This application is a divisional of application Ser. No. 12/787,241 (filed (May 25, 2010), which is a continuation in part of application Ser. No. 12/744,472 (filed May 24, 2010), which is the national stage of International Application No. PCT/US2009/030762 (filed Jan. 12, 2009), which claims the benefit of Provisional Application No. 61/020,684 (filed Jan. 11, 2008) and Provisional Application No. 61/029,337 (filed Feb. 16, 2008).

FIELD OF THE DISCLOSURE

The present disclosure relates to multifunctional copolymers of polyoxazolines, methods of synthesis and intermediate compounds useful in producing such polyoxazoline derivatives, and conjugates of these polyoxazolines with therapeutic, diagnostic and biological-targeting molecules produced using such polyoxazoline derivatives.

BACKGROUND

Polymer-modified therapeutics have proven to be of great utility in modem pharmaceutical science. Because of the success of polymer-modified therapeutics, it is of interest to expand the range of polymers suitable for such applications, especially to provide polymers having properties not possessed by polymers of the prior art. A need exists for water-soluble, non-toxic polymers which can be used to prepare desired conjugates with target molecules. A need also exists for such polymers with multiple functionalities for use in preparing desired conjugates with multiple molecules (such as, but not limited to, targeting moieties, therapeutic moieties and diagnostic moieties). Using such polymers with multiple functionalities would allow the production of conjugates containing one or more diagnostic and/or therapeutic moiety or conjugates containing a mixture of distinct diagnostic and/or targeting moieties. The present disclosure provides heterofunctional polyoxazoline compounds which provide ready coupling to a range of molecules, such as but not limited to, targeting, therapeutic and/or diagnostic moieties.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A shows the efficacy of Irinotecan in a mouse xenograft model using human colorectal cancer cells.

FIG. 1B shows the efficacy of POZ-Irinotecan in a mouse xenograft model using human colorectal cancer cells.

FIG. 2A shows the efficacy of Irinotecan in a mouse xenograft model using platinum resistant ovarian cancer cells.

FIG. 2B shows the efficacy of POZ-Irinotecan in a mouse xenograft model using platinum resistant ovarian cancer cells.

FIG. 3A shows the efficacy of Irinotecan in a mouse xenograft model using oropharyngeal carcinoma cancer cells.

FIG. 3B shows the efficacy of Folate-POZ-Irinotecan in a mouse xenograft model using oropharyngeal carcinoma cancer cells.

DETAILED DESCRIPTIONDefinitions

As used herein, the term â€œPOZâ€ or â€œPOZ polymerâ€ refers to a polymer of 2-substituted-2-oxazoline containing a repeating unit having the structure â€”[N(COR₂)CH₂CH₂]_nâ€” in which R₂is independently selected for each repeating unit from an unsubstituted or substituted alkyl, alkenyl, aralkyl or heterocyclylalkyl group and n is from 3-1000; in one embodiment, the unsubstituted or substituted alkyl, alkenyl, aralkyl or heterocyclylalkyl groups comprise from 1-10 carbon atoms.

As used herein, the term â€œPMOZâ€ refers to POZ with the repeating unit having the structure â€”[N(COCH₃)CH₂CH₂]_nâ€”.

As used herein, the term â€œPEOZâ€ refers to POZ with the repeating unit having the structure â€”[N(COCH₂CH₃)CH₂CH₂]_nâ€”.

As used herein, the term M-POZ, M-PMOZ or M-PEOZ refers to the polymers above in which the nitrogen of the initiating monomer unit is bound to methyl.

As used herein, the term â€œPOZ derivativeâ€ or â€œpolyoxazoline derivativeâ€ refers to a structure comprising a POZ polymer, the POZ polymer having at least one functional group capable of forming a linkage, directly or indirectly, with a chemical group on a target molecule, a linker or a branching moiety.

As used herein, the term â€œtarget moleculeâ€ refers to any molecule having therapeutic or diagnostic application or a targeting function, wherein the target molecule is capable of reacting with a functional group on a POZ polymer or a POZ derivative of the present disclosure, including, but not limited to, a drug, a diagnostic agent, a targeting molecule, an organic small molecule, an oligonucleotide, a carbohydrate, a polypeptide, an antibody or antibody fragment or a protein.

As used herein, the term â€œtarget molecule-POZ conjugateâ€ refers to a conjugate of a POZ derivative of the present disclosure and at least one target molecule.

As used herein, the term â€œhydrolytically stable target molecule-POZ conjugateâ€ refers to a conjugate of a POZ derivative of the present disclosure and at least one target molecule, such that all the chemical linkages in the conjugate are hydrolytically stable.

As used herein, the term â€œhydrolytically stableâ€ refers to a linkage that is stable in aqueous solutions under physiological conditions; in one embodiment, such linkages are stable for at least 12 hours, 24 hours, 48 hours, 96 hours, 192 hours or greater; in an alternate embodiment such linkages are stable indefinitely.

As used herein, the term â€œhydrolytically unstableâ€ refers to a linkage that is not stable in aqueous solutions under physiological conditions.

As used herein, the term â€œphysiological conditionsâ€ refers to an aqueous solution having a pH from 6-8 and a temperature from 30-42 degrees Celsius.

As used herein, the term â€œfunctional groupâ€ refers to those groups that will react with a corresponding group, including those groups that will react readily with electrophilic or nucleophilic groups, in contrast to those groups that require strong catalysis or impractical reaction conditions in order to react.

As used herein, the term â€œlinkâ€ , â€œlinkedâ€ â€œlinkageâ€ or â€œlinkerâ€ when used with respect to a POZ derivative described herein, or components thereof, refers to groups or bonds that normally are formed as the result of a chemical reaction and typically are covalent linkages.

As used herein, the term â€œprotectedâ€ with respect to hydroxyl groups, amine groups, sulfhydryl groups and other reactive groups refers to forms of these functionalities which are protected from undesirable reaction with a protecting group known to those skilled in the art such as those set forth in Protective Groups in Organic Synthesis, Greene, T. W.; Wuts, P. G. M., John Wiley & Sons, New York, N.Y., (3rd Edition, 1999) which can be added or removed using the procedures set forth therein. Examples of protected hydroxyl groups include, but are not limited to, silyl ethers such as those obtained by reaction of a hydroxyl group with a reagent such as, but not limited to, t-butyldimethyl-chlorosilane, trimethylchlorosilane, triisopropylchlorosilane, triethylchlorosilane; substituted methyl and ethyl ethers such as, but not limited to methoxymethyl ether, methylthiomethyl ether, benzyloxymethyl ether, t-butoxymethyl ether, 2-methoxyethoxymethyl ether, tetrahydropyranyl ethers, 1-ethoxyethyl ether, allyl ether, benzyl ether; esters such as, but not limited to, benzoylformate, formate, acetate, trichloroacetate, and trifluoracetate. Examples of protected amine groups include, but are not limited to, various alkyloxycarbonyls, amides such as, formamide, acetamide, trifluoroacetamide, and benzamide; imides, such as phthalimide, and dithiosuccinimide; and others. Examples of protected sulfhydryl groups include, but are not limited to, thioethers such as S-benzyl thioether, and S-4-picolyl thioether; substituted S-methyl derivatives such as hemithio, dithio and aminothio acetals; and others.

As used herein, the terms â€œpreventionâ€ , â€œpreventâ€ , â€œpreventingâ€ , â€œsuppressionâ€ , â€œsuppressâ€ and â€œsuppressingâ€ as used herein refer to a course of action (such as administering a compound or pharmaceutical composition) initiated prior to the onset of a symptom, aspect, or characteristics of a disease or condition so as to prevent or reduce such symptom, aspect, or characteristics. Such preventing and suppressing need not be absolute to be useful.

As used herein, the terms â€œtreatmentâ€ , â€œtreatâ€ and â€œtreatingâ€ as used herein refers a course of action (such as administering a compound or pharmaceutical composition) initiated after the onset of a symptom, aspect, or characteristics of a disease or condition so as to eliminate or reduce such symptom, aspect, or characteristics. Such treating need not be absolute to be useful.

As used herein, the term â€œin need of treatmentâ€ as used herein refers to a judgment made by a caregiver that a patient requires or will benefit from treatment. This judgment is made based on a variety of factors that are in the realm of a caregiver's expertise, but that includes the knowledge that the patient is ill, or will be ill, as the result of a disease or condition that is treatable by a method or compound of the disclosure.

As used herein, the term â€œin need of preventionâ€ as used herein refers to a judgment made by a caregiver that a patient requires or will benefit from prevention. This judgment is made based on a variety of factors that are in the realm of a caregiver's expertise, but that includes the knowledge that the patient will be ill or may become ill, as the result of a disease or condition that is preventable by a method or compound of the disclosure.

As used herein, the terms â€œindividualâ€ , â€œsubjectâ€ or â€œpatientâ€ as used herein refers to any animal, including mammals, such as mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, or primates, and humans. The term may specify male or female or both, or exclude male or female.

As used herein, the term â€œtherapeutically effective amountâ€ as used herein refers to an amount of a compound, either alone or as a part of a pharmaceutical composition, that is capable of having any detectable, positive effect on any symptom, aspect, or characteristics of a disease or condition. Such effect need not be absolute to be beneficial.

As used herein, the term â€œalkylâ€ , whether used alone or as part of a substituent or linking group, includes straight hydrocarbon groups comprising from one to twenty carbon atoms. Thus the phrase includes straight chain alkyl groups such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, decyl, undecyl, dodecyl and the like. The phrase also includes branched chain isomers of straight chain alkyl groups, including but not limited to, the following which are provided by way of example: â€”CH(CH₃)₂, â€”CH(CH₃)(CH₂CH₃), â€”CH(CH₂CH₃)₂, â€”C(CH₃)₃, â€”C(CH₂CH₃)₃, â€”CH₂CH(CH₃)₂, â€”CH₂CH(CH₃)(CH₂CH₃), â€”CH₂CH(CH₂CH₃)₂, â€”CH₂C(CH₃)₃, â€”CH₂C(CH₂CH₃)₃, â€”CH(CH₃)CH(CH₃)(CH₂CH₃), â€”CH₂CH₂CH(CH₃)₂, â€”CH₂CH₂CH(CH₃)(CH₂CH₃), â€”CH₂CH₂CH(CH₂CH₃)₂, â€”CH₂CH₂C(CH₃)₃, â€”CH₂CH₂C(CH₂CH₃)₃, â€”CH(CH₃)CH₂CH(CH₃)₂, â€”CH(CH₃)CH(CH₃)CH(CH₃)CH(CH₃), â€”CH(CH₂CH₃)CH(CH₃)CH(CH₃)(CH₂CH₃), and others. The phrase also includes cyclic alkyl groups such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl and such rings substituted with straight and branched chain alkyl groups as defined above. The phrase also includes polycyclic alkyl groups such as, but not limited to, adamantyl norbornyl, and bicyclo[2.2.2]octyl and such rings substituted with straight and branched chain alkyl groups as defined above.

As used herein, the term â€œalkyleneâ€ , whether used alone or as part of a substituent group, includes any group obtained by removing a hydrogen atom from an alkyl group; an alkylene group forms two bonds with other groups.

As used herein, the term â€œalkenylâ€ , whether used alone or as part of a substituent group, includes an alkyl group having at least one double bond between any two adjacent carbon atoms.

As used herein, the term â€œalkynylâ€ , whether used alone or as part of a substituent group, includes an alkyl group having at least one triple bond between any two adjacent carbon atoms.

As used herein, the term â€œunsubstituted alkylâ€ , â€œunsubstituted alkenylâ€ , and â€œunsubstituted alkynylâ€ refers to alkyl, alkenyl and alkynyl groups that do not contain heteroatoms.

The phrase â€œsubstituted alkylâ€ , â€œsubstituted alkenylâ€ , and â€œsubstituted alkynylâ€ refers to alkyl, alkenyl and alkynyl groups as defined above in which one or more bonds to a carbon(s) or hydrogen(s) are replaced by a bond to non-hydrogen or non-carbon atoms such as, but not limited to, a halogen atom in halides such as F, Cl, Br, and I; and oxygen atom in groups such as carbonyl, carboxyl, hydroxyl groups, alkoxy groups, aryloxy groups, and ester groups; a sulfur atom in groups such as thiol groups, alkyl and aryl sulfide groups, sulfone groups, sulfonyl groups, and sulfoxide groups; a nitrogen atom in groups such as amines, amides, alkylamines, dialkylamines, arylamines, alkylarylamines, diarylamines. N-oxides, imides, enamines imines, oximes, hydrazones, and nitriles; a silicon atom in groups such as in trialkylsilyl groups, dialkylarylsilyl groups, alkyldiarylsilyl groups, and triarylsilyl groups; and other heteroatoms in various other groups. Other alkyl groups include those in which one or more bonds to a carbon or hydrogen atom is replaced by a bond to an oxygen atom such that the substituted alkyl group contains a hydroxyl, alkoxy, aryloxy group, or heterocyclyloxy group. Still other alkyl groups include alkyl groups that have an amine, alkylamine, dialkylamine, arylamine, (alkyl)(aryl)amine, diarylamine, heterocyclylamine, (alkyl)(heterocyclyl)-amine, (aryl)(heterocyclyl)amine, or diheterocyclylamine group.

As used herein, the term â€œunsubstituted arylâ€ refers to monocyclic or bicyclic aromatic hydrocarbon groups having 6 to 12 carbon atoms in the ring portion, such as, but not limited to, phenyl, naphthyl, anthracenyl, biphenyl and diphenyl groups, that do not contain heteroatoms. Although the phrase â€œunsubstituted arylâ€ includes groups containing condensed rings such as naphthalene, it does not include aryl groups that have other groups such as alkyl or halo groups bonded to one of the ring members, as aryl groups such as tolyl are considered herein to be substituted aryl groups as described below. Unsubstituted aryl groups may be bonded to one or more carbon atom(s), oxygen atom(s), nitrogen atom(s), and/or sulfur atom(s) in the parent compound, however.

As used herein, the term â€œsubstituted aryl groupâ€ has the same meaning with respect to unsubstituted aryl groups that substituted alkyl groups had with respect to unsubstituted alkyl groups. However, a substituted aryl group also includes aryl groups in which one of the aromatic carbons is bonded to one of the non-carbon or non-hydrogen atoms, such as, but not limited to, those atoms described above with respect to a substituted alkyl, and also includes aryl groups in which one or more aromatic carbons of the aryl group is bonded to a substituted and/or unsubstituted alkyl, alkenyl, or alkynyl group as defined herein. This includes bonding arrangements in which two carbon atoms of an aryl group are bonded to two atoms of an alkyl, alkenyl, or alkynyl group to define a fused ring system (e.g. dihydronaphthyl or tetrahydronaphthyl). Thus, the phrase â€œsubstituted arylâ€ includes, but is not limited to tolyl, and hydroxyphenyl among others.

As used herein, the term â€œunsubstituted aralkylâ€ refers to unsubstituted or substituted alkyl, alkenyl or alkynyl groups as defined above in which a hydrogen or carbon bond of the unsubstituted or substituted alkyl, alkenyl or alkynyl group is replaced with a bond to an aryl group as defined above. For example, methyl (CH₃) is an unsubstituted alkyl group. If a hydrogen atom of the methyl group is replaced by a bond to a phenyl group, such as if the carbon of the methyl were bonded to a carbon of benzene, then the compound is an unsubstituted aralkyl group (i.e., a benzyl group).

As used herein, the term â€œsubstituted aralkylâ€ has the same meaning with respect to unsubstituted aralkyl groups that substituted aryl groups had with respect to unsubstituted aryl groups. However, a substituted aralkyl group also includes groups in which a carbon or hydrogen bond of the alkyl part of the group is replaced by a bond to a non-carbon or a non-hydrogen atom.

