WO1995006484A1

WO1995006484A1 - Lipopeptide compounds

Info

Publication number: WO1995006484A1
Application number: PCT/EP1994/002838
Authority: WO
Inventors: Wolfgang Rapp; Günther Jung; Karl-Heinz Wiesmüller
Original assignee: Rapp Polymere Gmbh
Priority date: 1993-08-31
Filing date: 1994-08-26
Publication date: 1995-03-09
Also published as: DE4329309A1

Abstract

Lipopeptide compounds have the formula (I), in which X stands for OR?1, SR1, NR1R2¿, (a), COOR4 or R-X¿1?, in which R stands for a polymer matrix and X1 for a divalent linker group, R?1, R2, R3 and R4¿ represent independently from each other a hydrogen atom, a benzyl or a C¿1?-C6-alkyl group; n is a number from 5 to 500; Y is a m-valent group, m being equal to at least 2; o stands for m-1; when o = 1, Z is an auxiliary agent, a peptide-auxiliary agent conjugate or a precursor thereof; when o > 1, Z is an auxiliary agent, a peptide, a peptide-auxiliary agent conjugate and/or a precursor thereof, whereas the auxiliary agent fraction may not be higher than the peptide fraction. Also disclosed are the salts of said lipopeptide compounds, which are particularly suitable for producing antibodies, vaccines or for affinity chromatography.

Description

-Lipopeptide compounds

The invention relates to lipopeptide compounds, their use and pharmaceutical compositions containing these compounds.

EP-A-0 000 330 describes lipopeptides of the formula:

R.— O— O— CH

• I •

R, - C0-0- CH

I CH,

I.

S I

CH.

I.

R, CO— NH— CH— CO— X. _ _R

• ^• = R or S

wherein R _j and R _{2 are,} inter alia, a saturated or unsaturated aliphatic hydrocarbon radical having 11 to 21 carbon atoms, R ^{3 is} a hydrogen atom and X is a peptide-bound natural aliphatic amino acid or an amino acid sequence of 2 to 10 natural amino acids. These lipopeptides have an immunopotentiating effect and stimulate, for example, the proliferation of B lymphocytes determined in vitro on the basis of tymidine incorporation.

EP-A-0 210 412 describes membrane anchor-active substance conjugates of the formula:

R -C0-0-CH,

R'-CO-O-CH *

I ^{2 n}

A

⁽ "| -2. ^, MIT)

R "-CO-NH-CH * -CO-X

I. where A is, inter alia, a sulfur atom, n = 0 to 5; = 1 or 2; R, R ', R "represent an alkyl group having 7 to 25 carbon atoms and X is an active ingredient or a _S pacer active ingredient group. These compounds are used for the production of antibodies and synthetic vaccines, in genetic engineering to facilitate cell fusion, for the production of cell markers , used for affinity chromatography, for liposome preparations, for medical purposes and as an additive to food or feedstuffs in the human and animal sector, and as an additive to culture media for microorganisms and for cell cultures.

The main disadvantage of these known lipopeptides is that they are not sufficiently water-soluble for most applications.

Surprisingly, it has now been found that the lipopeptide compounds based on a polyethylene glycol unit described in more detail below do not have this disadvantage, but rather have good water solubility or are notable for good solvability.

The present invention therefore relates to lipopeptide compounds of the formula I:

_X _ (_ _C H ₂ - CH ₂ 0 - * - "_ _! CH ₂ - CH ₂ - Y - Z ₀ in

X for OR ¹ ; SR ¹ ; NR ^] R ² ; NR ^] R ² R ³ ; COOR ⁴ or RX _j - stands, where

R represents a polymer matrix and X _{j represents} a divalent linker group, R ¹ , R ² , R ³ and R ⁴ independently of one another represent a hydrogen atom, a benzyl or a C _j -C ₆ alkyl group, n represents a number in the range from 5 to 500 stands,

Y is an m-valent group, where m is at least 2, o is m-1, where, if o = 1, Z is an adjuvant, a peptide

Adjuvant conjugate or a precursor thereof. when o> 1, Z stands for an adjuvant, a peptide and / or a peptide-adjuvant conjugate or a precursor thereof, preferably at least one radical Z being an adjuvant, a peptide-adjuvant conjugate or a precursor thereof and wherein the The adjuvant portion cannot be larger than the peptide portion, and the salts thereof. The alkyl group may be a methyl, ethyl, n-propyl, isopropyl, n-butyl, iso-butyl, t-butyl group, etc.

