CN105085613B

CN105085613B - Novel thiostrepton analogue and preparation method and application thereof

Info

Publication number: CN105085613B
Application number: CN201410216093.1A
Authority: CN
Inventors: 王守锋; 郑庆飞; 刘�文
Original assignee: Shanghai Institute of Organic Chemistry of CAS
Current assignee: Shanghai Institute of Organic Chemistry of CAS
Priority date: 2014-05-20
Filing date: 2014-05-20
Publication date: 2021-06-08
Anticipated expiration: 2034-05-20
Also published as: CN105085613A

Abstract

The invention provides a novel thiostrepton analogue and a preparation method and application thereof. In particular, the present invention provides methyl substitution of the quinacrid moiety of the thiostrepton side chain to obtain thiostrepton analogs. Compared with thiostrepton, the obtained thiostrepton analogue has obviously improved antibacterial activity and water solubility.

Description

Novel thiostrepton analogue and preparation method and application thereof

Technical Field

The invention belongs to the field of medicines and the field of biotechnology engineering, and particularly relates to a novel thiostrepton analogue and a preparation method and application thereof.

Background

Thiopeptoids are a class of natural products of macrocyclic polypeptides that are rich in sulfur elements, with highly modified structures, and about 100 natural members have been discovered to date [ chem.rev.,2005,105,685; plos One2012,7, e45878 ]. Such antibiotics selectively act on bacterial ribosomes, thereby inhibiting the synthesis of their proteins [ bioorg.med.chem.lett.,2004,14, 5573; nat. prod.,2009,72, 841; j.am.chem.soc.,2008,130,12102 ]; in addition, their strong antimalarial, antitumor and immunosuppressive activities have been reported sequentially [ angelw. chem. int. ed.,2012,51,12414 ]. How to overcome the defects of physicochemical properties (low bioavailability caused by poor water solubility and the like) by structural derivation so as to meet the requirements of clinical application becomes one of the key points of the current research of the thiopeptide antibiotics.

Thiostrepton (Thiostrepton), one of the members discovered earlier in the family of thiopeptide antibiotics, has long been widely used industrially because of its excellent antibacterial activity. However, due to its complex structure, its molecular structure was not finally determined by means of X-ray diffraction techniques until 1989. Structurally, thiostrepton has a special quinaldine side ring in addition to the characteristic macrocyclic skeleton of thiopeptoid antibiotics, so that the synthetic route is very complicated. Even today, where organic synthetic chemistry is highly developed, obtaining products by chemical total synthesis is a formidable challenge. The chemical total synthesis of thiostrepton was first reported until 2004 [ angelw.chem.int.ed.2004, 43,5087; angew. chem. int. ed.2004,43,5092], but the whole synthesis process is cumbersome to operate and has low yield.

In order to improve the antibacterial activity and other characteristics of thiostrepton, the development of various derivatives or analogs of thiostrepton has been attempted in the art, but the thiostrepton analogs with higher activity and the like have not been successfully developed so far. Therefore, there is an urgent need in the art to develop various novel thiostrepton analogs.

Disclosure of Invention

The object of the present invention is to provide a novel thiostrepton analogue with higher activity.

Another object of the present invention is to provide a preparation method and use of the thiostrepton analog.

Another object of the present invention is to provide a mutant synthetic element for preparing thiostrepton analogs, and a preparation method and a use thereof.

In a first aspect of the present invention, there is provided a thiostrepton analog, or a pharmaceutically acceptable salt thereof, characterized in that the thiostrepton analog has a structure represented by formula I:

in the formula, R₁Is unsubstituted or substituted C1-C6 alkyl, wherein said substitution means having a substituent selected from the group consisting of: halogen, NH₂、OH、NO₂Or CF₃；R₂Is H or F; r₃Is H or F. Preferably, R is an unsubstituted C1-C6 alkyl group. The C1-C6 alkyl refers to C1, C2, C3, C4, C5 and C6 alkyl.

In another preferred embodiment, R is₁Is unsubstituted or substituted methyl, ethyl or propyl, wherein said substitution means having a substituent selected from the group consisting of: halogen, NH₂、OH、NO₂Or CF₃. Preferably, said R is₁Is unsubstituted methyl, ethyl or propyl.

In another preferred embodiment, R is₁Is unsubstituted or substituted methyl. Preferably, said R is₁Is unsubstituted methyl.

In another preferred embodiment, the thiostrepton analog has a structure represented by formula Ia:

in a second aspect of the present invention, there is provided a pharmaceutical composition comprising a thiostrepton analog of the first aspect of the present invention or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

In a third aspect of the invention, there is provided the use of a thiostrepton analogue of the first aspect of the invention, or a pharmaceutically acceptable salt thereof, to prepare a medicament for treating or preventing a disease associated with a subject in need thereof

(a) For the preparation of antimicrobial compositions;

(b) a composition for inhibiting the growth of microorganisms;

(c) for the preparation of a composition for the treatment of microbial or bacterial infections; and/or

(d) Can be used as feed additive.

