HK1020739B - Novel lantibiotic related to actagardine, and processes for the preparation and use thereof - Google Patents

Description

Novel acagdine-related wool sulfur antibiotic and preparation method and application thereof

The invention relates to a novel lantibiotic associated with acagatdine (actagardine), in particular to a novel lantibiotic named Ala⁰-a lantibiotic of acagdine, a process for its preparation, chemical derivatives derived from the lantibiotic and the use of the lantibiotic as a medicament.

Various lantibiotics have been described. Lantibiotics are polycyclic peptide antibiotics characterized by containing lanthionine or methyllanthionine. They are natural substances obtained by microorganisms and are used as antibacterial active compounds, preservatives or enzyme inhibitors for the treatment of human diseases (G.Jung, Angew.chem.int.Ed.Engl., 1991, 30, 1051-1068).

Various antibiotics are used to treat bacterial infectious diseases. However, pathogens are becoming more and more resistant to these drugs used, and the greatest threat currently facing is the so-called multi-resistant microorganisms which not only become resistant to individual groups of antibiotics, such as β -lactam antibiotics or glycopeptides or macrolides, but also have several resistances at the same time. Even pathogens exist that become resistant to all commercially available antibiotics. Infectious diseases caused by these microorganisms cannot be treated. There is therefore a great need for new drugs that can be used to treat resistant microorganisms. Although the literature describes thousands of antibiotics, most are too toxic to be used as drugs.

Acacadidine is the lantibiotic first described in 1977 by s.somma et al, antimicrobial and chemotherapy 11, 396-797, the structure of which has only recently been correctly elucidated (n.zimmermann et al, europe journal of biochemistry 1995, 228, 786-797).

Surprisingly, it has been found that both strains of Actinoplanes Ricproceed and Actinoplanes gabapentin produce at least one novel antibiotic, for example Ala⁰Acagdine, the novel antibiotic not only has high antibacterial activity, but also has high drug resistance. According to the provisions of the terms of the Budapest treaty, an isolated strain of Actinoplanes leucotrichum, Ligulia, was deposited at 24/9/1997 in Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (German Collection of microorganisms and cell cultures GmbH), MASCHERDER WEG 1B, 38124 BRAUNSCHWEIG, Germany (hereinafter referred to simply as 'DMS'), with the deposit number DSM 11797. An isolated strain of actinoplanes calibardador was deposited on DMS at 24/9 of 1997 under the provisions of the terms of the Budapest treaty with the deposit number DSM 11796.

Accordingly, the present invention provides a compound represented by formula 1 and physiologically tolerable salts thereof,

wherein R is an amino acid group. R may be a substituted or unsubstituted amino acid group wherein the amino group is in the alpha to omega-position and is present in the D or L form. Particularly preferred are substituted or unsubstituted alpha-amino acids in the D or L form.

Preferably, R is a natural amino acid group selected from the group consisting of Ala, Gly, Glu, Phe, Pro, Thr, Cys, Met, Trp, Tyr, Asn, Gln, Asp, His, Ile, Leu, Lys, Arg, Ser and Val. Particularly preferred amino acids are Ala, Ile, Lys, Phe, Val, Glu, Asp, His, Leu, Arg or Ser, and most preferred amino acid is Ala⁰。

R may also be a substituted or unsubstituted diaminoalkanoic acid group, such as 2, 4-diaminobutyric acid (Dab).

In addition, the invention also relates to a test formula C₈₄H₁₂₉N₂₁O₂₅S₄(Ala⁰-acalidine) and their pharmacologically tolerated salts, which compounds are obtainable by fermentative cultivation of actinoplanes ligulariae, DSM11797 or actinoplanes gabapentin, DSM11796 or one of their variants and/or mutants in a culture medium until the culture medium is enriched with the compound Ala⁰Acagdine, which is then isolated to give the compound.

The invention further relates to a composition of empirical formula C₈₄H₁₂₉N₂₁O₂₅S₄(Ala⁰-acalidine) and their pharmacologically tolerated salts, which chemical derivatives are obtained by fermentative cultivation of actinoplanes ligulariae, DSM11797 or actinoplanes gabapentin, DSM11796 or a variant and/or mutant thereof in a culture medium until the culture medium is enriched with the compound Ala, and⁰acagdine, which is then isolated and converted into its chemical derivative.

Preferred chemical derivatives are: ile⁰-，Lys⁰-，Phe⁰-，Val⁰-，Glu⁰-，Asp⁰-，His⁰-，Leu⁰-，Arg⁰-and Ser⁰-acagdine. Ala may be prepared by methods known to those skilled in the art⁰-conversion of acaradine to said chemical derivative.