As used herein, the term â€œunsubstituted heterocyclylâ€ refers to both aromatic and nonaromatic ring compounds including monocyclic, bicyclic, and polycyclic ring compounds such as, but not limited to, quinuclidyl, containing 3 or more ring members of which one or more is a heteroatom such as, but not limited to, N, O, and S. Although the phrase â€œunsubstituted heterocyclylâ€ includes condensed heterocyclic rings such as benzimidazolyl, it does not include heterocyclyl groups that have other groups such as alkyl or halo groups bonded to one of the ring members, as compounds such as 2-methylbenzimidazolyl are â€œsubstituted heterocyclylâ€ groups as defined below. Examples of heterocyclyl groups include, but are not limited to: unsaturated 3 to 8 membered rings containing 1 to 4 nitrogen atoms such as, but not limited to pyrrolyl, pyrrolinyl, imidazolyl, pyrazolyl, pyridyl, dihydropyridyl, pyrimidyl, pyrazinyl, pyridazinyl, triazolyl, tetrazolyl; saturated 3 to 8 membered rings containing 1 to 4 nitrogen atoms such as, but not limited to, pyrrolidinyl, imidazolidinyl, piperidinyl, piperazinyl; condensed unsaturated heterocyclic groups containing 1 to 4 nitrogen atoms such as, but not limited to, indolyl, isoindolyl, indolinyl, indolizinyl, benzimidazolyl, quinolyl, isoquinolyl, indazolyl, benzotriazolyl; unsaturated 3 to 8 membered rings containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms such as, but not limited to, oxazolyl, isoxazolyl, oxadiazolyl; saturated 3 to 8 membered rings containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms such as, but not limited to, morpholinyl; unsaturated condensed heterocyclic groups containing 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, for example, benzoxazolyl, benzoxadiazolyl, benzoxazinyl (e.g. 2H-1,4-benzoxazinyl etc.); unsaturated 3 to 8 membered rings containing 1 to 3 sulfur atoms and 1 to 3 nitrogen atoms such as, but not limited to, thiazolyl, isothiazolyl, thiadiazolyl (e.g. 1,2,3-thiadiazolyl, 1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl, etc.); saturated 3 to 8 membered rings containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms such as, but not limited to, thiazolodinyl; saturated and unsaturated 3 to 8 membered rings containing 1 to 2 sulfur atoms such as, but not limited to, thienyl, dihydrodithiinyl, dihydrodithionyl, tetrahydrothiophene, tetrahydrothiopyran; unsaturated condensed heterocyclic rings containing 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms such as, but not limited to, benzothiazolyl, benzothiadiazolyl, benzothiazinyl (e.g. 2H-1,4-benzothiazinyl, etc.), dihydrobenzothiazinyl (e.g. 2H-3,4-dihydrobenzothiazinyl, etc.), unsaturated 3 to 8 membered rings containing oxygen atoms such as, but not limited to furyl; unsaturated condensed heterocyclic rings containing 1 to 2 oxygen atoms such as benzodioxolyl (e.g. 1,3-benzodioxoyl, etc.); unsaturated 3 to 8 membered rings containing an oxygen atom and 1 to 2 sulfur atoms such as, but not limited to, dihydrooxathiinyl; saturated 3 to 8 membered rings containing 1 to 2 oxygen atoms and 1 to 2 sulfur atoms such as 1,4-oxathiane; unsaturated condensed rings containing 1 to 2 sulfur atoms such as benzothienyl, benzodithiinyl; and unsaturated condensed heterocyclic rings containing an oxygen atom and 1 to 2 oxygen atoms such as benzoxathiinyl. Heterocyclyl group also include those described above in which one or more S atoms in the ring is double-bonded to one or two oxygen atoms (sulfoxides and sulfones). For example, heterocyclyl groups include tetrahydrothiophene, tetrahydrothiophene oxide, and tetrahydrothiophene 1,1-dioxide. Preferred heterocyclyl groups contain 5 or 6 ring members. More preferred heterocyclyl groups include morpholine, piperazine, piperidine, pyrrolidine, imidazole, pyrazole, 1,2,3-triazole, 1,2,4-triazole, tetrazole, thiomorpholine, thiomorpholine in which the S atom of the thiomorpholine is bonded to one or more O atoms, pyrrole, homopiperazine, oxazolidin-2-one, pyrrolidin-2-one, oxazole, quinuclidine, thiazole, isoxazole, furan, and tetrahydrofuran.

As used herein, the term â€œsubstituted heterocyclylâ€ has the same meaning with respect to unsubstituted heterocyclyl groups that substituted alkyl groups had with respect to unsubstituted alkyl groups. However, a substituted heterocyclyl group also includes heterocyclyl groups in which one of the carbons is bonded to one of the non-carbon or non-hydrogen atom, such as, but not limited to, those atoms described above with respect to a substituted alky and substituted aryl groups and also includes heterocyclyl groups in which one or more carbons of the heterocyclyl group is bonded to a substituted and/or unsubstituted alkyl, alkenyl, alkynyl or aryl group as defined herein. This includes bonding arrangements in which two carbon atoms of an heterocyclyl group are bonded to two atoms of an alkyl, alkenyl, or alkynyl group to define a fused ring system. Examples, include, but are not limited to, 2-methylbenzimidazolyl, 5-methylbenzimidazolyl, 5-chlorobenzthiazolyl, 1-methyl piperazinyl, and 2-chloropyridyl among others.

As used herein, the term â€œunsubstituted heterocyclylalkylâ€ refers to unsubstituted or substituted alkyl, alkenyl or alkynyl groups as defined above in which a hydrogen or carbon bond of the unsubstituted or substituted alkyl, alkenyl or alkynyl group is replaced with a bond to a heterocyclyl group as defined above. For example, methyl (CH₃) is an unsubstituted alkyl group. If a hydrogen atom of the methyl group is replaced by a bond to a heterocyclyl group, such as if the carbon of the methyl were bonded to carbon 2 of pyridine (one of the carbons bonded to the N of the pyridine) or carbons 3 or 4 of the pyridine, then the compound is an unsubstituted heterocyclylalkyl group.

As used herein, the term â€œsubstituted heterocyclylalkylâ€ has the same meaning with respect to unsubstituted heterocyclylalkyl groups that substituted aryl groups had with respect to unsubstituted aryl groups. However, a substituted heterocyclylalkyl group also includes groups in which a non-hydrogen atom is bonded to a heteroatom in the heterocyclyl group of the heterocyclylalkyl group such as, but not limited to, a nitrogen atom in the piperidine ring of a piperidinylalkyl group.

General Description

Polyoxazolines (POZ) are polymers prepared from 2-substituted-2-oxazoline monomers. These polymers are water soluble and have been reported to be nontoxic in mammalian model systems. POZ is generally prepared by reaction of the appropriate stoichiometric amount of 2-alkyl-2-oxazoline with an electrophilic initiator, such as methyl triflate (CH₃â€”OSO₂â€”CF₃) or a strong acid such as triflic acid or p-toluenesulfonic acid, followed by termination with a nucleophile such as, but not limited to, hydroxide, a thiol or an amine. The polymer produced is conveniently described in shorthand with the initiating group designated by the leftmost group and the terminating group designated by the rightmost group, with the 2-alkyl-2-oxazoline component in the middle. Therefore, when this shorthand description is used in the current specification, it is intended that the left side of the designation presents the â€œinitiator endâ€ and the right side of the designation presents the â€œtermination endâ€ , unless designated otherwise.

For example, when the 2-alkyl-2-oxazoline is 2-methyl-2-oxazoline, methyl triflate is used as the initiator and hydroxide is used as the terminator, the following POZ is produced:

CH₃â€”[N(COCH₃)CH₂CH₂]_nâ€”OH

The polymer above is conveniently described in shorthand notation as M-PMOZ-OH, in which the methyl initiator is designated by the leftmost M, PMOZ represents polymethyloxazoline with the methyl of the repeating unit designated by the M of PMOZ, and the terminating hydroxyl is designated by the â€”OH.

Another commonly used monomer is 2-ethyl-2-oxazoline, which with methyl triflate initiation and hydroxide termination would provide the following POZ polymer:

CH₃â€”[N(COCH₂CH₃)CH₂CH₂]_nâ€”OH

The polymer above is conveniently described in shorthand notation as M-PEOZ-OH, in which the methyl initiator is designated by the leftmost M, PEOZ represents polymethyloxazoline with the ethyl of the repeating unit designated by the E of PEOZ, and the terminating hydroxyl is designated by the â€”OH.

The degree of polymerization, n, for well characterized polymers can range from approximately 3 to about 1000.

The polymerization process is referred to as a living, cationic polymerization since initiation with an electrophile produces an oxazolinium cation that then reacts in a chain reaction with additional monomer units to produce a growing, â€œlivingâ€ cation.

[Image Omitted]

One can predict the products of termination by assuming that the living cation can be represented in the following non-cyclic form (for polymerization of 2-methyl-2-oxazoline initiated with methyl triflate), although in reality the cyclic form is certainly the most important:

CH₃â€”[N(COCH₃)CH₂CH₂]_nâ€”N(COCH₃)CH₂CH₂⁺

In the current discussion we will represent this cation as M-PMOZ⁺. As noted above, this POZ cation can be â€œterminatedâ€ by reacting with nucleophiles such as hydroxide, thiols or amines.

Oxazoline polymerization can also be initiated with functional electrophiles. For example the electrophilic initiator ethyl 3-bromopropionate has been used to initiate 2-ethyl-2-oxazoline polymerization. Termination with hydroxide gives the following polymer:

HO₂Câ€”CH₂CH₂â€”[N(COCH₂CH₃)CH₂CH₂]_nâ€”OH

Yet another route to preparing polyoxazolines with functional groups is to copolymerize a monomer such as 2-ethyl-2-oxazoline with an oxazoline monomer having a functional group in the 2-position (F. C. Gaertner, R. Luxenhofer, B. Blechert, R. Jordan and M. Essler, J. Controlled Release, 2007, 119, 291-300). For example, Jordan and colleagues have prepared oxazolines with acetylenes and protected aldehydes, carboxylic acids and amines in the 2-position. Copolymerization of these functional monomers with 2-ethyl-2-oxazoline gives random copolymers with multiple pendent or side-chain functional groups. For example, initiation with methyl triflate of polymerization of 2-ethyl-2-oxazoline and 2-pentynyl-2-oxazoline, followed by termination with piperazine (NHC₄H₈NH) gives the following random copolymer:

CH₃â€”{[N(COCH₂CH₃)CH₂CH₂]_nâ€”[N(COCH₂CH₂CH₂â€”CCH)CH₂CH₂]_m}_ranâ€”NC₄H₈NH

The subscript â€œranâ€ indicates that the polymer is a random copolymer. Values of n are typically around 20-30 while m is around 2-5.

These copolymers with pendent functional groups and a terminal functional group are useful in that the pendent and terminal functional groups can be â€œchemically orthogonalâ€ functional groups. Chemically orthogonal functional groups are those functional groups that will not react with each other but will react selectively with other functional groups. For example, the molecule above has two functional groups, a terminal secondary amine and pendent acetylenes. The acetylene will not react with the amine but will, for example, react with an azide group (â€”N₃). Similarly, the amine will not react with acetylene or azide but will react with, for example, an isothiocyanate group (â€”NCS). Jordan has used this copolymer to couple an azide-functionalized RGD peptide to the acetylene group, and an isothiocyanate-functionalized metal chelator to the amine. The RGD peptide is known to target tumors, and a diagnostic or therapeutic radionuclide can bind to the chelating group. The final conjugate can be used to image or treat tumors (R. Luxenhofer, M. Lopez-Garcia, A. Frannk, H. Kessler and R. Jordan, Proceedings of the American Chemical Society, PMSE Prepr. 2006, 95, 283-284).

One problem hindering use of the above piperazine- or piperidine-terminated polyoxazolines is that they are difficult to purify. This difficulty arises because contaminating water present during termination leads to nucleophilic attack by water and consequent formation of secondary amine impurity (O. Nuyken, G. Maier, A. Gross, Macromol. Chem. Phys. 197, 83-95, 1996). Since the products from piperazine and piperidine termination always contain a tertiary amine, ion-exchange chromatography cannot be used to remove the contaminating secondary amine.

Still another limitation of the pendent polyoxazolines illustrated above is that these compounds possess a single terminal functional group. Consequently, this structural configuration limits the number of drug or targeting moieties that can be attached to the terminus, whereas effective use of such compounds for therapeutic diagnostic and targeting applications may require multiple loading of these moieties. The polymers of the current disclosure avoid this limitation by providing for multiple copies of each of two chemically reactive and orthogonal functional groups.

Novel Heterofunctional Polyoxazoline Derivatives

The present disclosure avoids the limitations of the prior art by providing heterofunctional polyoxazoline derivatives of two 2-substituted-2-oxazolines comprising at least two functional groups which are chemically reactive and chemically orthogonal to one another. The heterofunctional polyoxazoline derivatives may contain additional functional groups as well. In certain embodiments, all functional groups are chemically orthogonal to one another, while in other embodiments, the additional functional groups may be chemically orthogonal to at least one other functional group present on the heterofunctional polyoxazoline derivative.

Single-Arm Heterofunctional Polyoxazoline Derivatives

In one embodiment, the heterofunctional polyoxazoline derivative is represented by the general structure:

R₁â€”{[N(COX)CH₂CH₂]_oâ€”[N(COY)CH₂â€”CH₂)]_m}_aâ€”Z

wherein:
R₁is an initiating group;
X is a pendent moiety containing a first functional group;
Y is a pendent moiety containing a second functional group;
Z is a terminating nucleophile; in certain embodiments Z is inert (i.e., does not contain a functional group); in other embodiments, Z contains a third functional group;
a is ran which indicates a random copolymer or block which indicates a block copolymer; and
o and m are each an integer independently selected from 1-50.

Exemplary initiating groups include, but are not limited to, hydrogen, alkyl, substituted alkyl, aralkyl, or substituted aralkyl groups. In a particular embodiment, the initiating group is a methyl group. The R₁group is selected to lack a functional group. Additional exemplary initiating groups are disclosed in PCT Application No. PCT/US2008/078159, which is hereby incorporated by reference for such teaching.

As shown by the general structure above, the heterofunctional polyoxazoline derivative comprises at least two functional groups. The first and second functional groups are present on the pendent moieties X and Y, respectively. In certain embodiment, a third functional group is present on the Z group; however, the presence of the third functional group is optional and in some embodiments Z may be inert (i.e., lacking the third functional group).

X and Y are pendent moieties bearing first and second functional groups, respectively.

In certain embodiments, X and Y may contain a linking portion that links the first and second functional groups to the polyoxazoline derivative. Exemplary linking portions include alkylene groups. In certain cases, the alkylene group is a C₁-C₁₅alkylene group. The linking portions of X and Y may be the same or may be different. For example, both X and Y may contain a C₅alkylene group as the linking portion or X may contain a C₅alkylene group as the linking portion and Y may contain a C₈alkylene group as the linking portion.

The first and second functional groups are chemically orthogonal to one another. The first and second functional groups include, but are not limited to, alkyne, amine, oxyamine, aldehyde, ketone, acetal, ketal, maleimide, ester, carboxylic acid, activated carboxylic acid (such as, but not limited to, N-hydroxysuccinimidyl (NHS) and 1-benzotriazineyl active ester), an active carbonate, a chloroformate, alcohol, azide, vinyl sulfone, or orthopyridyl disulfide (OPSS), provided that the selection is made for X and Y so that the first and second functional groups are chemically orthogonal to one another.

As discussed above, Z may contain a third functional group or be inert. In those embodiments where Z contains a third functional group, the third functional group may be chemically orthogonal to one or both of the first and second functional groups. Exemplary third functional groups include, but are not limited to, alkyne, amine, oxyamine, aldehyde, ketone, acetal, ketal, maleimide, ester, carboxylic acid, activated carboxylic acid (such as, but not limited to, N-hydroxysuccinimidyl (NHS) and 1-benzotriazineyl active ester), an active carbonate, a chloroformate, alcohol, azide, vinyl sulfone, or orthopyridyl disulfide (OPSS).

In a particular embodiment, Z contains the third functional group and is represented by the structure â€”Sâ€”Uâ€”W, wherein S is a sulphur atom, U is a linking group and W is the third functional group. In this embodiment, representative U groups include alkylene groups. In a particular embodiment, U is â€”(CH₂)_pâ€” where p is an integer selected from 1 to 10. In a particular embodiment, W may be a carboxylic acid, a protected carboxylic acid, an active ester, an amine or a protected amine. Furthermore, W may be selected from the groups described above for Z.

As discussed herein, the third functional group may be chemically orthogonal to one or both of the first functional groups.

Embodiments of the foregoing include but are not limited to:

R₁â€”{[N(COX)CH₂CH₂]_oâ€”[N(COY)CH₂â€”CH₂)]_m}_aâ€”Sâ€”(CH₂)_pâ€”NH₂

and

R₁â€”{[N(COX)CH₂CH₂]_oâ€”[N(COY)CH₂â€”CH₂)]_m}_aâ€”Sâ€”(CH₂)_pâ€”CO₂H.

In an alternate embodiment, Z lacks the third functional group and is represented by the structure â€”S-T-V, wherein S is a sulphur atom, T is a linking group and V is an inert group. In this embodiment, representative T groups include alkylene groups. In a particular embodiment, T is â€”(CH₂)_pâ€” where p is an integer selected from 1 to 10. V may be any inert group. In a particular embodiment, V is â€”CO₂CH₃or â€”C₄H₅.

Embodiments of the foregoing include but are not limited to:

R₁â€”{[N(COX)CH₂CH₂]_oâ€”[N(COY)CH₂â€”CH₂)]_m}_aâ€”Sâ€”(CH₂)_pâ€”C₆H₅

and

R₁â€”{([N(COX)CH₂CH₂]_oâ€”[N(COY)CH₂â€”CH₂)]_m}_aâ€”Sâ€”(CH₂)_pâ€”CO₂CH₃.

In an alternate embodiment, the heterofunctional polyoxazoline derivative is represented by the general structure below.

R₁â€”{([N(COX)CH₂CH₂]_oâ€”[N(COR₂)CH₂â€”CH₂)]_nâ€”[N(COY)CH₂â€”CH₂)]_m}_aâ€”Z

wherein:
R₁, X, Y, Z, o, m and a are as defined above;

- R₂lacks a functional group and is independently selected for each repeating unit from an unsubstituted or substituted alkyl, an unsubstituted or substituted alkenyl, an unsubstituted or substituted aralkyl or an unsubstituted or substituted heterocyclylalkyl group.

In this embodiment, an additional 2-alkyl-2-oxaxoline is introduced as a third co-monomer. This third 2-alkyl-2-oxaxoline co-monomer lacks a functional group and provides a chemically unreactive spacer between the first and second functional groups present on X and Y, respectively. Such a configuration prevents the first and second functional groups from being sterically hindered.

In certain embodiment, o and m are not zero and the heterofunctional polyoxazoline derivative contains first and second functional groups on X and Y respectively. The first and second functional groups may be selected from the groups described above. As above, the first and second functional groups are chemically orthogonal to one another.

The Z group, as discussed above may contain a third functional group or be inert. In those embodiments where Z contains a third functional group, the third functional group may be chemically orthogonal to one or both of the first and second functional groups. When Z contains the third functional group, the third functional group may be selected from the groups described above.