In a fourth aspect of the invention, there is provided a method of non-therapeutically inhibiting the growth of or killing microorganisms in vitro comprising the steps of: use of a thiostrepton analogue according to the first aspect of the invention, or a pharmaceutically acceptable salt thereof, at a site in need of treatment.

In a fifth aspect of the present invention, there is provided a method of preparing a pharmaceutical composition, comprising the steps of: combining a thiostrepton analog of the first aspect of the invention, or a pharmaceutically acceptable salt thereof, with a pharmaceutically acceptable carrier to form a pharmaceutical composition.

In a sixth aspect of the invention, there is provided a method of treating a bacterial infection in an animal comprising the steps of: administering to an animal in need of treatment a thiostrepton analog of the first aspect of the invention or a pharmaceutically acceptable salt thereof or a pharmaceutical composition of the second aspect of the invention.

In another preferred embodiment, the animal comprises: mammals, livestock (e.g., cattle, pigs, sheep, etc.), poultry (e.g., chickens, ducks, geese, etc.), aquaculture animals.

In a seventh aspect of the present invention, there is provided a method of preparing a feed composition, characterised by the steps of: the thiostrepton analogue or the pharmaceutically acceptable salt thereof in the first aspect of the invention is used as a feed additive and feed raw materials to be mixed, so as to form a feed composition containing the thiostrepton analogue or the salt thereof.

In another preferred embodiment, the feed composition is used in livestock (e.g., cattle, pigs, sheep, etc.), poultry (e.g., chickens, ducks, geese, etc.), or aquaculture.

In an eighth aspect of the invention, there is provided a mutant strain of Streptomyces laurentii (Streptomyces laurentii) which is useful for producing a thiostrepton analog according to the first aspect of the invention, wherein the thioadenosylmethionine transferase (TsrT) is inactivated or knocked out in said strain.

In a ninth aspect of the invention, there is provided a process for the preparation of a thiostrepton analogue according to the first aspect of the invention, comprising the steps of:

(a) feeding Streptomyces laurentii (Streptomyces laurentii) according to claim 7 with a mutant synthon

Mutating the strain, thereby producing the thiostrepton analog of claim 1; and

(b) isolating said thiostrepton analog from the fermentation product.

In another preferred embodiment, the mutant synthetic element has a structure represented by formula II:

in the formula R₁、R₂、R₃As described above; r₄Is unsubstituted or substituted C1-C5 alkyl or C7-C8 aryl. In another preferred embodiment, R is₄Selected from the group consisting of methyl, ethyl, n-propyl, benzyl and n-butyl.

In another preferred embodiment, the mutant synthetic element has a structure represented by formula IIa:

in another preferred embodiment, the mutant synthetic element has the structure of formula IIb:

in a tenth aspect of the present invention, there is provided a method for preparing a mutant synthetic element having a formula II, comprising the steps of:

(a) a compound of the formula III with an alcohol R₁OH is reacted to generate a compound shown in a formula IV;

(b) a compound of formula IV and RCH₂CHO reacts to generate a mutant synthetic element shown in a formula II;

in the formula, R₁、R₂、R₃、R₄As described above.

In another preferred embodiment, R is₁CH₂CHO is propionaldehyde.

In another preferred embodiment, the alcohol R in step (a)₄OH is optionally selected from the group consisting of: methanol, ethanol, n-propanol or n-butanol.

In another preferred embodiment, the reaction of step (a) is carried out under the action of thionyl chloride.

In another preferred example, the reaction in the step (b) is carried out under the action of trifluoroacetic acid, ferric sulfate and hydrogen peroxide.

In an eleventh aspect of the invention, there is provided a compound having a structure represented by formula II,

in the formula, R₁、R₂、R₃、R₄As described above.

In another preferred embodiment, the compounds are useful as intermediates in the preparation of compounds of formula I.

In a twelfth aspect of the invention, there is provided a use of a mutant synthetic element of formula II for the preparation of a thiostrepton analogue of the first aspect of the invention, or a pharmaceutically acceptable salt thereof.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

The following drawings are included to illustrate specific embodiments of the invention and are not intended to limit the scope of the invention.

FIG. 1 shows a chemical synthesis route for the mutant synthetic element 4-propionyl-quinoline-2-carboxylic acid methyl ester.

