[0001] FIELD OF INVENTION [0002] The present invention relates to polymeric derivatives of vancomycin. More particularly, the invention relates to multi-loaded polymer conjugates containing vancomycin.

BACKGROUND OF THE INVENTION [0003] Vancomycin is an antibiotic which was initially discovered in the 1950's, see U. S. Patent No. 3,067, 099. It is usually reserved for use in the treatment of severe gram positive infections such as those caused by Staphylococcus aureus and when traditional antibiotics have failed. Over the years, there have been several proposals for improving one or more attributes of vancomycin, usually by continuous infusion. In another example, prodrugs of vancomycin have been proposed as a way of increasing the solubility and circulating life of the drug.

[0004] Prodrugs include chemical derivatives of a biologically-active parent compound which, upon administration, will eventually liberate the active parent compound in vivo. The use of prodrugs allows the artisan to modify one or more properties such as the onset and/or duration of action of a biologically-active compound in vivo. Prodrugs are often biologically inert or substantially inactive forms of the active compound. The rate of release of the active drug is influenced by several factors including the rate

of hydrolysis of the linker. which joins the parent biologically active compound to the prodrug carrier.

[0005] Polymer conjugates of vancomycin have also been proposed as potential prodrugs. For example, commonly-assigned U. S. Patent No.

6,180, 095 discloses benzyl elimination (BE) systems as part of a tripartite polymer-based prodrug platform. These BE prodrug systems are designed inter alia to releasably attach polymers such as polyethylene glycol (hereinafter PEG) to hydroxyl or amine residues on small molecules. After administration to a patient, the prodrugs break down in a predictable fashion. First, the polymer portion hydrolyzes at a predictable, predetermined rate due to the presence of selected bifunctional linkers which contain the desired"trigger". Once the polymer portion has been hydrolyzed, the BE system is initiated or triggered and rapidly releases the parent compound. Commonly assigned U. S. Patent Nos. 5,965, 119 and 6,303, 569 disclose related tripartate prodrug systems containing trimethyl lock triggers. The disclosure of each of the above-mentioned commonly- assigned patents is incorporated herein by reference.

[0006] In spite of the fact that vancomycin is listed among the various biologically active compounds having an available amino group for attachment of the prodrug platform in each of the foregoing commonly- assigned patents, further advances have been sought to refine and improve prodrugs of vancomycin. For example, unlike many biologically active compounds, vancomycin has two amino groups, i. e. the sugar amino (V3) and N-methyl amino (Xi), which are available for polymeric substitution. Thus, control of the substitution reactions involving these amino groups is desirable. Such control is especially useful when branched polymers are employed for delivering higher payloads of drug. The multiple attachment points on the polymer and the two potential points of attachment on the vancomycin raise a distinct possibility that cross-linking and side reactions will occur. Such conjugates can be less desirable than homogeneous multi-loaded conjugates. Separation of the desired

conjugates from the unwanted cross-linked conjugates can be difficult and adds to the expense of the product. There is a need not only to attach polymer to the amino groups of vancomycin selectively but also to increase the loading of vancomycin on polymers so that the full benefit of new and altered pharmacokinetic (PK) profiles can be realized. It would be desirable therefore to provide methods for providing these multi-loaded conjugates.

The present invention addresses this need.

SUMMARY OF THE INVENTION [0007] In one aspect of the invention, there is provided a method of preparing vancomycin-polymer conjugates. The method includes reacting a vancomycin compound of the formula: wherein: R11 and R12 are each independently selected from among hydrogen, CI-6 alkyls, C312 branched alkyls, C3-8 cycloalkyls, C16 substituted alkyls, C3-8 substituted cycloalkyls, aryls, substituted aryls, aralkyl, C1-6 heteroalkyls, substituted C16 heteroalkyls, C16 alkoxyalkyl, phenoxyalkyl and C1-6 heteroalkoxys ;

R13 is OH, NH-aryl, NH-aralkyl, or NH-C112 alkyl ; and w is 1 or 2 ; with a branched polymer residue containing at least one leaving group capable of reacting with the sugar amino group of a vancomycin compound in the presence of at least about a twenty-fold molar excess of triethylamine and a sufficient amount of dimethylformamide.

[00081 In an alternative method, the branched activated polymers are reacted with V3 protected vancomycin compounds to allow the branched polymers to be attached to the vancomycin derivatives through the N-methyl amino group. Suitable protecting groups include lower molecular weight polymer derivatives which preferably block the V3 position while the vancomycin derivative is reacted with the branched polymer derivative in the presence of at least about a 5 to 10 fold molar excess amount of dimethylaminopyridine (DMAP) and a sufficient amount of a solvent such as 1- (3-dimethylamino-propyl)-3-ethyl carbodiimide (EDC), or a mixture of solvents such as dichloromethane (DCM) and dimethylformamide (DMF).