In the compounds according to the invention, part of the polyoxyethylene units can be replaced by polyoxypropylene units. One or more polyoxypropylene blocks can be present in addition to one or more polyoxyethylene blocks. The polyoxypropylene units can also be statistically distributed. Generally no more than about 30 mole percent polyoxypropylene units are present.

By installing the polyoxypropylene units, it is possible to make the solubility of the lipopeptide compounds temperature-dependent. At temperatures below body temperature, for example at 20 to 25 ° C, lipopeptide compounds with polypropylene oxide units are water-soluble, while at body temperature they are largely water-insoluble. This has the advantage that a depot effect or a stronger immune response can be achieved.

The lipopeptide compounds according to the invention comprise at least one radical Z, which can stand for an adjuvant, a peptide or a peptide-adjuvant conjugate. If there is only one residue Z in the molecule (o = 1), Z stands for an adjuvant or a peptide-adjuvant conjugate. If, on the other hand, there are several Z residues (O> 1), Z stands for an adjuvant, a peptide and / or a peptide-adjuvant conjugate. At least one Z radical here means an adjuvant or a peptide-adjuvant conjugate, ie at least one adjuvant radical is present in the molecule. The remaining residues Z then represent a peptide, the number of adjuvant molecules not greater than the number of peptide residues is.

According to the invention, any peptide can be bound to the Y radical or synthesized on the Y radical. In general, however, the peptides comprise no more than 50 amino acids.

The peptides and, as far as possible, the adjuvants are peptide, i.e. via an amide bond, whether starting from the C or N terminus, or via other acid or base stable linkers (e.g. crotonic acid, 2-nitro-4-hydroxybenzoic acid and derivatives thereof).

The adjuvants are, in particular, immunostimulants which are generally not or only slightly soluble in water. Tripalmitoyl-S-glyceryl-cysteine (Pam ₃ Cys) of the formula:

^C 15 ^H 31 ^C0 ° ~ CH ₂ C ₁₅ H ₃₁ COO - CH

to use. This compound is bound via the carboxyl group.

The group Y serves to bind the radical (s) Z to the polyoxyethylene unit. Y stands in particular for O, NH, S, -OCO-, -O-Alk-COO- or -NH-Alk-COO-, where Alk is a C _j -C ₆ alkylene group, or the others below for X _j specified residues.

If two or more Z radicals are present in the molecule, Y represents a tri- or polyvalent group which is derived from a tri- or polyfunctional compound. The functional groups must be such that they on the one hand can react with the terminal hydroxyl group of the polyoxyethylene unit or a functional equivalent thereof and on the other hand with a functional group of the adjuvant or the peptide.

Y is preferably a trivalent or tetravalent group which is derived from a diaminocarboxylic acid, aminodicarboxylic acid, dihydroxycarbonic acid, hydroxydicarboxylic acid, tricarboxylic acid, a diamino alcohol, aminodiol, triamine, triol, trithio alcohol, triisocyanate, tetrol or tetraisocyanate.

Lysine, glycerin or thioglycerin are preferred.

Y is preferably built up from several trivalent groups, in such a way that one or both free valences of the first trivalent group is connected to a valence of the next trivalent group, so that a tree-like structure of group Y e.g. with the following structures:

oσ! e-r

y

These structures can be bonded directly (Y '= chemical bond) or via a suitable linker group (Y' = Y as defined in claim 4) to the polyoxyethylene unit.

In this way it is possible to provide a plurality of adjuvant molecules and a plurality of peptide molecules in a molecule of the formula I, the ratio of adjuvant to peptide being freely variable within the scope specified above.

A particularly preferred embodiment is accordingly the compounds of the formula IV:

X- CH ₂ CH ₂ 0 -ni - CH ₂ -CH ₂ -Y - A (IV)

wherein A stands for a tree-like radical from 1 to 15 trifunctional compounds, and Y 'stands for a chemical bond or for the meanings given in claim 4 for Y, the resulting free valences with a peptide and optionally with at least one adjuvant is busy.

When X is RX--, X- is a divalent linker group that binds the polyethylene glycol part of the molecule to the polymer matrix. X _j preferably represents 0, NH, S, C00-, -O-alk-COO- or -NH-alk-COO-, where alk is a C _j -C ₆ alkylene group,

- O - CO - CH = CH - CH ₂ - O - NH - CO - CH = CH - CH ₂ - O -

O - CO - CH ₂ - O - CH = CH - CH ₂ - O - or - NH - CO - CH ₂ - O - CH = CH - CH ₂ - 0 -.

