WO2017068051A1 - Dendron à base de peg et son procédé de production - Google Patents

Dendron à base de peg et son procédé de production Download PDF

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Publication number
WO2017068051A1
WO2017068051A1 PCT/EP2016/075246 EP2016075246W WO2017068051A1 WO 2017068051 A1 WO2017068051 A1 WO 2017068051A1 EP 2016075246 W EP2016075246 W EP 2016075246W WO 2017068051 A1 WO2017068051 A1 WO 2017068051A1
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formula
dendron
compound
hetero
group
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PCT/EP2016/075246
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English (en)
Inventor
Ema Zagar
Jasmina TURNSEK
David PAHOVNIK
Vladimira GABERC
Simona Jevsevar
Viktor Menart
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Lek Pharmaceuticals D.D.
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Publication of WO2017068051A1 publication Critical patent/WO2017068051A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G83/00—Macromolecular compounds not provided for in groups C08G2/00 - C08G81/00
    • C08G83/002—Dendritic macromolecules
    • C08G83/003—Dendrimers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
    • A61K47/542—Carboxylic acids, e.g. a fatty acid or an amino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
    • C08G69/02—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids
    • C08G69/08—Polyamides derived from amino-carboxylic acids or from polyamines and polycarboxylic acids derived from amino-carboxylic acids
    • C08G69/10—Alpha-amino-carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G69/00—Macromolecular compounds obtained by reactions forming a carboxylic amide link in the main chain of the macromolecule
    • C08G69/40—Polyamides containing oxygen in the form of ether groups
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
    • C08G83/00—Macromolecular compounds not provided for in groups C08G2/00 - C08G81/00
    • C08G83/002—Dendritic macromolecules
    • C08G83/003—Dendrimers
    • C08G83/004—After treatment of dendrimers

Definitions

  • the present invention relates to the synthesis of biodegradable dendrons based on poly(ethylene glycol) (PEG) and to a process for preparing the same.
  • PEG poly(ethylene glycol)
  • the present invention also relates to the use of these reagents for conjugation of biomolecules and to conjugates for use as medicament.
  • Biopharmaceuticals are very effective, both in replacement therapy when a particular protein cannot be produced by the body as well as in inhibiting therapy (e.g. antibodies for cancer treatment).
  • the first generation of biopharmaceuticals lacked features which are highly desirable for optimal drugs.
  • Their main shortcomings are sub- optimal physicochemical and pharmacokinetic properties which result from the physico- chemical instability of proteins, their limited solubility, proteolytic instability, a relatively short half-life and immunogenicity.
  • the applicability of low molar mass therapeutic proteins up to 50-60 kDa is limited also by rapid elimination from the circulation (antibodies for cancer and chronic inflammatory diseases).
  • Proteins and peptides produced by genetic engineering are used as very effective drugs, i.e., biopharmaceuticals, for the treatment of many pathophysiological states for nearly three decades.
  • biopharmaceuticals i.e., biopharmaceuticals
  • the rapid development of recombinant DNA technology enables the industrial production of recombinant human proteins, which significantly increases the availability of biological medicaments.
  • several technologies have been developed aimed at improving the properties of the first generation biopharmaceuticals with intention to improve the pharmacokinetic properties (biodistribution, metabolism and excretion of protein from the body) based on various factors, e.g., to increase the size of the protein
  • Such technologies include genetic fusion to immunoglobulins or serum proteins (albumin) and post-production modification - that is the conjugation of proteins with natural or synthetic polymers, e.g., PEGylation, polysialylation, HESylation, glycosylation, PASylation (preparation of protein conjugates with polyethylene glycol, polysialic acid, hydroxyethyl starch, oligosaccharides, polyaminoacids).
  • PEGylation polysialylation
  • HESylation glycosylation
  • PASylation preparation of protein conjugates with polyethylene glycol, polysialic acid, hydroxyethyl starch, oligosaccharides, polyaminoacids.
  • new drug delivery systems for proteins like nanoparticles, microparticles and microspheres have also been studied to improve the properties of biopharmaceuticals.
  • One successful technology for improving the properties of the first generation biopharmaceuticals is a post-production modification or conjugation of proteins with poly(ethylene glycol) (i.e., PEGylation), which is based on increasing the size or hydrodynamic volume of the protein. Due to enlarged hydrodynamic volume of PEG- protein conjugate a half-life of conjugated protein is significantly prolonged, which results in more effective and cheaper treatments. In addition, the frequency of drug administration can be reduced, which is a key advantage for patients who need an invasive drug administration by injection.
  • the attachment of PEG to protein can significantly increase the solubility of poorly- soluble (hydrophobic) proteins and, thus, their bioavailability, whereas the protein surface masked with PEG chains significantly reduces immunogenicity, toxicity and proteolytic degradation of proteins.
  • the pharmacokinetics of the protein/PEG conjugate highly depends on the architecture of the PEG. It was found that PEG with a branched architecture is much more efficient than a linear PEG of comparable molecular weight, meaning that branched conjugates show a longer half-life in vivo as compared to the linear analogues.
  • PEG-protein conjugates The drawback of PEG-protein conjugates is that PEG is non-biodegradable and, therefore, large PEG macromolecules accumulate in the body, especially in the case of long-term therapies. Accumulated PEG can cause unpredictable toxic effects and/or immune response. In some cases the attached PEG can also reduce protein activity.
  • an object of the present invention was to overcome the above mentioned drawbacks. Specifically, an object of the present invention was to provide a dendron on the basis of PEG which is biodegradable, has a defined branched structure, narrow molecular weight distribution and mono-functionality, which can efficiently increase the half-life of biomolecules after being conjugated therewith.
  • the present invention provides a dendron having the following general formula (I)
  • L -O- or -XCO-CHR 2 -NH- or -NH- wherein R is hydrogen, C1-C8 (hetero)alkyl or C1-C8 (hetero)alkyl substituted with a substituted or unsubstituted (hetero)aryl; X is -O- or -NH-
  • R and R 1 are independently a subunit of formula (II) or formula (III): -CO-(CH 2 )a-(OCH2CH 2 )b-OCH3
  • f 1 to 5
  • f, R and R 1 in formulae (I) and (III) are selected independently, wherein the dendron of formula (I) contains 0 to 30 subunits according to formula (III), wherein the dendron has a weight average molecular weight in the range of 10 to 500 kDa.
  • the present invention provides a process of producing a dendron, comprising the steps of: i) providing a compound of formula (V)
  • L -O- or -OCO-CHR 2 -NH- wherein R is hydrogen, C1-C8 (hetero)alkyl or C1-C8 (hetero)alkyl substituted with a substituted or unsubstituted (hetero)aryl; deprotecting the amino functions of the compound of formula (V); optionally reacting the so obtained deprotected compound with a compound of formula (VII)
  • step iii) can be carried out more than one time after removing the protecting groups PG , iv) optionally removing the protecting groups PG , v) reacting the so obtained deprotected compound either after step ii) or after step iv) with a compound of formula (VI);
  • L -O- or -OCO-CHR 2 -NH- wherein R is hydrogen, C1-C8 (hetero)alkyl or C1-C8 (hetero)alkyl substituted with a substituted or unsubstituted (hetero)aryl,
  • R' and R 1 are independently a subunit of formula (II) or of formula (III):
  • the present invention provides a conjugate obtainable by a reaction of a dendron according to the invention and a biomolecule comprising peptides, polypeptides and proteins.
  • the present invention provides a conjugate of the present invention for use as a medicament.
  • FIG. 1 shows the 1H NMR spectrum of the protected 2 nd generation dendron (6)
  • FIG. 2 shows a MALDI-TOF MS spectrum of the protected 2 nd generation dendron (6) according to one embodiment of the invention.
