Derived from lipopeptide proceeded for their drugs preparation the container and their use

The present invention relates to antibiotic derivatives of the complex lipopept idic. Has 1437, a process for their preparation and their use as a medicament.

Patent European language provides lipopeptide of amino acid sequence highly homologous, but with different fatty acid residues (lipid fraction), which are synthesized by Actinoplanes SPs. during fermentation and are excreted into the culture medium, and a method for the isolation of the lipopeptides from the culture medium, their purification and use of lipopeptides as pharmacologically active substances, especially against gram-positive bacteria.

The aim of the present invention is new derivatives of lipopeptide has complex 1437, having:

a lower toxicity than that of lipopeptides having natural 14217.

This is achieved according to the invention are derivatives of formula I.

Accordingly, the invention provides:

1. Derivatives of the lipopeptide has 1437 of formula I

R¹ is OH or NH ",

2 ^ ,

R is an aliphatic acyl group in c_grams - C.₂₂ , with straight or branched chain, saturated or unsaturated, which may be interrupted phenyl or cycloalkyle. or by an oxygen atom,

and pharmaceutically acceptable salts thereof.

2. A process for the preparation of a compound of formula I, characterized in that comprises reacting a compound of formula II

R¹ has the abovementioned meaning and

R^J represents an amino protecting group, known in peptide chemistry, preferably the tert-butoxycarbonyl protecting group (Boc or), benzyloxycarbonyl (Z-, of Cbz), fluorenylmethoxycarbonyl (Fmoc chemistry) or allyloxycarbonyl (alloc-),

with a carboxylic acid of formula III

R² OH-III wherein R has the abovementioned meanings, 2....

or with a derivative, group activated carbonyl, of this carboxylic acid.

3. Drugs containing a lipopepti.de derivative of formula I thus çpa ' a pharmaceutical carrier.

4, The use of a lipopeptide derivative of formula I, for the manufacture of a medicament against bacterial infection.

In the following, the invention is described in detail, in particular in its embodiments préféréis.

The starting compounds (compounds of formula II) are obtained from fermentation products protected, e.g. from 1437 has β [I-, R.¹ THE OH=,

R² =(HM ^)₂ HC (HC₂ )₇ THE CHCH=HM₂ Coextruded] and 9 a-fluorenylmethyl chloroformate, with formation of the corresponding compound, wherein

©

R³cooch - -₂ (û)

and subsequent enzymatic removal of the fatty acid radical, by means ofActinoplanes utahensis which Bacillus thuringiensis NRRL 12052 (J Antibiotics. 1988, 1093).

Si. used the carboxylic acids from zu -

slipper lll themselves as an acylating agent, it is carried out properly in the presence of a condensing agent, for example a carbodiimide such as n, n-' a-cycloalkylaned -

Said V

carbcdiinride. The activation of the carboxylic acids of formula II can be carried out according to usual methods in chemistry? s-peptide, as described for example in Generating chemiluminescence in-unsererZeit 27, 274 - 286 (19, 93). As activated derivatives, it is appropriate to use acid halides, e.g. acid chlorides, anhydrides or mixed anhydrides, e.g. with formic acid esters, azides, activated esters such as esters of P-nitrophenyl, pentafluorophenyl, 4.6-dimethoxy-a 1, 3, 5 triazine and 2-yl, or esters with n-hydroxysuccinimide or the 1 a-hydroxy benzotriazole, which are obtained with carbodiimides as coupling reagents, or thioesters, for example with the 2 a-mercaptobenzotriazole. other suitable coupling reagents are n, non - carbonyldiimidazole or those based on uronium or phosphonium salts, such as BOP stack, HBTU, PyBOP, or TOTUTBTU (tetrafluoroborate of o-a [a cyano (ethoxycarbonyl) methylidene-amino 1, 1, 3, 3 a-tetramethyl] alkylthiouronium).

In general, the reaction of the compounds of formula II with a carboxylic acid of formula III, or an activated derivative thereof, is carried out in the presence of an inert solvent, such as dichloromethane or dimethylformamide, of:

preferably in the presence of a tertiary base such as pyridine or 1' ethyldiisopropylamine. when using substituted benzoyl chlorides, can: also operate in the presence of water and with the addition of base such as pyridine or sodium carbonate. The reaction may be performed in a temperature range from:

of -20 to + 50 °c, preferably between -10 et + 30 °c.