Antibiotic Ala⁰Acagdine is different from the known substances represented by structural formula in literature. In addition to acagdine, the literature describes certain secondary components of acagdine (U.S. Pat. No. 4,022,884, 5/10/1976, and A. Malabarba et al, J. antibiotics, 1985, 38, 1506-containing 1511), however, the polarity, amino acid composition, antimicrobial activity or physical properties of the secondary components and acagdine are different from those of the compounds of the present invention.

Actinoplanes Riculiasia, strain DSM11797, produces acagatdine and the by-products described in the literature in culture broth containing glucose, starch or glycerol. Surprisingly, the same strain produces the antibiotic NH according to the invention in very high yields in a medium containing non-digestible mannitol₂-R-acagdine, wherein R is the radical of a natural amino acid, in particular alanine, but does not produce the known compounds, the produced acagdine also being in trace amounts.

The invention further relates to a process for the preparation of a compound of formula 1, which comprises culturing the microorganism Actinoplanes Rickia, DSM11797 or Actinoplanes gabapentin, DSM11796 or a variant or mutant thereof in a liquid medium, isolating and purifying the compound of formula 1 and optionally converting the compound into a pharmacologically tolerable salt thereof.

The method comprises culturing Actinoplanes Rickiana, DSM11797 or Actinoplanes gabapentin, DSM11796, mutants and/or variants thereof in a medium comprising a carbon source and a nitrogen source, inorganic salts and trace elements under aerobic conditions.

Preferably, the culturing is carried out at a temperature of 20 to 35 ℃ and a pH of 4 to 10.

In addition, the present invention relates to a method for preparing the compound represented by formula 1, which comprises the reaction of the compound acagdine with an amino acid.

For example, the activated amino acid ester can be reacted with the terminal amino group of acagdine. Preferably, a protecting group such as tert-butyloxycarbonyl (Boc-) is attached to the amino nitrogen of the amino acid to prevent the activated amino acid ester from reacting with itself. The activated ester is, for example, an N-hydroxysuccinate ester of each amino acid. The protecting group is removed and the reaction mixture is purified.

Actinoplanes have orange-yellow basal mycelium, but no aerial mycelium. It forms sporangia characteristic of actinoplanes. The cell wall contains meso-diaminopimelic acid and glycine as characteristic amino acids, and also contains xylose and arabinose as sugars; these are all characteristic of Actinoplanes.

According to the invention, the strain DSM11797 or the strain DSM11796 can be replaced by the compounds according to the invention if mutants and variants of the strain DSM11797 or of the strain DSM11796 are capable of synthesizing the compounds according to the invention. Such mutants can be prepared by known physical methods, for example by irradiation with radiation such as ultraviolet radiation, X-ray radiation or chemical mutagens such as Ethyl Methane Sulphonate (EMS); 2-hydroxy-4-Methoxybenzophenone (MOB) or N-methyl-N' -nitro-N-nitroso-guanidine (MNNG).

Mutants and variants which are capable of producing the antibiotic of the invention can be screened by measuring the biological activity of the active compound accumulated in the culture broth, for example by measuring its antibacterial activity.

Preferred carbon sources for aerobic fermentation should be assimilable, but non-digestible carbohydrates and sugar alcohols such as mannitol, inositol and carbohydrate-containing natural products such as soy flour. Suitable nitrogen sources are: amino acids, peptides and proteins and their degradation products such as peptones or tryptones, further meat extracts, ground seeds such as corn, wheat, beans, oats, soybeans or cotton plants, distillation residues from ethanol production, meat meal or yeast extracts, ammonium salts and nitrates. The inorganic salts in the medium can be, for example, the chlorides, carbonates, sulfates or phosphates of alkali metals or alkaline earth metals, iron, zinc, cobalt and manganese.

Ala (0) -acagdine is particularly well produced by using a culture broth containing about 0.5-5%, preferably 1-3% mannitol, 0.5-5%, preferably 1-3% soybean meal and a solution of trace elements at a concentration of 0.1-0.5%, preferably 0.2-0.3%. The microelement solution contains CaCl₂Iron (III) citrate, MnSO₄，ZnCl₂，CuSO₄Sodium tetraborate, CoCl₂And sodium molybdate.