  • FIG. 3 shows the 1H NMR spectrum of the 2 nd generation dendron (7) with amine
  • FIG. 4 shows a MALDI-TOF MS spectrum of the 2 nd generation dendron (7) with amine functional groups and benzyl protected carboxyl groups according to one embodiment of the invention.
  • FIG. 5 shows the 1 H NMR spectrum of the 2 nd generation dendron (8) with PEGylated amine functional groups and benzyl protected carboxyl groups according to one embodiment of the invention.
  • FIG. 6 shows the 1 H NMR spectrum of the 2 nd generation dendron (9) with PEGylated amine functional groups and free carboxyl groups according to one embodiment of the invention.
  • FIG. 7 shows the 1 H NMR spectrum of the PEG reagent (10) with aldehyde functional group according to one embodiment of the invention.
  • Full curves are RI detector responses, dashed lines are
  • the present invention provides a dendron having the following general formula (I)
  • R' and R 1 are independently a subunit of formula (II) or formula (III):
  • f 1 to 5
  • f, R and R 1 in formulae (I) and (III) are selected independently, wherein the dendron of formula (I) contains 0 to 30 subunits according to formula (III), wherein the dendron has a weight average molecular weight in the range of 10 to 500 kDa.
  • Y is the functional group that enables covalent attachment of the dendron to a biomolecule.
  • Y is selected from -COOH (carboxylic acid), -OH (alcohol) and -CHO (aldehyde), protected or activated forms thereof.
  • Y is -CHO.
  • Y is an aldehyde, it can react with terminal amino functions of a biomolecule to form covalent imine bonds.
  • Y is an alcohol it can react with terminal carboxylic functions of a biomolecule to form covalent ester bonds.
  • Y is a carboxylic acid, it can react with terminal amino functions or alcohol functions of a biomolecule to form covalent amide or ester bonds, respectively.
  • -COOH is present in an activated form thereof.
  • the carboxylic acid is activated by an activating agent such as a carbodiimide and/or a triazol, thus attaching an activating group to the carboxyl function.
  • activating agents are DCC (dicyclohexylcarbodiimide), DIC (diisopropylcarbodiimide), HOBt (1-hydroxy-benzo-triazole), HOAt (l-hydroxy-7-aza- benzotriazole), BOP (benzotriazol-l-yloxy)tris(di-methylamio)phosphonium
  • the activating agent is selected from DCC, HOBt, NHS and mixtures thereof.
  • the above mentioned functional groups defining Y are in a protected form, in order to avoid an unwanted reaction prior the desired coupling with a biomolecule, i.e. for a better handling of the dendron.
  • Common protecting groups for -CHO, -OH or - COOH can be used.
  • the protected -CHO, -OH or -COOH groups must be deprotected under conditions that keep the dendron intact. Suitable protecting groups
  • aldehyde function can be protected for example as cyclic or acyclic ⁇ , ⁇ -acetal, as ⁇ S- acetal or as cyanohydrin, preferably as cyclic or acyclic ⁇ , ⁇ -acetal.
  • Suitable ⁇ , ⁇ -acetals are for example 1,3-dioxolane, 1,3-dioxane, 5,5-dimethyl- l,3- dioxane, 1,3-dioxepane, or a derivative thereof, or dimethoxy acetal, diethoxy acetal, diisopropoxy acetal.
  • a suitable S, S- acetal is 1,3-dithiane, while a suitable cyanohydrin is O-trimethylsilyl cyanohydrin.
  • the aldehyde protecting groups is a cyclic ⁇ , ⁇ -acetal selected from 1,3- dioxolane, 1,3-dioxane, 5,5-dimethyl- 1,3-dioxane, 1,3-dioxepane and derivatives thereof.
  • the alcohol function can be protected for example as silyl ether, as alkyl ether, as alkoxymethyl ether, as tetrahydropyranyl ether, as methylthiomethyl, as ester, as carbonate, preferably as silyl ether, alky ether, carbonate or tetrahydropyranyl ether, more preferably as silyl ether, carbonate or tetrahydropyranyl ether.
  • Suitable silyl ethers are for example trimethylsilyl (TMS), triethylsilyl (TES), triphenylsilyl (TPS), tri-isoproylsilyl (TIPS), thexyldimethylsilyl (TDS), tert-butyl- diphenylsilyl (TBDPS), tert-butyldimethylsilyl (TBDMS), di-tert-butylmethylsilyl (DTBMS), diethylisopropylsilyl (DEIPS), dimethylisopropylsilyl (DMIPS), di-tert- butylsilylene (DTBS).
  • TMS trimethylsilyl
  • TES triethylsilyl
  • TPS triphenylsilyl
  • TIPS tri-isoproylsilyl
  • TDS thexyldimethylsilyl
  • TDPS tert-butyl- diphenylsilyl
  • Suitable alkyl ethers are for example methyl ether, tert-butyl ether, benzyl ether, p- methoxybenzyl (PMB) ether, 3,4-dimethoxybenzyl (DMB) ether, trityl ether, allyl ether.
  • Suitable alkoxymethyl ethers are for example methoxymethyl (MOM), 2-methoxy- ethoxymethyl (MEM), benzyloxymethyl (BOM), p-methoxybenzyloxymethyl
  • Suitable esters are for example acetate ester, benzoate ester, pivalate ester, methoxy- acetate ester, chloroacetate ester, levulinate ester.
  • Suitable carbonates are for example benzyl carbonate (RO-Cbz), p-nitrobenzyl carbonate (RO-C0 2 PNB), tert-butyl carbonate (RO-Boc), 2,2,2-trichloroethyl carbonate (RO-Troc), 2-(trimethylsilyl)ethyl carbonate (RO-Teoc), allyl carbonate (RO-Aloc).
  • RO-Cbz benzyl carbonate
  • RO-C0 2 PNB p-nitrobenzyl carbonate
  • RO-Boc tert-butyl carbonate
  • RO-Troc 2,2,2-trichloroethyl carbonate
  • RO-Troc 2-(trimethylsilyl)ethyl carbonate
  • RO-Aloc allyl carbonate
  • the carboxylic acid function can be protected for example as alkyl ester and derivatives thereof, as alkoxyalkyl ester, as ester cleaved by ⁇ -elimination reactions, silyl ester, preferably as alkyl ester and derivatives thereof, silyl ester or ester cleaved by ⁇ - elimination reactions, more preferably as alkyl ester and derivatives thereof.
  • Suitable esters are for example methyl ester, tert-butyl ester, benzyl ester, allyl ester, phenacyl ester.
  • Suitable alkoxyalkyl ester are for example methoxymethyl (MOM), 2-methoxyethoxy- methyl (MEM), 2-(trimethylsilyl)ethoxymethyl (SEM), methylthiomethyl (MTM).
  • Suitable esters cleaved by ⁇ -elimination reactions are for example 2,2,2-trichloroethyl esters, 2-(trimethylsilyl)ethyl (TMSE) ester, 2-tosylethyl ester, 9-fluorenylmethyl (Fm) ester, 2-cyanoethyl ester, l,3-dithianyl-2-methyl (Dim) ester, 2-(2'-Pyridyl)ethyl (Pet) ester.
  • TMSE trimethylsilyl)ethyl
  • Fm 9-fluorenylmethyl
  • Fm 9-fluorenylmethyl
  • 2-cyanoethyl ester 2-cyanoethyl ester
  • Dim 2-(2'-Pyridyl)ethyl
  • Pet 2-(2'-Pyridyl)ethyl
  • Suitable silyl esters are for example triethylsilyl (TES) ester, tert-butyldimethylsilyl (TBS) ester, tert-butyldiphenylsilyl ester, trimethylsilyl (TMS) ester, triphenylsilyl (TPS) ester, tri-isoproylsilyl (TIPS) ester.
  • TES triethylsilyl
  • TBS tert-butyldimethylsilyl
  • TPS tert-butyldiphenylsilyl ester
  • TMS trimethylsilyl
  • TPS triphenylsilyl
  • TIPS tri-isoproylsilyl
  • n in the dendron of formula (I) is 1 to 5, preferably 2 to 4, most preferably 2.
