AU2003204343B2

AU2003204343B2 - Vitamin B12 derivatives and methods for their preparation

Info

Publication number: AU2003204343B2
Application number: AU2003204343A
Authority: AU
Inventors: John Mcewan; Gregory Russell-Jones
Original assignee: Access Pharmaceuticals Australia Pty Ltd
Current assignee: Access Pharmaceuticals Australia Pty Ltd
Priority date: 1998-06-12
Filing date: 2003-05-23
Publication date: 2007-03-15
Anticipated expiration: 2019-06-11

Description

Our Ref:7825513 P/00/011 Regulation 3:2

AUSTRALIA

Patents Act 1990

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT Applicant(s): Biotech Australia Pty Limited 28 Barcoo Street Roseville New South Wales 2069 Australia Address for Service: Invention Title: DAVIES COLLISON CAVE Patent Trade Mark Attorneys Level 10, 10 Barrack Street SYDNEY NSW 2000 Vitamin B 1 2 derivatives and methods for their preparation The following statement is a full description of this invention, including the best method of performing it known to me:- 5020 WO 99/65930 PCT/AU99/00462 VITAMIN B 1 2 DERIVATIVES AND METHODS FOR THEIR PREPARATION TECHNICAL FIELD The present invention generally relates to novel derivatives of vitamin B2 carrier molecules for the delivery of therapeutic substances by administration of a complex comprising these substances linked to vitamin B 1 2 (VB 12 or an analogue thereof. The invention also generally relates to novel methods for preparing VB 1 2 derivatives. More particularly, the invention relates to reactions of the 5'OH group of VB 1 2 with electrophiles to prepare these VB 1 2 derivatives.

BACKGROUND OF THE INVENTION An oral delivery mechanism for peptides is described in International application PCT/AU86/0299 (WO87/02251) based on recent work undertaken by one of the current inventors. The mechanism utilises at least one carrier molecule to which an active substance is linked to transport the active substance from the intestinal lumen into the circulatory system. VB 1 2 and analogues thereof function as ideal carrier molecules by using the natural

VB

12 uptake system, mediated by the binding of VB 1 2 to intrinsic factor to transport the active substance/VB 1 2 complex. Once delivered into the lymphatic drainage system or serum, the complex substantially retains the bioactivity of the native active substance.

More recently conjugates of VB 12 with drugs, cytotoxins and MRI agents, have been used in the detection and treatment of tumour cells. For normal cellular uptake of vitamin B12 (cobalamin, Cbl, VB12), the vitamin must first bind to the plasma protein transcolbamin II (TCII). Following binding of Cbl to TCII the resultant TCII-Cbl complex binds with high affinity to receptors on the surface of cells and is internalized by the cell via a process called receptor-mediated endocytosis (RME). Once inside the cell the Cbl is enzymatically modified to form two coenzymes, which are in turn used for two essential metabolic pathways. One pathway involves the methylation of homocysteine in the de novo synthesis WO 99/65930 PCT/AU99/00462 of methionine, and is catalyzed by methionine synthase. The other pathway involves the rearrangement of methylmalonyl CoA to succinyl CoA, and is catalyzed by methylmalonyl CoA mutase. It has recently been shown that the in vitro proliferation of human and murine leukemia cells is dependent upon both TCII and Cbl (McLean, G. Quadros, E. B., Rothenberg, S. Morgan, A. Schrader, J. and Ziltener, H. 1997 Antibodies to transcobalamin II block in vitro proliferation of leukemic cells, Blood, 89, 235-242). Several workers have now concentrated on utilizing Cbl conjugates for both radio-imaging and for targeted cancer chemotherapy (Smeltzer, C. Pinson, P. Munger, J. West, F. G., and Grissom, C. 1999 Cytotoxicities of two new cobalamin bioconjugates. Proceedings Ninth International Symposium on Recent Advances in Drug Delivery Systems, pp 232-3; Canon, Munger, J. West, F. and Grissom, C. 1999 Synthesis and uptake of radiolabeled cobalamin bioconjugate, Proceedings Ninth International Symposium on Recent Advances in Drug Delivery Systems, pp 230-1; Pinson, P. Munger, J. West, F. and Grissom, C. 1999 Synthesis of two doxorubicin-cobalamin bioconjugates, Proceedings Ninth International Symposium on Recent Advances in Drug Delivery Systems, pp 228-9).

In order for VB 2 to co-transport pharmaceuticals across the intestinal epithelial cell layer and into the circulatory system the pharmaceuticals must first be covalently linked to the VB 2 molecule.

Similarly, in order that VBl2 can target an anti-tumour agent to a tumour, the agent must also be covalently linked to the VB 12 molecule. For this to occur, the VB, 2 molecule itself must first be modified to provide a suitable functional group for conjugation. A carboxylic acid derivative of

VB

1 2 is readily achieved by mild acid hydrolysis of the propionimide side chains of the corrin ring structurel (see Figure This hydrolysis results in the formation of the and "e" monocarboxylic acids of VB 2.2 The isolated monocarboxylic acid derivatives can then be conjugated directly to amino groups of proteins or peptides using commercial carbodiimides such as l-ethyl-3-( 3 -(dimethylamino)propyl)carbodiimide (EDAC) or dicyclohexylcarbodiimide (DCC) thereby linking the peptide to VB 1 2 via a peptide bond.l.