The cultivation is carried out aerobically, i.e.for example by shaking or stirring the flask or the fermenter under submerged cultivation, if appropriate with the introduction of air or oxygen. The fermentation can be carried out in wide-mouth bottles or round-bottom shake bottles or glass fermentation tanks or V with various volumes₂A is carried out in stainless steel tanks. The fermentation temperature is about 20 to 35 ℃, preferably about 25 to 30 ℃. The pH value is 4-10, preferably 5.5-8.5. Under these conditions, the culture period of the microorganism is generally 20 to 300 hours, preferably 24 to 140 hours. Advantageously, the cultivation is carried out in several stages, i.e.in a liquid medium, first one or more precultures are carried out and then transferred, for example in a volume ratio of 1: 10, into the production medium, i.e.the main medium. The preculture is carried out, for example, by transferring the mycelium into a culture medium and culturing for about 20 to 120 hours, preferably 24 to 72 hours. The mycelium is obtained by, for example, culturing the strain in a solid or liquid medium such as yeast-malt agar or oat flour agar for 1 to 40 days, preferably 3 to 10 days.

According to the invention, the course of the fermentation and antibiotic formation can be monitored by methods known to the person skilled in the art, for example by bioanalytical determination of the biological activity or by chromatographic methods such as Thin Layer Chromatography (TLC) or High Performance Liquid Chromatography (HPLC).

Both the mycelium and the culture filtrate contain the antibiotic Ala (0) -acagdine, usually in the major amount in the culture filtrate. The fermentation broth can therefore be conveniently separated by filtration or centrifugation. The filtrate was extracted using an adsorbent resin as a solid phase. The extraction of the mycelium is conveniently carried out with methanol or acetone, however, other solvents may be used.

The extraction can be carried out over a wide pH range, however, it is advantageous to carry out it in a neutral or weakly acidic medium, preferably between pH3 and pH 7. The extract may be concentrated and dried, for example, in vacuo.

One method of isolating the antibiotic of the invention is to isolate the solution by methods known per se.

Another purification method is chromatographic purification on an adsorbent resin, for example Diaion^HP-20(Mitsubishi Casei Corp.，Tokyo)，Amberlite^XAD7(Rohm and Haas，USA)，Amberchrom^CG, (Toso Hass, Philadelphia, USA) or the like. Many reverse phase supports are very suitable, e.g. RP₈And RP₁₈Generally, it has been disclosed in cases such as those involving High Performance Liquid Chromatography (HPLC).

The purification of the antibiotics of the invention may further comprise the use of a so-called normal phase chromatography support, for example silica gel or Al, in a manner known per se₂O₃Or other carrier.

A selective separation method is the use of molecular sieve methods, for example Fractogel in a manner known per se^TSK HW-40，Sephadex^G-25, and the like. It is also possible to obtain Ala (0) -acagdine from Ala (0) -acagdine-rich material by crystallization. For example, organic solvents and mixtures thereof, anhydrous or with water, are suitable for this purpose. Another method for separating and purifying the antibiotic of the present invention comprises the use of an anion exchanger, preferably having a pH of 4 to 10, and a cation exchanger, preferably having a pH of 2 to 5. Buffer solutions with added organic solvents are particularly suitable for this purpose.

Ala (0) -acagdine, its already mentioned chemical derivatives and its various chemical equivalents can be converted into its corresponding pharmacologically tolerable salts by methods known to the person skilled in the art.

The various chemical equivalents of the compounds of the invention are those compounds which differ slightly chemically, i.e., they have the same activity as the compounds of the invention or can be converted into the compounds of the invention under mild conditions. The mentioned equivalents comprise, for example, esters, amino derivatives, complexes or addition compounds of the invention.

Pharmacologically tolerated salts according to the invention are understood as being organic or inorganic salts, such as those described in Remington's pharmaceutical Sciences (17th Edition, page 1418 (1985)). Possible salts are, in particular, alkali metal salts, ammonium salts, alkaline earth metal salts, salts of physiologically tolerated amines and organic or inorganic acids, for example HCl, HBr, H₂SO₄Salts of maleic acid, fumaric acid.

The physicochemical and spectroscopic properties of the antibiotics of the invention can be summarized as follows:

ala (0) -acagdine:

appearance:

colorless material dissolved in methanol and water. Stable in neutral and weakly basic media, but unstable in strongly acidic and strongly basic solutions.