If lipopeptide compounds are desired in which the polymer matrix is split off, X _j must be such that the molecule can be split off from the polymer matrix in a customary manner, for example by acidic or basic hydrolysis or by catalytic hydrogenation. Suitable group X _j are known to the person skilled in the art. In such a case care is taken that the residues Z are not split off at the same time. This can be done using suitable linker groups Y, for example catalytically or photochemically cleavable linkers such as crotonic acid, 4-hydroxy-2-nitrobenzoic acid and derivatives thereof.

The type of polymer used is not critical. The polymer only has to be such that it has suitable functional groups or is appropriately functionalized so that the remaining part of the molecule can be bound via group X. The polymer can be linear and optionally have a molecular weight in the range from 200 to 10 million, but it can also be crosslinked. Suitable polymers are, for example, polystyrene or functionalized derivatives thereof, such as chloromethylstyrene,

Hydroxymethylstyrene or aminomethylstyrene, polyacrylic acid, polyvinyl alcohol, polyamino acids, such as polylysine and polyaminocaproic acid, polyacrylic acid, polyvinylpyrrolidone, polyamides, polyamines, polysaccharides etc.

If the polymer matrix is not split off, polyvinyl alcohol, polyacrylic acid, polyamino acids, polyvinylpyrrolidone or polysaccharides are preferably used.

The lipopeptide compounds according to the invention are produced using known methods.

1. Starting from soluble polymers and POE or functionalized derivatives of POE: the soluble polymer is reacted with the adjuvant and / or peptide or the peptide is built up directly on the soluble carrier using the liquid phase technique.

2. Starting from one of the abovementioned polymers: One of the abovementioned polymers, if appropriate after suitable functionalization, is reacted with polyoxyethylene or with a derivative thereof or the POE polymer is built up directly on the support. The product obtained is then, optionally after functionalization, reacted with the adjuvant and / or the peptide, or the peptide is built up on the support by the customary methods of solid-phase synthesis. Alternatively, the product obtained is reacted with at least one trifunctional or multifunctional compound. Finally, the adjuvant and the peptide (or the adjuvants and the peptides) are bound or built up step by step directly on the carrier.

The lipopeptide compounds according to the invention are notable for their water solubility. The lipopeptide compounds attached to the cross-linked carrier are distinguished by good solvation in aqueous systems. They can therefore be used in the form of an aqueous preparation. The lipopeptide compounds according to the invention are particularly useful for the production of antibodies, for the production of vaccines or for affinity chromatography.

The present invention also relates to a pharmaceutical composition which contains at least one of the compounds according to the invention together with customary auxiliaries and additives. The agent is preferably in aqueous form for injection.

The following examples illustrate the invention:

example 1

Production of Pam ₃ CvsPOE O CH ₃ (POE = polyoxyethylene)

3 g of α-methoxy-ω-amino-POE (MW.3000) are in

25 ml of methylene chloride dissolved. To this solution are added 1.5 mmol Pam ₃ Cys, 1.5 mmol dicyclohexylcarbodiimide, and 1.5 HOBt. This solution is stirred for 24 hours at room temperature and then the dicyclohexylurea is filtered off. The further purification is carried out by column chromatography.

In the adjuvant test, the water-soluble lipopeptide shows a clear adjuvant effect, see FIG. 1 Example 2

Production of Pam ₃ Cvs Ser POE 0 CH ₃ 3 g α-methoxy-ω-amino-POE (MW.3000) are made in 25 ml

Dissolved methylene chloride. 1.5 mmol Pam ₃ Cys Ser (tBu), 1.5 mmol dicyclohexylcarbodiimide, and 1.5 HOBt are added to this solution. This solution is stirred at room temperature for 24 h and then the dicyclohexylurea is filtered off. After the tBu protective group has been cleaved off with TFA, further purification is carried out by column chromatography.