FIG. 2 is a diagram of quinoline-2-carboxylic acid methyl ester¹H NMR(500MHz,CDCl₃)。

FIG. 3 is a drawing showing a process for producing quinoline-2-carboxylic acid methyl ester¹³C NMR(125MHz,CDCl₃)。

FIG. 4 is a scheme showing the preparation of methyl 4-propionyl-quinoline-2-carboxylate¹H NMR(500MHz,CDCl₃)。

FIG. 5 is a scheme showing the preparation of methyl 4-propionyl-quinoline-2-carboxylate¹³C NMR(125MHz,CDCl₃)。

FIG. 6 is a drawing showing the preparation of 12' -methyl-thiostrepton¹H NMR(400MHz,CDCl₃：CD₃OD＝4:1)。

FIG. 7 is a drawing showing the preparation of 12' -methyl-thiostrepton¹³C NMR(100MHz,CDCl₃：CD₃OD＝4:1)。

FIG. 8 shows gHMBC (400MHz, CDCl) of 12' -methyl-thiostrepton₃：CD₃OD＝4:1)。

FIG. 9 is gCOSY (400MHz, CDCl) for 12' -methyl-thiostrepton₃：CD₃OD＝4:1)。

FIG. 10 shows gHSQC (400MHz, CDCl) of 12' -methyl-thiostrepton₃：CD₃OD＝4:1)。

FIG. 11 shows the results of HPLC analysis of 12' -methyl-thiostrepton obtained by feeding mutant SL1102, wherein FIG. 11A shows the results of fermentation of wild-type Streptomyces laurentiii ATCC 31255; FIG. 11B shows the result of negative control fermentation of mutant SL 1102; FIG. 11C shows the results of fermentation of mutant SL1102 with methyl 4-propionyl-quinoline-2-carboxylate to produce 12' -methyl-thiostrepton.

FIG. 12 shows the experimental results of animal models of zebra fish juvenile fish and adult fish infected with Mycobacterium marinum. In FIG. 12, A shows the effect of different antibiotics after infection of young fish with Mycobacterium marinum (tail vein injection); b shows the survival curves of the young fishes under the action of different antibiotics (10 fishes in each group); panel C shows the tissue section condition of the infected part after adult fish is infected with mycobacterium marinum (intraperitoneal injection) and different antibiotics are administered for treatment; panel D shows the survival curves of adult fish (20 per group) under the action of different antibiotics.

Description of the symbols

In fig. 1, TFA: trifluoroacetic acid

In fig. 11, TSR: thiostrepton; 12'-Me-TSR 12' -methyl-thiostrepton.

Detailed Description

The present inventors have made extensive and intensive studies on the cloning of thiostrepton biosynthetic gene clusters and on the biosynthetic mechanism, and have surprisingly found that, when H is substituted for 4-acetyl of thiostrepton with methyl group, the produced thiostrepton derivative has not only improved bioactivity as compared with the parent compound but also improved water solubility by 1-fold, using a mutation biosynthesis method. The present invention has been completed based on this finding.

It is worth mentioning that in the course of the studies of structural modification of thiostrepton by the present inventors, it was found that when the water solubility of thiostrepton analog is increased, the biological activity is generally difficult to be improved, and even greatly reduced.

Active ingredient

As used herein, the terms "active ingredient of the invention", "compound of the invention" and "thiostrepton analog of the invention" are used interchangeably and all refer to a methyl-substituted thiostrepton, such as 12' -methyl-thiostrepton shown in formula I.

It is to be understood that the term also includes various crystalline forms, pharmaceutically acceptable salts, hydrates or solvates of the compounds of the present invention.

One preferred class of thiostrepton analogs has the structural formula shown in formula I below:

in the formula, R₁、R₂、R₃、R₄As described above.

as used herein, the term "12 '-Me-TSR" denotes 12' -methyl-thiostrepton, the structural formula of which is shown in formula Ia.

As used herein, the term "pharmaceutically acceptable salt" refers to a salt of a compound of the present invention with an acid or base that is suitable for use as a pharmaceutical. Pharmaceutically acceptable salts include inorganic and organic salts. One preferred class of salts is that formed by reacting a compound of the present invention with an acid. Salts may be formed from cations with charged groups (e.g., amino groups) on the compounds of the present invention. Suitable cations include hydrogen ions, sodium ions, potassium ions, magnesium ions, calcium ions, and ammonium ions. Suitable bases for salt formation include, but are not limited to: hydroxides of alkali metals and alkaline earth metals (e.g. NaOH, KOH), oxides of alkali metals and alkaline earth metals, carbonates of alkali metals and alkaline earth metals (e.g. Na)₂CO₃) Ammonia, and the like.

The present inventors confirmed the structure of the compound not only by extensive fermentation of methyl-substituted thiostrepton but also by isolation and purification. In addition, antibacterial activity experiments prove that the antibacterial activity of the methyl-substituted thiostrepton is obviously improved compared with that of thiostrepton; water-solubility experiments prove that the methyl-substituted thiostrepton has obviously improved water solubility compared with the thiostrepton.