Experimental formula (II): c₈₄H₁₂₉N₂₁O₂₅S₄

Molecular weight: 1961.21

¹H-and¹³C-NMR: see tables 1 and 2

Maximum ultraviolet absorption (log ε): 280nm (3.71), 288nm (shoulder peak)

Table 1: ala⁰Of acagdine¹H-NMR spectroscopic data

Amino acids HNH α H β H γ Others

Ala⁰ 8.010 2.891 1.383

Ala¹ 8.564 4.727 3.397 -

2.603

Ser² 8.264 4.350 3.651 OH：5.102

Gly³ 8.595 3.968 - -

3.263

Trp⁴ 8.147 4.475 3.311 H5：7.144；H6：

7.554；H7：6.983；

H8：7.061；H9：

7.333, respectively; indole:

10.740

2.978

Val⁵ 7.454 4.558 2.048 0.909

0.856

Ala⁶ 8.487 4.704 2.578

2.960

Abu⁷ 8.324 4.557 3.596 1.176

Leu⁸ 7.636 4.626 1.418 1.482 δ0.841

1.482 Y-Me：0.860

Abu⁹ 7.604 4.747 3.570 1.207

Ile¹⁰ 8.378 3.763 1.605 1.063 δ-Me：0.860

β-Me：0.879 1.642

Glu¹¹ 8.262 3.690 2.178 2.319

Ala¹² 7.325 4.553 2.559 -

2.866

Gly¹³ 8.140 3.538 - -

4.167

Abu¹⁴ 7.860 4.381 3.336 1.059

Val¹⁵ 7.778 4.104 2.042 0.875

0.873

Ile¹⁶ 7.589 3.867 1.875 1.513 δ-Me：0.833

β-Me：0.889 1.115

Ala¹⁷ 7.577 4.488 2.594

2.878

Ala¹⁸ 8.181 4.036 1.232

Ala¹⁹ 8.342 4.447 2.934

3.069

table 2: ala⁰Of acagdine¹³C-NMR spectroscopic data

Amino acids CO C α C β C γ others

Ala⁰ 169.51 48.35 17.35

Ala¹ 169.27 50.80 34.21 -

Ser² 170.48 55.04 61.23

Gly³ 168.90 43.44 -

Trp⁴ 54.28 27.62 110.41，123.28，

127.08，111.28，

120.83，136.00，

118.10，118.22

Val⁵ 171.20 56.73 31.50 17.76

19.02

Ala⁶ 170.14 53.73 32.79

Abu⁷ 57.83 43.95 19.96

Leu⁸ 171.45 51.01 41.66 24.03 22.38

22.62

Abu⁹ 171.59 55.77 46.38 20.10

Ile¹⁰ 170.96 60.00 35.71 24.75 11.59

β-Me：14.70

Glu¹¹ 55.86 24.49 30.98 173.75

Ala¹² 170.78 55.47 35.26

Gly¹³ 170.03 44.18

Abu¹⁴ 168.33 55.00 55.64 6.94

Val¹⁵ 170.63 60.03 30.02 19.15

18.564

Ile¹⁶ 170.66 59.64 35.69 24.55 10.62

β-Me：15.53

Ala¹⁷ 169.79 53.13 35.70

Ala¹⁸ 171.52 48.89 15.34

Ala¹⁹ 47.16 51.53

Unspecified 169.01

169.83

170.33

171.01

As a result of amino acid analysis, the amino acid of acagatdine was increased by one Ala (1Ser, 2Gly, 1Trp, 2Val, 1Leu, 2Ile, 1Glu, 1Ala, 1 lanthionine (Ala-S-Ala) and 3. beta. -methyllanthionine (Abu-S-Ala)).

Further, the compound of the invention is found to have strong antibacterial activity; table 3 summarizes by way of example the Minimum Inhibitory Concentrations (MIC) of Ala (0) -acaradine.

Table 3: in vitro Activity of Ala (0) -acagatdine against gram-Positive and anaerobic bacteria in Serial dilution assays

Microorganisms	Ala (0) -acagatdine MIC values (. mu.g/ml)
		Staphylococcus aureus SG511 Staphylococcus aureus 285 Staphylococcus aureus 503 Staphylococcus aureus FH1982 Staphylococcus aureus 701E Staphylococcus aureus 707E Staphylococcus aureus 9Tub Staphylococcus aureus 8236 Staphylococcus epidermidis ZH2c Staphylococcus epidermidis 6098W Staphylococcus epidermidis 763 Staphylococcus epidermidis 5747IIW Staphylococcus epidermidis 291 Staphylococcus epidermidis 799 enterococcus Md8B enterococcus urinaria VR1 enterococcus VR2 Staphylococcus pyogenes VR3 Staphylococcus pyogenes 308A Staphylococcus pyogenes 77A Propionibacterium acnes 6919 Propionibacterium acnes 6922 Clostridium tetani 9406 Clostridium perfringens 194	6.256.253.1312.512.512.56.256.256.2512.56.256.2512.56.256.255050256.250.1951.01.08.00.5