3 Elimination of protective groups R, with formation of compounds I, takes place according to methods known in the literature; for example, the BOC group is removed with trifluoroacetic acid, the radical Z with HBr/glacial acetic acid or by catalytic hydrogenation, the alloc-group with a nucleophilic compound promoted Pd catalyst, or the Fmoc group with secondary amines, for example piperidine.

" 2 Is preferred compounds of formula I in which R represents

a ch-saturated aliphatic acyl radical₃ (HM₂ >_{the n} COEXTRUDED,

a saturated branched aliphatic acyl radical, preferably (HM₃ )₂ HC (HC₂ )_{the n} C0 or CH₃ HM₂ HC (HC₃ ) (HM₂ )_{the n} C0,

an unsaturated aliphatic acyl group which may have one or more double bonds, a double bond under the configuration the trans or the cis, preferably

H₂ C.=HC (HC₂ )_{the n} C0, (HM₃ )₂ HC (HC₂ )_{the n} HC=HC (HC₂ )_{the n} C0, HC₃ (HM₂ HM=HM)_{the n} (HM₂ )_{the n} COEXTRUDED, HC₃ (HM₂ >_{the n} HC=HC (HC₂ )_{the n} HC=HC (HC₂ )_{the n} COEXTRUDED, HM, (HM)=CH-CH-CO, HC (HC,)=(HM)CH-CH-COEXTRUDED, HR [HM₂ THE C - (CH2₃ )=THE CHCH₂ ]_{the n} COEXTRUDED,

a saturated or unsaturated having one or more triple bonds, preferably hoc network (HM₂ ) COEXTRUDED,

HM₃ (HM₂ >_{the n} <>C. (HM₂ )_{the n} COEXTRUDED, HC₃ (HM₂ )_{the n} C.* ACTIVATED C-C=C-(HM₂ )_{the n} COEXTRUDED,

an aliphatic acyl group interrupted by phenyl or cycloalkyl radicals, preferably

C_OH₅ (HM₂ )_{the n} C0, HC₃ (HM₂ )_{the n} -<2>"^C0 '

CMS^hM₂ )" - ^ CH-GB₂ )_b. - ^ - C0 AT.

C_O HR.₁ - - (HC₂ )_{the n} - {]>- (CH2_J )_{the n} - ^ - C0., ℮Η, (℮Η,). - (η) - (℮Η,),, ℮ 0.

an acyl radical interrupted by an oxygen atom, preferably chch.j (HM₂ )_{the n} the O ^ VCOs, HC₃ (HM₂ )_{the n} THE O ^ A V - (CH2₂ )_{the n} ^ c°, n represents integers between 0 and 20.

Particularly preferred are the compounds which contain an acyl residue in^C₁₂^{- C.} 15^straight or branched chain, such as the groups tetradecanoyl, tridecanoyl, 12 a-méthyltridécanoyle, an acyl residue in c ^ - ^ c-g-unsaturated, containing one or more double or triple bonds, such as the groups iC e -10 a-pentadécénoyle, trans 9- hexadécénoyle, hr [HM₂ THE C - (CK20₃ )=THE CHCH₂J₃ CO or an aliphatic acyl radical interrupted by 1 - 3 phenyl and/or further by an oxygen atom, such as the radicals

CHj (^HM ii)_{the n} @ - " -^C0 •^HM the j (^HM i) no. - ^ - C0 at.

℮η, (℮η, μ ^ - ^ (℮η,). - ^ - ℮ο.

HM_J (HM₂ )_{the R} , - ^ ->(HM₂ )_{the n} - {^ - COEXTRUDED.

c., hr₅ (℮η,),, 0 Η η (℮η,),, - ^ - (℮η,)_b. - C0 AT,

in which

n represents integers from 0 and 8.

Are particularly preferred compounds which contain an aliphatic acyl radical interrupted by

3 phenyl groups, such as the radical

^<CH 2> < ^Eyelash 2> Ή (ζ ^) - -<^HM 2> N.^C0

wherein

n represents integers from 0

and 2.