Besides the antibacterial active ingredients of the compounds of the invention used for preparing medicines and being used as feed additives for preparing feed compositions, the compounds of the invention can also be used as intermediates for preparing other thiostrepton analogs, and can be used for further chemical derivation to generate other thiostrepton analogs.

Mutant synthons

As used herein, the term "mutant synthetic element" refers to a compound involved in the biosynthesis of methyl-substituted thiostrepton of the present invention, wherein the compound has the structure depicted in formula II. The compounds are used in the synthesis or preparation of the thiostrepton analogs of the present invention, and may be prepared by themselves.

The invention also provides a method for preparing the mutant synthetic element shown in the formula II, which comprises the following steps:

in the formula, R₁、R₂、R₃、R₄As described above.

In another preferred embodiment, R is₁CH₂CHO is propionaldehyde.

Starting strain

As used herein, the term "starting strain of the invention" or "starting microorganism of the invention" refers to Streptomyces laurentii (Streptomyces laurentiii) with the accession number ATCC 31255. The starting strain of the present invention was stored in the American type culture library (ATCC) under the accession number Streptomyces laurentiii ATCC 31255. It is understood that the starting strain includes not only the strain numbered Streptomyces laurentii ATCC31255 but also strains derived therefrom.

Engineered bacterial strains

The invention also provides engineering bacteria which can be used for producing the compound.

In a preferred embodiment of the present invention, there is provided a mutant strain of Streptomyces laurentii (Streptomyces laurentii) producing a thiostrepton analog of the present invention, wherein a thioadenosylmethionine transferase gene (tsrT) in a thiostrepton biosynthetic gene cluster is knocked out in said strain. The preparation method of the engineering strain can be seen in Chinese patent application, application number: 2011103591762.

preparation of active ingredients

The present invention provides a process for the preparation of a compound of formula I, comprising the steps of:

feeding the genetically engineered strain with a compound of formula II;

extracting the compound of formula I from the culture medium.

Centrifuging to remove supernatant, soaking thalli in acetone, filtering to remove insoluble substances, concentrating the filtrate under reduced pressure, draining, performing coarse separation on the obtained paste by using a normal phase column preloaded by 100-plus-200-mesh silica gel, further purifying by using HPLC preparation, and collecting the effluent of the compound in the formula I to finally obtain the target product.

The present inventors confirmed the structure of the compound by extensive fermentation and isolation and purification of the compound of formula I; antibacterial activity experiments prove that the antibacterial activity of the active compound is obviously improved compared with the antibacterial activity of thiostrepton; water solubility experiments prove that the water solubility of the active compound is obviously improved compared with the thiostrepton.

Pharmaceutical compositions and methods of administration

The compounds of the present invention have excellent bacteriostatic (antibacterial) activity and are therefore useful as antibiotics, and their properties are useful for the preparation of injections.

The compounds of the present invention can be administered to a mammal (e.g., a human) orally, rectally, parenterally (intravenously, intramuscularly or subcutaneously), topically, etc. The compounds may be administered alone or in combination with other pharmaceutically acceptable compounds. It is noted that the compounds of the present invention may be administered in combination.

Solid dosage forms for oral administration include capsules, tablets, pills, powders and granules. In these solid dosage forms, the active compound is mixed with at least one conventional inert excipient (or carrier), such as sodium citrate or dicalcium phosphate, or with the following ingredients: (a) fillers or extenders, for example, starch, lactose, sucrose, glucose, mannitol and silicic acid; (b) binders, for example, hydroxymethylcellulose, alginates, gelatin, polyvinylpyrrolidone, sucrose and acacia; (c) humectants, for example, glycerol; (d) disintegrating agents, for example, agar-agar, calcium carbonate, potato or tapioca starch, alginic acid, certain complex silicates, and sodium carbonate; (e) slow solvents, such as paraffin; (f) absorption accelerators, e.g., quaternary ammonium compounds; (g) wetting agents, such as cetyl alcohol and glycerol monostearate; (h) adsorbents, for example, kaolin; and (i) lubricants, for example, talc, calcium stearate, magnesium stearate, solid polyethylene glycols, sodium lauryl sulfate, or mixtures thereof. In capsules, tablets and pills, the dosage forms may also comprise buffering agents.

Solid dosage forms such as tablets, dragees, capsules, pills, and granules can be prepared using coatings and shells such as enteric coatings and other materials well known in the art. They may contain opacifying agents and the release of the active compound or compounds in such compositions may be delayed in release in a certain part of the digestive tract. Examples of embedding components which can be used are polymeric substances and wax-like substances. If desired, the active compound may also be in microencapsulated form with one or more of the above-mentioned excipients.

Liquid dosage forms for oral administration include pharmaceutically acceptable emulsions, solutions, suspensions, syrups or tinctures. In addition to the active compounds, the liquid dosage forms may contain inert diluents commonly employed in the art such as, for example, water or other solvents, solubilizing agents and emulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, propylene glycol, 1, 3-butylene glycol, dimethylformamide and oils, in particular, cottonseed, groundnut, corn germ, olive, castor and sesame oils or mixtures of such materials and the like.