It is particularly noteworthy that the compounds of the invention not only have about two-fold higher activity against gram-positive microorganisms than acaradine, but at the same time have no cross-resistance at all with conventional antibiotics such as β -lactams (penicillins, cephalosporins), aminoglycosides (streptomycins), macrolides (erythromycin), quinolones (ciprofloxacin), sulfonamides or glycopeptides (vancomycin) and others. It is further emphasized that the compounds have a strong inhibitory effect on anaerobes which cause chronic, and indeed life-threatening, infections.

Ala (0) -acagdine is particularly suitable for the treatment of this disease.

At the active concentrations and those mentioned above, Ala (0) -acagdine was well tolerated and no cytotoxicity or other toxicity was observed.

The invention therefore also relates to the use of the compounds according to the invention as medicaments and to the use of said compounds for the preparation of a medicament for the treatment and/or prophylaxis of bacterial infections.

In addition, the invention also relates to medicaments containing the compounds of the invention.

The medicament is prepared by mixing at least one compound shown as a formula 1 with a physiological auxiliary agent and/or excipient and preparing into a proper dosage form.

The medicament of the present invention may be administered enterally (orally), parenterally (intramuscularly or intravenously), rectally or topically. The dosage form may be a solution, a powder (tablet, capsule including microcapsules), an ointment (cream or gel), or a suppository. Possible adjuvants for the preparation of the dosage forms are customary pharmaceutical conventional liquid or solid fillers and extenders, solvents, emulsifiers, lubricants, flavoring agents, coloring agents and/or buffer substances. The dosage is 0.1-1000 mg/kg body weight, preferably 0.2-100 mg/kg body weight. An effective daily dosage unit contains the compound of the invention in an amount of at least, for example, 30 to 3000 mg, preferably 50 to 1000 mg.

The invention is explained in more detail below by means of the operating examples and the content of the claims.

Example 1: preparation of a mycelium suspension of the production Strain

100ml of a culture solution (1 liter of water containing 10g of starch, 10g of glycerol, 10g of glucose, 2.5g of corn steep liquor, 5g of peptone and 2g of yeast extract, pH 6.0 before sterilization) was charged into a 500ml Erlenmeyer flask, inoculated with the strain and cultured on a shaker at 28 ℃ for 72 hours, the rotation speed of the shaker being 140 rpm. 120ml of the culture broth was uniformly dispersed in 500ml of a medium in a sterile Erlenmeyer flask, wherein the medium contained 2.0g/l oat flour extract, and 15g/l agar was added thereto for solidification and decantation. The culture was carried out at 28 ℃ for 10 to 14 days. The mycelium was then picked from the Erlenmeyer flask and immediately reused either in 50% glycerol at-22 ℃ or in 10% dimethylsulfoxide at-140 ℃.

Example 2: preparation of the production Strain cultures or precultures in Erlenmeyer flasks

The culture grown on the slant of the test tube or an agar culture was inoculated into 100ml of the culture solution in a 500ml sterile Erlenmeyer flask as described in example 1, and shake-cultured in the dark at 28 ℃ with a shaker rotation speed of 140 rpm. The yield of the compound represented by formula 1 reached the highest value after 72 hours of culture. Submerged cultures grown for 72 hours (inoculum size about 5%) from the same broth were sufficient to inoculate both 10-liter and 100-liter fermenters.

Example 3: preparation of Ala (0) -acagdine

The fermentation conditions in a 10 liter fermentor were as follows:

culture medium: 2% soybean powder and 2% mannitol

Culturing time: 24 or 28 hours

The culture temperature is as follows: 28 deg.C

Stirring speed: 200rpm

Ventilation volume: 5 liters of air/min

Foam formation was inhibited by repeated dropping of several drops of an ethanol solution of the polyol. The yield reached a maximum after 48 hours.

Example 4: isolation of antibiotic Ala (0) -acagdine

The 27 l culture obtained in example 3 was centrifuged and the clear culture was filteredLoading the liquid sample into a column with a capacity of 3L and an adsorbent resin MIC Gel as the column filler^CHP 20P. Column size: width x height: 11.3cm × 30 cm. The column was eluted with a solvent gradient of 5% to 50% aqueous isopropanol. The column eluate was collected at 2 liters per fraction. The fractions containing Ala (0) -acagdine, checked by HPLC analysis, were collected, concentrated in vacuo and lyophilized (4 g).

