It was isolated from the culture liquid of the bacterium Streptomyces roseosporus as the most active component of a mixture of compounds with a 13-membered amino acid cycle. Daptomycin showed high activity in vitro against gram-positive bacteria - enterococci, including vancomycin-resistant (VRE), staphylococci, including methicillin-resistant (MRSA), streptococci and corynebacteria. Daptomycin is currently actively used as a drug for the treatment of various infections [Patel S, Saw S. Daptomycin. 2020 Apr 13. StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing; 2020 Jan-. Available from http://www.ncbi.nlm.nih.gov/books/NBK470407]. The formulation of the daptomycin injection formulation has been improved in order to improve its stability and dissolution rate while maintaining the same efficacy [Frankenfeld C, Mittal S, Melendez Y, Mendez-Vigo L, Lamp KC, Keller KN, Bertolami SR. Daptomycin: a comparison of two intravenous formulations. Drug Des Devel Ther. 2018 Jun 29;12:1953-1958].

An invention is known relating to the improvement of the purification method of daptomycin (patent RU 2348647 C2, publ. 10.03.2009). An improved method of using lipopeptide antibiotics, in particular daptomycin, bacteria, including strains resistant to other antibiotics, is presented in patent RU2363489C9, publ. 2010-03-20).

Other depsipeptide compounds are also known that have an antibacterial effect, however, some of them do not meet the requirements for obtaining drugs based on them. These are compounds such as enniatines [Sy-Cordero AA, Reagse CJ, Oberlies NH. Revisiting the enniatins: a review of their isolation, biosynthesis, structure determination and biological activities. J Antibiot (Tokyo). 2012 Nov;65(11):541-9.], beauvericin [Wang Q, Xu L. Beauvericin, a bioactive compound produced by fungi: a short review. Molecules. 2012 Feb 24;17(3):2367-77], ramoplanin A2 [Farver DK, Hedge DD, Lee SC. Ramoplanin: a lipoglycodepsipeptide antibiotic. Ann Pharmacother. 2005 May;39(5):863-8.], fusaricidins [Reimann M, Sandjo LP, Antelo L, Thines E, Siepe I, Opatz T. A new member of the fusaricidin family - structure elucidation and synthesis of fusaricidin E. Beilstein J Org Chem. 2017 Jul 20;13:1430-1438], acyl depsipeptides [Li Y, Lavey NP, Coker JA, Knobbe JE, Truong DC, Yu H, Lin YS, Nimmo SL, Duerfeldt AS. Consequences of Depsipeptide Substitution on the ClpP Activation Activity of Antibacterial Acyldepsipeptides. ACS Med Chem Lett. 2017 Oct 19;8(11):1171-1176. doi:10.1021/acsmedchemlett.7b00320.].

The teixobactin compound, discovered in 2015 as a metabolite of the soil P-proteobacterium Eleftheria terrae [Ling, L.L., Schneider, T., Peoples, A.J., Spoering, A.L., Engels, I., Conlon, B.P., Mueller, A., Schaberle, T.F. , Hughes, D.E., Epstein, S., Jones, M., 2015. A new antibiotic kills pathogens without detectable resistance. Nature 517 (7535), 455-459], and subsequently synthesized [Karas JA, Chen F, Schneider-Futschik EK, Kang Z, Hussein M, Swarbrick J, Hoyer D, Giltrap AM, Payne RJ, Li J, Velkov T. Synthesis and structure-activity relationships of teixobactin. Ann N Y Acad Sci. 2020 Jan; 1459(1): 86-105. doi: 10.1111/nyas.14282], is a promising candidate for drug development [Teixobactin and preparation method thereof (patent CN107266531A)].

The closest analogue of the invention is surotomycin, an antibacterial agent, a semi-synthetic analogue of daptomycin (patent RU 2666605 C2, publ 06.11.2014), which is a related compound.

The problem solved by the claimed invention is the presentation of new depsipeptide compounds with antibacterial activity.

Disclosure of the essence of the invention

The present invention provides a compound of the general structural formula:

where R represents the following radicals:

или

or

In another aspect of our invention, the use of the claimed compound for the preparation of an antibacterial agent is provided.