  • the PEG spacer between the functional group Y and the core of the dendron is a chain having 10 to 700 units, i.e. n is 10 to 700.
  • n is 10 to 500, more preferably 20 to 230.
  • the core of the dendron is formed by an alpha amino acid bearing an amino alkyl side chain.
  • the length of the alkyl side chain is defined by z, which is an integer of 1 to 5, preferably 2 to 4. More preferably, z is 3, the core of the dendron corresponding to ornithine, or 4, the core of the dendron corresponding to lysine. Most preferably, z is 4 (lysine).
  • the amino acid core of the dendron can be in the L or D stereoisomeric form.
  • it is in the (natural) L form.
  • the PEG spacer comprising the functional group Y is attached to the amino acid core of the dendron via a group L which is -0-, -XCO-CHR - NH- or -NH-, wherein R 2 is hydrogen, C1-C8 (hetero)alkyl or C1-C8 (hetero)alkyl substituted with a substituted or unsubstituted (hetero)aryl and X is -O- or -NH-.
  • L is -O- or -XCO-CHR 2 -NH-, more preferably -XCO-CHR 2 -NH-.
  • the amino acid -XCO-CHR 2 -NH- as L is defined as above.
  • R 2 is selected from the group consisting of hydrogen, -CH 3 , -CH(CH 3 ) 2 , -CH(CH 3 )CH 2 CH 3 , -CH 2 - CH(CH 3 ) 2 , -CH 2 CH 2 SCH 3 , -CH 2 -Ph, more preferably R 2 is hydrogen or -CH 3 .
  • X -0-.
  • the PEG spacer is connected to the amino acid core of the dendron via the carboxyl terminus, this means that the PEG spacer is attached to the core of the dendron via an ester, an amino acid linker or an amide.
  • the branches of the dendron of formula (I) develop from the amino acid core of the dendron.
  • the branches are represented in formula (I) by R' and R 1 which are
  • f 1 to 5, wherein f, R and R 1 in formulae (I) and (III) are selected independently. More precisely, the branches of the dendron are represented by the subunit of formula (III) while the subunit of formula (II) represents inert termini of the branches, as explained below.
  • R' and R 1 The definition of R' and R 1 is to be understood as follows.
  • the subunit of formula (II) describes a methoxy PEG which is inert towards common functional groups such as carboxylic acid, amino function, alcohol, thiol.
  • the subunit of formula (II) hence describes the inert ends of the branches of the dendron of formula (I).
  • a is 1 to 5, preferably 2 to 4.
  • b is 10 to 700, more preferably 10 to 500, most preferably 20 to 230.
  • n a value for b of 230 corresponds to a weight of the PEG of about 10 kDa.
  • the subunit of formula (III) derives from an alpha amino acid, wherein it bears an alkyl amino side chain.
  • the length of the amino alkyl chain is defined in subunit (III) by f which is 1 to 5, preferably 2 to 4, most preferably 2.
  • the subunit of formula (III) comprises two amino functions to which the residues R' and R 1 are attached which themselves are independently a subunit of either formula (II) or (III).
  • each subunit of formula (III) comprises two branching points which enable the growth of a dendron at each branching cycle.
  • dendron means a repetitively branched molecule starting from a core containing a focal point to which a single chemically addressable group (Y) is attached by a PEG linker.
  • the chemically addressable group is the only group in a dendron that can be used for a coupling reaction e.g. to a biomolecule.
  • branching cycles generations can be carried out at branching points during the synthesis of the dendron leading to the growth of the same.
  • inert termini are present at which no further branching cycles can take place and which are chemically inert.
  • the core of the dendron is represented by the amino acid core in formula (I).
  • the chemically addressable group Y is attached to the focal point of the core via a (PEG) linker, as schematically shown below.
  • the amino acid core bears two amino groups which function as branching points.
  • a branching cycle can be carried out. Specifically, at each branching point and at each branching cycle a subunit of formula (III) (branch) can be introduced.
  • the subunit of formula (III) bears itself two amino groups which act each as branching points for a subsequent branching cycle.
  • the branching points of the last branching generation i.e. the structurally outermost branching points, are "capped" with termini represented by the subunit of formula (II) which are chemically inert and do not comprise branching points.
  • the subunit of formula (II) is attached only to the last generation of branches of the dendron which are defined by the subunit of formula (III).
  • the subunit of formula (II) is attached only to the last generation of branches of the dendron which are defined by the subunit of formula (III).
  • the amino acid core of the dendron is present, and R and R 1 are two subunits of formula (II). This means that the amino functions (branching points) of the amino acid core are capped with the inert termini represented by the subunit of formula (II).
  • the amino acid core of the dendron is attached via its two amino functions to a subunit of formula (III) each.
  • R and R 1 are the both methoxy PEG subunits of formula (II).
  • R and R 1 are the both methoxy PEG subunits of formula (II).
  • the two amino functions (branching points) of each subunit of formula (III) are capped with the inert termini represented by the subunit of formula (II). That is, in a dendron of second generation there are two subunits of formula (III) and four subunits of formula (II), the latter of which are attached to the subunits of formula (III).
  • z and f can be the same or different.
  • z and f are the same.
  • z and f are both 3 or 4.
  • the weight of the dendron of formula (I) can be controlled by the length of the PEG units, i.e. the subunits of formula (II) and the PEG linker between Y and L, as well as by the number of generations of branches.
  • the dendron of the present invention comprises 0 to 30 subunits according to formula (III). 30 subunits of formula (III) correspond to a dendron of the 5 th generation.
  • the dendron of the present invention is preferably a dendron of the 1 st , 2 nd , 3 rd , 4 th or 5 th generation, more preferably of the 2 nd , 3 rd or 4 th generation.
  • the dendron branches are grown by forming peptide bonds between amino acid building blocks, the peptide bonds can be cleaved enzymatically, resulting in biodegradability of the dendron of formula (I).
  • the weight average molecular weight of the dendron of formula (I) is in the range of 10 to 500 kDa, preferably 20 to 400 kDa, more preferably 30 to 250 kDa.
  • the weight average molecular weight of the dendron can be determined for example via SEC-MALS.
  • the number of subunits of formula (III) and the length of the PEG chains are not limited to specific combinations, as long as the weight average molecular weight of the dendron of formula (I) is in the range as defined above. This means that for short PEG units higher branch generations can be provided, whereas longer PEG units can only be used to produce lower generations.
  • m is 2 to 4
  • z and f are independently 3 or 4.
  • z in the dendron of formula (I) is 4.
  • the dendron of formula (I) has the general formula (IVa)
  • n and b are independently 10 to 500, preferably 20 to 230, and L is -O- or - XCO-CHR 2 -NH-, wherein R 2 is hydrogen or a CI to C8 alkyl, preferably -CH 3 , X is - O- or -NH-.
  • L is -XCO-CHR 2 -NH-, wherein R 2 is hydrogen or a CI to C4 alkyl, preferably -CH 3 , and X is -0-.
  • the dendron of formula (I) has the general formula (IV)
  • n and b are independently 10 to 500, preferably 20 to 230. Most preferably, n is 180 to 230 and b is 100 to 130.
  • the dendron of formulae (I), (IVa) or (IV) has a polydispersity index (PDI) of 1 to 2, preferably 1 to 1.7, more preferably 1.1 to 1.4.
  • PDI polydispersity index
  • the polydispersity index (PDI) is defined as the ratio of the weight average molecular weight (M w ) to the number average molecular weight (M n ).
  • M w can be measured by SEC-MALS as mentioned above, M n is generally determined by Gel Permeation Chromatography.