Embodiments of the foregoing include but are not limited to:

R₁â€”{([N(COX)CH₂CH₂]_oâ€”[N(COR₂)CH₂â€”CH₂)]_nâ€”[N(COY)CH₂â€”CH₂)]_m}_aâ€”Sâ€”(CH₂)_pâ€”NH₂

and

R₁â€”{[N(COX)CH₂CH₂]_oâ€”[N(COR₂)CH₂â€”CH₂)]_nâ€”[N(COY)CH₂â€”CH₂)]_m}_nâ€”Sâ€”(CH₂)_pâ€”CO₂H.

Embodiments of the foregoing include but are not limited to:

R₁â€”{([N(COX)CH₂CH₂]_oâ€”[N(COR₂)CH₂â€”CH₂)]_nâ€”[N(COY)CH₂â€”CH₂)]_n}_aâ€”Sâ€”(CH₂)_pâ€”C₆H₅

and

R₁â€”{[N(COX)CH₂CH₂]_oâ€”[N(COR₂)CH₂â€”CH₂)]_nâ€”[N(COY)CH₂â€”CH₂)]_n}_aâ€”Sâ€”(CH₂)_pâ€”CO₂CH₃.

In still another alternate embodiment, the heterofunctional polyoxazoline derivatives are defined by the general structure:

R₁â€”{[N(COX)CH₂CH₂]_oâ€”[N(COR₂)CH₂â€”CH₂)]_n}_aâ€”Sâ€”Uâ€”W

wherein:
R₁, X, R₂, o, n and a are as defined above;
S is a sulphur atom;
U is a linking group; and
W is the third functional group.

In this embodiment, representative U groups include alkylene groups. In a particular embodiment, U is â€”(CH₂)â€” where p is an integer selected from 1 to 10. In this embodiment, the second functional group is lacking. In these embodiments, W contains a third functional group and the third functional group is chemically orthogonal to the first functional group on X. The first functional group may be selected from the groups described above. The third functional group W may be selected from the groups described above for X. In a particular embodiment, W may be a carboxylic acid, a protected carboxylic acid, an active ester, an amine or a protected amine.

Embodiments of the foregoing include but are not limited to:

R₁â€”{[N(COX)CH₂CH₂]_oâ€”[N(COR₂)CH₂â€”CH₂)]_n}_aâ€”Sâ€”(CH₂)_pâ€”CO₂H

and

R₁{[N(COX)CH₂CH₂]_oâ€”[N(COR₂)CH₂â€”CH₂)]_n}_aâ€”Sâ€”(CH₂)_pâ€”NH₂.

These heterofunctional polyoxazoline derivatives described herein may be prepared by terminating the POZ cation with a mercapto-ester (such as â€”Sâ€”CH₂CH₂â€”CO₂CH₃) or mercapto-protected amine (such as â€”Sâ€”CH₂CH₂â€”NH-tBoc). These heterofunctional polyoxazoline derivatives provide for effective, large-scale purification by ion-exchange chromatography (to remove secondary amines), and they provide chemically orthogonal functional groups X and W (â€”CO₂H or â€”NH₂) for attachment of one or more target molecules, such as targeting, diagnostic or therapeutic moieties.

Multi-Arm Heterofunctional Polyoxazoline Derivatives

The present disclosure also provides for multi-armed heterofunctional polyoxazoline derivatives. The multi-armed heterofunctional polyoxazoline derivatives may contain from 2 to 8 polyoxazoline chains. In a particular embodiment, the multi-armed heterofunctional polyoxazoline derivatives contain 2 or 4 polyoxazoline chains. The multi-armed heterofunctional polyoxazoline derivatives may be represented by the general formula:

{R₁-{[N(COX)CH₂CH₂]_oâ€”[N(COR₂)CH₂â€”CH₂)]_nâ€”[N(COY)CH₂â€”CH₂)]_m}_aâ€”K_kâ€”}_dâ€”R-Q_q-Z

wherein:
R₁is an initiating group;
R₂lacks a functional group and is independently selected for each polyoxazoline chain from a substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, a substituted or unsubstituted aralkyl or a substituted or unsubstituted heterocyclylalkyl group;
X is a pendent moiety bearing a first functional group;
Y is a pendent moiety bearing a second functional group;
K is a linking moiety linking each polyoxazoline chain to a branching moiety R;
Q is a linking moiety linking the branching moiety R to Z;
R is a branching moiety capable of forming linkages with Z, either directly or through linking group Q, and with each polyoxazoline chain, either directly or through the linking group K;
Z is a moiety containing a third functional group or an inert group;
A is independently selected for each polyoxazoline chain from ran which indicates a random copolymer or block which indicates a block copolymer;
d is an integer selected from 2-8;
k is an integer independently selected for each polyoxazoline chain from one or zero;
q is an integer selected from one or zero;
o is an integer independently selected for each polyoxazoline chain from 1-50;
m is an integer independently selected for each polyoxazoline chain from 0-50;
n is an integer independently selected for each polyoxazoline chain from 0-1000; and
wherein at least two of the first, second and third functional groups may be chemically orthogonal to one another.

In the general structure above, each POZ chain may be the same or may be different. Furthermore, the pendent moieties X and Y may be the same or may be different for each POZ chain.

Exemplary R₁groups include, but not limited to, hydrogen, alkyl, substituted alkyl, aralkyl, or substituted aralkyl groups. In a particular embodiment, the initiating group is a methyl group. The R₁group is selected to lack a functional group. Additional exemplary initiating groups are disclosed in PCT Application No. PCT/US2008/078159, which is hereby incorporated by reference for such teaching.

X and Y are pendent moieties bearing first and second functional groups, respectively. In certain embodiments, X and Y may contain a linking portion that links the first and second functional groups to the polyoxazoline derivative. Exemplary linking portions include alkylene groups. In certain cases, the alkylene group is a C₁-C₁₅alkylene group. The linking portions of X and Y may be the same or may be different. For example, both X and Y may contain a C₅alkylene group as the linking portion or X may contain a C₅alkylene group as the linking portion and Y may contain a C₈alkylene group as the linking portion.

Z may contain a third functional group or be inert. In those embodiments where Z contains a third functional group, the third functional group may be chemically orthogonal to one or both of the first and second functional groups.

The first, second and third functional groups include, but are not limited to, alkyne, amine, oxyamine, aldehyde, ketone, acetal, ketal, maleimide, ester, carboxylic acid, activated carboxylic acid (such as, but not limited to, N-hydroxysuccinimidyl (NHS) and 1-benzotriazineyl active ester), an active carbonate, a chloroformate, alcohol, azide, vinyl sulfone, or orthopyridyl disulfide (OPSS), provided that the selection is made so that at least two of the first, second and third functional groups are chemically orthogonal to one another.

Q is optional and can be any group capable of forming linkages with both the R and Z and will be different depending on the chemistry of the R and Z. Representative Q groups include, but are not limited to, substituted and unsubstituted alkylene groups. In a specific embodiment, Q is â€”(CH₂)_pâ€”, where p is independently selected from 1-10.

K is optional and can be any group capable of forming linkages with both the POZ chain and R and will be different depending on the chemistry of the POZ chain and R. Representative K groups include substituted and unsubstituted alkyl, alkenyl or alkynyl groups. In a specific embodiment, K is â€”(CH₂)_pOâ€”, â€”(CH₂)_pâ€”COâ€”, â€”Sâ€”(CH₂)_pCONHâ€”, â€”Sâ€”(CH₂)_oCO, â€”(CH₂)_pâ€”NHCSOâ€”, â€”(CH₂)â€”NHCO₂â€”, â€”NHâ€”(CH₂)_p, or â€”NHCO₂â€”, where p is an integer from 0-10. K may be the same or different for each POZ chain.

R is a branching moiety capable of forming linkages with both POZ chains and Z, either directly or through linking groups K and Q, respectively. R may be selected from a nitrogen, an aryl group, or â€”CR₃â€”, where R₃is hydrogen or a substituted or unsubstituted alkyl, a substituted or unsubstituted alkenyl, or a substituted or unsubstituted aralkyl group. In a specific embodiment, R is â€”NHâ€”CHâ€”(CH₂)₃â€”CH₂â€”NHâ€” or â€”(CH₂)₄â€”CHâ€”COâ€”NHâ€”(CH₂)₄, â€”CHâ€”NHâ€”COâ€”CHâ€”(CH₂)₄â€”.

In the general structure above, at least two of the first, second and third functional groups must be present. Therefore, when m or n is 0 for each POZ chain, Z cannot be inert.

Several embodiments of the multi-armed heterofunctional polyoxazoline derivative falling under the general structure above are presented below.

Example 1

[Image Omitted]

In this example, d is 2, k is 1, K is â€”Sâ€”(CH₂)_pâ€”COâ€”, where p is an integer from 1-10, for each polyoxazoline chain, q is 0, R is NHâ€”CHâ€”(CH₂)₃â€”CH₂â€”NH and the first and second functional groups are chemically orthogonal to one another; Z may be chemically orthogonal to one or both of the first and second functional groups.

Example 2

[Image Omitted]

In this example, d is 2, k is 1, K is â€”Sâ€”(CH₂)_pâ€”COâ€”, where p is an integer from 1-10, for each polyoxazoline chain, q is 0, R is NHâ€”CHâ€”(CH₂)₃â€”CH₂â€”NH, m is 0, Z is CO₂H and the first and third functional groups may be chemically orthogonal to one another.

Example 3

[Image Omitted]

In this example, d is 4, k is I and K is â€”Sâ€”(CH₂)₂â€”COâ€”NH for each polyoxazoline chain, q and m are 0, R is â€”(CH₂)₄â€”CHâ€”COâ€”NHâ€”(CH₂)₄â€”CHâ€”NHâ€”COâ€”CHâ€”(CH₂)₄â€” and the first and third functional groups may be chemically orthogonal to one another.

Example 4

[Image Omitted]

In this example, d, k, K, q, m and R are as in Example 3 and Z is CO₂H and the first and third functional groups may be chemically orthogonal to one another.

In all of the above embodiments, the polyoxazoline polymers contained in the heterofunctional polyoxazoline derivatives may be random or block copolymers. As used herein, a block copolymer includes those copolymers that have block configurations separated by a random copolymer sequence. Such random and block copolymers may be produced by controlling the introduction of various intermediates during the synthesis process as described below.

Furthermore, in all the embodiments discussed above, one or more of the functional groups may be charged species. For example, one or more of the functional groups may be a carboxylic acid having a negative charge or an amine having a positive charge. As such, the functional groups may form ionic linkages with one or more target molecules.

Methods of Synthesis

In one embodiment, the polymers of the present disclosure are prepared by co-polymerization of appropriate 2-substituted-2-oxazolines containing functional groups. For example, preparation of a water-soluble co-polymer bearing alkyne groups and acetal groups, in addition to a simple 2-alkyl-2-oxazoline can be synthesized using the following oxazoline monomers:

[Image Omitted]

The polymerization is initiated by an electrophile such as, but not limited to, methyl triflate, methyl tosylate, p-toluenesulfonic acid, or triflic acid. The co-polymerization can be terminated by a nucleophilic reagent as discussed herein. If terminal functionality is desired, a functionalized terminating agent, such as but not limited to, methyl thiolacetate can be used. For a non-reactive termination terminus, a nucleophile such as an alkyl mercaptan can be used. Similarly a terminating hydroxyl group is also unreactive to many reagents and can therefore be useful in this application. Preferred solvents for the polymerization are chlorobenzene or acetonitrile. The preferred temperature range is from about 40Â° C. to about 120Â° C. The time required for the polymerization is dependent on the temperature, the desired molecular weight, and the solvent and can range from about 1 h to about 100 h. In certain embodiments, it is desirable to limit the polymerization reaction to the time required to substantially complete the polymerization reaction. In one embodiment, the progress of the polymerization reaction is monitored using MALDI and/or GPC.

The polymerization can be conducted in several ways. In one embodiment, a mixture of the appropriate oxazoline components can be reacted with the initiator in a preferred solvent with stirring. This reaction yields a random copolymer when the oxazoline components are equally reactive with one another or a block copolymer when one or more of the oxazoline components are less reactive towards one another. In an alternate embodiment, the polymer may also be synthesized in blocks by initiating polymerization with an appropriate initiator in a preferred solvent using only one oxazoline component. The reaction may be monitored with MALDI or GPC to determine when the reaction is substantially complete. When polymerization of the first block is complete, a second oxazoline component is added to reinitiate the polymerization with the incipient living cation at the terminus of the polymer chain; monitoring of the polymerization reaction may be carried out as described. The solvent may be the same as in the first polymerization reaction or different; the reaction temperature and other variables may also be adjusted as desired. Upon completion of the polymerization of the second block, a third oxazoline component is added to reinitiate the polymerization with the living cation at the terminus of the polymer chain; monitoring of the polymerization reaction may be carried out as described. The solvent may be the same as in the first or second polymerization reactions or different; the reaction temperature and other variables may also be adjusted as desired. When the third polymerization step is completed, the polymerization may be terminated by the addition of a terminating agent. The oxazoline components may contain functional groups, or may lack a functional group. The sequential polymerizations can be done in any order. In another embodiment, random copolymerization of two of the oxazoline monomers, followed by continued polymerization of a third block can also be done to produce a polymer having both random and block configurations. In one embodiment, one of the oxazoline monomers lacks a functional group.

Once the desired polymerization process is completed, the polymer is precipitated, such as in ethyl ether, several times and dried under vacuum. The polymer may be further characterized by standard techniques such as but not limited to MALDI, NMR, and GPC.

Work with polyethylene glycol has shown that it is frequently necessary in modification of target molecules to utilize polymers of molecular weights (MWs) of 20,000 Da or higher and molecular weight distributions, or polydispersities (PDs), of less than 1.1. There has been a great deal of work showing that MWs and PDs in the above range cannot be achieved for POZ chains with conventional techniques. As is known in the art PD values will vary with MW; in general, as the molecular weight increases the PD value also increases. It is generally seen that as the molecular weight of growing POZ chains reaches approximately 5,000 Da, the polydispersity increases appreciably. Side reactions, including, but not limited to, chain transfer, begin to grow in importance. The prior art techniques described above when used to generate POZ chains of high MW produce POZ derivatives with unacceptable PD values. The polyoxazoline derivatives of the present disclosure may be produced using polyoxazoline chains that are manufactured using novel methods that result in polyoxazoline derivatives with low PD values and a decreased amount of impurities produced by unwanted side reactions, such as, but not limited to, chain transfer. In one embodiment, the polyoxazoline chains are manufactured to minimize unwanted side reactions, such as, but not limited to, chain transfer, allowing the production of POZ derivatives of increased purity with low PD values. Therefore, the POZ derivatives of the present disclosure may be produced with increased purity and with low PD values suitable for use in pharmaceutical applications. Such methods are described in PCT Application No. PCT/US2008/078159, which is hereby incorporated by reference for such teaching.

Use of the Heterofunctional Polyoxazoline Derivatives

The heterofunctional polyoxazoline derivatives of the present disclosure possess two or more chemically orthogonal functional groups and thus allow for attachment of two or more different target molecules to the polymer.

The novel heterofunctional polyoxazoline derivatives prepared as described above are intended for formation of conjugates with various target molecules, such as, but not limited to, therapeutic, diagnostic and targeting moieties (target molecule-POZ conjugates). Various combinations of target molecules may be incorporated into the target molecule-POZ conjugate. In one embodiment, the target molecule-POZ conjugate contains a therapeutic moiety and a targeting moiety, a diagnostic moiety and a targeting moiety or a diagnostic moiety, a therapeutic moiety and a targeting moiety. Target molecules include, but are not limited to, polypeptides such as, but not limited to, interferons (including alpha, beta and gamma), growth hormone, peptide hormones (including luteinizing-hormone-releasing hormone, LHRH), interleukins, enzymes, antibodies (including antibody fragments and monoclonal antibodies), blood factors (including GCSF, erythropoietin, and Factor VIII) and insulin. Target molecules further include, but are not limited to, carbohydrates, oligonucleotides and small-molecule therapeutics. Exemplary targeting moieties include, but are not limited to, folate, LHRH peptide and glucosamine, Exemplary small molecule therapeutics include, but are not limited to gemcitabine, irinotecan, zidovudine, and diclofenac.

The heterofunctional polyoxazoline derivatives of the present disclosure, possessing functional groups that have orthogonal chemistries, are ideally suited for preparation of polyoxazoline derivatives linked to target molecules with distinct functions. Such an approach is possible due to the presence of two or more functional groups on the polyoxazoline derivative that are chemically orthogonal to one another. As a result, target molecules with different linkage chemistries can be incorporated into the polyoxazoline derivative in a controlled, directed manner. Such an approach is capable of producing targeted polyoxazoline therapeutic and or diagnostic conjugates. Exemplary active groups, binding partners on target molecules and linkages formed there between are disclosed in PCT Application No. PCT/US2008/078159, which is hereby incorporated by reference for such teaching. Exemplary reaction chemistries for the attachment of various target molecules are described herein and exemplified in the Examples section. However, the teaching of the present disclosure should not be limited to such reactions chemistries as the present disclosure provides the requisite teaching to couple various target molecules to the heterofunctional polyoxazoline derivatives of the present disclosure.

In one embodiment, the target molecule may a therapeutic and/or diagnostic agent and a targeting agent. The linkages to the target molecule may be hydrolytically stable or hydrolytically unstable or a combination thereof. However, in one embodiment, all linkages in the polyoxazoline derivative itself are hydrolytically stable. For example, an ester linkage to a target molecule would be hydrolytically unstable in vivo, while an amide linkage would be stable for an extended period of time. The described polyoxazoline derivatives provide a wide range of options in linking target molecules to the described polymer structure. Depending on the number of functional groups present, the number of target molecules linked to the polyoxazoline derivative can be varied from 1 to at least 100.