In addition to these inert diluents, the compositions may also contain adjuvants such as wetting agents, emulsifying and suspending agents, sweetening and flavoring agents.

Suspensions, in addition to the active compounds, may contain suspending agents as, for example, ethoxylated isostearyl alcohols, polyoxyethylene sorbitol and sorbitan esters, microcrystalline cellulose, aluminum methoxide and agar, or mixtures of these substances, and the like.

Compositions for parenteral injection may comprise physiologically acceptable sterile aqueous or anhydrous solutions, dispersions, suspensions or emulsions, and sterile powders for reconstitution into sterile injectable solutions or dispersions. Suitable aqueous and nonaqueous carriers, diluents, solvents or vehicles include water, ethanol, polyols and suitable mixtures thereof.

Dosage forms for topical administration of the compounds of the present invention include ointments, powders, patches, sprays, and inhalants. The active ingredient is mixed under sterile conditions with a physiologically acceptable carrier and any preservatives, buffers, or propellants which may be required if necessary.

The compounds of the invention may be administered alone or in combination with other active ingredients, such as antibiotics.

When the pharmaceutical composition is used, a safe and effective amount of the compound of the present invention is suitable for mammals (such as human beings) to be treated, wherein the administration dose is a pharmaceutically-considered effective administration dose, and for an individual with a body weight of 60kg, the daily administration dose is usually 1 to 1000mg, preferably 20 to 500 mg. Of course, the particular dosage will also take into account factors such as the route of administration, the health of the individual, etc., which are within the skill of the skilled practitioner.

Feed additive and feed composition

The invention also provides the application of the compound in the field of feed. The compound of the invention has excellent antibacterial and bacteriostatic properties, and is very suitable to be used as a feed additive.

The present invention also provides a method of preparing a feed composition comprising the steps of: the compound of the present invention or a pharmaceutically acceptable salt thereof is mixed as a feed additive with a feed material to form a feed composition containing a thiostrepton analog (or a salt thereof).

The main advantages of the invention include:

(a) provides a novel thiostrepton analogue with the structure of formula I and the advantages of higher activity and the like.

(b) Based on the thiostrepton analogue, the invention is beneficial to preparing other types of thiostrepton derivatives.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the Laboratory Manual (New York: Cold Spring Harbor Laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are by weight.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the practice of the present invention. The preferred embodiments and materials described herein are exemplary only, and are not intended to limit the scope of the invention.

EXAMPLE 1 Synthesis of mutant Synthesis of Meta-4-propionyl-quinoline-2-carboxylic acid methyl ester

1. Synthesis of quinoline-2-carboxylic acid methyl ester

10mmol of quinoline-2-carboxylic acid is dissolved in 10ml of methanol, 0.92ml of thionyl chloride is slowly dropped in an ice-water bath, the temperature is slowly raised to the room temperature, and heating reflux is carried out for 12 hours. The reaction was cooled to room temperature, carefully poured into a saturated solution of sodium bicarbonate, and then extracted several times with dichloromethane. The extracts were combined, dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure and column separated to give 1.59 g of product in 85% yield.

The quinoline-2-carboxylic acid methyl ester was identified by NMR as follows:

¹H NMR(500MHz,CDCl₃)δ832(d, J ═ 8.5Hz,1H),8.21(d, J ═ 8.5Hz,1H),7.89(dd, J ═ 8.0,1.0Hz,1H),7.80(ddd, J ═ 8.5,7.0,1.5Hz,1H),7.66(ddd, J ═ 8.0,7.0,1.0Hz,1H),4.10(s,3H) (fig. 2).

¹³C NMR(125MHz,CDCl₃) δ 166.0,147.9,147.6,137.4,130.8,130.4,129.4,128.7,127.6,121.1,53.3 (fig. 3).

Synthesis of 2.4-propionyl-quinoline-2-carboxylic acid methyl ester

0.38 g of ferrous sulfate was added to a 50ml solution of propionaldehyde containing 10.0mmol of quinoline-2-carboxylic acid methyl ester and 0.97ml of trifluoroacetic acid. Under the condition of ice water bath, 5.5ml of 30% hydrogen peroxide is slowly added, 5.5ml of 30% hydrogen peroxide is added again after 30 minutes, and the reaction is continued for 90 minutes. The reaction was quenched with a 5% sodium thiosulfate solution and the remaining trifluoroacetic acid was neutralized by the addition of saturated sodium bicarbonate solution. The aqueous phase was extracted several times with ethyl acetate, the extract was dried over anhydrous sodium sulfate, filtered, concentrated under reduced pressure, and separated by column to give 1.95g of methyl 4-propionyl-2-quinolinate, with a yield of 80%.