Example 5: high Pressure Liquid Chromatography (HPLC) analysis of Ala (0) -acagdine

Column: nucleosil (I) and (II) and (III) and^100-5C18AB，250/4

mobile phase: 10mM phosphate buffer with acetonitrile concentration of 32%,

pH7

Flow rate: 1 ml/min

UV absorption detection was performed at 210 nm.

The retention time of Ala (0) -acagdine was found to be 16 minutes 50 seconds, the retention time of acagdine itself was found to be 11 minutes 20 seconds.

Example 6: concentration of Ala (0) -acagdine

3g of the product obtained according to example 4 were applied to a column having a capacity of 3 liters and packed with Fractogel^TSK HW-40s (width × height ═ 10cm × 50 cm). 50% aqueous methanol was pumped through the column at a flow rate of 50 ml/min and the column eluate was collected in fractions (65 ml). 140mg of the antibiotic Ala (0) -acagdine were found to be obtained mainly from fractions 24 to 28.

Example 7: final purification of Ala (0) -acagdine

The concentrated antibiotic Ala (0) -acagdine (280mg) obtained according to example 6 was applied to a Nucleosil column^12C18AB-HPLC column (width. times. height. 3.2 cm. times.25 cm), gradient method using 5% to 30% acetonitrile and 0.05% trifluoroethyl fluorideAnd (4) acid. The fractions analyzed by HPLC (see example 5) were mixed accordingly, concentrated in vacuo and lyophilized to give 185mg of Ala (0) -acagdine with a purity of 98%.

Ala (0) -acagdidine molecular weight determined by ESI + mass spectrometry: m + H⁺＝1962.6。

Example 8: obtaining Lys (0) -acagdine

94.5mg (0.05mmol) of acaradine were dissolved in 10ml of anhydrous dimethyl sulfoxide (DMF) and 22mg (0.05mmol) of di-Boc-lysine-O-N-hydroxysuccinimide to which 100. mu.l of Triethylamine (TEA) was added, and the mixture was allowed to stand at room temperature. The entire course of the reaction was monitored by HPLC analysis (see example 5). After 96 hours the reaction was terminated by removing DMF and TEA under high vacuum. The reaction product was purified by preparative HPLC using a gradient method with a 0.05% strength trifluoroacetic acid solution (TFA) with acetonitrile concentrations of 25% to 50%. The dimensions of the column are: height is 25mm and width is 250 mm; carrier: select B^. After lyophilization of the fractions containing the reaction product, 33mg (0.015mmol) of di-Boc-Lys (0) -acagdine were obtained.

The Boc protecting group was completely removed with 60% strength TFA. To do this, 25mg (0.011mmol) of the protected derivative are dissolved at room temperature in 5ml of 60% strength TFA. After 90 minutes, the removal reaction was complete. In a preparative HPLC column (10 mm. times.250 mm, LiChrospher) as described above^) The same gradient was used to purify the free lysyl-acagdine. The purified material was lyophilized to give 14mg (0.007mmol) of Lys (0) -acagdine.

The molecular weight of the final product was checked by mass spectrometry. Molecular weight is (M + H)⁺: 2019, corresponding to empirical formula C₈₇H₁₃₆O₂₅N₂₂S₄。

Example 9: preparation of Ile (0) -acagdine

189mg (0.1mmol) of acaradine was reacted with 33mg (0.1mmol) of Boc-Ile-O-N-hydroxysuccinimide ester as described in example 8. 210mg of Boc-Ile (0) -acagdine were obtained.

Removal of the Boc protecting group and final purification gave 84mg (0.042mmol) of Ile (0) -acagdine. The molecular weight determined by mass spectrometry was (M + H)⁺: 2004, corresponding to empirical formula C₈₇H₁₃₅O₂₅N₂₁S₄。

Example 10: preparation of N-alpha-aminobutyryl-acagatdine [ Abu (0) -acagatdine ]

94.5mg (0.05mmol) of acagdine were reacted with 16.3mg (0.05mmol) of p-nitrophenyl N-Boc- α -aminobutyric acid according to the method described in example 8, giving 54mg of N-Boc-Abu (0) -acagdine after 9 days, and 19mg (0.01mmol) of N- α -aminobutyryl-acagdine after removal of the protecting group.