The target molecule-POZ conjugates may be used in methods of treatment to treat various disease states. In one embodiment, the disease state to be treated is defined by the nature of the target molecules incorporated into the target molecule-POZ conjugate, such as for example, the therapeutic moiety and/or the targeting moiety. The examples described herein provide target molecule-POZ conjugates containing a therapeutic moiety and/or a targeting moiety and describe the use of such conjugates in the treatment of cancer and target molecule-POZ conjugates containing anti-viral (zidovudine) and anti-inflammatory (diclofenac, a nonsteroidal anti-inflammatory agent) compounds. As such, the present disclosure provides methods of treating cancer, viral infections and conditions and diseases treatable by NSAID compounds (including arthritis, osteoarthritis, back pain, sciatica, sprains, dental pain, post operative pain, period paid, headaches, migraine and inflammatory conditions as well as reducing the incidence of heart attack and stroke). These examples are provided for illustration only and are not intended to restrict the recited methods of treatment of the foregoing conditions and diseases.

In one embodiment, the present disclosure describes the use of target molecule-POZ conjugates to treat and/or prevent various diseases and conditions in a subject in need of such treatment and/or prevention. In one embodiment, the diseases and conditions include, but not limited to, cancer, viral infections arthritis, osteoarthritis, back pain, sciatica, sprains, dental pain, post operative pain, period paid, headaches, migraine and inflammatory conditions as well as reducing the incidence of heart attack and stroke.

The method of treatment and/or prevention comprises the step of administering a target molecule-POZ conjugate of the present disclosure to the subject. The method may also comprise identifying a subject in need of such treatment. In a specific embodiment, the target molecule-POZ conjugate is administered in a therapeutically effective amount. Such administration of the target molecule-POZ conjugate would thereby treat the disease or condition. As discussed above, the results of such methods need not be absolute to provide benefit in the treatment methods disclosed.

Other Polymers Containing Pendent Groups

The present disclosure also provides for other water soluble polymers containing at least one pendent functional group linked to a therapeutic moiety and a targeting moiety, a diagnostic moiety and a targeting moiety or a combination of a therapeutic moiety, a diagnostic moiety and a targeting moiety. In one embodiment, such water soluble polymers contain two pendent functional groups that are chemically orthogonal to one another as described herein. In those polymers where two or more pendent groups that are chemically orthogonal do not exist naturally, one or more of the existing functional groups may be modified using the techniques described herein to yield chemically orthogonal functional groups. The functional groups described above may be used in conjunctions with these water soluble polymers containing pendent functional groups. The provision of such water soluble polymers including a targeting moiety in addition to a therapeutic and/or diagnostic moiety is an advancement in the art.

Suitable water soluble polymers containing pendent functional groups include, but are not limited to, polyamino acid polymers, lipid-based polymers, polysaccharide polymers, polyacetals polymers, polyester polymers, poly(vinyl alcohol) polymers and poly(acrylate) polymers.

Exemplary polyamino acid polymers include, but are not limited to, poly(l-glutamic acid), poly(d-glutamic acid), poly(dl-glutamic acid), poly(l-aspartic acid), poly(d-aspartic acid), poly(dl-aspartic acid), poly(l-lysine), poly(d-lysine), poly(dl-lysine) and copolymers of the above listed polyamino acids with polyoxazoline polymers, including those described herein. Such polyamino acid polymers are described in U.S. Pat. Nos. 7,153,864, 5,977,163, 6,884,817 and 6,441,025 (each of which is incorporated by reference for such teachings). Such polyamino acid polymers contain numerous pendent functional groups, such as carboxyl functional groups, for linkage to the therapeutic, diagnostic moiety and/or targeting moieties described herein. Additional functional groups described herein may also be introduced into such polyamino acid polymers using the techniques described herein to yield additional functional groups for linkage to therapeutic, diagnostic moiety and/or targeting moieties described herein.

Exemplary polyacetals polymers include those polymers described in U.S. Pat. Nos. 5,811,510, 5,863,990 and 5,958,398 (each of which is incorporated by reference for such teachings). Such polyacetals polymers are produced by oxidative degradation of polysaccharides. Such polyacetals polymers contain acetal functional groups as pendent groups. Furthermore, terminal groups can be created to provide a hemiacetal group at the reduced end of the polyacetals, which can be readily and selectively transformed into a carboxylic acid group and further into a variety of other functional groups. A primary alcohol group at the non-reduced end can be selectively transformed into an aldehyde group and further into a variety of functional groups.

Polysaccharide polymers include polymers formed from repeating units of sugars (either mono- or di-saccharides) joined together by glycosidic bonds. These structures are often linear, but may contain various degrees of branching. As described above for polyacetal polymers, the reducing and non-reducing ends of the polysaccharide polymer can be modified to provide a variety of functional groups.

Polyester polymers include, but are not limited to, aliphatic polyester polymers and polyoxaesters. Such polyester polymers have been described that contain pendant hydroxyl, carboxyl, thiol or amino functional groups. Exemplary polyester polymers are described in US Patent Publication Nos. 20070225452 20040006199 and 20040002580 and methods for the synthesis of such polymers can be found in the U.S. Pat. Nos. 6,972,315 5,464,929 (each of which is incorporated by reference for such teachings).

Poly(vinyl alcohol) (PVA) polymers may also be used. PVA polymers are water soluble and contain OH groups as functional groups. The OH groups may also be converted into other functional groups as described herein.

Exemplary poly(acrylate) polymers include but are not limited to N-(2-hydroxypropyl)methacrylamide (HPMA), including copolymers with other acrylates such as, but not limited to, methacrylic acid and butyl methacrylate. Exemplary poly(acrylate polymers can be formed from a variety of acrylate starting materials as is known in the art. Such

Lipid based polymers include, but are not limited to phosphoryl choline polymers. Phosphorylcholine polymers include but are not limited to poly(2-acryoyloxyethyl phosphoryl choline) and poly(2-methacryoyloxyethyl phosphoryl choline). The foregoing may be combined with other polymers to produce copolymers; exemplary copolymers include copolymers of phosphorylcholine polymers with poly(acrylate) polymers, such as, but not limited to methacrylic acid and butyl methacrylate. Such polymers are described in the following articles: Macromol Biosci. 2004 Apr. 19; 4(4):445-53. Biomed Mater Eng. 2004; 14(4):345-54. Biomacromolecules. 2005 March-April; 6(2):663-70 and Bioconjugate Chem., 2007, 18 (1), pp 263-267.

The foregoing polymers may also contain additional polymer units selected from those described above. Furthermore, the polymers above may be in the form of block or random copolymers as described herein

EXAMPLESReagents

Reagents were acquired from EM Science, Oakwood Products, Fluka, Calbiochem, Chevron Phillips Chemicals International, or Aldrich and distilled before use. Chlorobenzene and oxazolines were distilled from calcium hydride. GPC was performed on an Agilent Technologies machine with an 1100 quaternary pump and RI detector. Two Phenogelâ„¢ GPC columns (Phenomenex, 5Î¼, 500AÂ° and 1000AÂ°, 300Ã—7.8 mm) were used in series in a column heater (60Â° C.). The mobile phase was 100% N,Nâ€²-dimethylformamide (DMF) at a flow rate of 1 mL/min. A calibration curve was generated with M-PEOZ-OH and H-PEOZ-COOH samples of different molecular weights as determined by MALDI (750, 1K, 2K, 5K, 10K, 20K, 30K and 40K). MALDI-TOF MS was performed with a Bruker, Microflexâ„¢ machine using dithranol as matrix. NMR was performed on a Varian 500 MHz machine.

Example 1Preparation of Random-Co-Polymer of with Two Pendent Groups

[Image Omitted]

A solution of monomers comprising 2-(4-pentynyl)-2-oxazoline (PtynOZâ€”0.274 g, 0.002 mol), T-methyl propionate-2-oxazoline (TMPOZâ€”0.463 g, 0.002 mol), and 2-ethyl-2-oxazoline (EOZâ€”1.62 mL, 0.016 mol) was prepared in chlorobenzene (10 mL). To this solution was added methyl triflate (MeOTfâ€”0.113 mL, 0.001 mol) at room temperature. After stirring for 30 minutes, the mixture was heated to 110Â° C. for 45 minutes. The mixture was cooled to 0Â° C. and then terminated using piperidine (0.30 mL, 0.003 mol). After stirring for 2 hours at room temperature, the mixture was dripped into diethyl ether, decanted, and dried in vacuo to give 2.4 g of a white powder in quantitative yield.

The product had a calculated Mn of 2400 Da and a Mn (as determined by GPC) of 2440 with a polydispersity index (PDI) of 1.14). The sample exhibited a high molecular weight shoulder of less than 3%. ¹H NMR spectra showed peaks corresponding to pendent groups (acetylene and methyl ester) in the proper ratio.

Example 2Preparation of Block-Co-Polymer Via Stepwise Addition of Monomer and Monomer Mixtures

[Image Omitted]

To a solution of PtynOZ (0.274 g, 0.002 mol, 2 eq) in chlorobenzene (7.5 mL) was added MeOTf (0.113 mL, 0.001 mol, 1 eq) at room temperature. The mixture was stirred for 15 minutes and then ethyl oxazoline (EOZ, 0.5 mL, 0.005 mol, 5 eq) was added. The mixture was heated to 110Â° C. and stirred for 17 minutes. To introduce the second block (homopolymer of EOZ), EOZ (1.01 mL, 0.01 mol, 10 eq) was added and the resulting mixture was stirred for 10 minutes at 110Â° C. The third block was introduced by the addition of a solution of T-methyl propionate oxazoline (TMPOZ, 0.463 g, 0.002 mol, 2 eq) and EOZ (0.5 mL, 0.005 mol, 5 eq). After stirring for 10 minutes at 110Â° C., the mixture was cooled to room temperature using an ice/water bath and terminated by the addition of piperidine (0.4 mL, 0.004 mol). The resulting mixture was allowed to stir overnight, and precipitated by addition to diethyl ether. The solution was decanted and the remaining material was dried in vacuo to give 1.8 g of the desired product as a white powder in 67% yield.

¹H NMR (Varian, 500 MHz, 10 mg/mL CDCl₃) showed the usual backbone peaks at 1.12 ppm (m, 3H, CH₃CH₂COâ€”); 2.30 ppm (m) and 2.41 (s) (total area 2H, CH₃CH₂COâ€”); and 3.46 ppm (m, 4H, â€”NCH₂CH₂Nâ€”). The pentynyl pendant group peaks appear at 1.86 ppm (m, 2H, â€”CH₂CH₂Câ‰¡CH), 1.98 ppm (s, 1H, â€”CH₂CH₂Câ‰¡CH), and 2.04 ppm (br s, 1H, â€”CH₂CH₂Câ‰¡CH). The TMP pendent group peaks show at 2.60-2.88 ppm (m, 7H, â€”C(â• O)CH(CH₃)CH₂SCH₂CH₂CO₂Me) and 3.67 ppm (s, 3H, CH₂CO₂Me). The ratio of Ptyn, TMP, and EOZ was determined as 2:1.7:20. GPC gave Mn=2510 Da with PDI of 1.14. MALDI provided Mn=2725 with PDI of 1.04.

Example 3Synthesis of Random H-(Ptyn)₄(EOZ)₂₀-T-CO₂H

[Image Omitted]

Triflic acid (HOTf, 0.177 mL, 0.002 mol) was added into a solution of 2-pentynyl-2-oxazoline (PtynOZ, 1.097 g, 0.008 mol, 4 eq) and 2-ethyl-2-oxazoline (EOZ, 4.04 mL, 0.04 mol, 20 eq) in chlorobenzene (20 mL). After stirring for 5 minutes at room temperature, the mixture was heated to 110Â° C. for 30 minutes followed by cooling to room temperature using an ice/water bath. In a separate flask, the terminating reagent was prepared by the dropwise addition of methyl 3-mercaptopropionate (0.87 mL, 0.008 mol) into a suspension of sodium hydride (60% in mineral oil, 0.24 g, 0.006 mol) in chlorobenzene (60 mL), at room temperature. This mixture was stirred for 2 hours, before the solution of H-(Ptyn)₄(EOZ)₂₀⁺ in chlorobenzene was slowly added. The resulting mixture was then stirred for 18 hours at room temperature. The organic solvent was evaporated with a rotary evaporator and the white residue was dissolved in water and the pH was adjusted to 12.0. After stirring for 1 hour, the mixture was acidified (pHËœ3) and purified by ion-exchange chromatography using SP Sepharose FF and DEAE Sepharose FF to give the desired product as a white powder (yield was 2.2 g, 42%).

¹H NMR (Varian, 500 MHz, 10 mg/mL CDCl₃) showed the usual backbone peaks at 1.13 ppm (m, 3H, CH₃CH₂COâ€”); 2.32 ppm (m) and 2.41 (s) (total area 2H, CH₃CH₂COâ€”), and 3.47 ppm (m, 4H, â€”NCH₂CH₂Nâ€”). The terminal group peaks appear at 2.63 ppm (m, 2H, â€”SCH₂CH₂CO₂H), 2.74 ppm (m, 2H, â€”CH₂SCH₂CH₂CO₂H), and 2.85 ppm (m, 2H, â€”SCH₂CH₂CO₂H). The pendent pentynyl group peaks appear at 1.85 ppm (m, 2H, â€”CH₂CH₂Câ‰¡CH) and 2.03 ppm (br s, 1H, â€”CH₂CH₂Câ‰¡CH). The ratio of Ptyn and EOZ was 4:20. GPC gave Mn=3100 Da and Mp=3140 Da with PDI of 1.05. MALDI provided Mn=2900 Da with PDI of 1.03.

Example 4Synthesis of Random H-(Ptyn)₄(EOZ)₂₀-T-NH₂

[Image Omitted]

Triflic acid (HOTf, 0.177 mL, 0.002 mol) was added into a solution of 2-pentynyl-2-oxazoline (PtynOZ, 1.097 g, 0.008 mol, 4 eq) and 2-ethyl-2-oxazoline (EOZ, 4.04 mL, 0.04 mol, 20 eq) in chlorobenzene (20 mL). After stirring for 5 minutes at room temperature, the mixture was heated to 110Â° C. for 30 minutes followed by cooling to room temperature using an ice/water bath. In a separate flask, the terminating reagent was prepared by the dropwise addition of N-Boc cysteamine (1.01 mL, 0.006 mol) into a suspension of sodium hydride (60% in mineral oil, 0.24 g, 0.006 mol) in chlorobenzene (60 mL), at room temperature. This mixture was stirred for 2 hours, before the solution of TH-(Ptyn)₄(PEOZ)₂₀⁺ in chlorobenzene was added dropwise. The resulting mixture was stirred for 18 hours at room temperature. Volatiles were removed using a rotary evaporator and the residue was dissolved in water. The pH of the solution was adjusted to 3.0. The resulting aqueous solution was passed through an Amberlite column and then an ion-exchange column using SP Sepharose FF. The aqueous solution was charged with NaCl (15% w/w) and extracted with dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated using a rotary evaporator to provide 4.73 g of H-(Ptyn)₄(EOZ)₂₀-T-NHBoc as a white powder in 87% yield.

¹H NMR (Varian, 500 MHz, 10 mg/mL CDCl₃) showed the usual backbone peaks at 1.12 ppm (m, 3H, CH₃CH₂COâ€”); 2.28 ppm (m) and 2.41 (s) (total area 2H, CH₃Cl₂COâ€”); and 3.47 ppm (m, 4H, â€”NCH₂CH₂Nâ€”). The terminal group peaks appear at 1.44 ppm (s, 9H, â€”NHBoc), 2.63 ppm (m, 2H, â€”SCH₂CH₂NHBoc), 2.71 ppm (m, 2H, â€”CH₂SCH₂CH₂NHBoc), and 3.30 ppm (m, 2H, â€”SCH₂CH₂NHBoc). The pendent group peaks show at 1.84 ppm (m, 2H, â€”CH₂CH₂Câ‰¡CH) and 2.04 ppm (br s, 1H, â€”CH₂CH₂Câ‰¡CH). The ratio of Ptyn and EOZ was determined to be 4:20. GPC gave Mn=3900 Da and Mp=4505 Da with PDI of 1.07.

H-(Ptyn)₄(EOZ)₂O-T-NHBoc (4.4 g) was dissolved in 3N methanolic HCl and then stirred for 1 hour at room temperature. Most of volatiles were removed using a rotary evaporator and the residue was dissolved in water and the pH adjusted to Ëœ12.5. The aqueous solution was charged with NaCl (15% w/w) and extracted with dichloromethane. The combined organic phases were dried over sodium sulfate, filtered, and concentrated using a rotary evaporator to provide 3.9 g of H-(Ptyn)₄(EOZ)₂₀-T-NH₂as a white powder. Deprotection of â€”NHBoc was confirmed by AKTA prime on SP Sepharose FF column media and by ¹H NMR spectra showing the disappearance of -Boc group peak at 1.44 ppm and the shifting of â€”CH₂NH₂from 3.30 ppm to 2.92 ppm. In addition, the comparison of integration shows that the polymer contains four pendant groups. GPC gave Mn=3067 Da and Mp=3927 Da with PDI of 1.16.