  • a second aspect of the present invention is a process of producing a dendron, comprising the steps of: i) providing a compound of formula (V)
  • z 1 to 5 and PG 1 is a protecting group for carboxyl, PG 2 is a protecting group for amino functions;
  • L -O- or -OCO-CHR 2 -NH- wherein R is hydrogen, C1-C8 (hetero)alkyl or C1-C8 (hetero)alkyl substituted with a substituted or unsubstituted (hetero)aryl, ii) deprotecting the amino functions of the compound of formula (V); iii) optionally reacting the so obtained deprotected compound with a compound of formula (VII)
  • step iii) can be carried out more than one time after removing the protecting groups PG , iv) optionally removing the protecting groups PG , v) reacting the so obtained deprotected compound either after step ii) or after step iv) with a compound of formula (VI),
  • R is hydrogen, C1-C8 (hetero)alkyl or C1-C8 (hetero)alkyl substituted with a substituted or unsubstituted (hetero)aryl, and R 1 are independently a subunit of formula (II) or of formula (III):
  • the amino functions are protected by protecting groups PG .
  • Suitable amino protecting groups are for example phthaloyl (Phth),
  • TCP tetrachlorophthaloyl
  • Dts dithiasuccinyl
  • Boc benzyloxycarbonyl
  • Cbz allyloxycarbonyl
  • Aloe 9-fluorenylmethoxycarbonyl
  • Fmoc 2-(trimethylsilyl)ethoxycarbonyl
  • Troc 2,2,2- trichloroethoxycarbonyl
  • phenylsulfonyl p-tolylsulfonyl (Ts)
  • DMPM p-methoxyphenyl
  • PMP p-methoxyphenyl
  • Tr Tr
  • Rh Tr
  • Rh 9-phenylfluorenyl
  • N- silyl derivatives N- silyl derivatives.
  • the protecting group PG is selected from the group consisting of methoxycarbonyl, ethoxycarbonyl, tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), allyloxycarbonyl (Aloe), 9-fluorenylmethoxycarbonyl (Fmoc), benzoyl (Bz) and benzyl (Bn).
  • the protecting group can be cleaved under acidic or basic conditions.
  • the protecting group can be cleaved under reductive conditions.
  • z is 1 to 5, preferably 2 to 4, more preferably 3 or 4, most preferably 4.
  • the compound of formula (V) can be present in form of the L or D stereoisomer, preferably L stereoisomer.
  • the group L is -O- or -OCO- CHR 2 -NH-, wherein R 2 is hydrogen, C1-C8 (hetero)alkyl or C1-C8 (hetero)alkyl substituted with a substituted or unsubstituted (hetero)aryl.
  • L is -O- or - OCO-CHR 2 -NH-, wherein R 2 is hydrogen, C1-C8 (hetero)alkyl or C1-C8 (hetero)alkyl substituted with a substituted or unsubstituted (hetero)aryl.
  • L is -O- or -OCO-CHR 2 -NH-, wherein R 2 is hydrogen or C1-C4 (hetero)alkyl.
  • R is selected from the group consisting of hydrogen, -CH 3 , -CH(CH ) 2 , -CH(CH 3 )CH 2 CH 3 , -CH 2 -CH(CH 3 ) 2 , -CH 2 CH 2 SCH 3 , -CH 2 -Ph, more preferably R 2 is hydrogen or -CH 3 .
  • L being -O- or -OCO-CHR -NH-, the carboxyl functional group is protected by a protecting group PG 1 .
  • the carboxylic acid function can be protected for example as alkyl ester and derivatives thereof, as alkoxyalkyl ester, as ester cleaved by ⁇ - elimination reactions, silyl ester, preferably as alkyl ester and derivatives thereof, silyl ester or ester cleaved by ⁇ -elimination reactions, more preferably as alkyl ester and derivatives thereof.
  • Suitable esters are for example methyl ester, tert- butyl ester, benzyl ester (Bn), allyl ester, phenacyl ester.
  • Suitable alkoxyalkyl ester are for example methoxymethyl (MOM), 2-methoxyethoxy- methyl (MEM), 2-(trimethylsilyl)ethoxymethyl (SEM), methylthiomethyl (MTM).
  • Suitable esters cleaved by ⁇ -elimination reactions are for example 2,2,2-trichloroethyl esters, 2-(trimethylsilyl)ethyl (TMSE) ester, 2-tosylethyl ester, 9-fluorenylmethyl (Fm) ester, 2-cyanoethyl ester, l,3-dithianyl-2-methyl (Dim) ester, 2-(2'-Pyridyl)ethyl (Pet) ester.
  • TMSE trimethylsilyl)ethyl
  • Fm 9-fluorenylmethyl
  • Fm 9-fluorenylmethyl
  • 2-cyanoethyl ester 2-cyanoethyl ester
  • Dim 2-(2'-Pyridyl)ethyl
  • Pet 2-(2'-Pyridyl)ethyl
  • Suitable silyl esters are for example triethylsilyl (TES) ester, tert-butyldimethylsilyl (TBS) ester, tert-butyldiphenylsilyl ester, trimethylsilyl (TMS) ester, triphenylsilyl (TPS) ester, tri-isoproylsilyl (TIPS) ester.
  • TES triethylsilyl
  • TBS tert-butyldimethylsilyl
  • TPS tert-butyldiphenylsilyl ester
  • TMS trimethylsilyl
  • TPS triphenylsilyl
  • TIPS tri-isoproylsilyl
  • the carboxyl is protected as benzyl ester (i.e. PG 1 is benzyl), allyl ester, phenacyl ester or 2,2,2-trichloroethyl ester.
  • PG 1 is a protecting group that can be cleaved off at the end of the formation of the branches of the dendron by hydrogenation or metal-catalysed deprotection, while the protecting groups PG are cleaved off either under acidic or basic conditions.
  • PG 1 is benzyl
  • L is -O- or -OCO-CHR 2 -
  • R is selected from the group consisting of hydrogen, -CH 3 , -CH(CH 3 ) 2 , - CH(CH 3 )CH 2 CH 3 , -CH 2 -CH(CH 3 ) 2 , z is 3 or 4
  • PG 2 is selected from the group consisting of of methoxycarbonyl, ethoxycarbonyl, tert-butoxycarbonyl (Boc), benzyloxycarbonyl (Cbz), allyloxycarbonyl (Aloe), 9-fluorenylmethoxycarbonyl (Fmoc), benzoyl (Bz) and benzyl (Bn).
  • the compound of formula (V) can be formed by a peptide bond formation by reacting a compound of formula PG 1 -OCO-CHR 2 -NH 2 or a corresponding ammonium salt thereof, such as trifluoroacetate, with the following compound of formula (Vila)
  • the carboxy function is activated by an activating (or coupling) agent such as a carbodiimide and/or a triazol, thus attaching an activating group to the carboxyl function .
  • an activating (or coupling) agent such as a carbodiimide and/or a triazol
  • activating agents are DCC (dicyclohexylcarbodiimide), DIC (diisopropylcarbodiimide), HOBt (1-hydroxy-benzotriazole), HOAt (l-hydroxy-7-aza- benzotriazole), BOP (benzotriazol-l-yloxy)tris(dimethylamio)phosphonium hexafluoro- phosphate), PyBOP (benzotriazol-l-yloxy)tris(pyrrolidino)phosphonium hexafluoro- phosphate, PyBroP (bromo)tris(pyrrolidino)phosphonium hexafluorophosphate), BroP (bromo)tris(dimethylamio)phosphonium hexafluorophosphate), HBTU (2-(lH- benzotriazole- 1 -yl)- 1,1,3 ,3-tetramethyluronium hexafluorophosphate), N-hydroxy
  • the activating reagent is selected from DCC, HOBt, NHS and mixtures thereof.
  • an organic alkaline substance preferably an amine
  • the organic alkaline substance are DBU (1,8-diaza- bicyclo[5.4.0]undec-7-en), trimethylamine (TEA) and DIPEA (diisopropylethylamine), in particular TEA.
  • the reaction can be carried out in an organic solvent, such as acetonitrile, DCM and DMF, preferably DCM.
