WO2011005980A1 - Nouveaux réactifs hétérobifonctionnels à base de polyéthylène glycol, leur préparation et leurs utilisations - Google Patents

Nouveaux réactifs hétérobifonctionnels à base de polyéthylène glycol, leur préparation et leurs utilisations Download PDF

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WO2011005980A1
WO2011005980A1 PCT/US2010/041394 US2010041394W WO2011005980A1 WO 2011005980 A1 WO2011005980 A1 WO 2011005980A1 US 2010041394 W US2010041394 W US 2010041394W WO 2011005980 A1 WO2011005980 A1 WO 2011005980A1
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branched
straight
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Daniel E. Levy
Samuel Zalipsky
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Intradigm Corporation
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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
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/333—Polymers modified by chemical after-treatment with organic compounds containing nitrogen
    • C08G65/33396—Polymers modified by chemical after-treatment with organic compounds containing nitrogen having oxygen in addition to nitrogen
    • 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
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • 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
    • C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
    • C08G65/02—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule from cyclic ethers by opening of the heterocyclic ring
    • C08G65/32—Polymers modified by chemical after-treatment
    • C08G65/329—Polymers modified by chemical after-treatment with organic compounds
    • C08G65/333—Polymers modified by chemical after-treatment with organic compounds containing nitrogen
    • C08G65/33331—Polymers modified by chemical after-treatment with organic compounds containing nitrogen containing imide group
    • C08G65/33337—Polymers modified by chemical after-treatment with organic compounds containing nitrogen containing imide group cyclic
    • C—CHEMISTRY; METALLURGY
    • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
    • C08L—COMPOSITIONS OF MACROMOLECULAR COMPOUNDS
    • C08L2203/00—Applications
    • C08L2203/02—Applications for biomedical use

Definitions

  • the invention relates to novel heterobifunctional polyethylene glycol reagents, methods of producing them and methods of using them.
  • PEG Polyethylene glycol
  • monofunctionalized PEG from a mixture of unmodified PEG, monofunctionalized PEG and bifunctionalized PEG.
  • bifunctional PEG reagents contain reactive groups on both ends of the PEG reagent. These bifunctional reagents may contain the same reactive group on both terminal ends of the PEG (i.e., homobifunctional PEG reagents) or different groups (i.e., heterobifunctional PEG reagents). Heterobifunctional PEG reagents provide advantages over homobifunctional PEG reagents in that each functional group of the heterobifunctional PEG reagent can form a covalent attachment with a different molecule on each terminus.
  • modified PEG reagents can be complicated and lengthy, particularly when different functionalities are desired on each end of the PEG reagent (i.e., heterobifunctional PEG reagents). Further, the coupling of the PEG reagent to biologically relevant target molecules can proceed with less than desirable levels of efficiency and target specificity.
  • the present invention provides a PEG reagent comprising a compound of Formula (I):
  • each Ri is independently selected from the group consisting of branched or straight-chain C 1 -C 6 alkyl, branched or straight-chain C 2 -C 6 alkenyl, and branched or straight-chain C 2 -C 6 alkynyl,
  • Ri can be taken together to form a 5- or 6-membered cyclic acetal or thioacetal
  • each Ri is independently, optionally substituted with one or more R 2 , or when both Ri are taken together to form a 5- or 6-membered cyclic acetal or thioacetal, the cyclic acetal or thioacetal is optionally substituted with one or more
  • each R 2 is independently selected from the group consisting of branched or straight-chain Ci-C ⁇ alkyl, branched or straight-chain C2-C6 alkenyl, branched or straight-chain C 2 -C 6 alkynyl, hydroxy, Ci-C 6 alkoxy, -CO 2 H, -CO 2 (RJi -CONH 2 , -CONH(RJ] -CON(RJi, carbocyclyl, heterocyclyl, aryl, aralkyl, heteroaryl, heteroaralkyl, nitro, cyano, and halo,
  • each carbocyclyl, heterocyclyl, aryl, aralkyl, heteroaryl, heteroaralkyl may be optionally substituted with one or more R5;
  • R3 is CO-CI 4 aryl, C5-C14 heteroaryl or C5-C14 heterocyclyl,
  • heteroaryl or heterocyclyl contains one or more heteroatoms selected from the group consisting of N, N(R 4 ), O, S, S(O), and S(O) 2 , and
  • R3 is optionally substituted with one or more R5;
  • R4 is selected from the group consisting of hydrogen, branched or straight- chain Ci-C 6 alkyl, branched or straight-chain C 2 -Ce alkenyl, branched or straight- chain C 2 -C 6 alkynyl, wherein one or more hydrogens in the alkyl, alkenyl or alkynyl chain may be replaced by one or more halogens;
  • R 5 is selected from the group consisting of branched or straight-chain Ci-C 6 alkyl, branched or straight-chain C 2 -Ce alkenyl, branched or straight-chain C 2 -Ce alkynyl, -CF 3 , -Rg-ORg-OH, -SH, -SR ⁇ protected OH (e.g., acyloxy), -NO 2 , -CN, - NH 2 , -NHRp-N(RJi, -NHCORg-NHCONH 2 , -NHCONHRg-NHCON(RJi, - NRCORg-NHCO 2 H, -NHCO 2 Rg-CO 2 Rg-CO 2 H, -CORg-CONH 2 , -CONHRg- CON(RJi, -S(O) 2 H, -S(O) 2 Rg-S(O) 3 H, -S(O) 3 Rg-S(O) 2 NH 2
  • RQs selected from the group consisting of hydrogen, branched or straight- chain Ci-C 6 alkyl, branched or straight-chain C 2 -Ce alkenyl, branched or straight- chain C 2 -C 6 alkynyl, carbocyclyl, heterocyclyl, aryl, aralkyl, heteroaryl, or heteroaralkyl and each RQs optionally substituted with one or more halogen, nitro, cyano, amino, -NH-(unsubstituted aliphatic), -N-(unsubstituted aliphatic)2, carboxy, carbamoyl, hydroxy, -O-(unsubstituted aliphatic), -SH, -S-(unsubstituted aliphatic), - CF 3 , -S(O) 2 NH 2 ainsubstituted aliphatic, unsubstituted carbocyclyl, unsubstituted heterocyclyl
  • A is selected from the group consisting of Ci-C 6 alkyl, C 2 -C 6 alkenyl, and C 2 - C O alkynyl;
  • A is optionally substituted with branched or straight-chain C 1 -
  • C 6 alkyl branched or straight-chain C 2 -C 6 alkenyl, branched or straight-chain C 2 -C 6 alkynyl, hydroxy, Ci-C 6 alkoxy, CO 2 H, CO 2 (Ci-C 6 alkyl), CONH 2 , CONH(Ci-C 6 alkyl), CON(Ci-C 6 alkyl) 2 , nitro, cyano, or halo;
  • each X is independently O or S;
  • j is an integer from O to 10;
  • n is an integer from 1 to 1,500.
  • Formula (I) has the following formula:
  • PEGs include PEG3400 and PEG8000.
  • the present invention provides a PEG reagent comprising a compound of Formula (II):
  • each Ri is independently selected from the group consisting of branched or straight-chain Ci-C 6 alkyl, branched or straight-chain C 2 -C 6 alkenyl, and branched or straight-chain C 2 -C 6 alkynyl,
  • Ri can be taken together to form a 5- or 6-membered cyclic acetal or thioacetal
  • each Ri is independently, optionally substituted with one or more R 2 , or when both Ri are taken together to form a 5- or 6-membered cyclic acetal or thioacetal, the cyclic acetal or thioacetal is optionally substituted with one or more
  • each R 2 is independently selected from the group consisting of branched or straight-chain Ci -C O alkyl, branched or straight-chain C2-C6 alkenyl, branched or straight-chain C 2 -C 6 alkynyl, hydroxy, Ci-C 6 alkoxy, -CO 2 H, -CO 2 (RJi -CONH 2 , -CONH(RJ] -CON(RJi, carbocyclyl, heterocyclyl, aryl, aralkyl, heteroaryl, heteroaralkyl, nitro, cyano, and halo,
  • each carbocyclyl, heterocyclyl, aryl, aralkyl, heteroaryl, heteroaralkyl may be optionally substituted with one or more R 7 ;
  • R 4 is selected from the group consisting of hydrogen, branched or straight- chain Ci-C 6 alkyl, branched or straight-chain C 2 -Ce alkenyl, branched or straight- chain C 2 -C 6 alkynyl, wherein one or more hydrogens in the alkyl, alkenyl or alkynyl chain may be replaced by one or more halogens;
  • R 6 is C 6 -Ci 4 aryl, C5-C14 heteroaryl or C5-C 14 heterocyclyl
  • heteroaryl or heterocyclyl contains one or more heteroatoms selected from the group consisting of N, N(R 4 ), O, S, S(O), and S(O) 2 , and
  • Re is optionally substituted with one or more R7;
  • R 7 is selected from the group consisting of branched or straight-chain Ci-C 6 alkyl, branched or straight-chain C 2 -C 6 alkenyl, branched or straight-chain C 2 -C 6 alkynyl, -CF 3 , -Rg-ORg-OH, -SH, -SRPprotected OH (e.g., acyloxy), -NO 2 , -CN, - NH 2 , -NHRp-N(RJl, -NHCOR 0-NHCONH 2 , -NHCONHRg-NHCON(RJ 2 , - NRCORf]-NHCO 2 H, -NHCO 2 Rf]-CO 2 Rg-CO 2 H, -CORg-CONH 2 , -CONHRg- CON(R[ ⁇ , -S(O) 2 H, -S(O) 2 RO-S(O) 3 H, -S(O) 3 Rg-S(O) 2
  • A is selected from the group consisting of Ci -C O alkyl, C 2 -C O alkenyl, and C 2 - C O alkynyl;
  • A is optionally substituted with branched or straight-chain C 1 - C ⁇ alkyl, branched or straight-chain C 2 -C 6 alkenyl, branched or straight-chain C 2 -C 6 alkynyl, hydroxy, Ci-C 6 alkoxy, CO 2 H, CO 2 R 6 , CO 2 (Ci-C 6 alkyl), CONH 2 ,
  • each X is independently O or S;
  • j is an integer from O to 10;
  • n is an integer from 1 to 1,500.
  • Formula (II) has the following formula:
  • PEG3400 While any PEG moiety can be used in the compounds of Formula (II), preferred PEGs include PEG3400 and PEG8000.
  • the present invention provides a PEG reagent comprising a compound of Formula (III):
  • Ri, R2, R4, R 6 , R7, R0X, j, and n are defined as in Formula (II);
  • each B is any natural amino acid, or an unnatural alpha or beta amino acid, wherein each is independently optionally substituted with one or more -CO 2 Re or R 7 ;
  • any -CO 2 H present in B is optionally substituted with R 6 to yield a -CO 2 R 6 moiety; and k is ⁇ in integer from 1 to 5.
  • Formula (III) has the following formula:
  • PEG3400 While any PEG moiety can be used in the compounds of Formula (III), preferred PEGs include PEG3400 and PEG8000.
  • the present invention provides a method for producing a heterobifunctional PEG according to Scheme I:
  • Ri, R 2 , R 3 , R 4 , R 5 , A, X, j and n are defined as in Formula (I);
  • Rs is selected from the group consisting of branched or straight-chain CI-C ⁇ alkyl, branched or straight-chain C 2 -Ce alkenyl, branched or straight-chain C 2 -C 6 alkynyl, C 6 -Cu aryl, C O -C I4 carbocycle, Cs-Ci 4 heteroaryl, and C5-C14 heterocycle, wherein each alkyl, alkenyl and alkynyl is optionally substituted with one or more R 2 ,
  • each heteroaryl or heterocycle contains one or more heteroatoms selected from the group consisting of N, N(R 4 ), O, S, S(O), and S(O)2, and
  • each aryl, carbocycle, heteroaryl, or heterocycle is optionally substituted with one or more R5;
  • Rg taken with the -OC(O)O- it is attached to, is any synthetically useful carbonate.