The technical result of the claimed invention is as follows.

Despite the successes associated with the use of existing antibacterial agents, there continues to be a need for new antibiotics. For many pathogens, repeated exposure to applied antibiotics results in selection of strain variants that are resistant to a wide range of antibiotics. The loss of antibiotic potency and efficacy determined by the emergence of resistance mechanisms leads to antibiotic ineffectiveness and therefore can lead to some life-threatening infections that are practically untreatable. As new antibiotics enter the market and begin to be used, microorganisms begin to evolve towards the development of resistance to these new drugs, effectively creating a need for new antibacterial agents to combat these emerging strains. The claimed invention expands the range of agents for combating bacterial infection, which can be used in the creation of antibacterial agents.

Brief description of explanatory materials

Rice. 1. Structure (I), along with NMR data. Proton, carbon and nitrogen chemical shifts at 30C in CDCl ₃ are shown in red blue and green, respectively.

Rice. 2. Different fragments of the structure of components (I) and (II) and NMR data for these fragments.

Rice. 3a - MS1 spectra of the molecular ion of compound (I) [M+H]+ and [M+Na]+.

Rice. 3b - MS2 spectrum of the molecular ion [M+H]+ at 1190.4234,

Rice. 3c - fragmentation scheme of the parent ion.

Rice. 4a - MS2 spectrum of the molecular ion of compound (II) [M+H]+ at 1154.4461,

Rice. 4b is a diagram of the fragmentation of the parent ion.

Table 1 - Minimum inhibitory concentrations (MICs) of compounds I and II.

Implementation of the invention

Example 1. Characteristics of the producer strain, production technology and determination of the authenticity of the obtained compounds.

The claimed substances of structural formulas (I) and (II) are obtained from the producer strain Streptomyces sp. KMM 9044 from the collection of marine microorganisms of the TIBOC FEB RAS, deposit certificate No. 16146-203 dated May 19, 2022, which belongs to the genus Streptomyces sp.

The Streptomyces sp.KMM 9044 strain was isolated from a soil sample by direct inoculation of a soil suspension on an SWM agar medium with the addition of sea water of the following composition (g/l): peptone - 5.0; yeast extract - 2.5; glucose - 1.0; K ₂ NPO ₄ - 0.2; _MgSO4 - 0.05; 500 ml distilled water, 500 ml sea water.

Streptomyces sp. KMM 9044 was grown on Petri dishes for 7-8 days at a temperature of 28°C, maintained on SY agar medium, stored for no more than three months at a temperature of about 4°C in a refrigerator. Long-term storage of the culture was carried out in liquid glycerol at -70°C.

On all the above media, the color of the colonies of the studied strain KMM 9044 is colorless or slightly yellowish. Colonies in the early stages of growth are opaque, convex, dense, smooth or slightly folded, usually grown into the agar. White aerial mycelium is formed on the Sucrose yeast medium within 5-7 days. Vegetative and aerial hyphae are weakly or intensively fragmented with age into rod-shaped immovable elements. The morphology of the studied strains is characteristic of the described species of streptomycetes.

Based on the analysis of the nucleotide sequences of the 16S rRNA gene fragments, the strain belongs to the genus Streptomyces (family Streptomycetaceae, class Actinobacteria) and is closest to the known species S. pseudovenezuelae DSM 40212 ^T (98.97% similarity), S. variegatus NRRL B-16380 ^T (98.96% ), S. cahuitamycinicus 13K301 ^T (98.96%), S. qaidamensis S10 ^T (98.83%), S cacaoi subsp.asoensis NRRL В-16592 ^T (98.76%), S. phaeoluteigriseus DSM 41896 ^T (98.76%), S. adustus WH-9 ^T (98.69%) and S. humidus NBRC 12877 ^T (98.69%).

The results of the analysis of the genomic assembly of Streptomyces sp.KMM 9044 showed that the strain has a genome size of 7.15 million bp. and HC-composition of DNA-71.1 mol%.