  • the solvent is a mixture of at least two organic solvents, such as DCM/DMF.
  • the amino functions are deprotected, i.e.
  • the protecting groups PG are cleaved off.
  • the reaction can be carried out in an organic solvent or in a mixture of an organic solvent and water. Suitable organic solvents are for example alcohol and ethers, e.g. methanol, ethanol, propanol, THF and dioxan, or other organic solvents like DCM.
  • the deprotection can be carried out under acidic, basic, or oxidizing conditions.
  • the second embodiment of the invention the
  • deprotection can be carried out in the presence of a catalyst, such as Pd/C and/or hydrogen.
  • a catalyst such as Pd/C and/or hydrogen.
  • the deprotection is preferably carried out under reducing conditions, for example with hydrogen in the presence of Pd/C in water, alcohol or a mixture of both as solvent.
  • the deprotection can be carried out under acidic, basic, or reducing conditions.
  • Boc as protecting group, the deprotection can be carried out with trifluoroacetic acid (TFA).
  • step (iii) After deprotection of the amino functions of formula (V), the so obtained compound having free amino functions can be optionally reacted in step (iii) with a compound of formula (VII)
  • AG is an activating group
  • PG is a protecting group for amino functions
  • f 1 to 5.
  • PG 3 is a protecting group for amino functions like PG 1 and is defined accordingly.
  • PG1 and PG can be identical or different, preferably they are identical.
  • f is an integer in the range of 1 to 5, preferably 2 to 4, more preferably 3 or 4, most preferably 4.
  • compound of formula (VII) comprises an activating group AG which is formed by reacting an activating agent with the carboxyl functional group of the amino acid on which basis compound of formula (VII) is formed.
  • the carboxyl function is activated by an activating (or coupling) agent such as a carbodiimide and/or a triazol, thus attaching an activating group AG to the carboxyl function.
  • activating agents are DCC (dicyclohexylcarbodiimide), DIC (diisopropylcarbodiimide), HOBt (1-hydroxy-benzotriazole), HOAt (l-hydroxy-7-aza- benzotriazole), BOP (benzotriazol-l-yloxy)tris(dimethylamio)phosphonium hexafluoro- phosphate), PyBOP (benzotriazol-l-yloxy)tris(pyrrolidino)phosphonium hexafluoro- phosphate, PyBroP (bromo)tris(pyrrolidino)phosphonium hexafluorophosphate), BroP (bromo)tris(dimethylamio)phosphonium hexafluorophosphate), HBTU (2-(lH- benzotriazole- 1-yl)- 1 , 1 ,3,3-tetramethyluronium hexafluorophosphate), N-hydroxy
  • the compound of formula (VII) is identical with the compound of formula (Vila) described above.
  • step iii) a peptide bond formation is carried out between the deprotected compound of formula (V) having free amino functions and the compound of formula (VII).
  • the peptide bond formation can be carried out according to known procedures.
  • the reaction can be carried out in an organic solvent, such as acetonitrile, DCM and DMF, preferably DCM.
  • the solvent is a mixture of at least two organic solvents, such as DCM/DMF.
  • an organic alkaline substance preferably an amine, is present in the mixture. Examples of the organic alkaline substance are DBU (l,8-diazabicyclo[5.4.0]undec-7-en),
  • TEA trimethylamine
  • DIPEA diisopropylethylamine
  • each free amino function of the deprotected compound of formula (V) is coupled to a compound of formula (VII) by a peptide bond.
  • step iii) represents the growth of the dendron.
  • Each step iii) which is carried out describes the growth of the dendron by one generation.
  • the compound of formula (VII) is independently selected in each step iii). This means that in each step iii) a different or an identical compound of formula (VII) is used.
  • the compound of formula (VII) used in the first and in the last step iii) is identical while in the second step iii) a different compound of formula (VII) is used.
  • the differences in the compounds of formula (VII) can reside in different AG, PG or f, preferably they reside only in f.
  • the same compound of formula (VII) is used.
  • the dendron is a dendron of more than one generation, i.e. if at least one step iii) is carried out, after the last step iii) the protecting groups PG at the branch ends of the dendron are removed in step iv).
  • the reaction can be carried out in an organic solvent or in a mixture of an organic solvent and water. Suitable organic solvents are for example alcohol and ethers, e.g. methanol, ethanol, propanol, THF and dioxan, or other organic solvents like DCM.
  • the deprotection can be carried out under acidic, basic, or oxidizing conditions.
  • the deprotection can be carried out in the presence of a catalyst, such as Pd/C and/or hydrogen.
  • a catalyst such as Pd/C and/or hydrogen.
  • the deprotection is preferably carried out under reducing conditions, for example with hydrogen in the presence of Pd/C in water, alcohol or a mixture of both as solvent.
  • the deprotection can be carried out under acidic, basic, or reducing conditions.
  • Boc the deprotection can be carried out with trifluoroacetic acid (TFA).
  • step v) either the deprotected compound of formula (V) or the deprotected branched dendron after steps iii) and iv) is reacted with a compound of formula (VI)
  • L -O- or -OCO-CHR 2 -NH- wherein R is hydrogen, C1-C8 (hetero)alkyl or C1-C8 (hetero)alkyl substituted with a substituted or unsubstituted (hetero)aryl,
  • R' and R 1 are independently a subunit of formula (II) or of formula (III):
  • Compound of formula (VI) describes an activated methoxy PEG (PEG-OMe).
  • the carboxyl function of the compound of formula (VI) is activated by an activating (or coupling) agent such as a carbodiimide and/or a triazol, thus attaching an activating group AG to the carboxyl function.
  • activating agents are DCC (dicyclohexylcarbodiimide), DIC (diisopropyl- carbodiimide), HOBt (1-hydroxy-benzotriazole), HOAt (l-hydroxy-7-aza-benzo- triazole), BOP (benzotriazol-l-yloxy)tris(dimethylamio)phosphonium hexafluoro- phosphate), PyBOP (benzotriazol-l-yloxy)tris(pyrrolidino)phosphonium
  • the activating agent is selected from DCC, HOBt, NHS and mixtures thereof.
  • a in the compound of formula (VI) a is 1 to 5, preferably 2 to 4, more preferably 2.
  • b in the compound of formula (VI) b is 10 to 700, preferably 10 to 500, more preferably 20 to 230, most preferably 100 to 130.
  • a value for b of 120 corresponds to a weight of the PEG of about 5 kDa.
  • step v) a peptide bond formation is carried out between free amino functions of either deprotected compound of formula (V) or the deprotected branched dendron after steps iii) and iv) and the compound of formula (VI).
  • the peptide bond formation can be carried out according to known procedures.
  • the reaction can be carried out in an organic solvent, such as acetonitrile, DCM and DMF, preferably DMF.
  • the solvent is a mixture of at least two organic solvents, such as
  • DCM/DMF DCM/DMF. Additionally it is preferred that an organic alkaline substance, preferably an amine, is present in the mixture. Examples of the organic alkaline substance are DBU (l,8-diazabicyclo[5.4.0]undec-7-en), trimethylamine (TEA) and DIPEA
  • DIPEA diisopropylethylamine
  • each free amino function of the deprotected compound of formula (V) or of the deprotected branched dendron after steps iii) and iv) is coupled to a compound of formula (VI) by a peptide bond.
  • the coupling of the free amino functions of either deprotected compound of formula (V) or the deprotected branched dendron after steps iii) and iv) with the PEG-OMe agent of formula (VI) has the objective to increase the size or hydrodynamic volume of the dendron and at the same time to render the branch termini inert under normal conditions, since the methyl ether in PEG-OMe is not reactive under physiological conditions towards other functional groups such as alcohol, amine or carboxylic acid.
  • the protecting groups PG 1 are removed from the compound of formula (VIII) in step vi).