Example 5Synthesis of Random H-(NHBoc)₄(EOZ)₂₀-T-CO₂H

[Image Omitted]

Triflic acid (HOTf, 0.177 mL, 0.002 mol) was added into a solution of T-NHBoc-2-oxazoline (NHBocOZ, 1.097 g, 0.008 mol, 4 eq) and 2-ethyl-2-oxazoline (EOZ, 4.04 mL, 0.04 mol, 20 eq) in chlorobenzene (20 mL). After stirring for 5 minutes at room temperature, the mixture was heated to 110Â° C. for 30 minutes followed by cooling to room temperature using an ice/water bath. In a separate flask, the terminating reagent was prepared by the dropwise addition of methyl 3-mercaptopropionate (0.87 mL, 0.008 mol) into a suspension of sodium hydride (60% in mineral oil, 0.24 g, 0.006 mol) in chlorobenzene (60 mL) at room temperature. This mixture was stirred for 2 hours before the solution of Hâ€”(NHBoc)₄(EOZ)₂₀⁺ in chlorobenzene was slowly added into it. The resulting mixture was stirred for 18 hours at room temperature. The organic solvent and other volatiles were removed using a rotary evaporator and the residue was dissolved in water and the pH of this aqueous solution was adjusted to 12.0. After stirring for 1 hour, the mixture was acidified (pHËœ3) and purified by ion-exchange chromatography using SP Sepharose FF and DEAE Sepharose FF to give the desired product as a white powder (yield was 1.4 g, 22%).

¹H NMR (Varian, 500 MHz, 10 mg/mL CDCl₃) showed the usual backbone peaks at 1.13 ppm (m, 3H, CH₃CH₂COâ€”); 2.31 ppm (m) and 2.41 (s) (total area 2H, CH₃CH₂COâ€”); and 3.47 ppm (m, 4H, â€”NCH₂CH₂Nâ€”). The terminal group peaks appear at 2.63 ppm (m, 2H, â€”SCH₂CH₂CO₂H), 2.73 ppm (m, 2H, â€”CH₂SCH₂CH₂CO₂H), and 2.84 ppm (m, 2H, â€”SCH₂CH₂CO₂H). The significant pendent group peaks show at 1.43 ppm (s, 9H, â€”NHBoc) and 3.28 ppm (m, 2H, â€”CH₂NHBoc). The ratio of NHBoc and EOZ was determined as 4:20. GPC gave Mn=3760 Da and Mp=3550 Da with PDI of 1.09. MALDI provided Mn=3130 with PDI of 1.03.

Example 6Synthesis of Random H-(TPA)₄(EOZ)₂₀-T-NHBoc

[Image Omitted]

Triflic acid (HOTf, 0.177 mL, 0.002 mol) was added into a solution of T-methyl propionate-2-oxazoline (TMPOZ, 1.85 g, 0.008 mol, 4 eq) and 2-ethyl-2-oxazoline (EOZ, 4.04 mL, 0.04 mol, 20 eq) in chlorobenzene (20 mL). After stirring for 5 minutes at room temperature, the mixture was heated to 110Â° C. for 30 minutes followed by cooling to room temperature using an ice/water bath. In a separate flask, the terminating reagent was prepared by the dropwise addition of N-Boc cysteamine (1.01 mL, 0.006 mol) into a suspension of sodium hydride (60% in mineral oil, 0.24 g, 0.006 mol) in chlorobenzene (60 mL) at room temperature. The mixture was stirred for 2 hours, and the solution of H-(TMP)₄(EOZ)₂₀⁺ in chlorobenzene was then slowly added into it. The resulting mixture was stirred for 18 hours at room temperature. The volatiles were then removed using a rotary evaporator and the residue was dissolved in water (pHËœ3). The resulting aqueous solution was passed through an Amberlite column and then an ion-exchange column with SP Sepharose FF media. The aqueous solution was charged with NaCl (15% w/w) and extracted with dichloromethane. The combined organic phases were dried over sodium sulfate, filtered, and concentrated using a rotary evaporator to provide 4.8 g of H-(TMP)₄(EOZ)₂₀-T-NHBoc as a white powder (78% yield).

¹H NMR (Varian, 500 MHz, 10 mg/mL CDCl₃) showed the usual backbone peaks at 1.12 ppm (m, 3H, CH₃CH₂COâ€”); 2.30 ppm (m) and 2.41 (s) (total area 2H, CH₃CH₂COâ€”); and 3.46 ppm (m, 4H, â€”NCH₂CH₂Nâ€”). The terminal group peaks appear at 1.44 ppm (s, 9H, â€”NHBoc), 2.61 ppm (m, 2H, â€”SCH₂CH₂NHBoc), 2.79 ppm (m, 2H, â€”CH₂SCH₂CH₂NHBoc), and 3.31 ppm (m, 2H, â€”SCH₂CH₂NHBoc). The pendent group peaks show at 2.60-2.90 ppm (m, 7H, â€”C(â• O)CH(CH₃)CH₂SCH₂CH₂CO₂Me) and 3.67 ppm (s, 3H, CH₂CO₂Me). The ratio of TMP and EOZ was determined as 3.3:20. GPC gave Mn=2340 Da and Mp=2200 Da with PDI of 1.08.

H-(TMP)₄(EOZ)₂₀-T-NHBoc (4.4 g) was dissolved in water and the pH adjusted to Ëœ12.5. After stirring for 1 hour at room temperature, purification was performed by ion-exchange chromatography using DEAE Sepharose FF. The aqueous solution was charged with NaCl (15% w/w) and extracted with dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated using a rotary evaporator to provide 2.0 g of H-(TPA)₄(EOZ)₂₀-T-NHBoc as a white powder in 32% yield. Hydrolysis of â€”CO₂Me was confirmed by AKTA prime on DEAE Sepharose FF and by ¹H NMR spectra (disappearance of â€”CO₂Me group peak at 3.67 ppm).

Example 7Synthesis of H-[(TPA)₄(EOZ)₂₀][(NHBoc)₄(EOZ)₂₀]-T-Bz

[Image Omitted]

Triflic acid (HOTf, 88.5 Î¼L, 0.001 mol) was added into a solution of T-methyl propionate-2-oxazoline (TMPOZ, 0.925 g, 0.004 mol, 4 eq) and 2-ethyl-2-oxazoline (EOZ, 2.02 mL, 0.02 mol, 20 eq) in chlorobenzene (12 mL). After stirring for 5 minutes at room temperature, the mixture was heated to 110Â° C. for 30 minutes and then a solution of T-NHBoc-2-oxazoline (NHBocOZ, 1.154 g, 0.004 mol, 4 eq) and 2-ethyl-2-oxazoline (EOZ, 2.02 mL, 0.02 mol, 20 eq) in chlorobenzene (12 mL) was added. After heating for an additional 30 minutes, the mixture was cooled to room temperature using an ice/water bath. The terminating reagent was prepared in a separate flask by the slow addition of benzyl mercaptan (0.35 mL, 0.003 mol) into a suspension of sodium hydride (60% in mineral oil, 0.08 g, 0.002 mol) in chlorobenzene (10 mL) at room temperature. After the mixture was stirred for 2 hours, the solution of living polymer species in chlorobenzene was added dropwise to the termination mixture. The resulting mixture was stirred for 18 hours at room temperature and then precipitated by addition to diethyl ether. The precipitated solution was filtered, and dried to give 4.8 g of polymer having methyl ester and â€”NHBoc as the pendent groups. The polymer was dissolved in water, passed through an Amberlite column and then an ion-exchange column with SP Sepharose FF packing. The resulting aqueous solution was charged with NaCl (15% w/w) and extracted with dichloromethane. The combined organic phases were dried over sodium sulfate, filtered, and concentrated to provide 3.72 g of H-[(TMP)₄(EOZ)₂₀][(NHBoc)₄(EOZ)₂₀]-T-Bz as a white powder in 78% yield.

¹H NMR (Varian, 500 MHz, 10 mg/mL CDCl₃) showed the usual backbone peaks at 1.12 ppm (m, 3H, CH₃CH₂COâ€”); 2.30 ppm (m) and 2.41 (s) (total area 2H, CH₃CH₂COâ€”); and 3.47 ppm (m, 4H, â€”NCH₂CH₂Nâ€”). The terminal group peaks appear at 2.56 ppm (m, 2H, â€”CH₂SCH₂Ar), 3.74 ppm (s, 2H, â€”SCH₂Ar), 7.27 ppm (m, 1H, â€”Ar), 7.34 ppm (m, 5H, â€”Ar). The TMP pendent group peaks show at 2.60-2.88 ppm (m, 7H, â€”C(â• O)CH(CH₃)CH₂SCH₂CH₂CO₂Me) and 3.67 ppm (s, 3H, CH₂CO₂Me). The â€”NHBoc pendent group peaks show at 1.42 ppm (s, 9H, â€”NHBoc) and 3.28 ppm (m, 2H, â€”CH₂NHBoc). The ratio of TMP, NHBoc, and EOZ was determined as 3.5:2:40. GPC gave Mn=4050 Da and Mp=4690 Da with PDI of 1.16.

H-[(TMP)₄(EOZ)₂₀][(NHBoc)₄(EOZ)₂₀]-T-Bz (0.8 g) was dissolved in water and the pH was adjusted to Ëœ13.0 using 0.5 M NaOH solution. After stirring for 1 hour, the mixture was extracted with dichloromethane and the combined organic phases were dried over sodium sulfate, filtered, and concentrated using a rotary evaporator. The resulting solution was precipitated by addition to diethyl ether. The diethyl ether solution was decanted and the remaining white powdery material was dried in vacuo to give the desired product, H-[(TPA)₄(EOZ)₂₀][(NHBoc)₄(EOZ)₂₀]-T-Bz in a quantitative yield. The hydrolysis was confirmed by ion exchange chromatography using a DEAE Sepharose FF. GFC and ¹H NMR showed the hydrolysis of the â€”CO₂Me group.

Example 8Synthesis of M-(PPtyn)₂(PEOZ)₁₈-T-CO₂H

[Image Omitted]

Methyl triflate (MeOTf, 0.556 mL, 0.005 mol) was added into a solution of 2-pentynyl-2-oxazoline (PtynOZ, 1.37 g, 0.01 mol, 2 eq) in chlorobenzene (20 mL). After stirring for 10 minutes at room temperature, 2-ethyl-2-oxazoline (9.09 mL, 0.09 mol, 18 eq) was added and the mixture was heated to 110Â° C. for 30 minutes followed by cooling to 0Â° C. To obtain a terminating reagent, methyl 3-mercaptopropionate (2.17 mL, 0.02 mol) was added dropwise into a suspension of potassium tert-butoxide (1.12 g, 0.01 mol) in chlorobenzene (10 mL) at 0Â° C. After the mixture was stirred for 2 hours in the cold, the solution of M-(PPtyn)(PEOZ)⁺ in chlorobenzene was added dropwise. The mixture was stirred in the cold for 4 hours and then stirred for 18 hours at room temperature. Water (100 mL) was added and the mixture was acidified (pHËœ0.3) by the addition of 5% aqueous HCl solution. Most of volatiles including chlorobenzene were removed using rotary evaporation. The resulting aqueous solution was treated with NaOH (1.0 g, 0.025 mol). After stirring for 1 hour, the mixture was acidified with 5% aqueous HCl solution and then extracted with dichloromethane. The combined organic phases were dried over sodium sulfate, filtered, concentrated, and precipitated by addition to ether. The ether was decanted and the residue was dried under vacuum. GFC showed that the crude product contains hydroxyl-terminated polymer (20%) and the desired acid-terminated polymer (80%). Further purification was performed by ion-exchange chromatography using DEAE Sepharose FF to give 5.6 g of the product in 61% yield. NMR (Varian, 500 MHz, 10 mg/mL CDCl₃) shows peaks for pendant pentynyl group at 1.85 ppm (m, 2H, â€”CH₂CH₂Câ‰¡CH); and 1.98 ppm (m, 1H, â€”CH₂CH₂Câ‰¡CH). GPC and GFC show a single main peak, with Mn 1830 Da and PD of 1.10.

Example 9Synthesis of {M-(PPtyn)₂(PEOZ)₁₈}₂-Lys-CO₂H

[Image Omitted]

A solution of M-(PPtyn)₂(PEOZ)₁₈-T-CO₂H (Mn=1830 Da, 2.0 g, 1.09 mmol) and 1-HOBT (0.351 g, 2.60 mmol) in acetonitrile (40 mL) was concentrated by rotary evaporation to dry azeotropically. The residue was dissolved in dry CH₂Cl₂(20 mL) and then DCC (0.322 g, 1.56 mmol) was added. After the mixture was stirred for 3 hours at room temperature, L-lysine ethyl ester dihydrochloride (0.129 g, 0.520 mmol) and DMAP (0.318 g, 2.60 mmol) were added. After stirring for 18 hours at room temperature, the mixture was added to ether (150 mL) to give a white precipitate. The compound was filtered and dried under vacuum to give {M-(PPtyn)₂(PEOZ)₁₈}₂-Lys-ethyl ester as a white powder. GPC and GFC showed the mixture of main product (98%, Mn=4540 Da, PD=1.04) and excess of acid polymer (2. The crude product was passed through a DEAE Sepharose FF column to remove the excess amount of acid polymer. The resulting aqueous solution was treated with NaOH (0.104 g, 2.60 mmol) for 1 hour. The mixture was acidified with 5% aqueous HCl solution and then extracted with dichloromethane. The combined organic phases were dried over sodium sulfate, filtered, concentrated, and precipitated by addition to ether. The ether was decanted and the residue was dried under vacuum to give 2.0 g of a white powder in 92% yield. ¹H NMR showed the completion of hydrolysis according to the disappearance of ethyl group peaks and peaks for lysine core at 1.42 ppm (br s, 2H, â€”C(â• O)NHCH₂CH₂CH₂â€”); 1.54 ppm (br s, 2H, â€”C(â• O)NHCH₂CH₂â€”); 1.85 ppm (m, 2H, â€”CH₂CH(CO₂H)NHâ€”); and 4.52 ppm (m, 1H, â€”CH₂CH(CO₂H)NHâ€”). MALDI provided Mn=4240 Da with PD of 1.01.

Example 10Synthesis of {M-(PPtyn)(PEOZ)}₂Lys-NHS

[Image Omitted]

N-hydroxysuccinimide (0.0235 g, 0.204 mmol) and DCC (0.0421 g, 0.204 mmol) were added into a solution of {M-(PPtyn)₂(PEOZ)₁₈}₂-Lys-CO₂H (Mn 4200 Da, 0.832 g, 0.198 mmol) in dichloromethane (4 mL) at 0Â° C. After stirring for 2 hours in the cold, the mixture was warmed to room temperature and stirred overnight. The white precipitate was removed by filtration and the solution was added to diethyl ether to give a white powder. The powder was collected by filtration and dried under vacuum (0.8 g, 88% yield). The attachment of maleimide was shown by ¹H NMR spectrum that shows the succinimidyl protons at 2.86 ppm (s, 2H) along with the usual backbone peaks. GPC showed 97% of the desired product (Mn=4550 Da, PD=1.04) and 3% of acid polymer.

To confirm product identity, the product (Mn 4550 Da, 0.103 g, 0.023 mmol) was treated with phenylethylamine (0.009 mL, 0.068 mmol) and triethylamine (0.009 mL, 0.068 mmol) in dichloromethane (3 mL). After stirring overnight, the mixture was filtered and added to diethyl ether. The white powder was isolated by filtration and dried under vacuum to provide the product in quantitative yield. According to GFC, the conversion yield was 99.7%.

Example 11Synthesis of POZ-4[{M-(PPtyn)₂(PEOZ)₁₈}₂-Lys]₂-Lys-ethyl ester

[Image Omitted]

A solution of M-{(PPtyn)₂(PEOZ)₁₈}₂Lys-T-CO₂H (Mn=4200 Da, 0.415 g, 0.0988 mmol) and 1-HOBT (0.0318 g, 0.235 mmol) in acetonitrile (15 mL) was concentrated using rotary evaporation. The residue was dissolved in dry CH₂Cl₂(3 mL) and then DCC (0.0291 g, 0.141 mmol) was added. After the mixture was stirred for 3 hours at room temperature, L-lysine ethyl ester dihydrochloride (0.0116 g, 0.047 mmol) and DMAP (0.0287 g, 0.235 mmol) were added. After stirring for 18 hours at room temperature, the mixture was filtered and added to ether (40 mL) to give a white precipitate. The precipitate was isolated by filtration and dried under vacuum to give the desired four-armed [{M-(PPtyn)₂(PEOZ)₁₈}-Lys]₂-Lys-ethyl ester as a white powder in quantitative yield. ¹H NMR (Varian, 500 MHz, 10 mg/mL CDCl₃) showed the peak for Lys-Lys-Lys core molecule at 1.28 ppm (t, 3H, CH₃CH₂Oâ€”); 1.42 ppm (m, 6H, â€”C(â• O)NHCH₂CH₂CH₂â€”); 1.54 ppm (m, 6H, â€”C(â• O)NHCH₂CH₂â€”); 1.85 ppm (m, 6H, â€”CH₂CH(CO₂H)NHâ€”); 4.14 ppm (br s, 2H, â€”CO₂CH₂CH₃) and 4.45 ppm (m, 3H, â€”CH₂CH(CO₂H)NHâ€”) and peaks for pendant pentynyl group at 1.85 ppm (m, 2H, â€”CH₂CH₂Câ‰¡CH); and 2.02 ppm (m, 1H, â€”CH₂CH₂Câ‰¡CH). GPC and GFC showed a mixture of main product (96%, Mn=7970 Da, PD=1.06) and excess of acid polymer (4%).