3 A second method of conjugation of peptides to VBl2 is by axial substitution of functional groups onto the Co atom of the corrin ring of the VB, 2 molecule (see Formula In this method, the axial CN ligand of VB, 2 can be replaced with a functionalised alkyl chain. This substituted functional group can then be used for conjugation to a peptide or protein using traditional chemical techniques. One major disadvantage of this method, however, is that the WO 99/65930 PCT/AU99/00462 resultant conjugate contains a light sensitive Co-C bond. Thus care must be taken not to expose solutions of the alkylcobalamins to visible light.

Early work by Toraya and Fukui 4 demonstrated the feasibility of conjugation to VB 1 2 via an ester linkage to the 5'OH of the ribose moiety of the nucleotide ligand. In their work Toraya and Fukui explored the possibility of using this chemistry to form an affinity ligand for purification of diol dehydrase. In order to form the 5'O-ester linkage the authors reacted VBi 2 with a 54 fold excess of succinic anhydride in a large volume of DMSO (VB12 at 5 mg/ml) plus a large excess of pyridine (128 fold These authors found that the linkage formed was not only unstable at basic pH, but was also ineffective in purifying the enzyme. Annunziato and co-workers 5 describe another method of linkage to the 5'OH of the ribose. These workers reacted p-maleimidophenyl isocyanate with VB 12 and subsequently used the activated VB 12 molecule to react with thiolated alkaline phosphatase. Subsequently, Habberfield and co-workers combined the work of Toraya and Fukui 4 with that of Annunziato et al., s as well as Russell-Jones et al.

3 6 and produced conjugates of G-CSF, EPO and consensus interferon to a 5'O-glutaroyl derivative of VB 12 The subsequent conjugates were claimed to be active following intraduodenal pump administration to rats of the conjugates pre-complexed to rat IF. In the method described by Habberfield and coworkers, 5 gm of cyanocobalamin (VB 1 2 1356 MW) was dissolved in 1,000 ml of DMSO and 200 gm of glutaric anhydride (116 MW) was added in 160 ml of pyridine. The product yield was around 65%. This represents a 468 molar excess of glutaric anhydride to VB 12 In the work of Toraya and Fukui, 4 these workers used 200 mg of cyanocobalamin dissolved in 40 ml DMSO plus 8 grams of succinic anhydride (100 MW) to couple to the hydroxyl group. This represents a 54 fold molar excess of anhydride, with a product yield of 90%. In the method of conjugation described by Russell-Jones and co-workers 3 6 the VBi 2 monoacid was prepared by treatment with acid for 72 hrs and subsequent purification on Dowex 1X8 and Dowex 1X2 to afford a yield of only about In order to link the VB 1 2 monoacid to some peptides and proteins further derivatization of the carboxyl group was often required.

Apart from the methods described by Toraya and Fukui 4 and Habberfield et a1 7 and Annunziato et al., 5 there are other methods which could be used to form covalent linkages to the 5'OH group of

VB

2 These methods are generally used in the preparation of affinity resins by modification of sugar residues resident in agarose. These methods include reaction with oxirane (1,4 butane-diol diglycidyl ether), benzoquinone or cyanuric chloride. These methods have been attempted in the synthesis of VB 12 derivatives, however, the yields were either so low as to make the process noncommercial, or the quantities of reagents employed were so high as to make them similarly non- Scommercial.

SThe present invention seeks to overcome, or at least alleviate one or more of the 00 5 abovementioned disadvantages of the prior art. In particular, the present invention seeks to provide methods for preparing derivatives of VB 1 2 carrier molecules which utilise the Sgroup of VB 1 2 for chemical bonding with spacer molecules. The present invention further Sseeks to provide VB 1 2 derivatives that are easy to make, obtainable in good to high yields O and are readily purified.

m' SSUMMARY OF THE INVENTION Surprisingly it has been found by the present inventors that VB, 2 derivatives, which are suitable for conjugation to polymers, nanoparticles and pharmaceutically active agents, are readily prepared by reaction of the 5'OH group on the ribose moiety of VB 2 with carbbnyl electrophiles.

According to an aspect of the present invention there is provided a method for preparing

VB

12 derivatives suitable for linking to a polymer, nanoparticle or therapeutic agent, protein or peptide comprising the steps of reacting the 5'OH group of VB, 2 or an analogue thereof with a bifunctional carbonyl electrophile to form an active intermediate, and subsequently reacting the intermediate with a nucleophilic spacer molecule to yield said VB 1 2 derivative.

According to another aspect of the present invention there is provided a method for preparing a VB 12 derivative suitable for linking to a polymer, nanoparticle or therapeutic agent, protein or peptide comprising the steps of reacting a carboxylic acid spacer molecule with a bifunctional carbonyl electrophile to form an active intermediate, and subsequently reacting the 5'OH group of VBl 2 with the active intermediate to yield said VBl2 derivative.