  • the removal of the protecting group PG 1 for a carboxyl group can be carried out in an organic solvent or in a mixture of an organic solvent and water.
  • Suitable organic solvents are for example alcohol and ethers, e.g. methanol, ethanol, propanol, THF and dioxan, or other organic solvents like DCM. Alternatively, it can be carried out in water.
  • the deprotection can be carried out under acidic, basic, or oxidizing conditions.
  • the deprotection can be carried out in the presence of a catalyst, such as Pd/C and/or hydrogen.
  • the deprotection is preferably carried out under reducing conditions, for example with hydrogen in the presence of Pd/C in water, alcohol or a mixture of both as solvent.
  • the deprotection can be carried out under reducing conditions, for example in the presence of a Pd catalyst such as Pd[PPh 3 ] and a phosphine, in water, alcohol, THF or a mixture thereof as solvent.
  • a Pd catalyst such as Pd[PPh 3 ] and a phosphine
  • step vii) the deprotected compound after step vi) having a free carboxyl function is reacted with a compound of formula (IX)
  • Y is the functional group that enables covalent attachment of the dendron to a polypeptide.
  • Y is selected from -COOH (carboxylic acid), -OH (alcohol) and -CHO (aldehyde), protected and activated forms thereof.
  • Y is -CHO.
  • Y is an aldehyde, it can react with terminal amino functions of a protein to form covalent imine bonds.
  • Y is an alcohol it can react with terminal carboxylic functions of a protein to form covalent ester bonds.
  • Y is a carboxylic acid it can react with terminal amino functions or alcohol functions of a protein to form covalent amide or ester bonds, respectively.
  • -COOH is present in an activated form thereof.
  • the carboxylic acid is activated by an activating agent such as a carbodiimide and/or a triazol, thus attaching an activating group to the carboxyl function.
  • activating agents are DCC (dicyclohexylcarbodiimide), DIC (diisopropylcarbodiimide), HOBt (1-hydroxy-benzotriazole), HOAt (l-hydroxy-7-aza- benzotriazole), BOP (benzotriazol- l-yloxy)tris(dimethylamio)phosphonium
  • the activating agent is selected from DCC, HOBt, NHS and mixtures thereof.
  • the above mentioned functional groups defining Y are in a protected form, in order to avoid an unwanted reaction prior the desired coupling with a protein, i.e. for a better handling of the dendron.
  • Common protecting groups for -CHO, -OH or -COOH can be used.
  • the protected -CHO, -OH or -COOH groups must be deprotected under conditions that keep the dendron intact. Suitable protecting groups are for example disclosed in P. J. Kocienski "Protecting Groups", 3 rd edition, Georg Thieme Verlag Stuttgart, 2004.
  • m in the compound of formula (IX) is 1 to 5, preferably 2 to 4, most preferably 2.
  • the PEG spacer is a chain having 10 to 700 units, i.e. n is 10 to 700.
  • n is 10 to 500, more preferably 20 to 230.
  • compound of formula (VIII) with a free carboxylic acid is obtained.
  • W -OH.
  • the peptide bond formation can be carried out according to known procedures.
  • the carboxyl function is activated by an activating (or coupling) agent such as a carbodiimide and/or a triazol, thus attaching an activating group to the carboxyl function.
  • activating agents are DCC (dicyclo- hexylcarbodiimide), DIC (diisopropylcarbodiimide), HOBt (1-hydroxy-benzotriazole), HOAt (l-hydroxy-7-aza-benzotriazole), BOP (benzotriazol-l-yloxy)tris(dimethylamio)- phosphonium hexafluorophosphate), PyBOP (benzotriazol-l-yloxy)tris(pyrrolidino)- phosphonium hexafluorophosphate, PyBroP (bromo)tris(pyrrolidino)phosphonium hexafluorophosphate), BroP (bromo)tris(dimethylamio)phosphonium hexafluoro- phosphate), HBTU (2-(lH-benzotriazole-l-yl)-l,l,3,3-tetramethyluronium
  • the activating reagent is selected from DCC, HOBt, NHS and mixtures thereof.
  • an organic alkaline substance preferably an amine
  • the organic alkaline substance are DBU (l,8-diazabicyclo[5.4.0]undec-7-en), trimethylamine (TEA) and DIPEA (diisopropylethylamine), in particular TEA.
  • the reaction can be carried out in an organic solvent, such as acetonitrile, DCM and DMF, preferably DCM.
  • the solvent is a mixture of at least two organic solvents, such as
  • the ester bond formation can be carried out according to known procedures and similarly to the peptide bond formation described above.
  • the carboxyl function is activated by an activating (or coupling) agent such as a carbodiimide and/or a triazol, thus attaching an activating group to the carboxyl function.
  • activating agents are EDC (l-Ethyl-3-(3- dimethylaminopropyl)carbodiimide), DCC (dicyclohexylcarbodiimide), DIC
  • the activating reagent is selected from DCC, HOBt, NHS and mixtures thereof. Additionally it is preferred that an organic alkaline substance, preferably an amine, is present in the mixture. Examples of the organic alkaline substance are DBU (1,8- diazabicyclo[5.4.0]undec-7-en), dimethylaminopyridine (DMAP), trimethylamine (TEA) and DIPEA (diisopropylethylamine), in particular TEA.
  • DBU 1,8- diazabicyclo[5.4.0]undec-7-en
  • DMAP dimethylaminopyridine
  • TEA trimethylamine
  • DIPEA diisopropylethylamine
  • an organic catalyst preferably a pyridinium 4-toluenesulfonate, such as 4-(dimethylamino)pyridinium 4-toluenesulfonate (DPTS)
  • DPTS 4-(dimethylamino)pyridinium 4-toluenesulfonate
  • Preferred combinations are DCC/DPTS and EDC/DMAP.
  • the reaction can be carried out in an organic solvent, such as acetonitrile, DCM and DMF, preferably DCM.
  • the solvent is a mixture of at least two organic solvents, such as DCM/DMF.
  • a further aspect of the present invention is a conjugate obtainable by a reaction of a dendron according to the present invention and a biomolecule comprising peptides, polypeptides and proteins.
  • the subject-matter of the present invention is further is a conjugate comprising a dendron of formula (I) and a biomolecule, wherein the dendron of formula (I) is linked via a peptide, ester or imine bond to the biomolecule.
  • a further subject of the present invention is a process for the preparation of a conjugate of the present invention, comprising the steps
  • biomolecules comprising an amino, hydroxyl or carboxyl comprising peptides, polypeptides and proteins can react with the inventive dendron, specifically with the single chemically addressable group Y of the dendron of formula (I). By this reaction a covalent linkage is formed.
  • biomolecules are peptides, polypeptides, proteins, antibodies and antibody derivatives, polysaccharides, steroids, nucleotides, oligonucleotides, polynucleotides, fats, polyelectrolytes, and mixtures thereof.
  • biomolecules are aspariginase, amdoxovir (DAPD), becaplermin, calcitonins, cyanovirin, denileukin, diftitox, erythropoietin (EPO), erythropoiesis stimulating protein (NESP), coagulation factors such as Factor V, Factor VII, Factor Vila, Factor VIII, Factor IX, Factor X, Factor XII, Factor XIII, von
  • Willebrand factor ceredase, cerezyme, alpha-glucosidase, collagen, cyclosporin, alpha defensins, beta defensins, exedin-4, granulocyte colony stimulating factor (G-CSF), thrombopoietin (TPO), alpha- 1 proteinase inhibitor, elcatonin, granulocyte macrophage colony stimulating factor (GM-CSF), fibrinogen, follicle stimulating hormone (FSH), human growth hormone (hGH), growth hormone releasing hormone (GHRH), GRO- beta, GRO-beta antibody, acidic fibroblast growth factor, basic fibroblast growth factor, CD-40 ligand, heparin, human serum albumin, interferons such as interferon alpha, interferon beta, interferon gamma, interferon omega, interferon tau, consensus interferon; interleukins and interleukin receptors such as interleukin-1 receptor, interleukin-2,
  • antibodies are known, e.g. antibodies directed against HER2 (e.g. antibodies directed against HER2 (e.g. antibodies directed against HER2).