The invention further comprises a process for the preparation of compounds of formula I, which is characterized qtie which comprises reacting a compound of formula II

R¹ has the abovementioned meaning and

3.

R represents a protecting group LFDA known from peptide chemistry, such as the tert-butoxycarbonyl protecting group (Boc or), benzyloxycarbonyl (Z-, of Cbz), fluorenylmethoxycarbonyl (Fmoc chemistry) or allyloxycarbonyl (alloc-),

with: a carboxylic acid of formula III

R2^O HR-III

2

in which R has the meanings already data.

As pharmaceutically acceptab3 AE. compounds of formula I, can be used in particular salts with mineral acids and organic acids, for example hydrochloric, sulfuric, acéticjue, citric, the P-toluenesulfonic, with inorganic bases organiqpjes bases, such as NaOH, KOH solution, MgtOH ^ / diethanolamine, 1' ethylenediamine, or with amino acids such as arginine, lysine, glutamic acid, like they are prepared by conventional processes.

By virtue of their valuable pharmacological properties, one or more of the lipopeptides according to the invention, or salts thereof, are suitable for use as medicaments.

8 the I ' r * f 9

The substances according to the invention have: a pharmacological activity, in particular as antibiotics against gram-positive bacteria, preferably against glycopeptide-resistant strains and strains of methicillin resistant (MRSA infections= The Staphylococcus; methicillin methicillin-resistant).

In the case of resistant strains of penicillin or methicillin (MRSA strain), which have developed resistances against antibiotics, frequently only glycopeptides such as vancomycin or teicoplanin have a sufficient therapeutic activity. However, strains that are resistant even to these antibiotics appear increasingly [FEMS Fertil. Toxicol, 98 109 to 116 (1992)]. One or more derivatives of lipopeptides according to the invention have a remarkable activity also against these germs to problems.

The L * invention also provides pharmaceutical compositions comprising one or more derivatives of lipopeptides according to the invention or their salts.

One or more derivatives of lipopeptides according to the invention, preferably one or more compounds having 3 phenyl radicals the acyl residue in the R, may in chestnut - " 2 ICSB be administered as such. It is preferred to use them in admixture with conventional excipients, carriers or diluents suitable as vehicles, may be used, in drugs for veterinary use, the compositions of conventional animal feed: or, in humans, all vehicles and/or pharmacologically acceptable adjuvants.

The medicaments according to the invention are in general administered orally or parenterally, but rectal administration is in principle also possible. The dosage form of liquid or solid dosage suitable are e.g. granules, powders, tablets, dragees, capsules (microwave), suppositories, syrups, emulsions, slurries, compositions for aerosols, drops or injectable solutions in the form of ampoules, as well as compositions with delayed release of active ingredient, in the manufacture of which are the usual vehicles and/or additives and adjuvants such as disintegrants, binders, coating agents, swelling agents, lubricants or glidants, taste correctors, sweeteners or solubilizers. Examples of adjuvants or vehicles frequently used, include magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, lactalbumin, gelatin, starch, vitamins, cellulose and its derivatives, animal or vegetable oils, polyethylene glycols and solvents, such as sterile water, alcohols, glycerol and polyols.

Examples of diluents, include polyglycols, ethanol and water. Buffer substances are e.g. organic compounds, such as n, n-'a-dibenzylethylenediamine, diethanolamine, 1' ethylenediamine, n-methylglucamine, N-benzylphénéthylamine, diethylamine, tris (bales) aminomethane, or inorganic compounds, such as a phosphate buffer, sodium bicarbonate, sodium carbonate. It can be administered in a form suitable substances as such, without vehicle or diluent. Suitable doses of the compounds of formula I or salts thereof Pharma S. C. had expressions of acceptable range from about 0.4 grams, preferably 0.5 grams, to 20 g per day at most, for an adult weighing about 75 kg. Keys can be administered dose uniquess or, in general, multiple doses, the dosage unit may contain the active substance in an amount of about 50 to 1,000 mg.

TST may optionally microencapsulating the dosage units for oral administration, in order to slow the release or extend it over a longer duration, as for example by coating or embedding of the substance

10 -

J

X

* - activates, in particle form, in suitable polymers or waxes, or the like.