The technology for obtaining streptocinnamides is based on the cultivation of the producer strain KMM 9044 and the extraction of the substance from the culture liquid (CL) of the indicated strain by the method of solid-phase extraction and liquid chromatography. It consists of the following steps.

1. Preparation of seed. Streptomyces sp. KMM 9044 was grown on Petri dishes for 7 days at a temperature of 28 C on a Sucrose yeast (SY) _medium with the following composition (g/L): sucrose, 40.0; yeast extract, 5.0; K ₂ HPO ₄ , 1.5 _{; O , 3.1 ; NaCl , 2.0 ; _} - 15.0, distilled water - up to 1 l, pH 6.8.

2. Growing seed material of the 1st generation (first inoculum). The culture was transferred from the surface of the agar medium to a 750 ml Erlenmeyer flask with 50 ml of SY nutrient medium. Cultivation was carried out at 28°C for 5 days on a thermostatically controlled shaker Innova 40 (New Brunswick Scientific) at 150 rpm.

3. Growing inoculum of the 2nd generation (second inoculum). The seed culture of the second generation was grown in the SY medium under similar conditions for 5 days, using the first inoculum for seeding in the amount of 5% vol.

4. Cultivation in a fermenter. Cultivation was carried out in a 2 L BIOSTAT A plus fermenter (Sartorius BBI Systems). Inoculation material in a volume of 10% (150 ml of culture per 1.5 L of medium) and 150 ml of sterile glucose solution at a concentration of 20 g/L (final concentration 2 g/L) were added to a fermenter containing 1350 ml of sterile SY medium. Cultivation was carried out at a temperature of 28°C. The concentration of dissolved oxygen (pO2) was maintained at 90%, the stirring speed was 300 rpm, the amount of sterile air supplied to the apparatus was 1.5 L/min, and the pH of the medium was maintained in the range of 6.8 by titration with 25% ammonia solution. Silicone defoamer (0.01%) was added at the end of the first day of cultivation. Fermentation time 43 hours.

5. Separation of QOL from biomass. The grown culture was centrifuged at 10,000 rpm (Beckman J2-21 centrifuge), after which the supernatant was filtered. Whatman glass microfiber GF/A and MF-Millipore MCE membrane glass fiber filters with a pore diameter of 0.45 µm were used. The resulting volume of QOL was 1.2 liters.

6. Preparation of the sorption cartridge. The cartridge was filled with polymeric sorbent LPS-500-H, fraction 70 µm (Technosorbent company) in bulk. A 45 ml cartridge was used; the sorbent weight was about 7.5 g.

7. Application of CL on a polymeric sorbent. The cartridge was washed with 150 ml of acetonitrile (ACN) and activated with 150 ml of distilled water using a Masterflex Easy-Load 11 peristaltic pump at a flow rate of 15 ml/min. QOL was applied at the same speed; when finished, the cartridge was washed with 150 ml of water. The cartridge can be reused, but not more than 5 times.

8. Elution from the cartridge using a flash chromatograph. The cartridge was attached to the eluent line of a PuriFlash 5.250 chromatograph and eluted at a rate of 15 ml/min with a mixture of solvents water - ACN (HPLC grade). The content of ACN in the eluent was set according to the following program: initial composition - 5%; 1 min - 45%, 10 min - 45%, 11 min - 100%, 22 min - 100%. The composition of the eluate was monitored using a UV detector at a wavelength of 290 nm. The UV profiles had two peaks: a major peak at 5–6 min, corresponding to the sum of non-target compounds, and a minor peak at 13–15 min, containing the target compounds Scm A and Scm B, whose UV spectrum has a maximum at 290 nm. This fraction of about 30 ml was collected using a fraction collector.