Example 12Conjugation of Glucosamine and Zidovudine onto Ht-(Ptyn)₄(EOZ)₂₀-T-CO₂H

After drying azeotropically with acetonitrile, H-(Ptyn)₄(EOZ)₂₀-T-CO₂H (0.18 g, 0.0619 mmol, Mn 2100 Da by MALDI) was dissolved in dichloromethane (2 mL). NHS (0.0071 g, 0.0619 mmol) and DCC (0.0128 g, 0.0619 mmol) were added at room temperature. After stirring overnight at room temperature, the mixture was filtered and precipitated by addition to diethyl ether. Diethyl ether solution was decanted and the residue was dried in vacuo to give the desired N-hydroxysuccinimide ester; i.e. H-(Ptyn)₄(EOZ)₂₀-T-SPA as a white powder in a quantitative yield. GFC shows â‰§5% purity and attachment of NHS was proved by ¹H NMR showing the succinimidyl protons at 2.86 ppm (s, 4H).

[Image Omitted]

D(+)-glucosamine hydrochloride (0.137 g, 0.0663 mmol) was dissolved in 2 mL of 0.1 N boric acid solution followed by the adjustment of pH to 8.5 using 0.1 N NaOH solution. H-(Ptyn)₄(EOZ)₂₀-T-SPA (0.19 g, 0.0663 mmol, Mn 3000 Da) was added as a solid. The mixture was stirred for 3 hours at room temperature while maintaining pH at 8.5 using 0.1 N NaOH. The mixture was acidified (pHËœ3) and extracted with dichloromethane. The combined organic phases were dried over anhydrous Na₂SO₄, filtered, concentrated, and dried in vacuo to give 0.14 g of pale yellow powder. The conjugation of glucosamine was confirmed by ¹H NMR (Varian, 500 MHz, 10 mg/mL DMSO-d6) that shows the 2-position proton on glucoseamide at 4.92 ppm (s, 1H). Ion exchange chromatography showed a substitution yield of 83%.

[Image Omitted]

H-(Ptyn)₄(EOZ)₂₀-T-Gluco (0.05 g, 0.0156 mmol, Mn 3200 Da) and the antiviral nucleoside zidovudine (AZT, 0.0167 g, 0.0625 mmol, 4 eq) were dissolved in water (2 mL). Sodium ascorbate (0.0012 g, 0.00625 mmol) and CuSO₄.5H₂O (0.0008 g, 0.00313 mmol) were added at room temperature. After stirring for 18 hours, water was removed using a rotary evaporator. The residue was dissolved in a 1:1 mixture of MeOH and CHCl₃and then precipitated by addition to diethyl ether. Diethyl ether solution was decanted and the residue was dried in vacuo to give the desired product as a white powder in a quantitative yield. The â€˜clickâ€™ coupling of the azide group on zidovudine to each acetylene pendant on the polymer chain was verified by NMR. ¹H NMR (Varian, 500 MHz, 10 mg/mL DMSO-d6) shows that the polymer chain contained an average of â‰§3.5 units of thymidines with the thymidyl proton peaks at 11.3 ppm (br s, 1H, â€”OH), 8.05 ppm (s, 1H, triazole), 7.81 ppm (s, 1H), 6.41 ppm (t, 1H), 5.31 ppm (m, 1H), 5.26 ppm (m, 1H), 4.18 ppm (br s, 1H), and 1.80 (s, 3H). The 2-position proton on glucoseamide shows at 4.92 ppm (s, 1H). GPC gave Mn=4700 Da and Mp=4830 Da with PDI of 1.07.

Example 13Conjugation of Irinotecan to H-(Ptyn)₄(EOZ)₂₀-T-NH₂

Irinotecan.HCl.3H₂O (0.200 g, 0.295 mmol) was dissolved in acetonitrile (15 mL) and dried by azeotropic distillation. The residue was dissolved in dichloromethane (6 mL) and 6-azidohexanoic acid (0.0928 g, 0.591 mmol) was added. After the addition of dimethyaminopyridine (DMAP) (0.0722 g, 0.591 mmol) and dicyclohexylcarbodiimide (DCC) (0.122 g, 0.591 mmol), the resulting mixture was allowed to stir overnight at room temperature. The mixture was precipitated by addition to diethyl ether. The ether solution was decanted and the remaining precipitate was dried to give 0.144 g of the desired product as a pale yellow powder (67% yield). ¹H NMR (Varian, 500 MHz, 10 mg/mL CDCl₃) Î´ 0.98 (t, 3H, C-9H), 1.41 (m, 2H, C-3H), 1.41 (m, 3H, C-18H), 1.41 (m, 2H, bipiperidyl ii), 1.58 (m, 2H, C-4H), 1.58 (2H, bipiperidyl H), 1.70 (m, 2H, C-2H), 1.93 (m, 4H, bipiperidyl H), 2.16 (m, 1H, C-1H), 2.28 (m, 1H, C-1H), 2.32 (m, 1H, bipiperidyl H), 2.50 (m, 2H, C-5H), 2.50 (m, 2H, bipiperidyl H), 2.75 (m, 2H, C-17H), 2.94 (m, 1H, bipiperidyl C-3â€²H), 3.17 (m, 3H, bipiperidyl H), 3.25 (t, 2H, C-1H, â€”CH₂N₃), 3.55 (br s, 2H, bipiperidyl C-1â€²H and C-5â€²H), 4.56 (m, 2H, bipiperidyl C-1â€²H and C-5â€²H), 5.25 (s, 2H, C-14H), 5.41 (d, 1H, C-11H), 5.68 (d, 1H, C-11H), 7.19 (s, 1H, C-27H), 7.60 (d, 1H, C-22H), 7.87 (s, 1H, C-20H), 8.23 (d, 1H, C-23H).

[Image Omitted]

Conjugation of Random H-(Ptyn)₄(EOZ)₂₀-T-NH₂and Irinotecan Azido Hexanoate

[Image Omitted]

H-(Ptyn)₄(EOZ)₂₀-T-NH₂(0.05 g, 0.0163 mmol, 1 eq., Mn 3070 Da), from a previous example, and irinotecan azido hexanoate (0.0473 g, 0.0652 mmol, 4 eq) were dissolved in water (2 mL). Sodium ascorbate (0.0013 g, 0.00652 mmol, 0.4 eq) and CuSO₄.5H₂O (0.0008 g, 0.00326 mmol, 0.2 eq) were added at room temperature. After stirring for 22 hours, water was removed using a rotary evaporator. The residue was dissolved in dichloromethane and then precipitated by addition to diethyl ether. Diethyl ether solution was decanted and the residue was dried in vacuo to give 0.7 g of the desired product as an off-white powder (72% yield). The â€˜clickâ€™ coupling of the azide group (on irinotecan azido hexanoate) to the acetylene pendants (on POZ) was verified by NMR. ¹H NMR (Varian, 500 MHz, 10 mg/mL CDCl₃) shows that the polymer chain contained an average of 4 units of irinotecans with the aromatic proton peaks of irinotecan at 7.14 ppm (t, 1H), 7.57 ppm (br s, 1H), and 7.85 ppm (br s, 1H). 8.16 ppm (br s, 1H), and new signals at 4.27 ppm (br s, 2H, adjacent to triazole â€”CH₂NR) and 7.40 ppm (br s, 1H, triazole). GPC gave Mn=4160 Da and Mp=4900 Da with PDI of 1.19.

Example 14Conjugation of Irinotecan to H-[(Ptyn)₁₀(EOZ)₂₀₀]-T-PA

[Image Omitted]

Synthesis of Random H-[(Ptyn)₁₀(EOZ)]₂₀₀-T-CO₂H

[Image Omitted]

Triflic acid (HOTf, 0.201 mL, 2.27 mmol) was added into a solution of 2-pentynyl-2-oxazoline (PtynOZ, 3.78 g, 0.0272 mol, 12 eq) and 2-ethyl-2-oxazoline (EOZ, 50.5 mL, 0.500 mol, 220 eq) in chlorobenzene (132 mL). After stirring for 5 minutes, the mixture was heated to 80Â° C. for 10 hours followed by cooling to room temperature. In a separate flask, the terminating reagent was prepared by the dropwise addition of methyl 3-mercaptopropionate (1.23 mL, 0.0114 mol) into a suspension of sodium hydride (60% in mineral oil, 0.272 g, 0.0068 mol) in chlorobenzene (34 mL). This mixture was stirred for 7 hours, before the solution of living polymer of H-(Ptyn)₁₀(EOZ)₂₀₀⁺ was added. The resulting mixture was then stirred for 18 hours. The solvent was removed by rotary evaporation to yield a white residue. This residue was dissolved in water and the pH adjusted to 12.0. The resulting aqueous solution was purified by ion-exchange chromatography using DEAE Sepharose FF. The aqueous solution was saturated with NaCl (15% w/w) and extracted with dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated using a rotary evaporator. The residue was precipitated by adding the dichloromethane concentrate to diethyl ether. The precipitated material was collected and dried in vacuo to give 22.8 g of desired product as a white powder (50% yield).

¹H NMR (Varian, 500 MHz, 10 mg/mL CDCl₃) showed the usual backbone peaks at 1.13 ppm (m, 3H, CH₃CH₂COâ€”); 2.32 ppm (m) and 2.41 (s) (total area 2H, CH₃CH₂COâ€”); and 3.47 ppm (m, 4H, â€”NCH₂CH₂Nâ€”). The terminal group peaks appear at 2.63 ppm (m, 2H, â€”SCH₂CH₂CO₂H), 2.74 ppm (m, 2H, â€”CH₂SCH₂CH₂CO₂H), and 2.85 ppm (m, 2H, â€”SCH₂CH₂CO₂H). The pendent pentynyl group peaks appear at 1.85 ppm (m, 2H, â€”CH₂CH₂Câ‰¡CH) and 2.03 ppm (br s, 1H, â€”CH₂CH₂Câ‰¡CH). The number of pendent, Ptyn, groups were determined as 8.5 by comparing the integrations of terminal acetylene proton and polymer backbone protons. GPC gave Mn=19,500 Da and Mp=20,800 Da with PDI of 1.07.

Synthesis of Azidoacetic Acid

To a 250 mL single neck round-bottomed flask was added deionized water (75 mL) and the flask was placed in an ice bath. Sodium azide (23.4 g, 0.360 mol) was added to give a colorless clear solution. Bromoacetic acid (20.0 g, 0.144 mol) was added as a solid. After stirring in the cold for 1 hour, the mixture was allowed to stir at room temperature for 16 hours. The mixture was acidified (pHËœ3) using 1.0 N HCl solution and extracted with diethyl ether (4Ã—100 mL) with readjustment of pH to 3 for each extraction. The combined organic phases were dried over Na₂SO₄(25 g) and filtered. Volatiles were removed using a rotary evaporator (250 mbar at 22Â° C.) to give 13.0 g (89.4% yield) of desired product as a colorless clear oil. ¹H-NMR (Varian, 500 MHz, 10 mg/mL CDCl₃) 3.96 ppm (s, 2H, N₃CH₂CO₂H).

Synthesis of Azido-Irinotecan.HCl

A 500 mL single neck round-bottomed flask was fitted with a rubber septum, a magnetic stir bar, and argon gas inlet. Irinotecan.HCl.3H₂O (9.00 g, 13.3 mmol) and MeCN (250 mL) were added to give a pale yellow suspension. All the volatiles were removed using a rotary evaporator. Azidoacetic acid (5.37 g, 53.2 mmol) was added and dichloromethane (266 mL) was added to give a clear yellow solution. 4-(Dimethylamino)pyridine (DMAP, 2.44 g, 20.0 mmol) was added and the mixture was cooled to 0Â° C. A solution of DCC (11.0 g, 53.2 mmol) in dichloromethane (10 mL) was added. After stirring in the cold for 1 hour, the mixture was allowed to stir at room temperature for 16 hours. The mixture was filtered and the filtrate was poured into 0.5 N HCl (100 mL) to keep the solution acidic (pHËœ3). The resulting solution was charged with NaCl (15.0 g, 15% w/v water) and two layers were separated. The organic layer was collected and the aqueous layer was extracted twice with dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to give a pale yellow powder containing Ëœ20% of irinotecan.HCl.

A 250 mL single neck round-bottomed flask with a magnetic stir bar was fitted with a rubber septum and then connected to an argon gas inlet. The crude product prepared above (9.1 g), azidoacetic acid (1.34 g, 13.3 mmol), and dichloromethane (200 mL) were placed in a flask and DMAP (1.95 g, 16.0 mmol) and DCC (2.74 g, 13.3 mmol) were added at room temperature. After stirring overnight, the aqueous workup described above was performed. After concentrating, the residue was dissolved in MeCN and filtered. After the filtrate was concentrated, the residue was dissolved in dichloromethane and precipitated by adding into diethyl ether. The precipitated material was collected and dried in vacuo to give 8.9 g (95% yield) of desired product as pale yellow powder. ¹H-NMR (Varian, 500 MHz, 10 mg/mL CDCl₃) showed a significant chemical shift change of proton on C5 from 7.64 ppm (s, 1H) to 7.21 ppm (s, 1H) and the azidoacetyl proton peak at 4.09 ppm (m, 2H, N₃CH₂C(â• O)Oâ€”) due to the conjugation of hydroxyl group of C4 with azidoacetic acid.

â€˜Clickâ€™ Addition of Azido-Irinotecan-HCl to [H-(Ptyn)₁₀(EOZ)₂₀₀]-T-PA

[Image Omitted]

A 1 L single neck round-bottomed flask was fitted with a rubber septum, a magnetic stir bar, and argon gas inlet. The reaction flask was charged with azido-irinotecan.HCl (4.65 g, 6.58 mmol), H-[(Ptyn)₁₀(EOZ)₂₀₀]-T-PA (12.5 g, 0.548 mmol, Mn 22800 Da), and H₂O (500 mL). Sodium ascorbate (0.434 g, 2.19 mmol) and CuSO₄.5H₂O (0.548 g, 2.19 mmol) were added. After stirring for 24 hours at room temperature, the mixture was quenched with 1.0 N HCl solution (5 mL). The resulting solution was passed through DowexÂ® (M4195, 100 mL) to remove copper ions and then passed through a silica gel (50 g) column using 0.01% HCl solution as an eluent. The filtrate was saturated with NaCl (15% w/v) and then extracted with dichloromethane (3Ã—500 mL). The combined organic phases were dried over Na₂SO₄, filtered, and concentrated. The residue was precipitated using dichloromethane and diethyl ether. The precipitated material was collected and dried in vacuo to give 16.0 g of desired product as pale yellow powder. The â€˜clickâ€™ reaction was also confirmed by ¹H-NMR (Varian, 500 MHz, 10 mg/mL CDCl₃) showing the newly formed triazole ring proton at 7.61 ppm (s, 1H). The content of irinotecan was assayed by reverse phase HPLC. The average drug loaded determined from multiple batches ranged from 15 to 17% w/w. Heavy metal analysis showed that the sample had <25 ppm of copper metal.

Example 15Conjugation of Synthesis of H-[(Irino.HCl)₁₀(EOZ)₁₉₀]-T-Folate

[Image Omitted]

Triflic acid (HOTf, 173.3 L, 1.96 mmol) was added to a solution of 2-pentynyl-2-oxazoline (PtynOZ, 3.76 g, 27.4 mmol, 14 eq) and 2-ethyl-2-oxazoline (EOZ, 46.61 g, 470.2 mmol, 240 eq) in chlorobenzene (124 mL). After stirring for 5 minutes at room temperature, the mixture was heated to 80Â° C. for 10 hours followed by cooling to room temperature. In a separate flask, the terminating reagent was prepared by the dropwise addition of N-Boc cysteamine (1.06 mL, 9.80 mmol) into a suspension of sodium hydride (60% in mineral oil, 0.235 g, 5.88 mmol) in chlorobenzene (60 mL), at room temperature. This mixture was stirred for 6 hours, and then the chlorobenzene solution of the living polymer H-(Ptyn)₁₀(PEOZ)₂₀₀⁺ was added. The resulting mixture was stirred for 18 hours at room temperature. The mixture was precipitated by addition into diethyl ether. The white precipitate was collected and washed with diethyl ether. After drying in vacuo, the white powdery material was dissolved in water. The pH of the solution was adjusted to 3.0. The resulting aqueous solution was purified by ion-exchange chromatography on an SP Sepharose FF column. The aqueous solution was saturated with NaCl (15% w/w) and extracted with dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate, filtered, and concentrated to provide 47.8 g of H-(Ptyn)₁₀(EOZ)₂₀₀-T-NHBoc as a white powder.

¹H-NMR (Varian, 500 MHz, 10 mg/mL CDCl₃) showed the usual backbone peaks at 1.12 ppm (m, 3H, CH₃CH₂COâ€”); 2.28 ppm (m) and 2.41 (s) (total area 2H, CH₃CH₂COâ€”); and 3.47 ppm (m, 4H, â€”NCH₂CH₂Nâ€”). The terminal group peaks appear at 1.43 ppm (s, 9H, â€”NHBoc), 2.63 ppm (m, 2H, â€”SCH₂CH₂NHBoc), 2.71 ppm (m, 2H, â€”CH₂SCH₂CH₂NHBoc), and 3.30 ppm (m, 2H, â€”SCH₂CH₂NHBoc). The pendent group peaks show at 1.84 ppm (m, 2H, â€”CH₂CH₂Câ‰¡CH) and 2.03 ppm (br s, 1H, â€”CH₂CH₂Câ‰¡CH). The number of pendent, Ptyn, groups was determined as 8.8 by comparing the integrations of terminal acetylene proton and polymer backbone protons. GPC gave Mn=21,100 Da and Mp=25,000 Da with PDI of 1.13.