Preferably, the pharmaceutical compositions are prepared and administered in dosage units, each unit containing as an active component a selected dose of one or more derivatives of lipopeptides according to the invention. In the case of solid dosage units, such as tablets, capsules and suppositoix AE *, this dose can be up to about 200 mg, but preferably about 0.1 to 100 mg, and in the case of injectable solutions in the form of ampoules, it may range up to about 200 mg, but preferably about 0.5 to 100 mg per day.

The daily dose administered is based on the body weight, age, sex and condition of the mammal. In some cases, it is nevertheless possible to administer daily doses higher or lower concentrations. Subsequent administration €ii the daily dose can be carried out both by single dosage, as a single unit dose or multiple unit dose weaker, that by multiple socket dose fragmented, at predetermined intervals.

The process of manufacturing the inventive medicaments by turning on an appropriate display one or more derivatives of lipopeptides according to the invention, in addition to customary carriers and optionally, additives and/or auxiliaries.

The compounds of formula I having 3 - Phe radicals

2

r is the acyl residue in nylnyl.e (e.g. 55, 56), which are:

particularly preferred, have further toxicological properties particularly advantageous. Thus, in the hemolysis test types, they have practically an indication of hemolysis, whereas all tested compounds, acyl radicals having aliphatic straight or branched chain, including the natural products, exhibit significant activity between 16 and 25%

ο ΐ / ; * 3

(table 1)

Table 1

Vitro hemolytic activity

2)

Fermentation product of I [R.¹ THE OH=,

R² =(HM₃ )₂ hC (HC₂ )₇ the CHCH=HM₂ coextruded].

For the measurement of the hemolytic activity, venous blood is used Rhesus, freshly obtained from. Blood are collected in small tubes heparinised, and is divided into aliquots of 200 μ 1 in 12 small tubes of polyethylene. Is added to an aliquot 200 μ 1 of distilled water, and using said mixture as a reference to 100%, and mixed in another aliquot with 1 200 μ of physiological saline (0.9% NaCl) (reference mixture to 0%). The remaining tubes is distributed in each case 200 μ 1 dilution of substances to 1,600, 800, 400, 200, 100, 50, 25, 12.5, 6.25 and 3,125 mg/1 in saline. Is rotated all tubes carefully and passed on incubated for 3 hours at 37 °c. Then added 5 ml of distilled water at reference mixture to 100% and 5 ml of physiological saline to each of the other mixed, and.

centrifuging the tubes during 5 minutes to 700 grams.

The hemolysis is determined by measuring the absorbance of the supernatant, in a spectrophotometer at a wavelength of 540 nm is assigned the value 100% to the absorption of the reference mixture to complete hydrolysis (distilled water).

Measured absorbances dilutions of test mixtures and the reference mixture to 0%, and they are indicated as a percentage of the maximum hemolysis inducible.

The present invention is illustrated by the examples the descriptive and non-limiting hereinafter. The percentages relate to the weight. In the case of liquid, the proportions relate to the volume, unless otherwise indicated.

The purity of the reaction products is determined by

.®

The HPLC analytical (Österreich Merck, the Darmstadt, LiChrospher 100rp and 8, 125 x 4 mm, elution system: water + trifluoroacetic acid, at pH 2.5, 0.1% of sodium sulfonate/acetonitrile, detection to light (BCP to 220 nm), the structure is demonstrated by electrospray mass spectroscopy (bio-Q-msec).

To simplify, is used in the following the term

2 "cyclopeptide with 1437" for the compound I wherein r=h.

Example 1

Derivative of the cyclopeptide with tridecanoyl 1437

[compound I, R.¹ AN HO=, R.² =HC₃ (CUj)₁₃ C0]

A method of coupling TOTU (tetrafluoroborate of o-to-one cyano [(ethoxycarbonydmethylidene-amino 1, 1, 3, 3 a-tetramethyl] alkylthiouronium):

a) an ACTivations L'tridécai>oic acid

113 Mg is dissolved (0,527 nmol) of tridecanoic acid in 3.75 ml of n, n-dimethylformamide (PMD), added 172.5 mg (0,526 mmol) of 1.25 g of a TOTU and êthyldiisopropylamine solution (0.5 mmol) in DMF

(0.4 mmol/gm). Dropping the solution for 1 hour at room temperature.