[0009] The present invention also includes the vancomycin-polymer conjugates such as those made by the above-mentioned processes.

Methods of treating mammals having conditions susceptible to vancomycin and related compound therapies are also provided.

[0010] For purposes of the present invention, the term"residue"shall be understood to mean that portion of a vancomycin compound or bifunctional spacer which remains after it has undergone a substitution reaction.

[0011] Methods of preparing the compositions of the invention and methods of treatment using the same are also provided.

[0012] For purposes of the present invention, the term"polymeric containing residue"or"PEG residue"shall each be understood to mean that portion of the polymer or PEG which remains after it has undergone a

reaction with a vancomycin compound such as those described herein as being of formula (I).

[0013] For purposes of the present invention, the term"alkyl"shall be understood to include straight, branched, substituted, e. g. halo-, alkoxy-, nitro-, C112 alkyls, C3 8 cycloalkyls or substituted cycloalkyls, etc. Positive integer shall mean an integer greater than or equal to one, preferably between 1 and 10 and more preferably 1.

[0014] For purposes of the present invention, the term"substituted" shall be understood to include adding or replacing one or more atoms contained within a functional group or compound with one or more different atoms.

[0015] For purposes of the present invention, substituted alkyls include carboxyalkyls, aminoalkyls, dialkylaminos, hydroxyalkyls and mercaptoalkyls ; substituted alkenyls include carboxyalkenyls, aminoalkenyls, dialkenylaminos, hydroxyalkenyls and mercaptoalkenyls ; substituted alkynyls include carboxyalkynyls, aminoalkynyls, dialkynylaminos, hydroxyalkynyls and mercaptoalkynyls ; substituted cycloalkyls include moieties such as 4-chlorocyclohexyl ; aryls include moieties such as napthyl ; substituted aryls include moieties such as 3-bromo-phenyl ; aralkyls include moieties such as toluyl ; heteroalkyls include moieties such as ethylthienyl ; substituted heteroalkyls include moieties such as 3-methoxy-thienyl ; alkoxy includes moieties such as methoxy; and phenoxy includes moieties such as 3-nitrophenoxy. Halo- shall be understood to include fluoro, chloro, iodo and bromo.

[0016] The term"sufficient amounts"for purposes of the present invention shall mean an amount which achieves a desired effect or therapeutic effect as such effect is understood by those of ordinary skill in the art.

[0017] An advantage of the compounds of the invention is that in certain preferred embodiments, the releasable polymer not only extends the circulating life of the vancomycin derivative, but it also provides a

means for controlling the rate of hydrolysis of the derivative. Thus, the artisan has the ability to include varied substituents that allow for modulation of the rate of hydrolysis of the prodrug to optimize the pK profile, reduce dose frequency and its related medical costs. The modifications described herein also allow one to maintain serum levels and prevent bacterial resistance of vancomycin from developing. Further, the conjugates of the present invention can be used on a prophylactic basis to provide protection against bacterial infection.

[0018] Other and further advantages will be apparent from the following description and drawings.

BRIEF DESCRIPTION OF THE DRAWINGS [0019] Figures 1-5 schematically illustrate methods of forming compounds of the present invention which are described in the Examples.

DETAILED DESCRIPTION OF THE INVENTION [0020] The vancomycin compounds included in the conjugates and methods of the present invention generally correspond to formula (I) :

wherein: Rll and R12 are independently selected from among hydrogen, C16 alkyls, C312 branched alkyls, C3-8 cycloalkyls, C1-6 substituted alkyls, C3-8 substituted cycloalkyls, aryls, substituted aryls, aralkyl, C1-6 hetero- alkyls, substituted C16 heteroalkyls, C16 alkoxyalkyl, phenoxyalkyl and Ci-f, heteroalkoxys ; R13 is OH, NH-aryl, NH-aralkyl, or NH-C112 alkyl ; and w is 1 or 2.

[0021] Ril is preferably hydrogen, R12 is preferably methyl and R13 is preferably OH. In alternative aspects of the invention, RI, can be -CH2 =CI , while R13 can be -HNCH2 <CI or-NH (CH2) 10.