  • trastuzumab examples include trastuzumab, VEGF (e.g. bevacizumab), EGF (e.g. cetuximab), CD20 (e.g. rituximab), TNF (e.g. infliximab, adalimumab).
  • VEGF e.g. bevacizumab
  • EGF e.g. cetuximab
  • CD20 e.g. rituximab
  • TNF e.g. infliximab, adalimumab
  • derivatives of antibodies are also known and comprise e.g. immune globulin Fc fusion proteins (e.g. etanercept) or Fab fragments (e.g. ranibizumab).
  • the biomolecule is a polypeptide, comprising at least one free amino or carboxyl group.
  • polypeptides examples include EPO, IFN- [alpha], IFN-[beta], IFN- [gamma], consensus IFN, Factor VII, Factor VIII, Factor IX, G-CSF, GM-CSF, hGH, insulin, FSH, PTH or mixtures thereof. Particularly preferred is G-CSF. Alternatively, EPO is particularly preferred.
  • the present invention preferably employs purified and isolated polypeptides having part or all of the primary structural conformation (i.e., continuous sequence of amino acid residues) and one or more of the biological properties (e.g., immunological properties and in vitro biological activity) and physical properties (e.g., molecular weight).
  • these polypeptides are also characterized by being the product of chemical synthetic procedures or of prokaryotic or eukaryotic host expression (e.g., by bacterial, yeast, higher plant, insect and mammalian cells in culture) of exogenous DNA sequences obtained by genomic or cDNA cloning or by gene synthesis.
  • yeast e.g., Saccharomyces cerevisiae
  • prokaryote e.g., Escherichia coli (E. coli)
  • E. coli prokaryote
  • the products of microbial expression in vertebrate (e.g., non-human mammalian and avian) cells are free of association with any human proteins.
  • polypeptides used in the invention may be glycosylated with mammalian or other eukaryotic carbohydrates or may be non-glycosylated.
  • G-CSF human granulocyte-colony stimulating factor
  • the amino acid sequence of the preferred G-CSF is shown in SEQ ID No: 1.
  • the amino acid sequence shown in SEQ ID No: 1 is preferably not glycosylated and is commercially available as Filgrastim.
  • G-CSF granulocyte-colony stimulating factor
  • the amino acid sequence of this preferred embodiment is the same as shown in SEQ ID No: 1, provided that Metl is absent.
  • the polypeptide of this preferred embodiment is glycosylated and is commercially available as Lenograstim.
  • EPO erythropoietin
  • Erythropoietin is an acid glycoprotein hormone of approximately 34 kDa.
  • Human erythropoietin is a 166 amino acid polypeptide that exists naturally as a monomer (Lin et al., 1985, PNAS 82, 7580-7584, EP 148 605 B2, EP 411 678 B2).
  • the EPO used in the context of the invention can be of any human or another mammalian source and can be obtained by purification from naturally occurring sources like human kidney, embryonic human liver or animal, preferably monkey kidney.
  • the EPO is recombinantly produced.
  • the EPO may be expressed in transgenic animals (e.g. in body fluids like milk, blood, etc.), in eggs of transgenic birds, especially poultry, preferred chicken, or in trans-genic plants or algae.
  • transgenic animals e.g. in body fluids like milk, blood, etc.
  • eggs of transgenic birds especially poultry, preferred chicken, or in trans-genic plants or algae.
  • polypeptides also can encompass analogues, agonists, antagonists, inhibitors, isomers, and pharmaceutically acceptable salt forms thereof.
  • polypeptides encompass synthetic, recombinant, native, glycosylated, and non-glycosylated forms, as well as biologically active fragments thereof, provided at least one free amino or carboxyl group is present.
  • biologically active particularly "biologically active fragment” is understood by the person skilled in the art. Particularly, a fragment or derivative is considered to be biologically active, if it retains the quality of biological activity of the parent molecule, even if the activity as such is increased or decreased. More particularly, biologically active means that the fragment or derivative should be therapeutically active if administered in a suitable dose.
  • inventive conjugates can be used as medicaments. Therefore, a further subject of the present invention is an inventive conjugate for use as a medicament.
  • the present invention also concerns a process for the preparation of a conjugate of the present invention, comprising the steps
  • the conjugation reaction can be carried out in an aqueous solution.
  • the solution preferably is buffered.
  • the pH may range from 3 to 10, preferably from 6 to 9.
  • the reaction can be carried out e.g. from 2 to 50° C, preferably from 20 to 30° C.
  • the conjugation reaction is allowed to proceed until substantially no further conjugation occurs, which can generally be determined by monitoring the progress of the reaction over time. Progress of the reaction can be monitored by withdrawing aliquots from the reaction mixture at various time points and analyzing the reaction mixture e.g.
  • the reaction time may vary, e.g. from 1 hour to 50 hours, preferably from 10 to 25 hours.
  • the reaction can be carried out under the use of a dehydrant agent, such as molecular sieves or a salt such as magnesium sulfate.
  • the peptide bond formation can be carried out under the same conditions and using the same activating agents as disclosed above for the production of the dendron of formula (I).
  • the carboxyl function is activated by an activating (or coupling) agent such as a carbodiimide and/or a triazol, thus attaching an activating group to the carboxyl function.
  • activating agents are EDC (l-Ethyl-3-(3- dimethylaminopropyl)carbodiimide), DCC (dicyclohexylcarbodiimide), DIC
  • the activating reagent is selected from DCC, HOBt, NHS and mixtures thereof. Additionally it is preferred that an organic alkaline substance, preferably an amine, is present in the mixture. Examples of the organic alkaline substance are DBU (1,8- diazabicyclo[5.4.0]undec-7-en), dimethylaminopyridine (DMAP), trimethylamine (TEA) and DIPEA (diisopropylethylamine), in particular TEA.
  • DBU 1,8- diazabicyclo[5.4.0]undec-7-en
  • DMAP dimethylaminopyridine
  • TEA trimethylamine
  • DIPEA diisopropylethylamine
  • an organic catalyst preferably a pyridinium 4-toluenesulfonate, such as 4-(dimethylamino)pyridinium 4-toluenesulfonate (DPTS)
  • DPTS 4-(dimethylamino)pyridinium 4-toluenesulfonate
  • Preferred combinations are DCC/DPTS and EDC/DMAP.
  • the reaction can be carried out in an organic solvent, such as acetonitrile, DCM and DMF, preferably DCM.
  • the solvent is a mixture of at least two organic solvents, such as DCM/DMF.
  • the ester bond formation can be carried out as described above.
  • composition comprising (a) a conjugate according to the present invention.
  • pharmaceutically acceptable excipient means an excipient that can be included in the compositions of the invention and that causes no significant adverse toxicological effects to a patient.
  • excipients examples include carbohydrates, antimicrobial agents, surfactants, buffers, acids, bases, antioxidants, inorganic salts, and mixtures thereof.
  • carbohydrate excipients examples include monosaccharides, such as fructose, maltose, galactose, glucose, D-mannose, sorbose, disaccharides, such as lactose, sucrose, trehalose; polysaccharides, such as starches; and alditols, such as mannitol, sorbitol.
  • an inorganic salt or buffer examples include citric acid, sodium chloride, potassium chloride, sodium sulfate, potassium nitrate, sodium phosphate monobasic, sodium phosphate dibasic, and combinations thereof.
  • surfactants examples include polysorbates, such as “Tween 20” and “Tween 80,” sorbitan esters; lipids, such as phospholipids such as lecithin, fatty acids and fatty acid esters; steroids, such as cholesterol; and chelating agents, such as EDTA.
  • the pharmaceutical composition of the present invention comprises all types of formulations, wherein those that are suited for injection are preferred.