  • carbamate (D) reacted with amine to yield carbamate (D).
  • the ester of (D) is hydrolyzed (e.g., under basic conditions) to yield carboxylic acid (E).
  • carboxylic acid (E) is converted to the heterobifunctional PEG of Formula (I) using standard techniques in the art.
  • the present invention provides a method for producing a heterobifunctional PEG according to Scheme II:
  • R 1 , R 2 , R 4 , R O , R 7 , A, X, j, and n are defined as in Formula (II);
  • R 8 is defined as in Scheme I.
  • the PEG mixture containing (G) is hydrolyzed (e.g., under basic conditions) and then subjected to ion exchange chromatography to yield the pure carboxylic acid (H).
  • Carboxylic acid (H) is then converted the heterobifunctional PEG of Formula (II) using standard techniques in the art.
  • the present invention provides a method for producing a heterobifunctional PEG according to Scheme III: Scheme III
  • R 1 , R 2 , R 4 , Re, R 7 , B, X, j, k, and n are defined as in Formula (III);
  • R 8 is defined as in Scheme I. 2
  • (B )k contains more than one carboxylic acid (i.e., when k is greater than 1 , and/or one or more B S are substituted with or naturally comprise a carboxylic acid)
  • certain carboxylic acids may be protected as, e.g., -CO 2 Rs esters, where appropriate.
  • the PEG mixture containing (J) is hydrolyzed (e.g., under basic conditions) and then subjected to ion exchange chromatography to yield the pure carboxylic acid (K).
  • Carboxylic acid (K) is then converted the heterobifunctional PEG of Formula (III) using standard techniques in the art.
  • (B)k contains more than one carboxylic acid (Le., when k is greater than 1, and/or one or more B ⁇ are substituted with or naturally comprise a carboxylic acid), some or all of the carboxylic acids may be activated as, e.g., -CO2R6 esters, where appropriate.
  • the present invention provides methods of using a heterobifunctional PEG to generate a vector of Formula (IV):
  • (X) and (Y) can be a small molecule, protein, polypeptide, peptide, monosaccharide, polysaccharide, antibody, polynucleotide, oligonucleotide, other polymeric species, polypeptide side chain or a biologically relevant targeting moiety, or a fragment, dimer, trimer or oligomer thereof.
  • (X) is typically conjugated via an imine, oxime or amine linkage (represented by a squiggly bond).
  • (X) and (Y) are simultaneously reacted with a heterobifunctional PEG. In other embodiments, (X) and (Y) are sequentially reacted with a heterobifunctional PEG. In yet other embodiments, (Y) is reacted with a heterobifunctional PEG and the acetal end of the resulting product is then hydro lyzed to an aldehyde prior to reacting with (X).
  • (X) is a cationic polymer. In some embodiments, (Y) is a targeting moiety. In one embodiment, (X) is a cationic polymer and (Y) is a targeting moiety.
  • the present invention provides methods of using a heterobifunctional PEG to generate a vector of Formula (V):
  • (X) and (Y) can be a small molecule, protein, polypeptide, peptide, monosaccharide, polysaccharide, antibody, polynucleotide, oligonucleotide, other polymeric species, polypeptide side chain or a biologically relevant targeting moiety, or a fragment, dimer, trimer or oligomer thereof.
  • (X) is typically conjugated via an imine, oxime or amine linkage (represented by a squiggly bond).
  • (X) and (Y) are simultaneously reacted with a heterobifunctional PEG. In other embodiments, (X) and (Y) are sequentially reacted with a heterobifunctional PEG. In yet other embodiments, (Y) is reacted with a heterobifunctional PEG and the acetal end of the resulting product is then hydro lyzed to an aldehyde prior to reacting with (X).
  • (X) is a cationic polymer. In some embodiments, (Y) is a targeting moiety. In one embodiment, (X) is a cationic polymer and (Y) is a targeting moiety.
  • the present invention provides methods of using a heterobifunctional PEG to generate a vector of Formula (VI):
  • O H V ⁇ comprising the s tep of re nd (Y), wherein (X) and (Y) can be a small molecule, protein, polypeptide, peptide, monosaccharide, polysaccharide, antibody, polynucleotide, oligonucleotide, other polymeric species, polypeptide side chain or a biologically relevant targeting moiety, or a fragment, dimer, trimer or oligomer thereof.
  • (X) is typically conjugated via an imine, oxime or amine linkage (represented by a squiggly bond).
  • (Y) is attached via a PEG or aminoethoxyalkyl moiety. In other embodiments (Y) is attached via an aminoethoxy ethyl moiety.
  • B can be any natural amino acid, or an unnatural alpha or beta amino acid, wherein each B is independently optionally substituted with one or more -CO 2 R O or R 7 . Further, any -CO2H present in B is optionally substituted with R ⁇ to yield a -CO2R 6 moiety.
  • a vector of Formula (VI) may comprise one or more (Y) moieties (attached by, for example, coupling via a -CO2R6 moiety).
  • the vector of Formula (VI) comprises 1-10 (Y) moieties.
  • the vector of Formula (VI) comprises 1-5 (Y) moieties.
  • the vector of Formula (VI) comprises 2-4 (Y) moieties.
  • the vector of Formula (VI) comprises 3 (Y) moieties.
  • (X) and (Y) are simultaneously reacted with a heterobifunctional PEG. In other embodiments, (X) and (Y) are sequentially reacted with a heterobifunctional PEG. In yet other embodiments, (Y) is reacted with a heterobifunctional PEG and the acetal end of the resulting product is then hydro lyzed to an aldehyde prior to reacting with (X).
  • (X) is a cationic polymer.
  • (Y) is a targeting moiety.
  • (X) is a cationic polymer and (Y) is a targeting moiety.
  • (Y) comprises a monosaccharide or polysaccharide optionally attached via a PEG or aminoethoxyalkyl moiety.
  • (Y) comprises N-acetyl galactosamine or an N-acetyl galactosamine beta amino ethoxy ethyl glycoside.
  • the present invention provides a method of using a heterobifunctional PEG to generate a vehicle for targeted nucleic acid delivery of Formula (VII):
  • (X) and (Y) are as described in the context of Formula (IV).
  • (X) is a cationic polymer.
  • (Y) is a targeting moiety.
  • (X) and (Y) are simultaneously reacted with heterobifunctional PEG. In other embodiments, (X) and (Y) are sequentially reacted with heterobifunctional PEG. In yet other embodiments, (Y) is reacted with a heterobifunctional PEG and the acetal end of the resulting product is then hydro lyzed to an aldehyde prior to reacting with (X).
  • the present invention provides a method of using a heterobifunctional PEG to generate a vehicle for targeted nucleic acid delivery of Formula (VIII):
  • (X) is typically conjugated via an imine, oxime or amine linkage (represented by a squiggly bond).
  • (X) and (Y) are as described in the context of Formula (IV).
  • (X) is a cationic polymer.
  • (Y) is a targeting moiety.
  • (X) and (Y) are simultaneously reacted with heterobifunctional PEG. In other embodiments, (X) and (Y) are sequentially reacted with heterobifunctional PEG. In yet other embodiments, (Y) is reacted with a heterobifunctional PEG and the acetal end of the resulting product is then hydro lyzed to an aldehyde prior to reacting with (X).
  • the present invention provides a method of using a heterobifunctional PEG to generate a vehicle for targeted nucleic acid delivery of
  • (X) is typically conjugated via an inline, oxime or amine linkage (represented by a squiggly bond).
  • (X) and (Y) are as described in the context of Formula (IV).
  • (X) is a cationic polymer.
  • (Y) is a targeting moiety.
  • (Y) is attached via a PEG or aminoethoxyalkyl moiety. In other embodiments (Y) is attached via an aminoethoxy ethyl moiety.
  • B can be any natural amino acid, or an unnatural alpha or beta amino acid, wherein each B is independently optionally substituted with one or more -CO2R0 or R 7 . Further, any -CO2H present in B is optionally substituted with R ⁇ to yield a -CO2R6 moiety.
  • a vehicle of Formula (IX) may comprise one or more (Y) moieties (attached by, for example, coupling via a -CO 2 R O moiety).
  • the vehicle of Formula (IX) comprises 1-10 (Y) moieties.
  • the vehicle of Formula (IX) comprises 1-5 (Y) moieties.
  • the vehicle of Formula (IX) comprises 2-4 (Y) moieties.
  • the vehicle of Formula (IX) comprises 3 (Y) moieties.
  • (X) and (Y) are simultaneously reacted with heterobifunctional PEG. In other embodiments, (X) and (Y) are sequentially reacted with heterobifunctional PEG. In yet other embodiments, (Y) is reacted with a heterobifunctional PEG and the acetal end of the resulting product is then hydro lyzed to an aldehyde prior to reacting with (X).
  • (Y) comprises a monosaccharide or polysaccharide optionally attached via a PEG or aminoethoxyalkyl moiety.
  • (Y) comprises N-acetyl galactosamine or an N-acetyl galactosamine beta
  • aliphatic refers to straight chain or branched hydrocarbons that are completely saturated or that contain one or more units of unsaturation.
  • aliphatic groups include substituted or unsubstituted linear or branched alkyl, alkenyl and alkynyl groups. Unless indicated otherwise, the term “aliphatic” encompasses both substituted and unsubstituted hydrocarbons.
  • Alkyl refers to both straight and branched saturated chains containing, for example, 1-3, 1-6, 1-9, or 1-12 carbon atoms.
  • Alkenyl refers to both straight and branched saturated chains containing, for example, 1-3, 1-6, 1-9, or 1-12 carbon atoms, and at least one carbon-carbon double bond.
  • Alkynyl refers to both straight and branched saturated chains containing, for example, 1-3, 1-6, 1-9, or 1-12 carbon atoms, and at least one carbon-carbon triple bond.
  • an "activating agent” refers to an agent which, for example, facilitates a reaction at a carboxylic acid or, for example, allows for more facile nucleophilic substitution on a carbon atom adjacent to a hydroxy group.
  • Activating agents are generally known in the art and are routinely used to convert, for example, carboxylic acids to active ester and hydroxyl groups to leaving groups.
  • carboxylic acid activating agents include, but are not limited to, N,NEcarbonyldiimidazole (CDI), N ⁇ NEHicyclohexylcarbodiimide (DCC), l-(3-dimethylaminopropyl)-3- ethylcarbodiimide hydrochloride (EDCI), l-[bis(dimethylamino)methylene]-lH- l,2,3-triazolo[4,5-b]pyridinium-3-oxid- e hexafluorophosphate (HATU), 1-hydroxy- 1,2,3-benzotriazole (HOBT), O-benzotriazol-l-yl-N,N,NCNaetramethyluronium tetrafluoroborate (TBTU), disuccinimidyl carbonate (DSC), O-(N-Succinimidyl)- 1,1,3,3-tetramethyluronium tetrafluoroborate (TSTU) and 4- nitro
  • hydroxy activating agents include, but are not limited to, methane sulfonyl chloride, trifluoroacetic anhydride, bis(4-nitrophenyl) carbonate, 4-nitrophenylchloroformate and disuccinimidylcarbonate (DSC).
  • Aryl refers to monocyclic or polycyclic aromatic carbon ring systems having five to fourteen members.
  • aryl groups include, but are not limited to, phenyl (Ph), 1-naphthyl, 2-naphthyl, 1-anthracyl and 2-anthracyl.
  • the term “aralkyl” refers to an alkyl group substituted by an aryl. Also explicitly included within the scope of the term “aralkyl” are alkenyl or alkynyl groups substituted by an aryl. Examples of aralkyl groups include benzyl and phenethyl.