[0022] While the above formula covers many of the more well known vancomycin-type compounds known to have biological activity, it is to be understood that the invention embraces not only these specific compounds, but also those vancomycin-based compounds know to artisans of ordinary skill to have a sugar amino and/or an N-methyl amino group. For example, the inventive processes described herein can also be carried out with the vancomycin derivatives described in, for example, EP 0 201 251,"The Role of Hydrophobic Substituents in the Biological Activity of Glycopeptide Antibiotics", J. Am. Chem Soc. 2000,122, 12608-12609 and U. S. Patent Nos. 4,495, 179,3, 067,099, 4,556, 008,4, 548,925 and 4,547, 488 to name but a few. The disclosure of each of the foregoing is incorporated herein by reference. In most preferred aspects of the invention, however, the vancomycin compound employed for the processes described herein is:

[0023] In accordance with a first aspect of the invention, methods are provided for preparing vancomycin-polymer conjugates by reacting a vancomycin compound of formula (I) as shown above with a branched polymer containing at least one leaving group capable of reacting with the sugar (V3) amino group of a vancomycin compound in the presence of at least about a 20-fold molar excess of triethylamine (TEA) and a sufficient amount of dimethylformamide (DMF). The ratio of vancomycin to branched polymer is based on the amount of leaving groups present on the branched polymer. Preferably, there is at least about a 1: 1 ratio of vancomycin to leaving groups.

[0024] Important aspects of this embodiment are the selection and amount of the base used in the reaction of the vancomycin compound with the activated polymer, e. g. the polymer residue containing the leaving group. Since the vancomycin compounds employed in the invention usually contain two amino groups, care must be taken during the reaction so as to avoid formation cross-linked conjugates and/or heterogeneous mixtures of vancomycin-polymer conjugates in which the polymer termini are attached at more than one of the sugar amino (V3) and N-methyl amino (Xi). It has been surprisingly found that when the preferred amount of at least about 20 equivalents and preferably at least

about 30 equivalents of TEA in combination with a sufficient amount of DMF, preferably in the presence of a sufficient amount of molecular sieves, it is possible to obtain a substantially homogeneous reaction product of a branched polymer containing a vancomycin compound attached to each terminal end thereof via the vancomycin V3 amino group.

[0025] For purposes of the present invention, the amount of the solvent DMF employed in the reaction is referred to as a"sufficient amount". As will be appreciated by those of ordinary skill, this amount will be an amount which is capable of at least dissolving the reactants. In most aspects of the invention, the amount of DMF employed will range form about 10 mL/g to about 500 mL/g and preferably from about 100 mL/g to about 200 mL/g based upon the vancomycin compound used.

[0026] The branched activated polymers which can be employed in this process are preferably selected from among those compounds described in commonly assigned PCT publication numbers W002/065988 and W002/066066, the disclosure of each being incorporated herein by reference. Within these general formulae, the following are preferred:

where Ri is a polymeric residue such as PEG; W is a bifunctional linker, such as O, amino acid, - (CH2) y, and-NH (CH2CH20) 2-; z is 0 or a positive integer, preferably 0,1, 2,3 or 4 ; y is a positive integer; and and D is one of

where B is a leaving group.

[0027] Suitable leaving groups identified herein as B can be selected without limitation from groups such as N-hydroxysuccinimidyl, N-hydroxy- benzotriazolyl, halogen, N-hydroxyphthalimidyl, p-nitrophenoxy, imidazolyl, thiazolidinyl thione, O-acyl ureas, pentafluorophenol or 2,4, 6-trichloro- phenol. Other suitable leaving groups will be apparent to those of ordinary skill.

[0028] For purposes of the present invention, leaving groups are to be understood as those groups which are capable of reacting with an amino group (nucleophile) found on the vancomycin compound.

[0029] Within the above guidelines, the following activated branched polymer linkers are more preferred:

wherein PEG is -O (-CH2CH2O) x- ; mPEG is Me-O (-CH2CH20) x-; n is a positive integer, and x is a positive integer selected from about 10 to about 2300.

[0030] Some of the preferred vancomycin conjugates which result from reacting the vancomycin compounds with the branched activated polymer linkers shown above include :

wherein D is one of

where Va is and all other variables if any, are as previously defined. [0031] More preferred V3-linked polymer conjugates of the invention include:

and

wherein Xis and PEG, mPEG, and Va are as shown above.

[0032] In another aspect of the invention there are provided higher payload, branched polymer-vancomycin conjugates in which the termini of the branched polymers are attached to the Xi or N-methyl amino group of the vancomycin compound. Such compounds can be formed by capping the V3 amino group of a vancomycin compound of formula (I) and thereafter reacting the V3 capped vancomycin compound with a branched activated polymer containing at least one leaving group capable of reacting with the N-methyl-amino group of the vancomycin compound under conditions sufficient to form branched polymer conjugates containing vancomycin molecules linked to each terminal through the vancomycin N-methyl amino group.

[0033] Further, in certain preferred aspects of the invention, a low molecular weight (e. g. less than about 10,000) releasable polymer residue, or small molecular weight protecting group is used to temporarily protect the sugar amino group (V3) in order to prepare the selective branched polymer-vancomycin N-methyl amino derivatives. These protecting groups can be removed once the Xi amino group (s) has/have been derivitized.

The protecting groups can be hydrolyzed either in vitro in a PBS or similar buffer followed by purification or in vivo based upon enzyme degradation.