The 4-propionyl-2-quinolinic acid methyl ester is subjected to NMR identification, and the identification result is as follows:

¹H NMR(500MHz,CDCl₃) δ 8.35 to 8.33(m,2H),7.82(ddd, J ═ 8.3,6.9,1.4Hz,1H),7.73 to 7.70(m,1H),4.10(s,3H),3.11(q, J ═ 5Hz,2H),1.29(t, J ═ 5Hz,3H) (fig. 4).

¹³C NMR(125MHz,CDCl₃) Delta 203.9,165.4,148.5,147.47,144.6,131.1,130.6,130.2,128.7,125.3,118.9,53.4,35.7,7.9 (FIG. 5)

LC-MS(EI⁺)m/z244.20[M+H]⁺.

The synthetic scheme for mutating and synthesizing the meta-4-propionyl-quinoline-2-carboxylic acid methyl ester is shown in figure 1.

NMR and LC-MS results show that the inventors successfully synthesized the mutant synthetic element 4-propionyl-quinoline-2-carboxylic acid methyl ester.

Example 2 construction of engineered Strain and analysis of fermentation products

For the process of constructing the mutant SL1102, refer to the Chinese patent publication No. CN 103102396A.

Analysis of fermentation products

The wild type Streptomyces laurentii ATCC31255 and the mutant SL1102 were fermented separately under the following conditions:

fermentation of wild-type Streptomyces laurentiii ATCC 31255; the mutant strain SL1102 is fermented without adding 4-propionyl-quinoline-2-carboxylic acid methyl ester; and mutant SL1102 is fermented by feeding 4-propionyl-quinoline-2-carboxylic acid methyl ester.

The fermentation products were analyzed by HPLC, respectively, and the results were as follows:

FIG. 11 shows an HPLC analysis of methylsulfocysin obtained by feeding mutant SL 1102. FIG. 11A is the result of fermentation of wild-type Streptomyces laurentiii ATCC 31255; FIG. 11B shows the results of fermentation of a negative control (no methyl 4-naphthoyl-quinoline-2-carboxylate) of mutant SL1102 (mutant. DELTA.TsrT); FIG. 11C shows the results of fermentation of mutant SL1102 with methyl 4-enoyl-quinoline-2-carboxylate to produce 12' -methyl-thiostrepton.

The result shows that the wild strain can normally produce TSR, and the mutant strain without the mutant synthetic element can not produce thiostrepton and 12' -methyl-thiostrepton; while the mutant fed with the mutant synthon has the ability to synthesize 12' -methyl-thiostrepton.

Example 312 fermentation, detection, isolation and purification and structural characterization of methyl-thiostrepton

The mutant strain Δ TsrT was inoculated into 50ml of a pre-fermentation medium (containing TSB15g/L, soluble starch 15g/L, sucrose 50g/L), and cultured at 28 ℃ for 48 hours. 5ml of the pre-fermented broth was inoculated into 50ml of a fermentation medium (containing TSB15g/L, calcium sulfate 15g/L, yeast extract 11g/L, glucose 50g/L, dipotassium hydrogenphosphate heptahydrate 0.05g/L, magnesium sulfate heptahydrate 0.1g/L, sodium sulfate 0.2g/L, zinc sulfate heptahydrate 0.001g/L, ferrous sulfate heptahydrate 0.002g/L, calcium carbonate 0.3g/L) and mutant synthetic methyl 4-propionyl-quinoline-2-carboxylate was added to a final concentration of 0.2mmol, and the mixture was cultured at 28 ℃ and 250 rpm. After culturing for 120 hours, the fermentation broth and mycelia were treated.

Centrifuging, removing supernatant, soaking thallus in acetone overnight, filtering to remove insoluble substances, and vacuum distilling the filtrate to obtain dark brown paste. The paste was roughly fractionated in the first step using a normal phase column pre-packed with 100-200 mesh silica gel under gradient elution conditions as shown in Table 1.

TABLE 1

Methylsulfothiomycin was present in the eluted fraction of 95% dichloromethane/5% methanol, and the eluate was drained under reduced pressure, dissolved in 10ml methanol and then subjected to HPLC preparation.

The preparation conditions of HPLC are as follows:

the instrument comprises the following steps: agilent1100HPLC system

Column: agilent ZORBAX SB-C18column (9.4x250mm, PN880975-202)

Detection wavelength: UV 254nm

Mobile phase: a ═ H₂O；B＝CH₃CN

Flow rate: 3 ml/min.

The mobile phase gradient ratio is shown in table 2.