  • aryl “aryl group” or “aryl ring” also refers to rings that are optionally substituted, unless otherwise indicated.
  • Carbocyclyl refers to monocyclic or polycyclic non- aromatic carbon ring systems, which may contain a specified number of carbon atoms, for example from 3 to 12 carbon atoms, which are completely saturated or which contain one or more units of unsaturation.
  • a carbocyclic ring system may be monocyclic, bicyclic or tricyclic.
  • a carbocyclyl ring may be fused to another ring, such as an aryl ring or another carbocyclic ring. Examples of carbocyclic rings could include cyclohexyl, cyclopentyl, cyclobutyl, cyclopropyl, cyclohexenyl,
  • Halo refers to a fluorine, chlorine, bromine or iodine substituent.
  • Heteroaryl refers to monocyclic or polycyclic aromatic ring systems having five to fourteen members and one or more heteroatoms.
  • One having ordinary skill in the art will recognize that the maximum number of heteroatoms in a stable, chemically feasible heteroaryl ring is determined by the size of the ring and valence.
  • the term “heteroaralkyl” refers to an alkyl group substituted by a heteroaryl. Also explicitly included within the scope of the term “heteroaralkyl” are alkenyl or alkynyl groups substituted by a heteroaryl. In general, a heteroaryl ring may have one to four heteroatoms.
  • Heteroaryl groups include, without limitation, 2-furanyl, 3-furanyl, N- imidazolyl, 2imidazolyl, 4-imidazolyl, 5-imidazolyl, 3-isoxazolyl, 4isoxazolyl, 5- isoxazolyl, 2-oxadiazolyl, 5-oxadiazolyl, 2-oxazolyl, 4-oxazolyl, 5-oxazolyl, 2- pyrrolyl, 3-pyrrolyl, 2-pyridyl, 3-pyridyl, 4-pyridyl, 2-pyrimidyl, 4-pyrimidyl, 5- pyriniidyl, 3-pyridazinyl, 2-thiazolyl, 4thiazolyl, 5-thiazolyl, 5-tetrazolyl, 2-triazolyl, 5-triazolyl, 2-thienyl, and 3-thienyl.
  • heteroaryl ring also refers to rings that are optionally substituted.
  • fused polycyclic heteroaryl and aryl ring systems in which a carbocyclic aromatic ring or heteroaryl ring is fused to one or more other rings include, tetrahydronaphthyl, benzimidazolyl, benzothienyl, benzofuranyl, indolyl, quinolinyl, benzothiazolyl, benzoxazolyl, benzimidazolyl, isoquinolinyl, isoindolyl, acridinyl, benzoisoxazolyl, and the like.
  • Heterocyclic or “heterocyclyl” refers to non-aromatic saturated or unsaturated monocyclic or polycyclic ring systems containing one or more heteroatoms and with a ring size of three to fourteen.
  • One having ordinary skill in the art will recognize that the maximum number of heteroatoms in a stable, chemically feasible heterocyclic ring is determined by the size of the ring, degree of unsaturation, and valence.
  • a heterocyclic ring may have one to four heteroatoms so long as the heterocyclic ring is chemically feasible and stable and may be fused to another ring, such as a carbocyclic, aryl or heteroaryl ring, or to another heterocyclic ring.
  • a heterocyclic ring system may be monocyclic, bicyclic or tricyclic. Also included within the scope of within the scope of the term “heterocyclic” or “heterocyclyl”, as used herein, is a group in which one or more carbocyclic rings are fused to a heteroaryl.
  • heterocyclic rings include, but are not limited to, 3- IH- benzimidazol-2-one, 3-lH-alkyl-benzimidazol-2-one, 2-tetrahydrofuranyl, 3- tetrahydrofuranyl, 2-tetrahydrothiophenyl, 3-tetrahydrothiophenyl, 2-morpholino, 3- morpholino, 4-morpholino, 2-thiomorpholino, 3-thiomorpholino, 4-thiomorpholino, 1-pyrrolidinyl, 2-pyrrolidinyl, 3-pyrrolidinyl, 1-piperazinyl, 2-piperazinyl, 1- piperidinyl, 2-piperidinyl, 3-piperidinyl, 4-piperidinyl, 4-thiazolidinyl, diazolonyl, N- substituted diazolonyl, 1 -phthalimidinyl, benzoxane, benzotriazol-1-yl,
  • heterocyclic ring, whether saturated or unsaturated, also refers to rings that are optionally substituted, unless otherwise indicated.
  • An aryl, aralkyl, heteroaryl, or heteroaralkyl group may contain one or more independently selected substituents.
  • suitable substituents on the unsaturated carbon atom of an aryl or heteroaryl group include halogen, -CF 3 , -Rp -ORp-OH, -SH, -SR ⁇ protected OH (such as acyloxy), -NO 2 , -CN, -NH 2 , -NHRO -N(RIJl, -NHCORO-NHCONH2, -NHCONHRO-NHCON ⁇ ft, -NRCORO-NHCO2H, -NHCO 2 RO-CO 2 RO-CO 2 H, -CORO-CONH 2 , -CONHRp-CON(R ⁇ , -S(O) 2 H, -S(O) 2 RO-S(O) 3 H, -S(O) 3 R0-S(O) 2 NH2 QS(O)H, -S(
  • An aliphatic group or a non-aromatic heterocyclic ring may contain one or more substituents.
  • Natural amino acid refers to any of the 20 amino acids found in nature. These amino acids are alanine, asparagine, aspartic acid, arginine, cysteine, glutamine, glycine, glutamic acid, histidine, isoleucine, lysine, leucine, phenylalanine, methionine, serine, proline, tryptophan, threonine, tyrosine, and valine. The following amino acid abbreviations are commonly used in the art:
  • Protecting group refers to a group used in organic synthesis to temporarily mask the characteristic chemistry of a select functional group.
  • Suitable protecting groups for the methods and compounds described herein include, but are not limited to, those described in standard textbooks, such as Greene, T. W. et al., Protective Groups in Organic Synthesis, Wiley, N. Y. (1999).
  • Unnatural amino acid refers to any amino acid other than the 20 amino acids found in nature (listed above). Examples of such amino acids include, but are not limited to, ⁇ -alanine, ⁇ -aminobutyric acid, ⁇ -amino- ⁇ -methylbutyrate,
  • aminocyclopropane-carboxylate amino isobutyric acid, amino norbornyl-carboxylate, L-N-methylglutamic acid, cyclohexylalanine, cyclopentylalanine, D-alanine, D- arginine, D-aspartic acid, D-cysteine, D-glutamine, D-glutamic acid, D-histidine, D- iso leucine, D-leucine, D-lysine, D-methionine, D-ornithine, D-phenylalanine, D- proline, D-serine, D-threonine, D-tryptophan, D-tyrosine, D-valine, D- ⁇ - methylalanine, D- ⁇ -methylarginine, D- ⁇ -methylasparagine, D- ⁇ -methylaspartate, D- ⁇ -methylcysteine, D- ⁇ -methylglutamine, D- ⁇ -methylhistidine
  • heterobifunctional PEG reagents containing terminal protected (or “masked") aldehyde and ester functionalities. These heterobifunctional PEGs are useful for a variety of reasons described above.
  • the protected aldehyde and ester functionalities provide different reactivities such that a reaction, e.g., with a cationic polymer, can occur selectively at one terminus over the other.
  • the present invention provides a PEG reagent comprising a compound of Formula (I):
  • each Ri is independently selected from the group consisting of branched or straight-chain C 1 -Ce alkyl, branched or straight-chain C 2 -C O alkenyl, and branched or straight-chain C2-C6 alkynyl,
  • Ri can be taken together to form a 5- or 6-membered cyclic acetal or thioacetal
  • each Ri is independently, optionally substituted with one or more R 2 , or when both Ri are taken together to form a 5- or 6-membered cyclic acetal or thioacetal, the cyclic acetal or thioacetal is optionally substituted with one or more
  • each R 2 is independently selected from the group consisting of branched or straight-chain Q-C 6 alkyl, branched or straight-chain C2-C6 alkenyl, branched or straight-chain C 2 -C 6 alkynyl, hydroxy, Ci-C 6 alkoxy, -CO 2 H, -CO 2 (RJl -CONH 2 , -CONH(RJ] -CON(RJi, carbocyclyl, heterocyclyl, aryl, aralkyl, heteroaryl, heteroaralkyl, nitro, cyano, and halo,
  • alkyl, alkenyl or alkynyl chain may be replaced by one or more halogens, and wherein each carbocyclyl, heterocyclyl, aryl, aralkyl, heteroaryl, heteroaralkyl may be optionally substituted with one or more Rs;
  • R 3 is C O -C I4 aryl, C 5 -C14 heteroaryl or C 5 -C14 heterocyclyl,
  • heteroaryl or heterocyclyl contains one or more heteroatoms selected from the group consisting of N, N(R 4 ), O, S, S(O), and S(O) 2 , and
  • R 3 is optionally substituted with one or more R 5 ;
  • R 4 is selected from the group consisting of hydrogen, branched or straight- chain C 1 -Ce alkyl, branched or straight-chain C 2 -Ce alkenyl, branched or straight- chain C2-C6 alkynyl, wherein one or more hydrogens in the alkyl, alkenyl or alkynyl chain may be replaced by one or more halogens;
  • R5 is selected from the group consisting of branched or straight-chain Ci-C 6 alkyl, branched or straight-chain C 2 -Ce alkenyl, branched or straight-chain C 2 -Ce alkynyl, -CF 3 , -R0-OR0-OH, -SH, -SR ⁇ protected OH (e.g., acyloxy), -NO 2 , -CN, - NH 2 , -NHRO-N(RJl, -NHCORO-NHCONH2, -NHCONHR 0-NHCON(RJi, -
  • RQs selected from the group consisting of hydrogen, branched or straight- chain C 1 -Ce alkyl, branched or straight-chain C 2 -Ce alkenyl, branched or straight- chain C 2 -Ce alkynyl, carbocyclyl, heterocyclyl, aryl, aralkyl, heteroaryl, or heteroaralkyl and each RQs optionally substituted with one or more halogen, nitro, cyano, amino, -NH-(unsubstituted aliphatic), -N-(unsubstituted aliphatic) 2 , carboxy, carbamoyl, hydroxy, -O-(unsubstituted aliphatic), -SH, -S-(unsubstituted aliphatic), - CF3, -S(O) 2 NH 2 , unsubstituted aliphatic, unsubstituted carbocyclyl, unsubstituted heterocycl
  • A is selected from the group consisting of Ci-C 6 alkyl, C 2 -C 6 alkenyl, and C 2 - C 6 alkynyl;
  • A is optionally substituted with branched or straight-chain Ci- C 6 alkyl, branched or straight-chain C 2 -C 6 alkenyl, branched or straight-chain C 2 -C 6 alkynyl, hydroxy, Ci-C 6 alkoxy, CO 2 H, CO 2 (Ci-C 6 alkyl), CONH 2 , CONH(Ci-C 6 alkyl), CON(Ci-C 6 alkyl) 2 , nitro, cyano, or halo; each X is independently O or S;
  • j is an integer from 0 to 10;
  • n is an integer from 1 to 1,500.
  • each Ri is independently a branched or straight-chain Ci-C 6 alkyl, or both Ri are taken together to form a 5- or 6- membered cyclic acetal, optionally substituted as discussed above.
  • Ri is methyl, ethyl, isopropyl or both Ri are taken together to form a 1,3-dioxolane ring or a 1,3-dioxane ring.
  • Ri is ethyl.
  • each R 2 is independently selected from the group consisting of branched or straight- chain Ci-C ⁇ alkyl, branched or straight-chain C 2 -C 6 alkenyl, and branched or straight-chain C 2 -C 6 alkynyl optionally substituted as discussed above.