[0034] The polymeric portion, i. e. that portion containing the Ri group, of such compounds corresponding to this embodiment is same as that set forth above with respect to the V3-linked conjugates described above. In this aspect, however, the D moieties are selected from among: wherein Vb is:

wherein J is H or a polymer residue containing a capping group.

[0035] Ri is a water soluble polymer residue which is preferably substantially non-antigenic. Preferably, it is a polyalkylene oxide (PAO) a polyethylene glycol (PEG) or residues corresponding thereto. In some preferred aspects of the invention, Ri further includes a capping group designated herein as A which allows the non-activated end to be capped or unavailable for further reaction. A non-limiting list of suitable PEG's include : <BR> <BR> <BR> <BR> <BR> A-O- (CH2CH20) X ;<BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> <BR> A-O-(CH2CH20) X-CH2C (O)-O-; A-O-(CH2CH2O)x-CH2CH2NR15-, A-O-(CH2CH2O)x-CH2CH2S-, -O-(CH2CH2O)x- -O-C (O) CH2-O-(CH2CH20) X-CH2C (O)-O-, -NR15CH2CH2-O-(CH2CH2O)x-CH2CH2NR15 and -SCH2CH2-0-(CH2CH20) X-CH2CH2 S-7 wherein

x is a positive integer representing the degree of polymerization and ranges from about 10 to about 2,300 ; Ri5 is selected from the group consisting of hydrogen, Cul-6 alkyls, 63-12 branched alkyls, C3-8 cycloaikyls, C16 substituted alkyls, C3-8 sub- stituted cycloalkyls, aryls substituted aryls, aralkyls, C16 heteroalkyls, substituted Ci. heteroalkyls, C16 alkoxyalkyl, phenoxyalkyl and Cul-6 heteroalkoxys.

[0036] A is a capping group such as a C16 alkyl, preferably methyl, or other PEG terminal activating groups, as such groups are understood by those of ordinary skill.

[0037] Also useful are polypropylene glycols, branched PEG derivatives such as those described in commonly-assigned U. S. Patent No.

5,643, 575, "star-PEG's"and multi-armed PEG's such as those described in Shearwater Corporation's 2001 catalog"Polyethylene Glycol and Derivatives for Biomedical Application". The disclosure of each of the foregoing is incorporated herein by reference. It will be understood that the water-soluble polymer can be functionalized for attachment to the bifunctional linkage groups if required without undue experimentation.

[0038] Although PAO's and PEG's can vary in average molecular weight, the polymer portion of the prodrug is broadly from about 2,000 Da to about 100,000 Da. In other aspects, the polymer has an average molecular weight of from about 5,000 Da to about 100,000 Da and is preferably from about 5,000 Da to about 40,000 Da. The average molecular weight of the polymer selected for inclusion in the prodrug must be sufficient so as to provide sufficient circulation of the prodrug before hydrolysis of the linker.

[0039] The polymeric substances included herein are preferably water-soluble at room temperature. A non-limiting list of such polymers include polyalkylene oxide homopolymers such as polyethylene glycol (PEG) or polypropylene glycols, polyoxyethylenated polyols, copolymers

thereof and block copolymers thereof, provided that the water solubility of the block copolymers is maintained.

[0040] In a further embodiment, and as an alternative to PAO's, the polymers are optionally selected from among one or more effectively non-antigenic materials such as dextran, polyvinyl alcohols, carbohydrate-based polymers, hydroxypropylmethylacrylamide (HPMA), polyglutamic acid, polyaspartic acid, polyhydroxyethyl aspartate (HEA), chitans and other like multifunctional non-antigenics as well as polyalkylene oxides, and/or copolymers thereof. See also commonly-assigned U. S.

Patent No, 6,153, 655, the contents of which are incorporated herein by reference. It will be understood by those of ordinary skill that the same type of activation is employed as described herein as for PAO's such as PEG. Those of ordinary skill in the art will further realize that the foregoing list is merely illustrative and that all polymeric materials having the qualities described herein are contemplated. For purposes of the present invention, "effectively non-antigenic"and"substantially non-antigenic"shall be understood to include all polymeric materials understood in the art as being substantially non-toxic and not eliciting an appreciable immune response in mammals.

10041] Preferably the substituents are reacted in an inert solvent such as dimethylformamide (DMF), methylene chloride (DCM), tetrahydrofuran (THF), acetonitrile (CH3CN), chloroform (CHCI3), or mixtures thereof. The reaction is preferably conducted at a temperature from 0°C up to about 22°C (room temperature).

METHODS OF TREATMENT [0042] Another aspect of the present invention provides methods of treatment for various medical conditions in mammals. The methods include administering to the mammal in need of such treatment, an effective amount of the prodrug, i. e. vancomycin, which has been prepared

as described herein. The compositions are useful for, among other things, treating vancomycin-sensitive infections.