TABLE 2

Time (min)	A％	B％
			0	40	60
20	40	60

Collecting the effluent of 12' -methyl-thiostrepton according to the elution condition of HPLC, and finally obtaining the target product. The target product was identified as follows:

¹H NMR(400MHz,CDCl₃:CD₃OD 4: 1: 8.83(s,1H),8.32(s,1H),8.31(s,1H),8.22(s,1H),7.60(s,1H),7.59(s,1H),7.31(s,1H),7.07(s,1H),6.89(d, J9 Hz,1H),6.73(d, J2.0 Hz,1H),6.56(s,1H),6.38(q, J6.5, 1H),6.37(dd, J9.6, 5.6Hz,1H),6.24(q, J7.0 Hz,1H),5.84(s,1H),5.80(d, J8, 1H),5.76(d, J6, J0 Hz,1H), 4.7, 4H, 5H, 4H, 6H, 6.6H, 6H, 4H, 5H, 4H, 5H, 4H, 6H, 4H, 6H, 1H, 6H, 4H, 6H, 4H, 1H, 4H, 1H, j ═ 3.1Hz,1H),4.43 to 4.39(m,1H),4.10(m,1H),3.83(q, J ═ 6.5Hz,1H),3.82 to 3.80(m,1H),3.68(m,1H),3.62 to 3.60(m,1H),3.17(t, J ═ 12.0Hz,1H),2.32(m,1H),2.96 to 2.93(m,2H),2.98(d, J ═ 4.6Hz,1H),1.92(m,1H),1.74(q, J ═ 6.6Hz,3H),1.64 to 1.60(m,2H),1.63(d, J ═ 7.0, 3H),1.55 to 1.53(m,1H),1.46 (J ═ 1.6 Hz,3H),1.64 to 1.60(m,2H),1.63(d, J ═ 7.0, 3H), 3H, 1.53(m, 1.6H), 1.6H, 3H), 3H, 1.6H, 3H, 1.6H, 1.7, 3H, 1.6H, 1H, 1.6H, 3H, 1H, 1.6H), 1.6H, 1H, 1.6H, 1.7, 3, 0.85(d, J ═ 6.5Hz,3H) (fig. 6).

¹³C NMR(100MHz,CDCl₃:CD₃OD 4:1) 174.1,173.9,172.6,170.9,170.7,170.2,169.5,168.6,166.9,166.7,166.1,163.4,162.8,162.7,162.6,162.3,161.4,160.3,157.4,155.1,153.8,150.7,150.5,146.9,144.0,134.7,133.4,132.7,132.6,130.2,129.2,129.0,128.5,128.4,126.3,124.5,123.4,118.9,105.0,104.3,104.0,79.5,77.9,72.2,70.2,68.2,68.1,67.0,66.6,64.8,59.5,58.2,56.3,53.6,52.3,50.1,39.0,35.7,31.1,29.3,23.3,23.0,20.3,19.5,19.4,19.3,16.3,16.2,16.0,12.1,11.0 (fig. 7).

HRMS(m/z)[M+H]⁺Measured value 1678.5130 (C)₇₃H₈₈N₁₉O₁₈S₅Calculated 1678.5153).

FIGS. 8, 9 and 10 are the gHMBC, gCOSY and gHSQC spectra of 12' -methyl-thiostrepton.

The identification result shows that the inventor successfully prepares 12' -methyl-thiostrepton.

Example 4 antibacterial Activity of Thioflavin analogs

The antibacterial activity of 12' -methyl-thiostrepton, 5' -fluoro-thiostrepton, 6' -fluoro-thiostrepton and thiostrepton is measured by the following method: in a 96-well plate, 12' -methyl-thiostrepton, 5' -fluoro-thiostrepton, 6' -fluoro-thiostrepton and thiostrepton dissolved in dimethyl sulfoxide were added to the medium to 0.256. mu.g/mL and stepwise diluted to 0.00025. mu.g/mL, respectively. Adding test bacteria to the culture medium to 10⁷-10⁸cfu/mL (according to 0.5McFarland standard) and incubated overnight. The minimum concentration at which the test bacteria cannot grow is the MIC.

The results of the Minimum Inhibitory Concentration (MIC) test showed that 12' -methyl-thiostrepton, 5' -fluoro-thiostrepton, 6' -fluoro-thiostrepton all had superior antibacterial activity to thiostrepton (see Table 3).

TABLE 3

The unit is mu g/mL;^aa gram-positive bacterium, a microorganism selected from the group consisting of,^bgram-negative bacteria

As can be seen from the table above, the thiostrepton analogue of the invention is obviously superior to natural TSR in the aspects of inhibiting bacillus subtilis, staphylococcus aureus and clinically separated multi-drug resistant bacteria (PRSP, MRSA, VRE and the like), and the biological activity of the thiostrepton analogue is more than twice of that of TSR. The inventor also discovers for the first time that thiostrepton and derivatives thereof have strong antibacterial action on polymyxin drug resistant gram negative bacteria, and people think that thiostrepton antibiotics have no bacteriostatic action on gram negative bacteria in early research.