Molecular peak (M + H)⁺: 1976 corresponding to empirical formula C₈₅H₁₃₁O₂₅N₂₁S₄。

Example 11: preparation of Gln (0) -acagdine

94.5mg (0.05mmol) of acagdine was reacted with 16.4mg (0.05mmol) of Boc-glutamine p-nitrophenyl ester as described in example 8 to remove the protecting group, yielding 38mg (0.019mmol) of Gln (0) -acagdine.

Molecular peak (M + H)⁺: 2019, corresponding to empirical formula C₈₆H₁₃₂O₂₆N₂₂S₄。

Example 12: preparation of Phe (0) -acagdine

94.5mg (0.05mmol) of alcafadine were reacted with 15.6mg (0.05mmol) of Boc-Phe-O-N-hydroxysuccinimide ester as described in example 8. After removal of the protective group, 26mg (0.013mmol) of Phe (0) -acagdine are obtained.

Molecular peakValue (M + H)⁺: 2038 corresponding to empirical formula C₉₀H₁₃₃O₂₅N₂₁S₄。

Example 13: preparation of Phe-Ala (0) -acagdine

94.5mg (0.05mmol) of alcafadine were reacted with 21.7mg (0.05mmol) of Boc-Phe-Ala-O-N-hydroxysuccinimide ester for 3 hours as described in example 8. After removal of the protective group, 37mg (0.018mmol) of Phe-Ala (0) -acagdine are obtained.

Molecular peak (M + H)⁺: 2019, corresponding to empirical formula C₉₃H₁₃₈O₂₆N₂₂S₄。

Example 14: preparation of D-Ala (0) -acagdine

94.5mg (0.05mmol) of acagdine were reacted with 14.5mg (0.05mmol) of Boc-D-Ala-O-N-hydroxysuccinimide ester for 24 hours as described in example 8 to give 47mg (0.024mmol) of D-Ala (0) -acagdine after removal of the protecting groups.

Molecular peak (M + H)⁺: 1961, corresponding to empirical formula C₈₄H₁₂₉O₂₅N₂₁S₄。

Tables 4-6 show the in vitro antibacterial activity (MIC values [ μ g/ml ]) of acaradine (Acta) and its compounds of the invention.

TABLE 4

The culture time is 24h	Acta	Ala-Acta	Ile-Acta	Gln-Acta	Phe-Acta	Phe-Ala-Acta	Lys-Acta	Abu-Acta
									Staphylococcus aureus SG511	20	5	1.2	20	0.6	5	1.2	1.2
Staphylococcus aureus SG511+ 10% serum	40	20	2.5	40	5	10	2.5	10
									Staphylococcus aureus Exp 54146	＞40	40	2.5	＞40	5	20	10	20
Staphylococcus pyogenes A561	＞40	＜＝0.04	＜＝0.04	5	＞40	＞40	＜＝0.04	＞40
									Enterococcus faecium M78L	10	5	5	10	＞40	10	5	＞40
Escherichia coli	＞40	＞40	＞40	＞40	＞40	＞40	＞40	＞40

TABLE 5

Inoculation time is 24h	Acta	Boc-Ala-Acta	Boc-Ile-Acta	Boc-Gln-Acta	Boc-Phe-Acta	Boc-Phe-Ala-Acta	Boc-Lys-Acta	Boc-Abu-Acta
									Staphylococcus aureus SG511	20	10	20	40	1.2	5	2.5	5
Staphylococcus aureus SG511+ 10% serum	40	40	40	＞40	10	10	10	10
									Staphylococcus aureus Exp 54146	＞40	40	＞40	＞40	5	10	10	＞40
Staphylococcus pyogenes A561	＞40	0.150	＞40	0.3	＜＝0.04	＞40	＜＝0.04	＞40
									Enterococcus faecium M78L	＞40	20	＞40	40	5	＞40	10	＞40
Escherichia coli	＞40	＞40	＞40	＞40	＞40	＞40	＞40	＞40