[0043] The amount of the prodrug administered will depend upon the vancomycin compound selected. Generally, the amount of prodrug used in the treatment methods is that amount which effectively achieves the desired therapeutic result in mammals. Naturally, the dosages of the various prodrug compounds will vary somewhat depending upon the parent compound, rate of in vivo hydrolysis, molecular weight of the polymer, etc.

In general, however, it is contemplated that the vancomycin prodrugs will be administered in amounts ranging from about 0.5 to about 60 mg/kg twice a week. Preferably, the vancomycin is administered in amounts ranging from about 0.5 to about 30 mg/kg per day. The ranges set forth above are based on the amount of vancomycin derivative. The ranges are also illustrative and those skilled in the art will determine the optimal dosing of the prodrug selected based on clinical experience and the treatment indication. Actual dosages will be apparent to the artisan without undue experimentation.

[0044] In one embodiment of this aspect of the invention, the method of treatment includes administering the prodrugs of the present invention in combination with an unmodified vancomycin derivative such as vancomycin HCI. The combination of prodrug and vancomycin derivative may be administered to a patient in need of the drug or such treatment as part of a single pharmaceutical dosage form (e. g. intravenous or parenteral injection/infusion or oral dosage form) or as part of a treatment regimen in which both of the vancomycin and vancomycin prodrug are administered as separate dosage forms to a patient in need thereof.

[0045] The prodrugs of the present invention can be included in one or more suitable pharmaceutical compositions for administration to mammals. The pharmaceutical compositions may be in the form of a solution, suspension, tablet, capsule or the like, prepared according to methods well known in the art. It is also contemplated that administration of

such compositions may be by the oral and/or parenteral routes depending upon the needs of the artisan. A solution and/or suspension of the composition may be utilized, for example, as a carrier vehicle for injection or infiltration of the composition by any art known methods, e. g. , by intravenous, intramuscular, subdermal injection and the like.

[0046] Such administration may also be by infusion into a body space or cavity, as well as by inhalation and/or intranasal routes. In preferred aspects of the invention, however, the prodrugs are parenterally administered to mammals in need thereof.

[0047] Another aspect of the invention is a method of treating vancomycin susceptible diseases in mammals using a combination of a vancomycin in unmodified or commonly available forms, e. g. vancomycin HCI, or other pharmaceutically acceptable salt, solvate or hydrate thereof, and a polymeric conjugate of the invention. The total amount of vancomycin administered to the patient in need thereof is an effective amount as mentioned above, based on the amount of vancomycin. The combination of prodrug and vancomycin derivative can be administered to a patient in need of the drug or such treatment as part of a single pharmaceutical dosage form (e. g. intravenous or parenteral injection/infusion or oral dosage form) or as part of a treatment regimen in which both of the vancomycin and vancomycin prodrug are administered as separate dosage forms to a patient in need thereof. Thus, the vancomycin and polymeric conjugate of the invention are administered either substantially concurrently in separate dosage forms or combined in a unit dosage form.

[0048] Since the present invention can relate to treatment with a combination of vancomycin dosage forms which can be administered separately, the invention also relates to combining separate pharmaceutical compositions in kit form. That is, a kit is contemplated wherein two separate unit dosage forms are combined: for example a vancomycin pharmaceutical composition and a separate pharmaceutical composition

containing a polymer conjugate of the invention. The kit will preferably include directions for the administration of the separate components. The kit form is particularly advantageous when the separate components must be administered in different dosage forms and/or are administered at different dosage intervals. Thus a kit may comprise, in separate containers in a single package, pharmaceutical compositions for use in a therapeutically effective amount of vancomycin or a pharmaceutical acceptable salt, solvate or hydrate thereof in a pharmaceutically acceptable carrier and in a second container a therapeutical effective amount of a polymer conjugate as described herein in the form of a pharmaceutical acceptable salt, solvate or hydrate thereof in a pharmaceutically acceptable carrier.

EXAMPLES [0049] The following examples serve to provide further appreciation of the invention but are not meant in any way to restrict the effective scope of the invention. The underlined and bold-faced numbers recited in the Examples correspond to those shown in the Figures 1-5.

[0050] General Procedures. All reactions were run under an atmosphere of dry nitrogen or argon. Commercial reagents were used without further purification. All PEG compounds were dried under vacuum or by azeotropic distillation from toluene prior to use. Polyethylene glycol (PEG) linkers : compounds 46 and 53 are made according to the procedures described in US patent"Polymeric prodrugs of amino-and hydroxyl-containing bioactive agents" (US 6,180, 095) and the paper"Drug delivery systems employing 1, 4- or 1, 6-elimination : releasable poly (ethylene glycol) conjugates of amine-containing compounds" (J. Med.