Example 5 animal model experiment of infection of Mycobacteria marinum in juvenile and adult zebra fish

The experimental results are shown in fig. 12, and fig. 12 shows an animal model experiment of the mycobacterium marinum infected by the zebra fish juvenile fish and the adult fish. FIG. 12A shows quantification of pathogenic bacteria proliferation in zebrafish larvae by green fluorescence optical density (stable GFP expression in M.marinus) using different antibiotics administered after infection of larvae with M.marinus (tail vein injection); b shows the survival curves of young fish (10 per group) under the action of different antibiotics. The experimental results show that the thiopeptide antibiotics have better in vivo antibacterial activity, especially the 12' -Me-TSR has obvious activity of inhibiting intracellular bacteria, and the 12' -Me-TSR can obviously prolong the survival time of juvenile fish to 15 days or even longer as seen in a graph B, the 12' -Me-TSR experimental group still has 40 percent of survival rate at the 12 th day, and juvenile fish of other experimental groups are all dead. C, the graph shows that after adult fish is infected with mycobacterium marinum (intraperitoneal injection), different antibiotics are used for administration treatment, and the proliferation condition of the mycobacterium marinum in adult fish bodies is visually detected through acid-fast staining of tissue sections of infected parts, so that in an animal model infected by the adult fish, the thiopeptide antibiotics have remarkable in-vivo antibacterial activity, and the 12' -Me-TSR has better effect than other antibiotics; panel D shows the survival curves of adult fish (20 per group) under the action of different antibiotics, 12' -Me-TSR has better effect than other antibiotics. Through the animal experiments, the inventor conducts intracellular antibacterial activity research on thiostrepton and derivatives thereof on the level of a marine mycobacterium-zebra fish infection model. Through statistical analysis of animal model survival and fluorescent quantitation of intracellular bacterial proliferation, the present inventors found that 12' -alkyl-thiostrepton has higher in vivo antibacterial activity than other derivatives.

Example 6

The results of the water solubility test for thiostrepton analogs are shown in Table 4.

TABLE 4

Unit is mug/mL

As can be seen from the data in the table, the water solubility of 12 '-methyl-thiostrepton (12' -Me-TSR) is improved by more than 1 time compared with thiostrepton. The improved water solubility can improve the bioavailability of the compound and facilitate administration.

EXAMPLE 8 pharmaceutical composition

Compound (I)

20g

Starch	140g
		Microcrystalline cellulose	60g

The materials are evenly mixed according to a conventional method and then are filled into common gelatin capsules to obtain 1000 capsules.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. A thiostrepton analog, or a pharmaceutically acceptable salt thereof, having a structure according to formula I:

wherein the formula I has a structure shown in formula Ia:

2. a pharmaceutical composition comprising the thiostrepton analog of claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

3. The use of a thiostrepton analog or a pharmaceutically acceptable salt thereof as claimed in claim 1, characterized in that the thiostrepton analog or the pharmaceutically acceptable salt thereof

(a) For the preparation of antimicrobial compositions;

(b) for the preparation of a composition for inhibiting the growth of microorganisms;

(c) for the preparation of a composition for the treatment of microbial infections; or

(d) Can be used as feed additive.

4. A method for non-therapeutically inhibiting the growth of or killing microorganisms in vitro comprising the steps of: use of a thiostrepton analog of claim 1, or a pharmaceutically acceptable salt thereof, at a site in need of treatment.

5. A method of preparing a pharmaceutical composition comprising the steps of: mixing the thiostrepton analog of claim 1, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier to form a pharmaceutical composition.

6. A method of preparing a feed composition, comprising the steps of: a thiostrepton analog or its pharmaceutically acceptable salt as feed additive and feed material as the claim 1 are mixed to form the feed composition containing thiostrepton analog or its salt.

7. A process for preparing a thiostrepton analog of claim 1, comprising the steps of:

(a) feeding a mutant strain of Streptomyces laurentii (Streptomyces laurentii) having a thioadenosylmethionine transferase TsrT inactivated or knocked out with a mutant synthesis element, thereby producing a thiostrepton analog of claim 1; and

(b) isolating said thiostrepton analog from the fermentation product;

wherein the mutant synthetic element has a structure shown in formula II:

wherein the mutant synthetic element of formula II has a structure represented by formula IIb:

8. a method of preparing a mutant synthetic element having the formula II, comprising the steps of:

in the formula, R₁Is unsubstituted methyl;

R₂is H; r₃Is H;

R₄is unsubstituted methyl.

9. An intermediate compound useful in the preparation of a thiostrepton analog of claim 1, wherein the compound has a structure represented by formula II,

10. use of an intermediate compound as claimed in claim 9, for the preparation of a thiostrepton analogue as claimed in claim 1, or a pharmaceutically acceptable salt thereof.