  • each R 2 is independently selected from the group consisting of branched or straight-chain Ci-C 6 alkyl, branched or straight-chain C 2 -C 6 alkenyl, and branched or straight-chain C 2 -C 6 alkynyl.
  • R 3 is C ⁇ -Ci4 aryl, C 5 -C14 heteroaryl optionally substituted as discussed above.
  • R3 is a C 6 -CiO aryl or a C5-C10 heteroaryl optionally substituted as discussed above.
  • R3 is a C 6 aryl, or C5 heteroaryl optionally substituted as discussed above.
  • R 3 is phenyl, pyridyl, pyrimidinyl, or naphthyl optionally substituted as discussed above.
  • R3 is phenyl optionally substituted as discussed above.
  • R 3 is phenyl optionally substituted with one or more nitro or one or more halo.
  • R 3 is trichlorophenyl, trifluorophenyl,
  • R 3 is para- nitrophenyl.
  • R5 is selected from the group consisting of -CF 3 , -Rp-ORp-OH, -SH, -SRPprotected OH ⁇ e.g., acyloxy), -NO 2 , - CN, -NH 2 , -NHRp-N(RJi, -NHCORg-NHCONH 2 , -NHCONHR 0-NHCON(R %, - NRCOR0-NHCO 2 H, -NHCO 2 Rf]-CO 2 Rp-CO 2 H, -CORp-CONH 2 , -CONHRp- CON(RJi, -S(O) 2 H, -S(O) 2 Rp-S(O) 3 H, -S(O) 3 Rp-S(O) 2 NH 2 , -S(O)H, -S(O)Rp- S(O) 2 NHRp-S(O) 2 N(RJi,
  • R5 is selected from the group consisting of -CO2R 0-CORO-CON(RJi 5 - S(O)RO-S(O) 2 RO-S(O) 3 Rp-CF 3 , nitro, cyano, and halo.
  • R 5 is selected from the group consisting of -CF3, nitro, cyano, and halo.
  • R5 is ortho- or para-nitro. In another embodiment, R5 is para-nitro.
  • R3s selected from the group consisting of hydrogen, branched or straight-chain C1-C 6 alkyl, branched or straight- chain C 2 -C 6 alkenyl, or branched or straight-chain C 2 -C 6 alkynyl optionally substituted as discussed above.
  • A is Ci-C ⁇ alkyl optionally substituted as discussed above. In other embodiments of Formula (I), A is C1-C 3 alkyl optionally substituted as discussed above. In another embodiment, A is CH 2 .
  • X is O.
  • j is an integer from 0 to 6. In other embodiments, ] is an integer from 1 to 3. In a particular embodiment, j is 1.
  • n is an integer from 5 to 1,000. In other words,
  • n is an integer from 20 to 500. In a particular embodiment, n is an integer from 50 to 250.
  • PEGs include PEG3400 and PEG8000.
  • the present invention provides a PEG reagent comprising a compound of Formula (II):
  • each Ri is independently selected from the group consisting of branched or straight-chain C 1 -C 6 alkyl, branched or straight-chain C 2 -C 6 alkenyl, and branched or straight-chain C2-C6 alkynyl,
  • Ri can be taken together to form a 5- or 6-membered cyclic acetal or thioacetal
  • each Ri is independently, optionally substituted with one or more R 2 , or when both Ri are taken together to form a 5- or 6-membered cyclic acetal or thioacetal, the cyclic acetal or thioacetal is optionally substituted with one or more R 2 ;
  • each R 2 is independently selected from the group consisting of branched or straight-chain Ci -C O alkyl, branched or straight-chain C2-C6 alkenyl, branched or straight-chain C 2 -C 6 alkynyl, hydroxy, Ci-C 6 alkoxy, -CO 2 H, -CO 2 (RJi -CONH 2 , -CONH(RJ] -CON(RJ 2 , carbocyclyl, heterocyclyl, aryl, aralkyl, heteroaryl, heteroaralkyl, nitro, cyano, and halo,
  • each carbocyclyl, heterocyclyl, aryl, aralkyl, heteroaryl, heteroaralkyl may be optionally substituted with one or more R 7 ;
  • R 4 is selected from the group consisting of hydrogen, branched or straight- chain Ci-C 6 alkyl, branched or straight-chain C2-C6 alkenyl, branched or straight- chain C 2 -C 6 alkynyl, wherein one or more hydrogens in the alkyl, alkenyl or alkynyl chain may be replaced by one or more halogens;
  • R 6 is C 6 -Ci 4 aryl, C5-C14 heteroaryl or C5-C 14 heterocyclyl
  • heteroaryl or heterocyclyl contains one or more heteroatoms selected from the group consisting of N, N(R 4 ), O, S, S(O), and S(O) 2 , and
  • R 6 is optionally substituted with one or more R7;
  • R 7 is selected from the group consisting of branched or straight-chain Ci-C 6 alkyl, branched or straight-chain C 2 -C 6 alkenyl, branched or straight-chain C 2 -C 6 alkynyl, -CF 3 , -Rp-ORP-OH, -SH, -SRPprotected OH (e.g., acyloxy), -NO 2 , -CN, - NH 2 , -NHRp-N(RJi, -NHCORg-NHCONH 2 , -NHCONHR0-NHCON(Rpi, - NRCORg-NHCO 2 H, -NHCO 2 R 0-CO 2 Rp-CO 2 H, -CORp-CONH 2 , -CONHRp- CON(RJi, -S(O) 2 H, -S(O) 2 RP-S(O) 3 H, -S(O) 3 RP-S(O) 2 NH 2
  • RQs selected from the group consisting of hydrogen, branched or straight-chain Ci -C O alkyl, branched or straight-chain C2-C6 alkenyl, branched or straight-chain C 2 -C 6 alkynyl, carbocyclyl, heterocyclyl, aryl, aralkyl, heteroaryl, or heteroaralkyl and each RQs optionally substituted with one or more halogen, nitro, cyano, amino, -NH-(unsubstituted aliphatic), -N-(unsubstituted aliphatic)2, carboxy, carbamoyl, hydroxy, -O-(unsubstituted aliphatic), -SH, -S-(unsubstituted aliphatic), - CF3, -S(O) 2 NH 2 Qinsubstituted aliphatic, unsubstituted carbocyclyl, unsubstituted heterocyclyl, unsubsti
  • A is selected from the group consisting of Ci -C O alkyl, C 2 -C O alkenyl, and C 2 - C 6 alkynyl;
  • A is optionally substituted with branched or straight-chain C 1 - C 6 alkyl, branched or straight-chain C 2 -C 6 alkenyl, branched or straight-chain C 2 -C 6 alkynyl, hydroxy, Ci-C 6 alkoxy, CO 2 H, CO 2 R 6 , CO 2 (Ci-C 6 alkyl), CONH 2 ,
  • each X is independently O or S;
  • j is an integer from O to 10;
  • n is an integer from 1 to 1,500.
  • each Ri is independently a branched or straight-chain Ci-C 6 alkyl, or both Ri are taken together to form a 5- or 6- membered cyclic acetal, optionally substituted as discussed above.
  • Ri is methyl, ethyl, isopropyl or both Ri are taken together to form a 1,3-dioxolane ring or a 1,3-dioxane ring.
  • Ri is ethyl.
  • each R2 is independently selected from the group consisting of branched or straight- chain Ci-C 6 alkyl, branched or straight-chain C 2 -C 6 alkenyl, and branched or straight-chain C 2 -C 6 alkynyl optionally substituted as discussed above.
  • each R 2 is independently selected from the group consisting of branched or straight-chain Ci-C 6 alkyl, branched or straight-chain C 2 -C 6 alkenyl, and branched or straight-chain C 2 -C 6 alkynyl.
  • R 6 is a Cs-C 6 aryl, a Cs-C 6 heteroaryl or a C 3 -C 6 heterocyclyl optionally substituted as discussed above.
  • R 6 is Cs-C 6 heterocyclyl optionally substituted as discussed above.
  • Re is a C3-C6 heterocyclyl optionally substituted as discussed above.
  • Re is C5 heterocyclyl optionally substituted as discussed above.
  • R O is a phthalimidyl, glutarimidyl, tetrahyrophthalimidyl, morbornene-2,3-dicarboximidyl, or succinimidyl optionally substituted as discussed above.
  • R 6 is an optionally sulfated succinimidyl.
  • Re is succinimidyl.
  • Re is a Ce aryl, or C 5 heteroaryl optionally substituted as discussed above.
  • R 6 is phenyl, pyridyl, pyrimidinyl, benzotriazolyl or naphthyl optionally substituted as discussed above.
  • R 6 is phenyl optionally substituted as discussed above.
  • Re is phenyl optionally substituted with one or more nitro or one or more halo.
  • Re is trichlorophenyl, trifluorophenyl, pentafluorophenyl, ortho- or para-nitrophenyl.
  • Re is para-nitrophenyl.
  • R 7 is selected from the group consisting of -CF 3 , -R0-OR0-OH, -SH, -SRPprotected OH (e.g., acyloxy), -NO 2 , - CN, -NH 2 , -NHRp-N(RJi, -NHCORg-NHCONH 2 , -NHCONHR P-NHCON(R %, - NRCORg-NHCO 2 H, -NHCO 2 Rg-CO 2 Rp-CO 2 H, -CORp-CONH 2 , -CONHRp- CON(RJi, -S(O) 2 H, -S(O) 2 RP-S(O) 3 H, -S(O) 3 Rp-S(O) 2 NH 2 , -S(O)H, -S(O)Rp- S(O) 2 NHRp-S(O) 2 N(RJi, -
  • R 7 is selected from the group consisting Of-CO 2 Rp-CORp-CON(RJi, -S(O)Rp-S(O) 2 Rp -S(O) 3 RP-CF 3 , nitro, cyano, and halo.
  • R 7 is selected from the group consisting Of-CF 3 , nitro, cyano, and halo.
  • Rg is phenyl
  • R 7 is ortho- or para-nitro.
  • Re is phenyl
  • R 7 is para-nitro.
  • RCIs selected from the group consisting of hydrogen, branched or straight-chain Ci-Ce alkyl, branched or straight- chain C 2 -C 6 alkenyl, or branched or straight-chain C 2 -C 6 alkynyl optionally substituted as discussed above.
  • A is Ci-C 6 alkyl optionally substituted as discussed above.
  • A is C1-C3 alkyl optionally substituted as discussed above. In a particular embodiment, A is CH2CH2.
  • X is O.
  • j is an integer from 0 to 6. In other embodiments, j is an integer from 1 to 3. In a particular embodiment, j is 1.
  • n is an integer from 5 to 1,000. In other words,
  • n is an integer from 20 to 500. In a particular embodiment, n is an integer from 50 to 250.
  • PEG3400 While any PEG moiety can be used in the compounds of Formula (II), preferred PEGs include PEG3400 and PEG8000.
  • Formula (II) has the following formula:
  • anoth eagent comprising a
  • R 1 , R2, R4, Re, R7, RpX, j, and n are defined as in Formula (II);
  • each B is any natural amino acid, or an unnatural alpha or beta amino acid, wherein each is independently optionally substituted with one or more -CO2R 6 or R 7 ;
  • any -CO 2 H present in B is optionally substituted with R 6 to yield a -CO 2 RO moiety;
  • k is an integer from 1 to 5.
  • the (B)k moiety comprises at least one natural amino acid and at least one unnatural alpha or beta amino acid, wherein each is independently and optionally substituted with one or more -CO2R6, -CO2H, or R 7 .
  • the (B) k moiety comprises at least two natural amino acids, wherein each is independently and optionally substituted with one or more
  • B is a natural amino acid optionally substituted with one or more -CO2R 6 , -CO2H, or R 7 .
  • B is an unnatural alpha or beta amino acid optionally substituted with one or more -CO 2 R O , - CO 2 H, or R 7 .
  • B is an unnatural alpha or beta amino acid comprising at least two carboxylic acid moieties.