Chem. , 1999, Vol. 42, No. 18, pages3657-3667). 13CNMRspectrawere obtained at 75.46 MHz using a Varian Mercury@300 NMR spectrometer and deuterated chloroform and pyridine as the solvents unless otherwise specified. Chemical shifts (8) are reported in parts per million (ppm)

downfield from tetramethylsilane (TMS). All vancomycin-polymer conjugation reactions were carried out in the presence of 4 Angstrom molecular sieves.

[0051] HPLC method. The reaction mixtures and the purity of intermediates and final products were monitored by a Beckman Coulter System Gold HPLC instrument. It employs a ZOBAX 300SB C8 reversed phase column (150 x 4.6 mm) or a Phenomenex Jupiter 300A C18 reversed phase column (150 x 4.6 mm) with a multiwavelength UV detector, using a gradient of 10-90 % of acetonitrile in 0.05 % trifluoroacetic acid (TFA) at a flow rate of 1 mL/min.

[0052] Example 1: Compound 40. To a solution of 40kDa PEG do- acid 6 (33.0 g, 0.823 mmol), aspartic acid methylester hydrochloride (0.65 g, 3.29 mmol), and DMAP (1.0 g, 8.23 mmol) in DCM (300 mL) cooled to 0 °C was added EDC (0.95 g, 4.94 mmol) and the mixture was allowed to warm to room temperature and stirred overnight, followed by partial removal of the solvent in vacuo. The product was precipitated with ethyl ether, collected by filtration, and crystallized from IPA (660 mL) to yield 40 (32 g, 96 %). 13C NMR (67.8 MHz, C5D5N) 8 170.41, 170.23, 169.24, 72.09- 69.32 (PEG), 52.33, 51.60, 47.56, 35.70.

[0053] Example 2: Compound 41. A solution of 40 (32 g, 0.79 mmol) and lithium hydroxide hydrate (0.27 g, 6.43 mmol) in water (200 mL) was stirred overnight at room temperature and the pH adjusted to 2.5 with 0. 1N HCI. This solution was extracted with DCM and the organic layer dried (anhydrous sodium sulfate), filtered, followed by partial removal of the solvent under reduced pressure. The product was precipitated with ethyl ether, collected by filtration, and crystallized from IPA (640 mL) to give 41 (31.5 g, 98 %)."C NMR (67.8 MHz, C5D5N) 8 170.99, 170.76, 169.28, 70.71-69. 74 (PEG), 47.36, 35.73.

[0054] Example 3: Compound 42. To a solution of 41 (30.0 g, 0.744 mmol), glycine methylester hydrochloride (0.75 g, 5.95 mmol), and DMAP (1.8 g, 14.75 mmol) in DCM (300 mL) cooled to 0 °C was added

EDC (1.71 g, 8.92 mmol) and the mixture was allowed to warm to room temperature and stirred overnight, followed by partial removal of the solvent in vacuo. The product was precipitated with ethyl ether, collected by filtration, and crystallized from IPA (600 mL) to give 42 (29.2 g, 97 %). 13C NMR (67.8 MHz, C5D5N) 8 170.37, 170.18, 169.82, 169.57, 169.31, 70.61- 69.93 (PEG), 51.78, 48.87, 40.84, 40.80, 36.77.

[0055] Example 4: Compound 43. A solution of 42 (29 g, 0.71 mmol) and lithium hydroxide hydrate (0.24 g, 5.71 mmol) in water (200 mL) was stirred overnight at room temperature, and the pH adjusted to 2.5 with 0. 1 N HCI. This solution was extracted with DCM and the organic layer dried (anhydrous sodium sulfate), filtered, followed by partial removal of the solvent under reduced pressure. The product was precipitated using ethyl ether, collected by filtration, and crystallized from IPA (640 mL) to give 43 (27.4 g, 94 %). 13C NMR (67.8 MHz, C5D5N) 8 170.44, 170.32, 170.11, 169.86, 72.16-69. 78 (PEG), 61.26, 49.22, 41.09, 37.30.

[0056] Example 5: Compound 44. To a solution of 43 (5.0 g, 0.123 mmol), 2-MT (0.18 g, 1.51 mmol), and DMAP (0.36 g, 2.96 mmol) in anhydrous DCM (50 mL) cooled to 0 °C was added EDC (0.28 g, 1.48 mmol) and stirred overnight, followed by partial removal of the solvent under reduced pressure. The product was precipitated with ethyl ether, collected by filtration, and crystallized from IPA (100 mL) to yield 44 (4.6 g, 92 %). 13C NMR (67.8 MHz, C5D5N) à 200.74, 200.71, 170.29, 170.17, 169.85, 169.64, 72.09-68. 39 (PEG), 55.39, 48.92, 46.32, 36.67, 28.72.