  • the amount of the conjugate in the composition will vary, but will preferably be a therapeutically effective dose when the composition is stored in a unit dosage form (e.g., a vial).
  • the pharmaceutical preparations of the present invention are preferably administered via injection and are therefore generally liquid solutions or suspensions.
  • the invention should be illustrated by the following examples.
  • Size-exclusion chromatography coupled to a multi-angle light-scattering detector (SEC/MALS).
  • the separation of fully protected dendrons was carried out in 0.05 M LiBr/DMAc (N,N- dimethylacetamide) using an Agilent 1260 HPLC chromatograph, a PolarGel-L analytical column (7.5 mm x 300 mm) with a precolumn (Agilent Techn.).
  • the PolarGel-L column has a wide pore size distribution, which covers the molar masses up to 30kDa.
  • MALS with 18 angles detector
  • RI differential refractive index detector
  • the nominal eluent flow rate was 1 mL/min, the injection volume was typically 100 ⁇ , and the mass of the samples injected onto the column was typically 200 ⁇ g.
  • HO-PEG-CHO hydroxyl-poly(ethylene glycol)-aldehyde
  • dendrons with PEGylated amino functional groups and free carboxyl group as well as branched PEG reagent with aldehyde functional group 0.05 M NaN0 3 /H 2 0 as a mobile phase was used.
  • RP-HPLC Preparative Reversed-Phase Liquid-Adsorption Chromatography
  • Solvent A 5 mM aqueous ammonium acetate time (min) solvent A (vol. %) solvent B (vol. %
  • MALDI-TOF Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
  • MALDI-TOF mass spectra were recorded on a Bruker UltrafleXtreme MALDI-TOF- TOF mass spectrometer (Bruker Daltonik, Bremen, Germany). Samples were dissolved either in THF or 30% mixture of ACN in 0.1% TFA. Matrices used were DHB (2,5- dihydroxybenzoic acid) or DCTB (iraw5 , -2-[3-(4-ieri-butylphenyl)-2-methyl-2- propenylidene]malononitrile) with NaTFA or TFA as cationizer. Solutions of sample/matrix/cationizer were spotted on the target plate using dried-droplet method. Spectra were recorded in a positive reflector mode. The calibration was made externally with a Peptide calibration standard II (Bruker Daltonics) using nearest-neighbor positions.
  • the present invention will be exemplarily demonstrated by the synthesis route leading to a 2 nd generation PEG dendron (10).
  • the synthesis route is summarized in the following Schemes 1-4.
  • Boc-glycine (1) (62 mmol), N,N " -dicyclohexylcarbodiimide (DCC) (67.8 mmol) and 4- (N,N'-dimethylamino)pyridinium 4-toluensulfonate (DPTS) (1.86 mmol) were dissolved in DCM (180 mL) and set on an ice-bath. Benzyl alcohol (BnOH) (67.6 mmol) was added drop-wise. Reaction mixture was stirred over night. Precipitated dicyclohexylurea (DCU) was filtered off and washed with DCM.
  • DCM N,N " -dicyclohexylcarbodiimide
  • DPTS 4- (N,N'-dimethylamino)pyridinium 4-toluensulfonate
  • Step E Coupling of 5 with Boc-lysine with activated carboxyl group to yield a 2 nd generation dendron
  • Step G Coupling of methoxy-poly(ethylene glycol) -succinimide activated carboxyl group (carboxylated-mPEG, NHS active ester) with amino terminal groups of the 2 nd generation dendron via amide bonds 7 (0.0204 mmol) was dissolved in DMF (2 mL) and N,N'-diisopropylethylamine (0.49 mmol) was added.
  • Step H Deprotection of benzyl protected carboxyl group of the PEGylated 2 nd generation dendron
  • Step I Coupling of hydroxyl-poly(ethylene glycol)-aldehyde (HO-PEG-CHO) with carboxyl group in focal point of the PEGylated 2 nd generation dendron via ester bond
  • a fresh solution of DCC (10 mg/mL) and DPTS (10 mg/mL) in DCM were separately prepared.
  • the reaction was performed in dry-box. 9 (0.0049 mmol) was dissolved in DCM (0.2 mL). Remaining reagents are added in two parts. First 2/3 of DCC solution (0.0036 mmol) and 2/3 of DPTS solution (0.001 mmol) were added to solution of 9 in DCM. After 5 minutes 2/3 of HO-PEG-CHO (0.0034 mmol) was added. After 24 hours the remaining 1/3 of DCC solution (0.0018 mmol), 1/3 of DPTS (0.0005 mmol) and 1/3 of HO-PEG-CHO (0.0017 mmol) were added to reaction mixture.

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Abstract

L'invention concerne un procédé de préparation de dendrons biodégradables à base de poly(éthylèneglycol) (PEG), ainsi que de nouveaux dendrons biodégradable à base de PEG. La présente invention concerne également des conjugués des dendrons biodégradables de la présente invention avec des biomolécules et des conjugués destinés à être utilisés en tant que médicaments.
PCT/EP2016/075246 2015-10-21 2016-10-20 Dendron à base de peg et son procédé de production WO2017068051A1 (fr)

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Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0148605B2 (fr) 1983-12-13 1998-12-23 Kirin-Amgen, Inc. Production d'érythropoiétine
EP0411678B2 (fr) 1984-12-04 2000-01-12 Genetics Institute, Inc. Méthode de production de l'érythropoiétine
WO2007082331A1 (fr) * 2006-01-20 2007-07-26 Starpharma Pty Limited Macromolecule modifiee
WO2008017122A1 (fr) * 2006-08-11 2008-02-14 Starpharma Pty Ltd Agent de contraste dendrimère de polylysine
WO2011140376A1 (fr) * 2010-05-05 2011-11-10 Prolynx Llc Libération contrôlée de médicament à partir de dendrimères
WO2012167309A1 (fr) * 2011-06-06 2012-12-13 Starpharma Pty Ltd Macromolécules

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0148605B2 (fr) 1983-12-13 1998-12-23 Kirin-Amgen, Inc. Production d'érythropoiétine
EP0411678B2 (fr) 1984-12-04 2000-01-12 Genetics Institute, Inc. Méthode de production de l'érythropoiétine
WO2007082331A1 (fr) * 2006-01-20 2007-07-26 Starpharma Pty Limited Macromolecule modifiee
WO2008017122A1 (fr) * 2006-08-11 2008-02-14 Starpharma Pty Ltd Agent de contraste dendrimère de polylysine
WO2011140376A1 (fr) * 2010-05-05 2011-11-10 Prolynx Llc Libération contrôlée de médicament à partir de dendrimères
WO2012167309A1 (fr) * 2011-06-06 2012-12-13 Starpharma Pty Ltd Macromolécules

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
LIN ET AL., PNAS, vol. 82, 1985, pages 7580 - 7584
LISA M. KAMINSKAS ET AL: "The Impact of Molecular Weight and PEG Chain Length on the Systemic Pharmacokinetics of PEGylated Poly l-Lysine Dendrimers", MOLECULAR PHARMACEUTICS, vol. 5, no. 3, 4 May 2008 (2008-05-04), pages 449 - 463, XP055159689, ISSN: 1543-8384, DOI: 10.1021/mp7001208 *
OHKAWA H ET AL: "SYNTHESIS OF MULTIACYL POLY(ETHYLENE GLYCOL) FOR THE CONJUGATION OFCYTOCHROME C TO PHOSPHOLIPID VESICLE", BIOCONJUGATE CHEMISTRY, ACS, WASHINGTON, DC, US, vol. 11, no. 6, 1 November 2000 (2000-11-01), pages 815 - 821, XP001015727, ISSN: 1043-1802, DOI: 10.1021/BC000025N *
P. J. KOCIENSKI: "Protecting Groups, 3rd ed.", 2004, GEORG THIEME VERLAG

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