13 the I, ΐ ς 9 ΐ R ^{ii F.} the I '>the I' the I - 4

b.) Coupling

Is suspended in 7.2 ml of anhydrous DMF

348 mg (0,264 ramole) derivative of Fmoc-ll (R.¹ THE OH=,

R³ =fluorenylmethoxycarbonyl; example 69) and; added 2.9 g of the solution for active) (0.25 mmol) in ice bath. The solution is stirred for 1.5 brownish formed hours at ambient temperature.

^C. ) Ielimination of the Fmoc protecting group

Which is cooled to 10 °c solution B), 6 ml of piperidine is added thereto and the mixture is stirred for 1 hour at room temperature. Diluted with 250 ml of water, and then lyophilized.

d) P-urification

Is suspended in 100 ml of acetonitrile/water (5:1) the residue lyophilized, adjusted to pH 2.0 HCl the mixture with 1.5 ml of n 2, and chromatography the clear solution on 90 g of RP-silica gel ^ (Merk,

item 9303), with water +•0.01% acetonitrile/CF ^ COON. elution sequence: 500 ml of mixture 3:1, 2:1 mixture of 500 ml, 600 ml of 1:1 mixture. The objective compound appears in the fraction 1:1 (Ntn having ultraviolet detection to 2, 20). Yield: 267 mg (78% of the theoretical amount). Purity: 72%.

The chromatography again the crude product on a medium-pressure column Büchi [250 g of SR₁₈ , elution with water/0.01% CF ^ COOH-acetonitrile (3:2)]. The product fractions are lyophilized.

Yield: 130 mg, purity: 96%.

C₅₈H_g3 NR₁₃O₂₀ [1292.5], MS: 1293.

Example 2

Derivative of the cyclopeptide with 4 a-octylbenzoyle 1437

The acid chloride method :

^has ) Coextrudeduplage

6.6 Mg is dissolved (0,005 mmol) of derivative-to-Fmoc-ll (example 69) in 200 mg of a mixture of pyridine and water (9:1) and, to -20 °c, added 25 mg (0.1 mmol) of chloride 4 a-octylbenzoyle. The solution is stirred for 4 hours at ambient temperature. 2 Ml of dioxane after addition, the solvent is removed under vacuum and the residue is dissolved in 0.2 ml of DMF.

b) the ELimination of the fmoc protecting group

0.2 Ml of piperidine are added to the solution ôl), and dropping; the mixture for 1 hour at the temperature_T- ambient. The solution is diluted with 5 ml of water, and then lyophilized.

c.) Purifying

The residue is chromatographed on 10 lyophilized RP-silica gel g ^ with water + 0.01% CF ^-COOH/acetonitrile. Sequence elution: 80 ml of 3:1 mixture, 80 ml of méalnge of 2:1, 80 ml of 1:1 mixture. The fraction 1:1, containing the product, is lyophilized.

Yield: 4.6 GTN (70% of the theoretical amount), purity: 85%

^C 60^H 89^NR 13° 20^11312.51 '^{IMS 1313} '

By doing the in the example 1, we obtain the compounds of formula I mentioned hereinafter, in which

R¹ An HO==, and which carry the substituents R² indicated in table 2. Yields are 60 and 85% of the theoretical amount; the degree of purity is between 75 and 98%.

16, 6 - i-VBE1> i-Α I ' has>η * η R.

By doing in the example 2, obtained the compounds of formula I shown below, wherein R¹ An HO=, and which carry the substituents mentioned in table 3. Yields are 70 and 85 1r of the theoretical amount; the degree of purity is between 30 and 98%.

18, 4 r * f the Q u-V-λ. i 4

En. by proceeding as in the example 1 (compounds 59 to 66) or as in the example 2 (67 and 68 compounds), we obtain the compounds of formula I shown below, in which R=the NH₂ , .and which carry the substituents R standing upward trend - 1, 2 in table 4 mobilit. Yields are 75 and 85% of the theoretical amount; the degree of purity is between 80 and 98%.

Table 4

Yield: 12.2 grams, MS: 1526.7.