  • B is selected from the group consisting of amino-malonic acid, amino-succinic acid, ⁇ -amino- glutaric acid, ⁇ -amino-glutaric acid, ⁇ -amino-adipic acid, ⁇ -amino-adipic acid, ⁇ - amino-pimelic acid, ⁇ -amino-pimelic acid, ⁇ -amino-suberic acid, ⁇ -amino-suberic- acid, ⁇ -amino-azelaic acid, ⁇ -amino-azelaic acid, ⁇ -amino-sebacic acid, ⁇ -amino- sebacic acid, aspartic acid, glutamic acid, and Glu-Glu (Le., a glutamic acid dimer).
  • B is amino-malonic acid, aspartic acid, glutamic acid, or Glu-Glu.
  • k is an integer from 1 to 3. In other embodiments, k is either 1 or 2. In yet other embodiments, k is 1.
  • PEG3400 While any PEG moiety can be used in the compounds of Formula (III), preferred PEGs include PEG3400 and PEG8000.
  • Formula (III) has the following formula:
  • heterobifunctional PEG reagents of Formula (II) and Formula (III) provide additional advantages. Among these are the ability to incorporate unique amino acids (such as beta-alanine) and the ability to incorporate termini bearing multiple carboxylate groups.
  • unique amino acids such as beta-alanine
  • termini bearing multiple carboxylate groups are useful for the quantification of the degree of PEG conjugation to peptides using amino acid analysis.
  • termini bearing multiple carboxylate groups is useful for improving the efficiency of purification by ion-exchange chromatography (particularly when larger PEGs are incorporated), and for presentation of clustered binding ligands to take advantage of multivalency opportunities.
  • This invention specifically contemplates the use of any chemically feasible salts of the heterobifunctional PEGs of the present invention.
  • Non-limiting examples of such salts include alkali metal (e.g., sodium and potassium) and alkaline earth metal (e.g., magnesium) salts of, for example, -OH, -SH, -CO 2 H, and -SO 3 H.
  • heterobifunctional PEG reagents of Formulas (I), (II), (III) and their precursors can be synthesized from the appropriate sized, unfunctionalized or partially functionalized, PEG.
  • the particular reactions may be carried out according to methods well-known in the literature.
  • the present invention provides a method for producing a heterobifunctional PEG according to Scheme I, II or III.
  • compounds of Formula (I) can be synthesized using any synthetic techniques known in the art.
  • some compounds of Formula (I) can be synthesized using Scheme I:
  • Ri, R 2 , R 3 , R 4 , R 5 , A, X, j and n are defined as in Formula (I);
  • Rs is selected from the group consisting of branched or straight-chain CI-C ⁇ alkyl, branched or straight-chain C 2 -Ce alkenyl, branched or straight-chain C 2 -C 6 alkynyl, C 6 -Cu aryl, C O -C I4 carbocycle, Cs-Ci 4 heteroaryl, and C5-C14 heterocycle, wherein each alkyl, alkenyl and alkynyl is optionally substituted with one or more R 2 ,
  • each heteroaryl or heterocycle contains one or more heteroatoms selected from the group consisting of N, N(R 4 ), O, S, S(O), and S(O)2, and
  • each aryl, carbocycle, heteroaryl, or heterocycle is optionally substituted with one or more R5;
  • Rg taken with the -OC(O)O- it is attached to, is any synthetically useful carbonate.
  • carbamate (D) reacted with amine to yield carbamate (D).
  • the ester of (D) is hydrolyzed (e.g., under basic conditions) to yield carboxylic acid (E).
  • carboxylic acid (E) is converted to the heterobifunctional PEG of Formula (I) using standard techniques in the art.
  • Rs is selected from the group consisting of branched or straight-chain C 1 -Ce alkyl, C 6 -C H aryl, or C 5 -C 14 heteroaryl optionally substituted as discussed above.
  • Rs is branched or straight-chain Ci-Ce alkyl optionally substituted as discussed above.
  • Rg is branched or straight-chain Ci-C 6 alkyl.
  • Rs is methyl
  • Rg is phenyl, aryloxy, or succinimidyl, each of which is optionally substituted with R 5 .
  • Rg is phenyl, succinimidyl, or aryloxy, each of which is optionally substituted with one or more electron withdrawing groups such as nitro, fluoro, chloro, cyano, sulpho, carboxy, amido, trifluoromethyl, or combinations thereof.
  • R 9 is sulpho- succinimidyl, 1 -oxybenzotriazolyl, nitrophenyl, or halo-substituted phenyl.
  • R9 is phenyl substituted with one or more fluorines, chlorines or nitro groups.
  • R9 is para-nitrophenyl, ortho- nitrophenyl, fluorophenyl or chlorophenyl.
  • Rg is para- nitrophenyl.
  • R 9 is succinimidyl.
  • PEG3400 While any PEG moiety can be used in Scheme I, preferred PEGs include PEG3400 and PEG8000.
  • P preferred PEGs include PEG3400 and PEG8000.
  • compounds of Formula (II) can be synthesized using any synthetic techniques known in the art.
  • some compounds of Formula (II) can be synthesized using Scheme II:
  • R 1 , R 2 , R 4 , Re, R 7 , A, X, j, and n are defined as in Formula (II);
  • R 8 is defined as in Scheme I. 2
  • (F) is first reacted with amine and then with amine NH 2 ACOORs to generate a mixture of bifuncti ein (G) is the major component (when A is CH 2 CH 2 ) of the product mixture.
  • the PEG mixture containing (G) is hydro lyzed (e.g., under basic conditions) and then subjected to ion exchange chromatography to yield the pure carboxylic acid (H).
  • Carboxylic acid (H) is then converted the heterobifunctional PEG of Formula (II) using standard techniques in the art.
  • Rs is selected from the group consisting of branched or straight-chain C 1 -C O alkyl, C 6 -Ci 4 aryl, or Cs-Ci 4 heteroaryl optionally substituted as discussed above.
  • Rg is branched or straight-chain Ci-C 6 alkyl optionally substituted as discussed above.
  • R 8 is branched or straight-chain Ci-C 6 alkyl.
  • Rg is ethyl
  • PEG3400 While any PEG moiety can be used in the compounds of Scheme II, preferred PEGs include PEG3400 and PEG8000.
  • P preferred PEGs include PEG3400 and PEG8000.
  • Ri, R.2, R 4 , Re, R 7 , B, X, j, k, and n are defined as in Formula (III);
  • R 8 is defined as in Scheme I.
  • (B) k contains more than one carboxylic acid (i.e., when k is greater than 1, and/or one or more B S are substituted with or naturally comprise a carboxylic acid)
  • certain carboxylic acids may be protected as, e.g., -CO 2 Rs esters, where appropriate.
  • the PEG mixture containing (J) is hydrolyzed (e.g., under basic conditions) and then subjected to ion exchange chromatography to yield the pure carboxylic acid (K).
  • Carboxylic acid (K) is then converted the heterobifunctional PEG of Formula (III) using standard techniques in the art.
  • (B)k contains more than one carboxylic acid (i.e., when k is greater than 1, and/or one or more BS are substituted with or naturally comprise a carboxylic acid), some or all of the carboxylic acids may be activated as, e.g., -CO 2 R0 esters, where appropriate.
  • PEG3400 While any PEG moiety can be used in the compounds of Scheme III, preferred PEGs include PEG3400 and PEG8000.
  • Scheme III Qllustrates the synthesis of Compound 10a. (See Examples 13- 15 for synthetic details).
  • GLU-GLU trimethyl ester hydrochloride to generate a mixture of bifunctional PEGs wherein (8a) is the major component of the product mixture.
  • the PEG mixture containing (8a) is hydrolyzed under basic conditions and then subjected to ion exchange chromatography to yield the pure tri-carboxylic acid (9a).
  • Carboxylic acid (9a) is then activated with disuccinimidyl carbonate to yield the heterobifunctional PEG (10a).
  • P preferred PEGs include PEG3400 and PEG8000.
  • heterobifunctional PEGs are not.
  • the acetal functionality provides a masked aldehyde which, when unmasked, is useful for the formation of imines and oximes through direct condensation.
  • the unmasked aldehyde is useful for the formation of amines via reductive animation, olefins via Wittig, aldol, Horner- Emmons or Julia-Lithgoe reactions or peptides via Ugi reactions.
  • any process capable of forming a chemical bond through reaction with an aldehyde will be applicable to the PEGs of the present invention.
  • the present invention provides novel classes of
  • heterobifunctional PEG reagents are advantageous in that they provide alternative methods of linking the PEG reagents to target biological molecules (e.g., via an aldehyde or activated ester moiety). In addition, such PEG reagents provide more efficient and specific means of coupling the PEG reagents to target biological molecules.
  • the heterobifunctional PEG reagents of the present invention provide two functional groups that can have different relative reactivities.
  • one functional group may be more reactive than the other.
  • one functional group may prefer, or even be selective, for a particular target molecule.
  • one functional group may selectively form a covalent bond with a target under certain reaction conditions.
  • the difference in reactivity between the functional groups facilitates the attachment of the PEG reagent to two different target molecules.
  • Bifunctional derivatization of the PEG reagent typically proceeds by coupling of the first (and usually the more reactive) functional group with a first target molecule, even in the presence of the second (and usually less reactive) functional group.
  • the second functional group can then be subsequently coupled to a second target molecule.
  • the PEG functional groups can be coupled to target molecules without activation. In other embodiments, the PEG functional groups are activated and then coupled to the target molecules.
  • the heterobifunctional PEGs of Formulas (I), (II) and (III) comprise functionalities particularly useful for coupling to target molecules (e.g., biologically relevant molecules).
  • target molecules e.g., biologically relevant molecules.
  • Compound 6, Compound 10 and Compound 10a comprise a protected aldehyde moiety and an activated ester moiety (either a nitrophenyl ester in Compound 6 or a succinimidyl ester in Compounds 10 and 10a). Accordingly, the activated ester portion of the heterobifunctional PEGs can first be reacted with a free amine on a first target molecule (see, e.g., Examples 9 and 11).
  • the activated ester can also react with free thiols or alcohols on the first target molecule under e.g., basic conditions.
  • the protected aldehyde moiety can be deprotected to yield a reactive aldehyde which serves as a site for coupling to a second target molecule.
  • the free aldehyde can be conjugated to amino (to yield an imine linkage) or aminooxy groups (to yield an oxime linkage) by direct condensation.
  • the aldehyde can be conjugated to an amino group by reductive amination to yield an amine linkage (see, e.g., Examples 10 and 12).
  • Such amino and aminooxy groups can be present on, but not limited to, lipids, polymers, pre-formed liposomes and pre-formed nanoparticles, peptides, proteins, enzymes, antibodies, aminoglycosides, proteoglycans,
  • glycosaminoglycans and the like.
  • additional reactions are useful for conjugation with aldehydes.
  • Such reactions include, but are not limited to, Wittig, Horner-Emmons, Julia-Lithgoe, Ugi, aldol reactions, and the like.
  • the aldehyde mediated coupling reactions are advantageous in that the deprotected aldehyde is able to selectively react with amine groups.
  • the aldehyde portion of the heterobifunctional PEGs of the present invention can selectively react with lysine residues present in polymers or polypeptides.
  • Such specificity is complementary to that seen in previously disclosed heterobifunctional PEGs comprising a vinyl sulfone which targets, for example, cysteine and lysine residues.
  • the heterobifunctional PEGs of the present invention can also be reacted with amine scaffolds in order to generate a "handle" for fatty acid attachment. While any amine scaffold may be used, amino-alcohols (such as l-amino-2,3-propanediol) are particularly useful.
  • the active ester component of the heterobifunctional PEG can be reacted with an amino scaffold.
  • the free hydroxyl groups on the scaffold portion of the resulting heterobifunctional PEG derivative can then be acylated with fatty acids.