H-(Ptyn)₁₀(EOZ)₂₀-T-NHBoc (47.8 g) was dissolved in 3N methanolic HCl and stirred for 1 hour at room temperature. Most of volatiles were removed and the residue was dissolved in water and the pH adjusted to Ëœ12.5. The resulting aqueous solution was passed through a DEAE Sepharose FF column. The aqueous solution was charged with NaCl (15% w/w) and extracted with dichloromethane. The combined organic phases were dried over sodium sulfate, filtered, concentrated, and precipitated by adding into diethyl ether. The white precipitate was filtered, washed with diethyl ether, and dried in vacuo to provide 30.5 g of H-[(Ptyn)₁₀(EOZ)₂₀₀-]-T-NH₂as a white powder. Deprotection of â€”NHBoc was confirmed by AKTA prime chromatography on an SP Sepharose FF column and by ¹H-NMR showing the disappearance of the -Boc group peak at 1.44 ppm and the shifting of â€”CH₂NH₂from 3.30 ppm to 2.92 ppm. In addition, integration shows that the polymer contains 8.6 pendent groups. GPC gave Mn=17,700 Da and Mp=20,200 Da with PDI of 1.11.

Synthesis of Folate-NHS

[Image Omitted]

Folic acid (1.00 g, 2.27 mmol) was dissolved in DMSO (11 mL). After addition of NHS (0.523 g, 4.54 mmol), TEA (0.63 mL, 4.54 mmol), and DCC (0.514 g, 2.49 mmol), the mixture was stirred at room temperature overnight in the dark. The mixture was precipitated in diethyl ether and filtered. The precipitate was washed three times with diethyl ether and dried in vacuo to give 1.35 g of yellow powder. No further purification was performed. ¹H NMR (Varian, 500 MHz, 10 mg/mL DMSO-d) showed the NHS group peak at 2.59 ppm (s, 4H).

Synthesis of H-[(Ptyn)₁₀(EOZ)₁₉₀]-T-Folate

[Image Omitted]

H-[(Ptyn)₁₀(EOZ)₁₉₀]-T-NH (5.46 g, 0.308 mmol) was dissolved in acetonitrile and the mixture was dried by azeotropic distillation. The residue was dissolved in DMSO (40 mL). Folate-NHS (0.830 g, 1.54 mmol) and TEA (85.9 Î¼L, 0.616 mmol) were added to this solution. The resulting mixture was stirred overnight at room temperature in the dark. The mixture was precipitated by addition into diethyl-ether. The yellow precipitate was collected on a glass frit and washed with diethyl ether. After drying in vacuo, the crude material was purified by SP Sepharose FF chromatography. The resulting aqueous solution was charged with NaCl (15% w/w) and extracted with dichloromethane. The combined organic phases were dried over anhydrous sodium sulfate, filtered, concentrated, and dried in vacuo to give 4.14 g of bright yellow solid. Conjugation of folate to the polymer was confirmed by ion-exchange chromatography (AKTA prime on both SP and DEAE Sepharose FF columns). GPC analysis showed Mn=19,200 Da and Mp=21,500 Da with PDI of 1.17.

â€˜Clickâ€™ Addition of Azido-Irinotecan.HCl to H-[(Ptyn)₁₀(EOZ)₁₉₀]-T-Folate

[Image Omitted]

A 500 mL single neck round-bottomed flask was fitted with a rubber septum, a magnetic stir bar, and argon gas inlet. To the flask was added the azido-irinotecan.HCl (0.920 g, 1.30 mmol), H-[(Ptyn)₁₀(EOZ)₁₉₀]-T-Folate (2.50 g, 0.130 mmol, Mn 19200 Da), and H₂O (200 mL). Sodium ascorbate (0.103 g, 0.521 mmol) and CuSO₄.5H₂O (0.130 g, 0.521 mmol) were added. After stirring for 24 hours at room temperature, the mixture was quenched with 1.0 N HCl solution (2 mL). The resulting solution was passed through DowexÂ® (M4195, 80 mL) to remove copper ions and then passed through a silica gel (50 g) column using 0.01% HCl solution as an eluent. The filtrate was charged with NaCl (15% w/v) and then extracted with dichloromethane (3Ã—250 mL). The combined organic phases were dried over anhydrous Na₂SO₄, filtered, and concentrated using a rotary evaporator. The residue was precipitated using dichloromethane and diethyl ether. The precipitated material was collected and dried in vacuo to give 2.3 g of desired product as pale yellow powder. The content of irinotecan based on multiple batch synthesis was determined to be 15 to 17 wt % by reverse phase HPLC analysis. â€˜Clickâ€™ reaction was also confirmed by ¹H-NMR (Varian, 500 MHz, 10 mg/mL CDCl₃) showing the newly formed triazole ring proton at 7.62 ppm (s, 1H). Heavy metal analysis showed that the sample had <25 ppm of copper metal.

Example 16Conjugation of Gemcitabine to H-[(Ptyn)₁₀(EOZ)₂₀₀]-T-PA

[Image Omitted]

Preparation of Compound 2, 5â€²-O-TBDPS-Gemcitabine:

Gemcitabine.HCl (3.00 gm, 10 mmol, 1 equiv.) was dissolved in 70 mL of anhydrous DMF (70 mL) and to this mixture was added 4-(dimethylamino)pyridine (DMAP, 1.24 gm, 10 mmol, 1.0 equiv.) and N,N-diisopropylethylamine (DIPEA 3.487 mL, 20 mmol, 2 equiv.). Molecular sieves (6 gm) were added to the solution. The solution was allowed to stir at room temperature for 1 hour. The solution was then filtered and to the filtrate tert-butylchloro-diphenylsilane (TBDPSCl, 5.30 mL, 20 mmol, 2 equiv.) was injected in a dropwise manner. The reaction mixture was allowed to stir at room temperature overnight. Methanol (20 mL) was next added to the reaction mixture, and allowed to stir at room temperature for an additional 1 hour. The solvent was then removed by rotary evaporation. The residue was dissolved in 100 mL of dichrolormethane (DCM), and this was added followed by washing with purified water (3 times). The DCM layer was dried over anhydrous sodium sulfate, filtered, concentrated to Ëœ10 mL, and then precipitated into hexanes (200 mL) with stirring. The white precipitate was filtered, and dried in vacuum. The crude mixture was dissolved in 240 mL solution of 0.1% TFA in water-acetonitrile (ACNâ€”1:1 v/v), and purified by reversed phase chromatography on an Ã„KTA Purifier system using a Waters SunFire Prep C8 OBD 30/250 Column. The mobile phase was 0.1% TFA in DI water and 0.1% TFA in ACN. The desired fraction that contained compound 2 was collected in each run, and pooled. ACN in the fraction was evaporated by rotary evaporation. Product 2 was precipitated out in the remaining water. The pH of aqueous mixture was adjusted to pH 9.6 by the addition of 1 N NaOH solution. DCM (5Ã—300 mL) was used to extract the product from the aqueous solution. The DCM solution was concentrated to Ëœ150 mL, and then dried over anhydrous sodium sulfate, and filtered. The filtrate was concentrated to 15 mL and added dropwise to hexanes (250 mL). The resulting precipitate was filtered and dried overnight under vacuum. The yield was 4.34 gm. ¹H-NMR (Varian, 500 MHz, 10 mg/mL CD₃OD) showed the peaks at 1.11 ppm (s, 9H, H-8), 3.95 ppm (m, 1H, H-6), 3.95 and 4.11 ppm (d, 1H each, H-4), 4.44 ppm (m, 1H, H-5), 5.65 ppm (d, 1H, H-2), 6.26 ppm (m, 1H, H-7), 7.43 ppm (m, 4H, Ar H-9), 7.44 ppm (m, 2H, Ar H-11), 7.74 ppm (d, 1H, H-3), 7.77 ppm (m, 4H, Ar H-10).

[Image Omitted]

Preparation of Compound 3,3â€²-O-(3-azidopropionyl)-5â€²-O-TBDPS-Gemcitabine

In a 250 mL round bottom flask was placed a solution of azidopropionic acid (1.43 gm, 12 mmol, 1.5 equiv., previously prepared by a method similar to azidoacetic acid described above) in DCM. The 5â€²-O-TBDPS-Gemcitabine (4.28 g, 93.55%, 8 mmol, 1 equiv.), and 4-(dimethylamino)pyridine (DMAP, 1.181 gm, 9.6 mmol, 1.2 equiv.) in DCM (100 mL) were added and allowed to stir under argon atmosphere for 15 min. To this solution dicyclohexyl carbodiimide was added (DCC 2.47 gm, 12 mmol, 1.5 equiv.). The solution was allowed to stir at room temperature for 50 min. The mixture was filtered and then diluted to 400 mL by addition of DCM. This solution was washed with 50 mM HCl (400 mL). The DCM solution was then dried over anhydrous sodium sulfate before it was filtered. The filtrate was evaporated to dryness, and then dried in vacuum. Reverse phase HPLC analysis of the crude mixture (using a C-18 column) indicated that the mixture contained 89% of desired compound 3, while 11% of TBDPS-Gemcitabine remained unreacted. The weight of the crude mixture was 5.5 gm. The crude mixture was next dissolved in 250 mL of a solution of 0.1% TFA in water-ACN (1:1 v/v) and then filtered. It was then purified in three separate runs using the Ã„KTA Purifier system fitted with a Waters SunFire Prep C8 OBD 30/250 Column. The mobile phase was 0.1% TFA in DI water and 0.1% TFA in ACN. The fraction that contains compound 3 was collected, pooled and concentrated. The concentrate contained a white precipitate that was extracted from the aqueous slurry with DCM (4Ã—300 ml). The DCM solution was dried over anhydrous sodium sulfate and then filtered. The filtrate was evaporated by rotary evaporation to dryness, and then was further dried under vacuum. Compound 3 was analyzed by reversed phase HPLC and ¹H-NMR. The yield was 3.24 gm.

¹H-NMR (Varian, 500 MHz, 10 mg/mL CD₃OD) showed the peaks at 1.11 ppm (s, 9H, H-8), 2.74 ppm (m, 2H, H-12), 3.63 (t, 2H, H-13), 3.98 and 4.12 ppm (d, 1H each, H-4), 4.34 ppm (m, 1H, H-5), 5.70 ppm (m, 1H, H-6), 5.81 ppm (d, 1H, H-2), 6.32 ppm (t, 1H, H-7), 7.44 ppm (m, 4H, Ar H-9), 7.49 ppm (m, 2H, Ar H-11), 7.69 ppm (m, 4H, Ar H-10), 7.84 ppm (d, 1H, H-3).

[Image Omitted]

Preparation of Compound 4,3â€²-O-(3-azidopropionyl)-Gemcitabine TFA:

To a solution of 3â€²-O-(3-azidopropionyl)-5â€²-O-TBDPS-Gemcitabine (3.24 gm, 5.4 mmol, 1.0 equiv.) in tetrahydrofuran (THF 80 mL) was added glacial acetic acid (852 Î¼L, 14.9 mmol, 2.75 equiv.) and 1 M solution of tetrabutylammonium fluoride in THF (TBAF 9.5 mL, 9.5 mmol, 1.75 equiv.). The solution was allowed to stir under argon at room temperature for 4 hours. THF was removed and the residue was dried under vacuum. The residue was rinsed with 200 mL of hexanes, and the solvent was decanted. The residue was again dried under vacuum. The crude mixture was then dissolved in 200 mL solution 0.1% TFA in 2:8 v/v ACN-water. The cloudy solution was filtered, and the filtrate purified by reversed phase chromatography in multiple runs by a Waters SunFire Prep C8 OBD 30/250 column using 0.1% TFA in deionized water and 0.1% TFA in ACN as mobile phases. The fractions that contained compound 4 were collected and pooled. ACN was evaporated at room temperature. The remaining aqueous solution was concentrated to 20 mL at 33 mbar and 35Â° C., and then lyophilized. The yield was 1.53 gm.

¹H-NMR (Varian, 500 MHz, 10 mg/mL CD₃OD) showed the peaks at 2.76 ppm (m, 2H, H-8), 3.63 (t, 2H, H-9), 3.81 and 3.95 ppm (d, 1H each, H-4), 4.28 ppm (m, 1H, H-5), 5.52 ppm (m, 1H, H-6), 6.13 ppm (d, 1H, H-2), 6.30 ppm (t, 1H, H-7), 8.15 ppm (d, 1H, H-3).

[Image Omitted]

â€˜Clickâ€™ Addition of Azidopropionyl Gemcitabine.TFA and H-[(Ptyn)₁₀(EOZ)₂₀₀]-T-PA

[Image Omitted]

A 250 mL single neck round-bottomed flask was equipped with a rubber septum, a magnetic stir bar, and an argon gas inlet. The reaction flask was charged with N₃-propionyl gemcitabine-TFA (0.880 g, 1.85 mmol), H-[(Irino)₁₀(EOZ)₂₀₀]-T-PA (4.21 g, 0.184 mmol, Mn 22,800 Da) and H₂O (170 mL). Sodium ascorbate (0.146 g, 0.738 mmol) and CuSO₄.5H₂O (0.184 g, 0.738 mmol) were added. After stirring for 24 hours at room temperature, the mixture was quenched with 1.0 NHC solution (1 mL). The resulting solution was passed through DowexÂ® (M4195, 80 mL) to remove copper and then passed through a silica gel (50 g) column using 0.01% HCl solution as eluent. The filtrate was charged with NaCl (15% w/v) and then extracted with dichloromethane (3Ã—200 mL). The combined organic phases were dried over anhydrous Na₂SO₄, filtered and concentrated. The residue was precipitated using dichloromethane and diethyl ether. The precipitated material was filtered and dried in vacuo to give 3.7 g of desired product as white powder. The content of gemcitabine was determined as 11 wt % by reverse phase HPLC. Heavy metal analysis showed that the sample had <25 ppm of copper metal. The â€˜clickâ€™ reaction was also confirmed by ¹H-NMR (Varian, 500 MHz, 10 mg/mL CDCl₃) showing the newly formed triazole ring proton at 7.62 ppm (s, 1H).

Example 17Conjugation of Irinotecan and LHRH Peptide to H-[(Ptyn)₅(EOZ)₄₅]-T-SPA

A 100 mg/mL solution of previously prepared H-[(Ptyn)₅(EOZ)₄₅]-T-SPA 5K (1.55 gm, 2.61Ã—10^âˆ’4mol, 2.70 equiv.; prepared as in Example 1) in 2 mM HCl (filtered through a 0.2 Î¼m syringe filter) was used. In a 50cc polypropylene tube, 15.5 mL of the polymer solution was added to 15 mL of a solution of (D-Lys⁶)-LHRH peptide (150 mg, 9.69Ã—10^âˆ’5mol, 1.0 equiv., Bachem, with peptide content 83.1%, Purity 97.4%, MW 1253 Da, sequence: Pyr-His-Trp-Ser-Tyr-D-Lys-Leu-Arg-Pro-Gly-NH₂, TFA salt) in 50 mM boric buffer at pH 9.6. The pH of the solution was adjusted to 9.4 with 0.1 N NaOH. The solution was allowed to stir at room temperature for 5 minutes. The solution pH was adjusted to and maintained at 10.8 by dropwise addition of 1.0 N NaOH. Following 5 hours of stirring, the solution pH was adjusted to 6.5 with 1 M acetic acid. The solution was concentrated to 10 mL by ultrafiltration under 70 psi pressure using a 50 mL Amicon stirred cell with a Millipore 44.5 mm PLBC regenerated cellulose membrane (NMWL 3,000). The concentrated solution was diluted to 50 mL by deionized water, and then was further concentrated to 15 mL. The concentrated solution was transferred into a 50 mL polypropylene tube. The solution pH was adjusted to 4.0 with 1 M acetic acid. Using an Ã„KTA Purifier system (Amersham Biosciences), the acidified mixture was loaded onto a 60.5 mL SP Sepharose HP column pre-equilibrated with 20 mM sodium acetate buffer at pH 4.0. Bound H-[(Ptyn)₅(EOZ)₄₅]-T-PA-(D-Lys⁶)LHRH was eluted with 20 mM sodium acetate buffer at pH 4.0 containing 1 M NaCl. Fractions that contain H-[(Ptyn)₅(EOZ)₄₅]-T-PA-(D-Lys⁶)LHRH were pooled, concentrated, and exchanged into deionized water by ultrafiltration under 70 PSI in an Amicon Stirred Ultrafiltration Cell (Millipore, 200 mL) using a ultrafiltration membrane (Millipore, PLBC, Dia. 63.5 mm, NMWL 3,000). The product solution in deionized water was analyzed by SEC and MALDI-TOF, and then lyophilized to form a fluffy off-white powder.

â€˜Clickâ€™ Addition of Azido-Irinotecan HCL and H-[(Ptyn)₅(EOZ)₄₅]-T-PA-(D-Lys⁶)LHRH

The click chemistry for attachment of azido-irinotecan HCL and H-[(Ptyn)₅(EOZ)₄₅]-T-PA-(D-Lys⁶)LHRH is the same as described above in Example 14.

Example 18Efficacy of POZ Irinotecan (H-(Irino.HCl)₁₀(EOZ)₂₀₀-T-PA) in a Mouse Xenograft Model Using Human Colorectal Cancer Cells (HT-29)

Female athymic (Ncr:Nu) mice were implanted subcutaneously with HT-29 tumor fragments and the tumors were allowed to reach a weight range of 162 to 188 mg. Each group of mice (n=10) was intravenously dosed every fourth day for a total of 3 doses of POZ-irinotecan or irinotecan at 40, 60, or 90 mg/kg. The control group received normal saline. The animals were weighed and the tumors measured twice weekly after administration of the first drug injection. All doses of POZ-irinotecan and irinotecan were well tolerated. The maximum loss in body weight for POZ irinotecan and irinotecan was 2% and 8%, respectively. The effects of the test compounds on tumor regression are shown in Table 1.