[0057] Example 6: Compound 45. A solution of 44 (4.5 g, 0.11 mmol), 3, 5-dimethyl-4-hydroxybenzylalcohol (0.35 g, 2.87 mmol), and DMAP (0.53 g, 2.87 mmol) in anhydrous DCM (50 mL) was refluxed for 18 hrs, followed by partial removal of the solvent under reduced pressure.

The PEG derivative was precipitated by the addition of ethyl ether, filtered, and crystallized from IPA (90 mL) to yield 45 (4.1 g, 91 %). 13C NMR (67.8 MHz, CDCI3) 5 170.69, 170.38, 169.96, 167.36, 167.06, 148.39, 138.72, 129.46, 126.56, 72. 15-69. 17 (PEG), 63.77, 49.01, 40.76, 36.83, 16.02.

[0058] Example 7: Compound 46. To a solution of 45 (4.0 g, 0.097 mmol) and DSC (0.80 g, 3.11 mmol) in anhydrous DCM (40 mL) and DMF (4 mL) cooled to 0 °C was added pyridine (0.25 g, 3.11 mmol). The reaction mixture was allowed to warm to room temperature and stirred for 12 hrs followed by partial removal of the solvent under reduced pressure.

The PEG linker was precipitated by the addition of ethyl ether, filtered, and crystallized from DCM/ethyl ether to yield 46 (2.8 g, 70 %). 13C NMR (67.8 MHz, CDCI3) 6 170. 73,170. 43,169. 97,168. 17,167. 18,166. 86, 151. 03, 147.96, 130.55, 130.38, 128.42, 71. 78-69.78 (PEG), 49.05, 40.78, 36.92, 25.15, 15. 98.

[0059] Example 8: Compound 47. To a solution of 1 (0.353 g, 0.238 mmol) and TEA (0.662 mL, 4.75 mmol) in anhydrous DMF (50 mL) was added 46 (2.2 g, 0.0528 mmol) and the resulting mixture stirred at room temperature for 12 hrs. The reaction mixture was filtered through celite and precipitated with ethyl ether (300 mL). Filtration gave crude product which was recrystallized twice from a mixture of DMF/ethanol (1: 1) to give 47 (2.0 g, 90 %). The structure of the compound is confirmed by 13C NMR.

[0060] Example 9: Compound 49. To a solution of 48 (4.0 g, 16.1 mmol) and triphosgene (1.92 g, 6.46 mmol) in anhydrous DCM (50 mL) cooled to 15 °C was added DIEA (7.6 mL, 43.6 mmol) dropwise over a period of 5 minutes, while maintaining the reaction temperature between 15 °C and 20 °C. This mixture was allowed to warm to room temperature over a period of one hour, followed by the addition of 3, 5-dimethyl-4- hydroxybenzaldehyde 25 (1.97 g, 16.13 mmol) and DMAP (2.4 g, 16.1 mmol) and then stirred at room temperature overnight. The mixture was washed with 0. 1 N HCI solution, the organic layer dried (anhydrous sodium sulfate), filtered, and the solvent removed from the filtrate under reduced pressure to give crude 49. 13C NMR (67.8 MHz, CDCI3) 8 191. 18,155. 64, 152.99, 152.82, 133.42, 132.06, 129.76, 78.99, 41.00, 40.17, 28.21, 16.12.

[0061] Example 10: Compound 50. To a solution of 49 (1.0 g, 2.36 mmol) in methanol (30 mL) cooled to 15 °C was added sodium borohydride

(0.1 g, 2.63 mmol) and the reaction mixture stirred at room temperature for one hour, followed by acidification with 0.1 N HCI solution. The solvent was removed from the filtrate in vacuo, and the residue was taken up in water (20 mL) and extracted with DCM. The organic layer was dried (anhydrous sodium sulfate), filtered, and the solvent removed from the filtrate under reduced pressure. The crude product was then purified by silica gel chromatography to yield 50 (0.9 g, 90 %). 13C NMR (67.8 MHz, CDCI3) 8 155.65, 153.95, 146.81, 138.01, 130.33, 126.56, 78.86, 63.89, 40.69, 39.96, 28.09, 15.92.

[0062] Example 11 : Compound 52. To a solution of 50 (0.9 g, 2.11 mmol) in DCM (57 mL) was added trifluoroacetic acid (3 mL) and the reaction mixture stirred for 1.5 hrs at room temperature, followed by removal of the solvent in vacuo to make 51. The residue was dissolved in anhydrous DCM (70 mL) and to this solution was added 43 (7.4 g, 0.18 mmol) and DMAP (1.26 g, 10.3 mmol). The mixture was cooled to 0 °C followed by the addition of EDC (0.56 g, 2.93 mmol), and the solution was allowed to warm to room temperature and stirred overnight followed by partial removal of the solvent under reduced pressure. The product was precipitated with ethyl ether, collected by filtration, and crystallized from DCM/ethyl ether to yield 52 (6.45 g, 85-%).'3C NMR (67.8 MHz, Ceci3) s 170.34, 170.23, 169.82, 153.96, 147.06, 146.91, 138.39, 138.02, 130.61, 130.55, 126.79, 72.30-69. 29 (PEG), 64.35, 64.26, 49.17, 39.19, 39.05, 16.16.