  • the protected aldehyde can then be deprotected and coupled to a target molecule as described above.
  • any biologically relevant targeting moiety/molecule may be used in the vectors of the present invention.
  • the targeting moiety is a small molecule, polypeptide, peptide, protein, antibody, or a fragment, dimer, trimer or oligomer thereof.
  • Suitable biologically relevant small molecule targeting moieties include, but are not limited to, vascular endothelial cell growth factor for targeting endothelial cells; FGF2 for targeting vascular lesions and tumors; transferrin for targeting tumors; melanotropin (alpha MSH) peptides for tumor targeting; ApoE and peptides for LDL receptor targeting; von Willebrand ⁇ Factor and peptides for targeting Coxsackie-adeno viral receptor (CAR) expressing cells; PDl and peptides for targeting Neuropilin 1 ; EGF and peptides for targeting EGF receptors expressing cells; folic acid and ligands for targeting folate receptors; RGD peptides for targeting integrin expressing cells and any other suitable targeting moiety.
  • target molecules include, but are not limited to, RGD, folate, LHRH, somatostatin, YIGSR, bombesin, hyaluronic acid, SLX, antibody fragments, N-acetyl galactosamine, mannose-6-phosphate, vitamin B 12, any cell penetrating peptide and any extracellular binding ligand. Further, any fragments or peptides of the above moieties having the same or similar targeting properties may be used as for their respective targets.
  • compounds of Formulas (II) and (III) can advantageously comprise a unique amino acid such as beta-alanine. Such an amino acid is useful in
  • the present invention provides methods of using a heterobifunctional PEG to generate a vector of Formula (IV):
  • (X) and (Y) can be a small molecule, protein, polypeptide, peptide, monosaccharide, polysaccharide, antibody, polynucleotide, oligonucleotide, other polymeric species, polypeptide side chain or a biologically relevant targeting moiety, or a fragment, dimer, trimer or oligomer thereof.
  • (X) is typically conjugated via an imine, oxime or amine linkage (represented by a squiggly bond).
  • (X) and (Y) are simultaneously reacted with a heterobifunctional PEG. In other embodiments, (X) and (Y) are sequentially reacted with a heterobifunctional PEG. In yet other embodiments, (Y) is reacted with a heterobifunctional PEG and the acetal end of the resulting product is then hydro lyzed to an aldehyde prior to reacting with (X).
  • (X) is a cationic polymer. In some embodiments, (Y) is a targeting moiety. In one embodiment, (X) is a cationic polymer and (Y) is a targeting moiety.
  • the present invention provides methods of using a heterobifunctional PEG to generate a vector of Formula (V):
  • (X) and (Y) can be a small molecule, protein, polypeptide, peptide, monosaccharide, polysaccharide, antibody, polynucleotide, oligonucleotide, other polymeric species, polypeptide side chain or a biologically relevant targeting moiety, or a fragment, dimer, trimer or oligomer thereof.
  • (X) is typically conjugated via an imine, oxime or amine linkage (represented by a squiggly bond).
  • (X) and (Y) are simultaneously reacted with a heterobifunctional PEG. In other embodiments, (X) and (Y) are sequentially reacted with a heterobifunctional PEG. In yet other embodiments, (Y) is reacted with a heterobifunctional PEG and the acetal end of the resulting product is then hydro lyzed to an aldehyde prior to reacting with (X).
  • (X) is a cationic polymer. In some embodiments, (Y) is a targeting moiety. In one embodiment, (X) is a cationic polymer and (Y) is a targeting moiety.
  • the present invention provides methods of using a heterobifunctional PEG to generate a vector of Formula (VI):
  • (X) and (Y) can be a small molecule, protein, polypeptide, peptide, monosaccharide, polysaccharide, antibody, polynucleotide, oligonucleotide, other polymeric species, polypeptide side chain or a biologically relevant targeting moiety, or a fragment, dimer, trimer or oligomer thereof.
  • (X) is typically conjugated via an imine, oxime or amine linkage (represented by a squiggly bond).
  • (Y) is attached via a PEG or aminoethoxyalkyl moiety. In other embodiments (Y) is attached via an aminoethoxy ethyl moiety.
  • B can be any natural amino acid, or an unnatural alpha or beta amino acid, wherein each B is independently optionally substituted with one or more -CO 2 R ⁇ or R 7 . Further, any -CO2H present in B is optionally substituted with R ⁇ to yield a -CO2R6 moiety.
  • a vector of Formula (VI) may comprise one or more (Y) moieties (attached by, for example, coupling via a -CO 2 Re moiety).
  • the vector of Formula (VI) comprises 1-10 (Y) moieties.
  • the vector of Formula (VI) comprises 1-5 (Y) moieties.
  • the vector of Formula (VI) comprises 2-4 (Y) moieties.
  • the vector of Formula (VI) comprises 3 (Y) moieties.
  • (X) and (Y) are simultaneously reacted with a heterobifunctional PEG. In other embodiments, (X) and (Y) are sequentially reacted with a heterobifunctional PEG. In yet other embodiments, (Y) is reacted with a heterobifunctional PEG and the acetal end of the resulting product is then hydro lyzed to an aldehyde prior to reacting with (X).
  • (X) is a cationic polymer.
  • (Y) is a targeting moiety.
  • (X) is a cationic polymer and (Y) is a targeting moiety.
  • (Y) comprises a monosaccharide or polysaccharide optionally attached via a PEG or aminoethoxyalkyl moiety.
  • (Y) comprises N-acetyl galactosamine or an N-acetyl galactosamine beta aminoethoxy ethyl glycoside.
  • (Y) is a targeting moiety
  • (Y) can be used to direct a vector of Formula (VI) to, for example, a particular area of the body, a particular tissue or tissue type, a particular organ, etc. Accordingly, the presence of more than one (Y) group may increase the targeting efficiency of the vector (i.e., the ability of the vector to reach a target).
  • the present invention provides a method of using a heterobifunctional PEG to generate a vehicle for targeted nucleic acid delivery of Formula (VII):
  • (X) is typically conjugated via an imine, oxime or amine linkage (represented by a squiggly bond).
  • (X) and (Y) are as described in the context of Formula (IV).
  • (X) is a cationic polymer.
  • (Y) is a targeting moiety.
  • (X) and (Y) are simultaneously reacted with heterobifunctional PEG. In other embodiments, (X) and (Y) are sequentially reacted with heterobifunctional PEG. In yet other embodiments, (Y) is reacted with a heterobifunctional PEG and the acetal end of the resulting product is then hydro lyzed to an aldehyde prior to reacting with (X).
  • the present invention provides a method of using a heterobifunctional PEG to generate a vehicle for targeted nucleic acid delivery of Formula (VIII):
  • (X) is typically conjugated via an imine, oxime or amine linkage (represented by a squiggly bond).
  • (X) and (Y) are as described in the context of Formula (IV).
  • (X) is a cationic polymer.
  • (Y) is a targeting moiety.
  • (X) and (Y) are simultaneously reacted with heterobifunctional PEG. In other embodiments, (X) and (Y) are sequentially reacted with heterobifunctional PEG. In yet other embodiments, (Y) is reacted with a heterobifunctional PEG and the acetal end of the resulting product is then hydro lyzed to an aldehyde prior to reacting with (X).
  • the present invention provides a method of using a heterobifunctional PEG to generate a vehicle for targeted nucleic acid delivery of
  • (X) is typically conjugated via an imine, oxime or amine linkage (represented by a squiggly bond).
  • (X) and (Y) are as described in the context of Formula (IV).
  • (X) is a cationic polymer.
  • (Y) is a targeting moiety.
  • (Y) is attached via a PEG or aminoethoxyalkyl moiety. In other embodiments (Y) is attached via an aminoethoxyethyl moiety.
  • B can be any natural amino acid, or an unnatural alpha or beta amino acid, wherein each B is independently optionally substituted with one or more -CO2R 6 or R 7 . Further, any -CO2H present in B is optionally substituted with Re to yield a -CO 2 Re moiety.
  • a vehicle of Formula (IX) may comprise one or more (Y) moieties (attached by, for example, coupling via a -CO2R0 moiety).
  • the vehicle of Formula (IX) comprises 1-10 (Y) moieties.
  • the vehicle of Formula (IX) comprises 1-5 (Y) moieties.
  • the vehicle of Formula (IX) comprises 2-4 (Y) moieties.
  • the vehicle of Formula (IX) comprises 3 (Y) moieties.
  • (X) and (Y) are simultaneously reacted with heterobifunctional PEG. In other embodiments, (X) and (Y) are sequentially reacted with heterobifunctional PEG. In yet other embodiments, (Y) is reacted with a heterobifunctional PEG and the acetal end of the resulting product is then hydro lyzed to an aldehyde prior to reacting with (X).
  • (Y) is a targeting moiety
  • (Y) can be used to direct a vehicle of Formula (XI) to, for example, a particular area of the body, a particular tissue or tissue type, a particular organ, etc. Accordingly, the presence of more than one (Y) group may increase the targeting efficiency of the vehicle (Le., the ability of the vehicle to reach a target).
  • (Y) comprises a monosaccharide or polysaccharide optionally attached via a PEG or amino ethoxy alky 1 moiety.
  • (Y) comprises N-acetyl galactosamine or an N-acetyl galactosamine beta
  • the heterobifunctional PEG regent used in the vectors above is a compound of Formula (I).
  • the heterobifunctional PEG reagent used in the vectors above is a compound of Formula (II).
  • the heterobifunctional PEG reagent used in the vectors above is a compound of Formula (III).
  • the heterobifunctional PEG reagent used in the vectors above is Compound 6.
  • the heterobifunctional PEG reagent used in the vectors above is Compound 10.
  • the heterobifunctional PEG reagent used in the vectors above is
  • Suitable nucleic acids include, but are not limited to, a recombinant plasmid; a replication-deficient plasmid; a mini-plasmid lacking bacterial sequences; a recombinant viral genome; a linear nucleic acid fragment encoding a therapeutic peptide or protein; a hybrid DNA/RNA double strand; double stranded DNA; an antisense DNA or chemical analogue thereof; a blunt, double blunt and overhanging double stranded DNA or RNA fragment comprising 5-200 base pairs; an antisense RNA or chemical analogue thereof; a linear polynucleotide that is transcribed as an antisense RNA or a ribozyme; a ribozyme; and a viral genome.
  • the blunt, double blunt and overhanging double stranded DNA or RNA comprises 15-30 base pairs. In certain embodiments, the blunt, double blunt and overhanging double stranded DNA or RNA comprises 15 base pairs. In certain embodiments, the blunt, double blunt and overhanging double stranded DNA or RNA comprises 16 base pairs. In certain embodiments, the blunt, double blunt and overhanging double stranded DNA or RNA comprises 17 base pairs. In certain embodiments, the blunt, double blunt and overhanging double stranded DNA or RNA comprises 18 base pairs. In certain embodiments, the blunt, double blunt and overhanging double stranded DNA or RNA comprises 19 base pairs.
  • the blunt, double blunt and overhanging double stranded DNA or RNA comprises 20 base pairs. In certain embodiments, the blunt, double blunt and overhanging double stranded DNA or RNA comprises 21 base pairs. In certain embodiments, the blunt, double blunt and overhanging double stranded DNA or RNA comprises 22 base pairs. In certain embodiments, the blunt, double blunt and overhanging double stranded DNA or RNA comprises 23 base pairs. In certain embodiments, the blunt, double blunt and overhanging double stranded DNA or RNA comprises 24 base pairs. In certain embodiments, the blunt, double blunt and overhanging double stranded DNA or RNA comprises 25 base pairs.