9. Purification of substance. Scm A and Scm B were purified by HPLC (Shimazu Necker chromatograph). The entire volume of the fraction after the cartridge was placed in a flask of a rotary evaporator (Buchi Rotavapor), concentrated by about 5 times. A solution of 0.1% acetic acid was added to the solution - half of the volume obtained (in the specific case, 30 ml was concentrated to 6 ml and 3 ml of acetic acid solution was added). In the presence of a precipitate, the solution is centrifuged at a speed of 10000 rpm, the supernatant is transferred into test tubes. Separation is carried out on a semi-preparative size column on a C18 sorbent, fraction 5 or 10 μm, with the same eluent composition, column thermostat temperature and UV detector wavelength, as in step 5. Eluent flow rates and sample volume eluted in one separation cycle are selected depending on column size. On a 9.2×150 mm column, separations were performed in 1.5 ml portions at a flow rate of 3 ml/min. The duration of each separation is 20 min. To clean the entire volume of the fraction, it is necessary to repeat the operation several times, in this case 6 times.

10. Obtaining a dry product from solutions of fractions. Fraction solutions are combined, poured into a flask and dried on a rotary evaporator with minimal heating or freeze-drying with a MiVac centrifuge. The total yield of the substance is 3-5 mg per 1 liter of QOL. The amount of the extracted product and the ratio of the Scm A and Scm B components in the samples may vary due to the influence of various factors on the production of the strain.

All stages of the technology can be scaled using the appropriate equipment and on the general principles used in dynamic sorption processes.

As a result, compounds Scm A(Z) (structural formula I) and Scm B(Z) (structural formula II) were obtained. Structural formulas were established from the results of NMR spectroscopy (Figs. 1, 2) and high resolution tandem mass spectrometry (Figs. 3, 4).

An analysis of the NMR spectra made it possible to directly elucidate the structure of a cyclic depsipeptide consisting of seven amino acid residues: threonine, two leucines, proline, three nonproteinogenic amino acids, and glyceric acid. O-acylated threonine is at the N-terminus of the peptide moiety, followed by leucine, then O-acetylated 4-acetoxy-5-methylproline, then leucine, N-hydroxyvaline, and finally proline at the C-terminus. Threonine is N-acylated with 2-methoxy-6,8-dichloro-7-hydroxycinnamic acid in the Z conformation and its side chain forms an ester bond with 3-hydroxy-4-chlorovaline. Finally, the cycle closes through glyceric acid, connecting proline carbonyl and chlorohydroxyvaline amide. It is important to note that the amide is modified with a hydroxyl group, which is directly observed in 1H NMR at 8.19 ppm. Two amino acids in the structure, 3-hydroxy-4-chlorovaline and trans-4-acetoxy-5-methyl-L-proline, are rare moieties in depsipeptides. The new antibiotic was named streptocinnamide A(Z).

Example 2

Confirmation of the antibacterial activity of the claimed compounds.

The strains of Micrococcus sp. KMM 1467 from the collection of marine microorganisms of the TIBOKh named after M. G.B. Elyakova, Arthrobacter sp.21022 (ATCC) and Mycobacterium smegmatis ATCC 700084 from the collection of Lytech Co. Ltd.

Minimal inhibitory concentrations (MICs) of bacteria were determined by serial two-fold dilutions (0.1-10000 ng/ml) in 2YT broth (BD, USA). The MIC was defined as the lowest concentration that interfered with the growth of a test organism in a 96-well plate after 16 hours of culture. For Mycobacterium smegmatis, MICs were determined after 60 hours of cultivation. The minimum bactericidal concentration (MBC) was defined as the lowest concentration preventing re-cultivation of the test organism after 24 h of incubation. Prolonged cultivation results in Scm degradation and inflated MIC values. The temporal course of bacterial growth was monitored at 800 nm using a Varioskan Flash multimode reader (Thermo Fisher Scientific, Waltham, MA, USA).

Both variants of the structures have antimicrobial activity, and for the reporter strain Micrococcus sp.KMM 1467, the values of the minimum inhibitory concentrations (MIC) were at the level of ng/ml: 6 ng/ml for Scm A and 2 ng/ml for Scm B, i.e. the values for the epoxy derivative are several times lower than those for the chlorine derivative.

Against other pathogens, the activity of the compounds was significantly lower. MICs were measured for Scm A and the values shown in Table 1 were obtained. 1.

Claims (5)

1. Compound of the general structural formula:

where R represents the following radicals:

or

2. The use of a compound according to claim 1 for the preparation of an antibacterial agent.

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