TABLE 6

The culture time is 18h	AIa-Acra	Ile-Acta	Boc-Phe-Acta	Lys-Acta	Boc-Lys-Acra
						Acagdine 011HT3	20	2.5	5	10	10
Acagdine 011HT3+ 50% serum	20	2.5	10	10	20
						Acagdine 011HT3+ 50% serum	40	5	40	10	40
Acagdine 011HT18	＞40	＞40	＞40	＞40	＞40
						Staphylococcus epidermidis 012GO20	＞40	＞40	＞40	＞40	＞40
Staphylococcus aureus 011HT1	1.1	1.2	10	0.6	10
						Staphylococcus aureus 011DU5	40	10	40	10	40
Staphylococcus aureus 011CB20	＞40	40	＞40	＞40
						Staphylococcus aureus 0121O64	＞40	＞40	＞40	＞40	＞40
Staphylococcus epidermidis 012GO42	＞40	＞40	＞40	＞40	＞40
						Staph.coag.negative012HT5	＞40	＞40	＞40	＞40	＞40
Staphylococcus pyogenes 02A1SJl	＜＝0.04	＜＝0.04	0.08	＜＝0.04	0.08
						Staphylococcus pyogenes 02A1UC1	＜＝0.04	＜＝0.04	＜＝0.04	＜＝0.04	＜＝0.04
Staphylococcus suppurative 02A1F16	＜＝0.04	＜＝0.04	＜＝0.04	＜＝0.04	＜＝0.04
						Strepto gr G02GOCB2	20	10	2.5	5	2.5
S.pnenmoniae 030BI2	2.5	1.2	1.2	2.5	1.2
						S.milleri02milGR12	40	＞40	40	40	5
S.miris 02mitGRl6	20	10	20	10	5
						Enterococcus faecalis 02D2HM9	＞40	40	40	＞40	＞40
Enterococcus faecalis 02D2uC5	20	40	40	40	40
						Enterococcus faecalis 02D2DU18	5	5	10	5	10
Enterococcus faecalis 02D2HT10	＞40	＞40	＞40	＞40	＞40

Claims (20)

1. A compound represented by formula 1 or a physiologically tolerable salt thereof,

wherein R is an amino acid group.

2. The compound of formula 1 according to claim 1, wherein R is a natural amino acid group.

The compound of formula 1 as set forth in claim 2, wherein the amino acid is alanine, isoleucine, lysine, phenylalanine or valine.

4. A compound of formula 1 according to claim 3, wherein the amino acid is alanine.

5. A compound of formula I according to claim 1, wherein R is Phe-Ala-.

6. The compound of formula 1 according to one of claims 1 to 5, wherein the amino nitrogen of the amino acid carries a removable protecting group.

7. Ala⁰-a compound of acagdine, obtained by the following process: carrying out fermentation culture on Actinoplanes ligularia, DSM11797 or Actinoplanes gabapentin or DSM11796 in culture medium until the culture solution is enriched with compound Ala⁰Acagdine, which is then isolated to give the compound.

8. Pharmacologically tolerable salts of the compounds as claimed in claim 7.

9. From Ala⁰-an amino derivative produced by the compound acagdine, obtained by fermentative culture of Actinoplanes ligulariae, DSM11797 or Actinoplanes gabapentin or DSM11796 in a culture medium until the culture broth is enriched with the compound Ala⁰Acagdine, which is then isolated and converted into its amino derivative or its pharmacologically tolerated salt.

10. A process for the preparation of a compound as claimed in one of claims 1 to 6, which comprises reacting acagdine with an amino acid or Phe-Ala-.

11. The process as claimed in claim 10, which further converts the compound into a pharmacologically tolerable salt thereof.

12. A process for the preparation of a compound according to one or more of claims 1 to 6, which comprises carrying out the fermentative culture of Actinoplanes lecularis, DSM11797 or Actinoplanes gabapentin or DSM11796 in a medium and isolating the compound of formula 1.

13. The process of claim 12, further comprising converting the compound into a pharmacologically tolerable salt thereof.

14. The process according to claim 12, wherein the actinoplanes libria, DSM11797 or actinoplanes gabapentin or DSM11796 is subjected to fermentative culture in a medium containing a carbon source, a nitrogen source, common inorganic salts and trace elements under aerobic conditions.

15. The method according to any one of claims 12 to 14, wherein the fermentation culture is carried out in a medium containing 0.5 to 5% of mannitol and 0.5 to 5% of soybean meal as a carbon source.

16. The method according to any one of claims 12 to 14, wherein the fermentation culture is carried out under aerobic conditions at a temperature of 20 to 35 ℃ and a pH of 4 to 10.

17. The method according to claim 15, wherein the fermentation culture is carried out under aerobic conditions at a temperature of 20 to 35 ℃ and a pH of 4 to 10.

18. Use of a compound according to one or more of claims 1 to 8 for the preparation of a medicament.

19. Use of a compound according to one or more of claims 1 to 8 for the manufacture of a medicament for the treatment and prevention of bacterial infectious diseases.

20. Pharmaceutical, characterized in that it contains at least one compound according to one of claims 1 to 8 and one or more physiologically acceptable excipients.