  • the blunt, double blunt and overhanging double stranded DNA or RNA comprises 26 base pairs. In certain embodiments, the blunt, double blunt and overhanging double stranded DNA or RNA comprises 27 base pairs. In certain embodiments, the blunt, double blunt and overhanging double stranded DNA or RNA comprises 28 base pairs. In certain embodiments, the blunt, double blunt and overhanging double stranded DNA or RNA comprises 29 base pairs. In certain embodiments, the blunt, double blunt and overhanging double stranded DNA or RNA comprises 30 base pairs.
  • Chemical modification may be useful in some embodiments of the invention to increase stability of the nucleic acid molecule used or to reduce cytokine production.
  • Incorporation of non-naturally occurring chemical analogues, such as 2 B O-Methyl ribose analogues of RNA, DNA, LNA and RNA chimeric oligonucleotides, and other chemical analogues of nucleic acid oligonucleotides, is one type of possible chemical modification.
  • flanking sequences at the 5 and/or 3Qnds
  • the inclusion of non-traditional bases as well as acetyl- methyl-, thio- and other modified forms of adenine, cytidine, guanine, thymine, and uridine.
  • Non-traditional nucleic acid bases that can be introduced into nucleic acids include, for example, inosine, purine, pyridin-4-one, pyridin-2-one, phenyl, pseudouracil, 2,4,6-trimethoxy benzene, 3- methyl uracil, dihydrouridine, naphthyl, aminophenyl, 5-alkylcytidines (e.g., 5- methylcytidine), 5-alkyluridines (e.g., ribothymidine), 5-halouridine (e.g., 5- bromouridine) or 6-azapyrimidines or 6-alkylpyrimidines (e.g.
  • 6-methyluridine 6-methyluridine
  • propyne quesosine, 2-thiouridine, 4-thiouridine, ⁇ vybutosine, wybutoxosine, A- acetyltidine, 5-(carboxyhydroxymethyl)uridine, 5'-carboxymethylaminomethyl-2- thiouridine, 5-carboxymethylaminomethyluridine, beta-D-galactosylqueosine, 1- methyladenosine, 1-niethy lino sine, 2,2-dimethylguanosine, 3-methylcytidine, 2- methyladenosine, 2-methylguanosine, N6-methyladenosine, 7-methylguanosine, 5- methoxyaminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5- methylcarbonyhnethyluridine, 5-methyloxyuridine, 5-methyl-2-thiouridine, 2- methylthio-N6-isopentenyladenosine,
  • oligonucleotides of the invention may be 2ED- substituted oligonucleotides, as described in U.S. Patent Nos. 5,623,065, 5,856,455, 5,955,589, 6,146,829, and 6,326,199, herein incorporated by reference in their entirety, in which 2 Substituted nucleotides are introduced within an oligonucleotide to induce increased binding of the oligonucleotide to a complementary target strand while allowing expression of RNase H activity to destroy the targeted strand. See also, Sproat, B. S., et al., Nucleic Acids Research, 1990;18:41, incorporated herein by reference in its entirety. Nucleic acid molecules comprising 2ED-methyl and ethyl nucleotides are also encompassed by the invention.
  • nucleic acid molecules of the invention comprise 2ED-methyl-, 2ED-allyl-, and 2 ED-dimethylallyl- substituted nucleotides.
  • At least one of the 2EHeoxyribofuranosyl moiety of at least one of the nucleosides of an oligonucleotide is modified.
  • a halo, alkoxy, aminoalkoxy, alkyl, azido, or amino group may be added.
  • PCT/US91/00243, application Ser. No. 463,358, and application Ser. No. 566,977, disclose that incorporation of, for example, a 2ED-methyl, 2ED-ethyl, 2ED-propyl, 2 B O-allyl, 2 ED-aminoalkyl or 2 EUeoxy-2 Efluoro groups on the nucleosides of an oligonucleotide enhance the hybridization properties of the oligonucleotide.
  • the nucleic acid molecules of the invention can be augmented to further include either or both a phosphorothioate backbone or a 2BO-Ci C 2 o-alkyl (e.g., 2ED-methyl, 2ED- ethyl, 2 ED-propyl), 2 ED-C 2 C 20 -alkenyl (e.g.
  • Suitable cationic polymers useful for forming a vector include, but are not limited to, linear or branched HK copolymers (copolymers of histidine and lysine), linear or branched polyethyleneimine (PEI), polylysine, linear or non-linear polyamidoamine, protamine sulfate, polybrine, chitosan, polymethacrylate, polyamines, spermine analogues and any other suitable polymer.
  • linear or branched HK copolymers copolymers of histidine and lysine
  • PEI linear or branched polyethyleneimine
  • polylysine linear or non-linear polyamidoamine
  • protamine sulfate polybrine
  • chitosan polymethacrylate
  • polyamines spermine analogues and any other suitable polymer.
  • a preferred cationic polymer is an HK copolymer.
  • the HK copolymer is synthesized from any appropriate combination of polyhistidine, polylysine, histidine and/or lysine.
  • the HK copolymer is linear. In certain preferred embodiments, the HK copolymer is branched.
  • the branched HK copolymer comprises a polypeptide backbone.
  • the polypeptide backbone comprises 1-10 amino acid residues, and more preferably 2-5 amino acid residues.
  • the polypeptide backbone consists of lysine amino acid residues.
  • the number of branches on the branched HK copolymer is one greater than the number of backbone amino acid residues.
  • the branched HK copolymer contains 1-11 branches.
  • the branched HK copolymer contains 2-5 branches.
  • the branched HK copolymer contains 4 branches.
  • the branch of the branched HK copolymer comprises 10-100 amino acid residues. In certain preferred embodiments, the branch comprises 10-50 amino acid residues. In certain more preferred embodiments, the branch comprises 15-25 amino acid residues. In certain embodiments, the branch of the branched HK copolymer comprises at least 3 histidine amino acid residues in every subsegment of 5 amino acid residues. In certain other embodiments, the branch comprises at least 3 histidine amino acid residues in every subsegment of 4 amino acid residues. In certain other embodiments, the branch comprises at least 2 histidine amino acid residues in every subsegment of 3 amino acid residues. In certain other embodiments, the branch comprises at least 1 histidine amino acid residues in every subsegment of 2 amino acid residues.
  • At least 50% of the branch of the HK copolymer comprises units of the sequence KHHH. In certain preferred embodiments, at least 75% of the branch comprises units of the sequence KHHH.
  • the HK copolymer branch comprises an amino acid residue other than histidine or lysine.
  • the branch comprises a cysteine amino acid residue, wherein the cysteine is a N-terminal amino acid residue.
  • lysine residues of the HK copolymer branch are replaced with alternative amino acids bearing positive charges.
  • suitable amino acids include, but are not limited to, arginine and ornithine.
  • suitable HK copolymers include, but are not limited to those found in U.S. Patent Nos. 6,692,911, 7,070,807 and 7,163,695, all of which are incorporated herein by reference.
  • any biologically relevant targeting moiety/molecule may be used in the vectors of the present invention.
  • the targeting moiety is a small molecule, polypeptide, peptide, protein, antibody, or a fragment, dimer, trimer or oligomer thereof.
  • Suitable biologically relevant small molecule targeting moieties include, but are not limited to, vascular endothelial cell growth factor for targeting endothelial cells; FGF2 for targeting vascular lesions and tumors; transferrin for targeting tumors; melanotropin (alpha MSH) peptides for tumor targeting; ApoE and peptides for LDL receptor targeting; von WillebrandS Factor and peptides for targeting Coxsackie-adenoviral receptor (CAR) expressing cells; PDl and peptides for targeting Neuropilin 1; EGF and peptides for targeting EGF receptors expressing cells; folic acid and ligands for targeting folate receptors; RGD peptides for targeting integrin expressing cells and any other suitable targeting moiety.
  • target molecules include, but are not limited to, RGD, folate, LHRH, somatostatin, YIGSR, bombesin, hyaluronic acid, SLX, antibody fragments, N-acetyl galactosamine, mannose-6-phosphate, vitamin B 12, any cell penetrating peptide and any extracellular binding ligand. Further, any fragments or peptides of the above moieties having the same or similar targeting properties may be used as for their respective targets.
  • Acetyl chloride (2.50 mL, 35.16 mmol) was added to 250 mL of methanol. This mixture was stirred for 5 min and then added to compound (1) (20.01 g, 5.81 mmol) as prepared in Zalipsky et al. (J. Bioactive and Compatible Polymers, Vol. 5, 227-231, (1990)). After stirring at room temperature for 24 hours, the resulting solution was concentrated to dryness. The residue was dissolved in methylene chloride (15 mL) and the resulting solution was added to ether (300 mL) with vigorous stirring.
  • the crude isolated solids were purified by ion exchange chromatography using a DEAE- Sephadex (7 g) column prepared in potassium tetraborate solution (50 g/500 mL water) and washed with water until neutral pH.
  • the column was eluted sequentially with water (200 mL), 6 mM ammonium bicarbonate (100 mL), 8 mM ammonium bicarbonate (100 mL), 10 mM ammonium bicarbonate (100 mL) and 48 niM ammonium bicarbonate (100 mL). All product fractions were combined and sodium chloride (20 g) was dissolved into the combined fractions.
  • the crude isolated solids were purified by ion exchange chromatography using a DEAE-Sephadex (15 g) column prepared in potassium tetraborate solution (50 g/500 mL water) and washed with water until neutral pH. The column was eluted sequentially with water (300 mL), 6 mM ammonium bicarbonate (400 mL) and 48 mM ammonium bicarbonate (200 mL). All product fractions were combined and sodium chloride (40 g) was dissolved into the combined fractions. The pH was adjusted to 5-6 with acetic acid and the product was extracted with methylene chloride (3 X 30 mL).
  • Coupling of the heterobifunctional PEGs of the present invention to peptides can generally be accomplished using the following protocol.
  • a heterobifunctional PEG can be coupled to RGD to yield, e.g., compound (12).
  • Compound 6 is added to a solution of compound (11) in DMSO. The mixture is then made basic and additional compound (11) is added in several portions over 4-24 hours. The reaction is monitored, preferably by RP-HPLC. When the reaction is complete, the reaction is diluted with phosphate buffer (20 mM, 2 ml, pH 8) and is dialyzed against, for example, phosphate buffer (20 mM, 1L-24H, IL-1.5H) followed by dialysis against DI water (2X, 1L-2H) using a 3500 MWCO membrane. The dialyzed solution is then lyophilized to give compound 12.
  • heterobifunctional PEGs of the present invention once conjugated to e.g., RGD, can be further conjugated to, for example, lipids, polymers, liposomes or nanoparticles. This can be accomplished in a one-pot synthesis where a) the aldehyde moiety on the heterobifunctional PEG is "liberated”; and b) the liberated aldehyde moiety is conjugated to a target molecule via reductive amination.
  • the aldehyde moiety on the heterobifunctional PEGs conjugated to proteins can generally be "liberated” or deprotected using standard conditions in the art ⁇ e.g., with aqueous acid).
  • aqueous acid conditions e.g., aqueous HCl or aqueous TFA. The hydrolysis is monitored, preferably by RP-HPLC.
  • the reaction is diluted with phosphate buffer (20 mM, 2 ml, pH 8) and is dialyzed against, for example, phosphate buffer (20 mM, 1L-24H, IL- 1.5H) followed by dialysis against DI water (2X, 1L-2H) using a 3500 MWCO membrane. The dialyzed solution is then lyophilized to give compound (14) or (15), respectively.
  • the reaction mixture is dialyzed against DI water (3X, 900 mL-2H) and then DI water (900 mL-16H) using a 3500 MWCO dialysis membrane.
  • the fractions containing compound (17) are combined and concentrated to ⁇ 10 mL using a 200 ml stir cell with a 5000 MWCO membrane.
  • the resulting solution is diluted with aqueous acetic acid (0.05%, 70 mL) and again concentrated to ⁇ 10 mL using a 200 mL stir cell with at 5000 MWCO membrane. The dilution and concentration steps are repeated an additional three times and the resulting solution is lyophilized giving pure compound (17) (19.9 mg) as a white powder.