Example 70

Derivative of the cyclopeptide with 9 a-fluorényiméthyloxycarbonyle 1437

The mixture is stirred 48 hours at 32 °c a mixture of 10 g of the product obtained in example 69 and 300 g of wet mycelium of Actinoplanes utahensis in potassium phosphate buffer 1 1. (100 mm, pH of 7.2, the EDTA 50 mm, 0.02% sodium azide/'d) sterile. Which is then separated from the biomass by centrifugation, the solution is filtered for securing the product on 500 g of gel IOM (-Mitsubishi) and the product is eluted with a mixture of water and methanol (1:1). The eluate is concentrated to remove methanol and the aqueous solution is chromatography on 500 g of SR ^ g with water + trifluoroacetic acid mixture to 0.05% and acetonitrile (2:1). The fractions containing the product are concentrated under vacuum and freeze-dried.

Yield: 6 grams, MS: 1318.4.

Example 71

4 Acids - [(2 - [4 - (2-phenylethyl) jphényléthyl)] benzoic PWTI1 sec.

33.9 G of triphenylphosphine is added to a solution of 2.2, 9 g of methyl 4 a-bromométhylbenzoate in 1,000 ml of toluene, and the mixture is heated to reflux. The reaction is fully, completed after 7 hours. The reaction mixture is allowed to cool and the product is isolated by filtration to the horn.

Yield: 47, 6g.

Step 2

Is 58.9 g of the product of step 1 suspended in 500 RIA of anhydrous tetrahydrofuran, cooled the suspension to 0 °c and added 120 ml of a solution of bis-trimethylsilyl amide 1 m in tetrahydrofuran, after 1 hour at room temperature, the mixture is further cooled down to 0 °c is added thereto and 19.3 g of stilbene 4-aldehyde. Then the mixture is agitated during 2.5 hours at 50 °c, cooled to 0 °c is isolated by filtration and fallopian tube which deposit on the product. Then the residue is washed with 0.5 1 of THF. The organic phase is diluted with 750 ml of ethyl acetate, then washed with 750 ml of a saturated solution of ammonium chloride, extracted the aqueous phase with 750 ml of ethyl acetate, and the organic phase is dried over sodium sulfate, then concentrated. The crude product is used in the next step.

Yield: 49.9 grams.

Step 3

Is suspended in 1,000 ml of methanol 26.7 g of the crude product, from step 2, with 5 g of 10% palladium-on-carbon. The hydrogenation is carried out during

3 hours at room temperature and under atmospheric pressure the catalyst is separated by hot filtration, the solution is concentrated under vacuum and product is purified by chromatography on silica gel with a mixture of heptane and ethyl acetate (10:1).

Yield: 7.4 grams.

Step 4

1.98 G of the is product of step 3 suspended in 60 ml of ethanol, and it is added to a solution of KOH in 10 ml 508 mg of water. The solution is heated at reflux for 1.5 hour. Ethanol is removed under vacuum, the residue is taken up in 500 ml of ethyl acetate and 200 ml of water, and; adjusted to pH 2 with HCl solution 2 n the agitation of the mixture during 0.5 hour, these phases are separated and extracted again the aqueous phase with 200 ml of ethyl acetate. The organic phases are combined, dried over sodium sulfate, then concentrated under vacuum.

Yield: 1.86 g of the desired compound.

Acid chloride :

Is 1.23 g of the compound of step 4 suspended in 10 ml of thionyl chloride. The mixture is then heated at reflux until the evolution of gas is completed. After cooling the mixture, concentrated in vacuo and evaporated twice the residue with 5 ml of toluene each time.

Yield: 1.35 g of a crystalline compound light gray.

Example 72

4 - Acid [(2-biphenyl 4 yl) ethyl] benzoic acid

Eshakeri 1

Operating as in step 2 of example 71, is reacted 6.4 g of phosphonium bromide (step 1 of example 71) Free: C. 1.82 g of biphenyl-4-aldehyde.

Yield: 5.8 grams

Step 2

5.8 G of the product is subjected to step 1 to hydrogenation as in step 3 in example 71, and product is purified by chromatography.

22 said V-VI|

. Yield: 97, 0 mg.

Step 3

Is saponified as in step 4 of example 71,950 mg of the product obtained in step 2.

Yield: 880 mg.

Step 4

Operating as in step 5 in example 71, is reacted 850 mg of the product of step 3 with thionyl chloride, to afford acid chloride.

Yield: 909 mg.