TABLE 1Summary of tumor growth parameters for HT-29 bearing micefollowing treatment with POZ-irinotecan versus irinotecanTumorMedianDays toRegression^bDuration of2 TimesTestDose^aPar-Com-Regression^cTumorT âˆ’ C^eCompound(mg/kg)tialplete(days)Weight^d(days)Control000NA8.0NAPOZ-irinotecan901725>60.0>52POZ-irinotecan601118>60.0>52POZ-irinotecan4000NA31.423.4Irinotecan9000NA24.816.8Irinotecan6000NA21.413.8Irinotecan4000NA16.88.8^aRefers to amount of active compound in each dose.^bTumor regression: smallest tumor size after the beginning of treatment relative to that observed on first day of treatment. Partial: <50% of size observed on day 1; Complete: unpalpable.^cInterval during which partial or complete tumor regression was observed.^dMedian number of days for tumor to triple in size from the original weight.^eDifference in the median of times for tumors to gain 2 times original size for the drug group minus that for the control group.NA = not applicable

Dose-related changes in HT-29 tumor weights (relative to control) following administration of irinotecan and POZ-irinotecan were observed as shown in FIGS. 1A and 1B, respectively. The observed anti-tumor effect persisted until the termination of the study. The irinotecan treated groups did not show a statistically significant decrease in tumor growth compared to the control group. Tumor growth delay (T-C value) was significantly higher at all doses of POZ-irinotecan compared to irinotecan at all 3 doses tested (p<0.001). A median duration of tumor regression of 25 and 18 days was observed at the 90 and 60 mg/kg dose, respectively, of POZ-irinotecan. Tumor regression was not observed with any of the irinotecan doses.

Example 19Efficacy of POZ Irinotecan (H-(Irino.HCl)₁₀(EOZ)₂₀₀-T-PA) in a Mouse Xenograft Model Using Platinum Resistant Ovarian Cancer Cells (A2780/DDPt)

Female athymic (Ncr:Nu) mice were implanted subcutaneously with A2780/DDPt tumor fragments and the tumors were allowed to reach a weight range of 163 to 189 mg. Each group of mice (n=10) was intravenously dosed every fourth day for a total of 3 doses of POZ-irinotecan or irinotecan at 40, 60, or 90 mg/kg. One group of animals was treated with cisplatin at a dose of 8 mg/kg. The control group received normal saline. The animals were weighed and the tumors measured twice weekly after administration of the first drug injection. All doses of POZ-irinotecan and irinotecan were well tolerated. The maximum loss in body weight for both groups was 2%. The effects of the test compounds on tumor regression are shown Table 2.

TABLE 2Summary of tumor growth parameters for A2780/DDPt bearingmice following treatment with POZ-irinotecan versus irinotecanDays toDose^a3 TimesT âˆ’ C^cTest Compound(mg/kg)Tumor Weight^b(days)Control08.4NAPOZ-irinotecan9036.327.9POZ-irinotecan6020.311.9POZ-irinotecan4012.74.3Irinotecan9018.610.2Irinotecan6016.48.0Irinotecan4013.04.6Cisplatin88.90.5^aRefers to amount of active compound in each dose.^bMedian number of days for tumor to triple in size from the original weight.^cDifference in the median of times for tumors to gain 3 times original size for the drug group minus that for the control group.NA = not applicable

Dose-related changes in A2780/DDPt tumor weights (relative to control) following administration of irinotecan and POZ-irinotecan were observed as shown in FIGS. 2A and 2B. The observed tumor growth delay profiles persisted for at least 32 days for POZ-irinotecan when compared to 14 days for irinotecan (indicated by dotted lines in the respective figures). The irinotecan treated groups did not show a statistically significant decrease in tumor growth compared to the control group. Tumor growth delay (T-C value) was significantly higher at the 90 mg/kg dose of POZ-irinotecan compared to irinotecan (p<0.001)

Example 20Efficacy of Folate-POZ-Irinotecan (H-(Irino.HCl)₁₀(EOZ)₁₉₀-T-Folate) in the Mouse Xenograft Model Using Oropharyngeal Carcinoma Cancer Cells (KB)

Female athymic (Ncr:Nu) mice were implanted subcutaneously with KB tumor fragments and the tumors were allowed to reach a weight range of 160 to 190 mg. Each group of mice (n=10) was intravenously dosed every fourth day for a total of 3 doses of Folate-POZ-Irinotecan or irinotecan at 40, 60, or 90 mg/kg. The control group received normal saline. The animals were weighed and the tumors measured twice weekly after administration of the first drug injection. All doses of POZ-irinotecan and irinotecan were well tolerated. The maximum loss in body weight for both groups was 2%.

Dose-related changes in KB tumor weights (relative to control) following administration of irinotecan and Folate-POZ-irinotecan were observed as shown in FIGS. 3A and 3B, respectively.

The observed anti-tumor effect persisted until the termination of the study. Tumor growth delay (T-C value) was significantly higher at all doses of Folate-POZ-irinotecan compared to irinotecan at all 3 doses tested (p<0.001). Complete tumor regression was seen in >50% of treated mice at all dose groups and at day 20 onwards and in 70% of the mice treated with the 40 and 60 mg/kg doses and 50% of the mice treated with the 90 mg/kg dose. Tumor regression was not observed with any of the irinotecan doses.

Example 20Conjugation of Diclofenac to H-[(Ptyn)₁₀(EOZ)₂₀₀]-T-PA

[Image Omitted]

Synthesis of Hydroxypropyl Diclofenac Ester

Sodium diclofenac (5.00 g, 15.7 mmol) and (dimethylamino)pyridine (DMAP 1.92 g, 15.7 mmol) were dissolved in dimethyl formamide (DMF, 13 mL). 3-Bromopropanol (1.56 mL, 17.3 mmol) was added and the resulting mixture was stirred for 18 hours at room temperature. The mixture was diluted with ethyl acetate (EtOAc, 75 mL) and filtered. The filtrate was washed twice with H₂O (50 mLÃ—2), and then with brine solution. The organic phase was collected, dried over Na₂SO₄, filtered, and concentrated using a rotary evaporator to give 2.5 g (45% yields) of the desired product. No further purification was performed. ¹H-NMR (Varian, 500 MHz, 10 mg/mL CD₃Cl) showed the peaks at 1.90 ppm (p, J=6.0 Hz, 2H, â€”OCH₂CH₂CH₂OH), 3.66 ppm (t, J=6.0 Hz, 2H, â€”OCH₂CH₂CH₂OH), 3.83 ppm (s, 2H, â€”CH₂C(â• O)Oâ€”), 4.32 ppm (t, J=6.5 Hz, 2H, â€”OCH₂CH₂CH₂OH), 6.55 ppm (d, J=7.5 Hz, 1H, Ar H), 6.87 ppm (s, 1H, â€”NHâ€”), 6.98 ppm (m, 2H, Ar H), 7.13 (t, J=8.0 Hz, 1H, Ar H), 7.23 ppm (d, J=7.0 Hz, 1H, Ar H), 7.35 ppm (d, J=8.0 Hz, 2H, Ar H).

Synthesis of Tosylpropyl Diclofenac Ester

Hydroxypropyl diclofenac ester (2.50 g, 7.06 mmol) was dissolved in dichloromethane (24 mL). p-Toluenesulfonyl chloride (p-TsCl 1.61 g, 8.47 mmol) and pyridine (1.14 mL, 14.1 mmol) were added and the resulting mixture was stirred overnight at room temperature. The reaction mixture was quenched with 5% HCl solution and two layers were separated. The aqueous layer was twice extracted with dichloromethane. The combined organic phases were dried over anhydrous Na₂SO₄, filtered, and concentrated using a rotary evaporator. The crude mother liquor was purified by silica gel column chromatography using dichloromethane as eluent to give 1.598 (44% yield) of the desired product as a waxy solid. ¹H-NMR (Varian, 500 MHz, 10 mg/mL CD₃Cl) showed the peaks at 2.01 ppm (p, J=6.0 Hz, 2H, â€”OCH₂CH₂CH₂OTs), 2.43 ppm (s, 3H, â€”CH₃on Tosyl), 3.73 ppm (s, 2H, â€”CH₂C(â• O)Oâ€”), 4.11 ppm (t, J=6.5 Hz, 2H, â€”OCH₂Cl₂CH₂OTs), 4.17 ppm (t, J=6.0 Hz, 2H, â€”OCH₂CH₂CH₂OTs), 6.54 ppm (d, J=7.5 Hz, 1H, Ar H), 6.83 ppm (s, 1H, â€”NHâ€”), 6.94 ppm (m, 1H, Ar H), 6.99 ppm (m, 1H, Ar H), 7.12 ppm (d, J=8.0 Hz, 1H, Ar H), 7.17 (d, J=7.5 Hz, 1H, Ar H), 7.32 ppm (d, J=7.5 Hz, 2H, Tosyl-Ar H), 7.34 ppm (d, J=8.0 Hz, 2H, Ar H), 7.77 ppm (d, J=8.5 Hz, 2H, Tosyl-Ar H).

Synthesis of Azidopropionyl Diclofenac Ester

Tosylpropyl diclofenac ester (1.37 g, 2.69 mmol) was dissolved in DMF (13 mL) and NaN₃(0.350 g, 5.39 mmol) was added. After stirring for 40 hours at room temperature, the mixture was diluted with diethyl ether (75 mL) and washed three times with H₂O (20 mLÃ—3). The diethyl ether solution was dried over Na₂SO₄, filtered, and concentrated to give a clear yellow oil. The crude product was purified by silica gel column chromatography eluting with CH₂Cl₂/hexanes (1:1) to give 0.765 g (75% yield) of the desired product as a clear oil. ¹H-NMR (Varian, 500 MHz, 10 mg/mL CD₃Cl) showed the peaks at 1.93 ppm (p, J=6.5 Hz, 2H, â€”OCH₂CH₂CH₂N₃), 3.35 ppm (t, J=7.0 Hz, 2H, â€”OCH₂CH₂CH₂N₃), 3.82 ppm (s, 2H, â€”CH₂C(â• O)Oâ€”), 4.24 ppm (t, J=6.5 Hz, 2H, â€”OCH₂CH₂CH₂N₃), 6.56 ppm (d, J=7.0 Hz, 1H, Ar H), 6.85 ppm (s, 1H, â€”NHâ€”), 6.96 ppm (m, 1H, Ar H), 6.98 ppm (m, 1H, Ar H), 7.13 (t, J=8.0 Hz, 1H, Ar H), 7.23 ppm (d, J=7.0 Hz, 1H, Ar H), 7.34 ppm (d, J=8.0 Hz, 2H, Ar H).

â€˜Clickâ€™ Addition of Azido Propionyl Diclofenac Ester to H-[(Ptyn)₁₀(EOZ)₂₀₀]-T-PA

[Image Omitted]

The reaction flask was charged with azidopropyl diclofenac ester (0.0166 g, 0.0439 mmol), H-[(Ptyn)₁₀(EOZ)₂₀₀]-T-PA (prepared as in Example 14, 0.10 g, 0.00439 mmol, Mn 22,800 Da), and H₂O (4 mL). After the solution was sparged with argon for 15 minutes, sodium ascorbate (0.0035 g, 0.0175 mmol) and CuSO₄.5H₂O (0.0044 g, 0.0175 mmol) were added. After stirring for 24 hours at room temperature, the reaction was quenched with 1.0 N HCl solution (0.1 mL). The resulting solution was passed through DowexÂ® (M4195, 8 mL) to remove copper ions and then passed through a silica gel (4 g) column using 0.01% HCl solution as an eluent. The filtrate was charged with NaCl (15% w/v) and then extracted with dichloromethane (3Ã—15 mL). The combined organic phases were dried over anhydrous Na₂SO₄, filtered, and concentrated using a rotary evaporator. The residue was dried in vacuo to give 0.111 g of desired product as a white powder. ¹H NMR (Varian, 500 MHz, 10 mg/mL CDCl₃) showed the usual backbone peaks at 1.12 ppm (m, 3H, CH₃CH₂COâ€”); 2.30 ppm (m) and 2.41 (s) (total area 2H, CH₃CH₂COâ€”); and 3.46 ppm (m, 4H, â€”NCH₂CH₂Nâ€”). The terminal group peaks appear at 2.73 ppm (m, 2H each, â€”SCH₂CH₂CO₂H, â€”CH₂SCH₂CH₂CO₂H, and â€”SCH₂CH₂CO₂H). The pendent diclofenac peaks appear at 1.97 ppm (m, 2H, â€”OCH₂CH₂CH₂-triazole 3.82 ppm (s, 2H, â€”CH₂C(â• O)Oâ€”), 4.16 ppm (br s, 2H, â€”OCH₂CH₂CH₂-triazole), 4.35 ppm (t, J=7.0 Hz, 2H, â€”OCH₂CH₂CH₂-triazole), 6.56 ppm (d, J=8.0 Hz, 1H, Ar H), 6.80 ppm (s, 1H, â€”NHâ€”), 6.98 ppm (m, 2H, Ar H), 7.13 (m, 1H, Ar H), 7.23 ppm (m, 1, Ar H), 7.35 ppm (d, J=8.0 Hz, 2H, Ar H). Heavy metal analysis showed that the sample had <25 ppm of copper metal. Comparison of integration of polymer backbone and pendent groups showed that each polymer backbone contains about 7.5 diclofenac molecules.

Prophetic ExampleExample 1Conjugation of Topotecan and Folate to H-[(Ptyn)₁₀(Et)₁₉₀]-T-NH₂

[Image Omitted]

Synthesis of Azidoacetic Acid

Azidoacetic acid was prepared as described in Example 1.

Synthesis of Azido-Topotecan.HCl

A 500 mL single neck round-bottomed flask is charged with a rubber septum, a magnetic stir bar, and argon gas inlet. 10-MEM-topotecan.HCl.3H₂O (13.3 mmol, prepared from topotecan and methoxyethoxymethyl chloridetriethylamine) and acetonitrile (MeCN 250 mL) are added to give a pale yellow suspended solution. All the volatiles are removed using a rotary evaporator to remove H₂O from 10-MEM-topotecan by azeotropic evaporation. Azidoacetic acid (5.37 g, 53.2 mmol) is added and then dichloromethane (266 mL) is added to give a clear yellow solution. 4-(dimethylamino)pyridine (DMAP, 2.44 g, 20.0 mmol) is added and the mixture is cooled down to 0Â° C. A solution of dicyclohexylcarbodiimide (DCC 11.00 g, 53.2 mmol) in dichloromethane (10 mL) is added slowly. After stirring in the cold for 1 hour, the mixture is allowed to stir at room temperature for 16 hours. The mixture is filtered using a filter paper and the filtrate is poured into 0.5 N HCl solution (100 mL) to keep the solution acidic (pHËœ3). The resulting solution is charged with NaCl (15.0 g, 15% w/v water) and two layers are separated. The organic layer is collected and the aqueous layer is twice extracted with dichloromethane. The combined organic phases are dried over anhydrous sodium sulfate, filtered and concentrated to give the product containing some 10-MEM-topotecan. A 250 mL single neck round-bottomed flask with a magnetic stir bar is fitted with a rubber septum and then connected to an argon gas inlet. The crude product prepared above (azidoacetic acid (1.34 g, 13.3 mmol), and dichloromethane (200 mL) are added into the flask. DMAP (1.95 g, 16.0 mmol) and DCC (2.74 g, 13.3 mmol) are added at room temperature. After stirring overnight, the aqueous workup as described above is performed. After concentrating, the residue is dissolved in MeCN and the methoxyethoxymethyl group is removed under reflux with cerium (III) chloride and filtered. After the filtrate is concentrated, the residue is redissolved in dichloromethane and precipitated by adding into diethyl ether. The precipitated material is collected and dried in vacuo to give the product as a powder. The structure is confirmed by ¹H-NMR (Varian, 500 MHz, 10 mg/mL CDCl₃).

Synthesis of Random H-[(Ptyn)₁₀(Et)₁₉₀]-T-NH₂

H-[(Ptyn)₁₀(Et)₁₉₀]-T-NH₂was prepared essentially as described in Example 15. Synthesis of Folate-NHS

Folate-NHS was prepared essentially as described in Example 15.

Synthesis of H-[(Ptyn)₁₀(Et)₁₉₀]-T-Folate

H-[(Ptyn)₁₀(Et)₁₉₀]-T-Folate was prepared essentially as described in Example 15. â€˜Clickâ€™ addition of azido-Topotecan.HCl to H-[(Ptyn)₁₀(Et)₁₉₀]-T-Folate

[Image Omitted]

A 500 mL single neck round-bottomed flask is fitted with a rubber septum, a magnetic stir bar, and argon gas inlet. To the flask is added the azido-topotecan.HCl (1.30 mmol), H-[(Ptyn)₁₀(Et)₁₉₀]-T-Folate (2.50 g, 0.130 mmol, Mn 19200 Da), and H₂O (200 mL). Sodium ascorbate (0.521 mmol) and CuSO₄.5H₂O (0.521 mmol) is added. After stirring for 24 hours at room temperature, the mixture is quenched with 1.0 N HCl solution (2 mL). The resulting solution is passed through DowexÂ® (M4195, 80 mL) to remove copper ions and then passed through SiO₂gel (50 g) column using 0.01% HCl solution as an eluent. The filtrate is charged with NaCl (15% w/v) and then extracted with dichloromethane (3Ã—250 mL). The combined organic phases are dried over anhydrous Na₂SO₄, filtered and concentrated. The residue is precipitated using dichloromethane and diethyl ether. The precipitated material is collected and dried in vacuo to give the product as a powder. The content of topotecan based on multiple batch synthesis is determined by reverse phase HPLC analysis. â€˜Clickâ€™ reaction is also confirmed by ¹H-NMR. Heavy metal analysis showed that the sample had <25 ppm of copper metal.