  • heterobifunctional PEGs of the present invention can be directly coupled with N-acetyl-galactosamine-beta- aminoethyl glycoside through the active ester terminus.
  • Example 12 Reductive Animation of Amines with the Aldehyde Group of Heterobifunctional PEGs Conjugated to N-acetyl-galactosamine-beta-aminoethyl glycoside
  • heterobifunctional PEGs of the present invention conjugated to N-acetyl-galactosamine-beta-aminoethyl glycoside, can be conjugated to an HK polymer (of the type described by Leng et al., Drug News Perspect 20(2), March 2007) through the acetal/aldehyde terminus.
  • Example 13 Preparation of PEG-8000 Mono-Diethoxypropylamine Carbamate GLU-GLU Carbamate Trimethyl Ester (8a)
  • the crude isolated solids were purified by ion exchange chromatography using a DEAE-Sephadex (15 g) column prepared in potassium tetraborate solution (50 g/500 mL water) and washed with water until neutral pH.
  • the column was eluted sequentially with water (500 mL), 6 mM ammonium bicarbonate (200 mL), 8 mM ammonium bicarbonate (200 mL), 10 mM ammonium bicarbonate (200 mL), 12 mM ammonium bicarbonate (200 mL), 14 mM ammonium bicarbonate (200 mL), 16 mM ammonium bicarbonate (200 mL) and brine (200 mL).
  • Example 16 Conjugation of Heterobifunctional PEGs with multiple N-acetyl- galactosamine-beta-aminoethoxyethyl glycoside units (Compound 19, carboxylic acid coupling)
  • Compound (9a) (203.4 mg, 0.024 mmol) and tetra-acetyl-galactosamine- beta-aminoethoxyethyl glycoside hydrochloride salt (18) (33.7 mg, 0.072 mmol) were dissolved in anhydrous dichloromethane (1 mL).
  • Diisopropylethylamine (37.5 ⁇ L, 0.215 mmol) was added followed by HBTU (37.2 mg, 0.072 mmol).
  • heterobifunctional PEGs of the present invention e.g., Compound 10a
  • heterobifunctional PEGs of the present invention can be directly coupled to three N-acetyl- galactosamine-beta-aminoethoxyethyl glycoside units through the three active ester termini giving the desired compound e.g., compound (19).
  • heterobifunctional PEGs of the present invention once conjugated to proteins or sugars (e.g., RGD, N-acetyl galactosamine), can be further conjugated to, for example, lipids, polymers, liposomes or nanoparticles. This can be accomplished in a one-pot synthesis where a) the hydroxyl moieties on the protected N-acetyl galactosamine are "liberated”; and b) the aldehyde moiety on the heterobifunctional PEG is "liberated”; and c) the liberated aldehyde moiety is conjugated to a target molecule via reductive amination.
  • proteins or sugars e.g., RGD, N-acetyl galactosamine
  • hydroxyl moieties on the heterobifunctional PEGs conjugated to sugars can generally be "liberated” or deprotected using standard conditions in the art (e.g., with aqueous base).
  • hydrolysis of the acetoxy groups of compound (19) is carried out under aqueous basic conditions (e.g., aqueous sodium hydroxide).
  • aqueous basic conditions e.g., aqueous sodium hydroxide.
  • the hydrolysis is monitored, preferably by RP-HPLC.
  • the aldehyde moiety on the heterobifunctional PEGs conjugated to proteins or sugars can generally be "liberated” or deprotected using standard conditions in the art (e.g., with aqueous acid).
  • hydrolysis of the acetal of compound (19) or (21) is carried out under aqueous acid conditions (e.g., aqueous HCl or aqueous TFA).
  • aqueous acid conditions e.g., aqueous HCl or aqueous TFA.
  • the hydrolysis is monitored, preferably by RP-HPLC.
  • the pH of the reaction mixture (now containing compound (22)) was adjusted to 5 using NaHaPO 4 (0.2 M in deionized water, 2 mL) and sodium hydroxide (1 M in deionized water, 65 ⁇ L).
  • An HK polymer (of the type described by Leng et al., Drug News Perspect 20(2), March 2007, 119 mg, 8.2 ⁇ mol) was then added followed by NaCNBH 3 (3 nig).
  • the reaction was monitored by RP-HPLC and was considered finished after 1.5 hrs.
  • the reaction mixture was dialyzed with a 3500 MWCO dialysis membrane against deionized water (I L, 2 x 1.5 hr, 1 x 16 hr).
  • MPB 5OmM phosphate pH 7.
  • heterobifunctional PEGs of the present invention once conjugated to e.g., aminoethoxyethanol, can be further conjugated to, for example, lipids, polymers, liposomes or nanoparticles. This can be accomplished in a one-pot synthesis where a) the aldehyde moiety on the heterobifunctional PEG is "liberated”; and b) the liberated aldehyde moiety is conjugated to a target molecule via reductive animation.
  • the aldehyde moiety on the heterobifunctional PEGs conjugated to proteins can generally be "liberated” or deprotected using standard conditions in the art (e.g., with aqueous acid).
  • hydrolysis of the acetal of compound (21) is carried out under aqueous acid conditions (e.g., aqueous HCl or aqueous TFA).
  • aqueous acid conditions e.g., aqueous HCl or aqueous TFA.
  • the hydrolysis is monitored, preferably by RP-HPLC.
  • the reaction was monitored by RP-HPLC and was considered finished after 2 hrs.
  • the reaction mixture was dialyzed with a 3500 MWCO dialysis membrane against deionized water (I L, 2 x 1.5 hr, 1 x 16 hr).
  • MPB MPA + 2 M NaCL).
  • the fractions containing mainly monoPEGylated product were combined and concentrated using a 200 mL Stir cell with 5000 MWCO membrane to ⁇ 10 ml.
  • the retentate was washed and concentrated down to ⁇ 10 ml four times with acetic acid (0.05% in deionized water, 75 mL) using the stir-cell. The final wash was lyopholized to give the desired compound (25) (56 mg) as a white powder.
  • GLU-GLU trimethyl ester hydrochloride (26) (677.6 mg, 1.91 mmol) was dissolved in ahydrous dichloromethane (15 mL). Diisopropylethylamine (1 mL, 5.74 mmol) and benzyl chloro formate (0.34 mL, 2.39 mmol) were added. The reaction was stirred at room temperature for 21 hours after which, it was diluted with dichloromethane (20 mL). The resulting solution was washed with aqueous hydrochloric acid (1 M, 3 X 10 mL), saturated aqueous sodium bicarbonate (2 X 10 mL) and brine (10 mL).
  • N-Benzylcarbamoyl GLU-GLU trimethyl ester (27) (615.6 mg, 1.36 mmol) was dissolved in methanol (10 mL).
  • Aqueous sodium hydroxide (6 M, 2.04 mL,
  • Pentaacetyl galactosamine (29) (7.01 g, 18.02 mmoles) was combined with anhydrous 1 ,2-dichloroethane (40 mL) and heated to 50 0 C.
  • Trimethylsilyl triflate (3.74 mL, 20.72 mmoles) was added to the heterogeneous mixture and the reaction was stirred at 50 0 C for 17.5 hours. After cooling to room temperature under nitrogen, triethylamine (3.41 mL) as added and the resulting solution was concentrated to % its original volume.
  • N-Beyzylcarbamoyl GLU-GLU tricarboxylic acid (28) (399.2 mg, 0.97 mmole) and 5-acetamido-2-(acetoxymethyl)-6-(2-(2-aminoethoxy)ethoxy)tetrahydro- 2H-pyran-3,4-diyl diacetate hydrochloride (32) (1.37 g, 2.92 mmole) were dissolved in anhydrous dichloromethane (40 mL). To the resulting mixture was added diisopropylethylamine (1.53 mL, 8.76 mmoles) and HBTU (1.11 g, 2.92 mmoles).
  • aminoethoxyethyl glycoside)tri-amide (34) (98.4 mg, 0.077 mmole) and 10% palladium on carbon (33.8 mg) were combined and placed under vacuum. Methanol (10 mL) added. The resulting mixture was degassed under vacuum and stirred under hydrogen for 19 hours. The reaction was then filtered, concentrated and dried under vacuum giving the desired product (35) (76.0 mg, 86% yield).
  • An analogue of Compound 19 can be synthesized from utilizing Compound 10.
  • the succinimidyl ester of Compound 10 is reacted with Compound 35 giving Compound 36.
  • Subsequent treatment with trifluoroacetic acid followed by reductive amination using sodium cyanoborohydride (as in Example 20) provides Compound 37.

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Abstract

La présente invention a pour objet de nouveaux réactifs hétérobifonctionnels à base de polyéthylène glycol, leurs procédés de production et leurs procédés d’utilisation.
PCT/US2010/041394 2009-07-09 2010-07-08 Nouveaux réactifs hétérobifonctionnels à base de polyéthylène glycol, leur préparation et leurs utilisations WO2011005980A1 (fr)

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US8426554B2 (en) 2010-12-29 2013-04-23 Arrowhead Madison Inc. In vivo polynucleotide delivery conjugates having enzyme sensitive linkages
US8883177B2 (en) 2011-06-28 2014-11-11 Nian Wu Pharmaceutical compositions for parenteral administration
CN107670050A (zh) * 2017-08-30 2018-02-09 重庆阿普格雷生物科技有限公司 基于pki‑587的抗癌中间体和聚乙二醇偶联抗癌药物、及其制备方法和应用
KR20180121925A (ko) * 2016-03-07 2018-11-09 애로우헤드 파마슈티컬스 인코포레이티드 치료용 화합물을 위한 표적화 리간드
CN113292616A (zh) * 2021-05-20 2021-08-24 内蒙古大学 一种单糖配体功能化的阳离子脂质类化合物及其制备方法与应用

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Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8426554B2 (en) 2010-12-29 2013-04-23 Arrowhead Madison Inc. In vivo polynucleotide delivery conjugates having enzyme sensitive linkages
WO2012109112A3 (fr) * 2011-02-08 2013-01-17 Wu Nian Conjugués de polymère-glucide-lipide
CN104704023A (zh) * 2011-02-08 2015-06-10 念·吴 聚合物-碳水化合物-脂质缀合物
US9175027B2 (en) 2011-02-08 2015-11-03 Nian Wu Polymer-carbohydrate-lipid conjugates
US8883177B2 (en) 2011-06-28 2014-11-11 Nian Wu Pharmaceutical compositions for parenteral administration
KR20180121925A (ko) * 2016-03-07 2018-11-09 애로우헤드 파마슈티컬스 인코포레이티드 치료용 화합물을 위한 표적화 리간드
KR102348425B1 (ko) 2016-03-07 2022-01-10 애로우헤드 파마슈티컬스 인코포레이티드 치료용 화합물을 위한 표적화 리간드
KR20220005633A (ko) * 2016-03-07 2022-01-13 애로우헤드 파마슈티컬스 인코포레이티드 치료용 화합물을 위한 표적화 리간드
KR102515329B1 (ko) 2016-03-07 2023-03-29 애로우헤드 파마슈티컬스 인코포레이티드 치료용 화합물을 위한 표적화 리간드
CN107670050A (zh) * 2017-08-30 2018-02-09 重庆阿普格雷生物科技有限公司 基于pki‑587的抗癌中间体和聚乙二醇偶联抗癌药物、及其制备方法和应用
CN107670050B (zh) * 2017-08-30 2019-06-07 重庆阿普格雷生物科技有限公司 基于pki-587的抗癌中间体和聚乙二醇偶联抗癌药物、及其制备方法和应用
CN113292616A (zh) * 2021-05-20 2021-08-24 内蒙古大学 一种单糖配体功能化的阳离子脂质类化合物及其制备方法与应用

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