[0063] Example 12: Compound 53. To a solution of 52 (6.4 g, 0.154 mmol) and DSC (1.26 g, 4.92 mmol) in a mixture of anhydrous DCM (120 mL) and DMF (12 mL) cooled to 0 °C was added pyridine (0.39 g, 4.92 mmol) and the mixture allowed to warm to room temperature and stirred overnight. The solvent was partially removed under reduced pressure and the product precipitated with ethyl ether, filtered, and crystallized from DCM/ethyl ether to give 53 (5.8 g, 89 %). 13C NMR (67.8 MHz, CDCI3) 8 170.26, 169.91, 168.25, 153.59, 151.15, 148.51, 131.31,

130.04, 128.53, 72.21-68. 18 (PEG), 49.40, 40. 96, 39.18, 37.19, 25.29, 16.11.

[0064] Example 13: Compound 54. Prepared in 79 % yield by reacting 1 with 53 as described for 47. The structure of the compound is confirmed by 43C NMR.

[0065] Example 14 : Compound 56. A solution of 55 (mw 40 kDa, 23 g, 0.575 mmol) and disuccinimidyl carbonate (DSC, 2.36 g, 9.2 mmol) in methylene chloride (DCM, 230 mL) and dimethylformamide (DMF, 23 mL) was cooled to 0 °C, followed by the addition of pyridine (0.75 mL, 9.2 mmol). This mixture was allowed to warm to room temperature overnight, followed by filtration through Celtes) and partial removal of the solvent from the filtrate under reduced pressure. The crude product was precipitated with ether, collected by filtration, and crystallized from 20% DMF/isopropanol (IPA) to yield 56 (20.1 g, 0.496 mmol, 86%). 13C NMR (67.8 MHz, C5D5N) 6 168.2, 151.1, 70.7-69. 6 (PEG), 68.0, 45.2, 25.2.

[0066] Example 15: Compound 57. To a solution of 50 (0.9 g, 2.11 mmol) in DCM (57 mL) was added trifluoroacetic acid (3 mL) and the reaction mixture stirred for 1.5 hrs at room temperature, followed by removal of the solvent in vacuo to make 51. The residue was dissolved in anhydrous DCM (70 mL) and to this solution was added 56 (7.2 g, 0.18 mmol) and DMAP (1.26 g, 10.3 mmol). The mixture was cooled to 0 °C followed by the addition of EDC (0.56 g, 2.93 mmol), and the solution was allowed to warm to room temperature with stirring overnight. After partial removal of the solvent under reduced pressure, the product was precipitated with ethyl ether, collected by filtration, and crystallized from DCM/ethyl ether to yield 52 (6. 45 g, 85 %). 13G NMR (67.8 MHz, CDCI3) 8 155.9, 153. 6, 146.8, 138.0, 130.4, 126.6, 72.2-68. 5 (PEG), 64.1, 63.5, 45.2, 40.7, 40.5, and 16.0.

[0067] Example 16: Compound 58. To a solution of 52 (6.2 g, 0.154 mmol) and DSC (1.26 g, 4.92 mmol) in a mixture of anhydrous DCM (120 mL) and DMF (12 mL) cooled to 0 °C was added pyridine (0.39 g,

4.92 mmol), and the mixture allowed to warm to room temperature and stirred overnight. The solvent was partially removed under reduced pressure and the product precipitated with ethyl ether, filtered, and crystallized from DCM/ethyl ether to give 58 (5.8 g, 89 %). 13C NMR (67.8 MHz, CDCI3) 8 168.2, 155.9, 153.4, 151.1, 148.4, 131.2, 129.9, 128.5, 72.1- 68.0 (PEG), 63.6, 45.3, 40.8, 40.5, 25.2 and 16.1.

[0068] Example 17: Compound 59. To a solution of 1 (0.550 g, 0.37 mmol) and triethylamine (TEA, 2.06 mL, 14.8 mmol) in DMF (50 mL) was added 58 (3 g, 0.074 mmol) and 5.5 g molecular sieves (4 A) and the mixture stirred at 30 °C for 5 hrs. The reaction mixture was filtered through celite, the PEG conjugate precipitated with ether, filtered, and crystallized from DMF/ethanol (50: 50) three times to give 59 (2.0 g, 0.0436 mmol, 59%).

[0069] Other embodiments of the invention will be apparent to one skilled in the art from a consideration of this specification or practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.