In the A juvan test, the water-soluble lipopeptide shows a clear adjuvant effect, see Fig. 2. (Melittin each 50 μg)

Example 3

Production of Pam ₃ Cys Peptidyl Lysyl POE O CH ₃ (Peptidyl = LLGILESRG _/ ARIT)

3 g of α-methoxy-ω-amino-POE (MW.3000) are dissolved in 25 ml of methylene chloride. In accordance with the liquid phase technique, Fmoc Lys (Boc) is bound to the soluble support and the Boc protective group is first selectively removed by TFA. 1.5 mmol Pam ₃ Cys, 1.5 mmol are added to this solution

Dicyclohexylcarbodiimide, and 1.5 HOBt. This solution is stirred for 24 hours at room temperature and then the dicyclohexylurea is filtered off. The further purification is carried out by fractional crystallization. After removal of the Fmoc protective group by piperidine, the peptide is built up according to the fmoc strategy in the liquid phase. After the synthesis is complete, the protective groups are removed by means of TFA and the lipopeptide conjugate is obtained by chromatography. The laser desorption mass spectrum of the conjugate clearly shows the desired compound (see FIG. 3). After foot pad

Immunization of mice without additional adjuvant shows a stimulation of the T helper cells.

Claims

Patent claims

1 . Lipopeptide compounds of formula I:

X- CH ₂ - CH ₂ 0 -) - ". _! CH ₂ - CH ₂ - Y - Z ₀ where

X for OR ¹ ; SR ¹ ; NO ^! R ² ; NR ^ R ³ ; COOR ⁴ or RX _j - stands, where

Adjuvant conjugate or a precursor stands if o> 1, Z stands for an adjuvant, a peptide and / or a peptide-adjuvant conjugate or a precursor thereof, where the adjuvant fraction cannot be greater than the peptide fraction, and the salts from that.

2. Compounds according to claim 1, part of the

Polyoxyethylene units is replaced by polyoxypropylene units.

3. Compounds according to claim 1 or 2 of formula I, wherein X- for O, NH, S, -COO-, -O-Alk-COO- or -NH-Alk-COO-, wherein Alk is a C _j -C ₆ -Alkylene group means

- 0 - CO - CH = CH - CH ₂ - O -

- NH - CO - CH = CH - CH ₂ - O -

- 0 - CO - CH ₂ - O - CH = CH - CH ₂ - 0 - or - NH - CO - CH ₂ - O - CH = CH - CH ₂ - O -.

4. Compounds according to any one of the preceding claims, wherein Y is O, NH, S, -OCO-, -O-Alk-COO- or -NH-Alk-COO, where Alk is a C _j -C ₆ alkylene group,

- 0 - CO - CH = CH - CH ₂ - O -

- NH - CO - CH = CH - CH ₂ - O -

- O - CO - CH ₂ - 0 - CH = CH - CH ₂ - O - or

- NH - CO - CH ₂ - O - CH = CH - CH ₂ - O -.

5. Compounds according to claim 4 of the formulas II or III:

R - X _! - CH ₂ CH ₂ 0 -) - _n . _! - CH ₂ -CH ₂ - Y - adjuvant (II) R - X _! - (- CH ₂ CH ₂ 0 t CH ₂ - CH ₂ - Y - peptide adjuvant (III).

6. Compounds according to any one of claims 1 to 3, wherein Y represents a trivalent or tetravalent group which is derived from a diaminocarboxylic acid, aminodicarboxylic acid, dihydroxycarboxylic acid, hydroxydicarboxylic acid, tricarboxylic acid, a diamino alcohol, aminodiol, triamine, triol, trithio alcohol, triisocyanate, tetrol or tetraisocyanate.

7. Compounds according to claim 6, wherein Y represents a trivalent group derived from lysine, glycerol or thioglycerol.

8. Compounds according to any one of claims 1 to 7, wherein Y is composed of several trivalent groups linked together in such a way that a tree-like structure of group Y results.

9. Compounds according to claim 7 of formula IV:

X - (- CH ₂ CH ₂ 0 - - _n - CH ₂ -CH ₂ -Y - A (IV)

where A stands for a tree-like radical from 1 to 15 trifunctional compounds, and Y 'for one chemical bond or for the meanings given for Y in Claim 4, the resulting free valences being populated with a peptide and possibly with at least one adjuvant.

10. Compounds according to any one of the preceding claims, wherein the adjuvant is an immunostimulant, in particular tripalmitoyl-S-glyceryl-cysteine (Pam ₃ Cyε) iεt.

11. Pharmaceutical agent containing at least one

Connection according to one of claims 1 to 10 together with customary auxiliaries and additives.

12. Use of at least one compound according to one of claims 1 to 10 for the production of antibodies, for the production of vaccines or for affinity chromatography.