There are also provided derivatives of VB 1 2 prepared by the methods of the present invention. These derivatives are ideally linked to a biocompatible polymer or associated with a nanoparticle. These polymers and nanoparticles may be mixed with pharmaceutically acceptable carriers and/or diluents to provide pharmaceutical compositions for therapeutic administration to subjects.

P \WPDOCS\CRN\RMHSpcc\7825513 doc-23102/2007 Thus according to an aspect of the present invention there is provided a method for preparing VB 1 2 derivatives suitable for linking to a polymer, nanoparticle or therapeutic 00 agent, protein or peptide comprising the steps of reacting the 5'OH group of VB 12 or an analogue thereof with a bifunctional carbonyl electrophile to form an active intermediate, and subsequently reacting the intermediate with a nucleophilic spacer molecule to yield said VB 1 2 derivative, wherein the bifunctional carbonyl electrophile is selected from the N group consisting of carbonyldiimidazole, phosgene, triphosgene, N,N'-disuccinimidyl 0 carbonate, carbonyl dipiperidine, 1,1'-carbonyldi(1,2,4-triazole), di(2-pyridyl)ketone or N di(l-benzotriazolyl)carbonate.

According to another aspect of the present invention there is provided a method for preparing a VB 1 2 derivative suitable for linking to a polymer, nanoparticle or therapeutic agent, protein or peptide comprising the steps of reacting a carboxylic acid spacer molecule with a bifunctional carbonyl electrophile to form an active intermediate, and subsequently reacting the 5'OH group of VB 1 2 with the active intermediate to yield said

VB

12 derivative, wherein the bifunctional carbonyl electrophile is selected from the group consisting of carbonyldiimidazole, phosgene, triphosgene, N,N'-disuccinimidyl carbonate, carbonyl dipiperidine, 1,1'-carbonyldi(1,2,4-triazole), di(2-pyridyl)ketone or di(lbenzotriazolyl)carbonate.

According to another aspect of the present invention there is provided a VB 2 derivative of the formula

VBI

2 -5'O-CO-NH-R'

(I)

or a salt thereof, wherein R' is hexyl, dodecyl, tetradecyl, hexadecyl, octadecyl, aminoethyl, aminobutyl, aminohexyl, aminododecanyl, t-butyl-Phe, succinylhydrazidyl, adipylhydrazidyl, Gly-OMe or Gly-OH.

According to another aspect of the present invention there is provided a VB 1 2 derivative of the formula (II):

VB

12 -5'O-CO-R' (II) P:\WPDOCS\CRNRMISSpc\7825513 doc-23/02/2007 00 "§or a salt thereof, wherein R' is -CH(R2)-NHR 3 and R 2 is Gly and R 3 is Boc or hydrogen, or R 2 is Phe and R is Boc or hydrogen.

OO

According to another aspect of the present invention there is provided a polymer or Snanoparticle suitable for therapeutic administration to a subject, said polymer or nanoparticle comprising a VB 1 2 derivative of the formula N

VB

1 2 -5'O-CO-NH-R' (I) 0or a salt thereof, wherein R' is Ci.

24 alkyl, C2-2 4 alkenyl, C 2 2 4 alkynyl, C 3 .8cycloalkyl, (C 3 N 8cycloalkyl)alkyl, amino, 2 alkyl)C(O)R 2 -(C2- 12 alkenyl)C(O)R 2

-NHC(O)-CI.

salkyl-C(O)NHNH 2 or -CH(R 3

)C(O)R

4 all of which optionally may be substituted by one or more groups selected from amino, amido, hydroxy, alkyl, halo, haloalkyl, carboxy, alkoxycarbonyl, acetoxy, sulfanyl, aryl, arylalkyl and alkylarylalkyl, R 2 is amino, hydroxy, Ci.

6 alkoxy or C 2 6 alkenyloxy, R 3 is an amino acid side chain or a derivative thereof, and R 4 is hydroxy, Cl-6alkoxy, an amino acid or a peptide.

According to another aspect of the present invention there is provided a polymer or nanoparticle suitable for therapeutic administration to a subject, said polymer or nanoparticle comprising a VB 1 2 derivative of the formula (II):

VB

1 2 -5'O-CO-R' (II) or a salt thereof, wherein R' is Cl.

24 alkyl or C 2 24 alkenyl optionally which may be substituted by one or more groups selected from amino, amido, hydroxy, alkyl, halo, haloalkyl, carboxy, alkoxycarbonyl, acetoxy, sulfanyl, aryl, arylalkyl and alkylarylalkyl, or R' is -CH(R2)-NHR 3

R

2 is an amino acid side chain or derivative thereof, and R 3 is hydrogen, an amine protecting group, an amino acid or a peptide.

These and other aspects of the invention are described below and in the claims that follow.

WO 99/65930 PCT/AU99/00462 Throughout this specification and the claims which follow, unless the text requires otherwise, the word "comprise", and variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

BRIEF DESCRIPTION OF THE FIGURE The present invention will now be described with reference to the Figure wherein: Figure 1 is a representation of a VB 12 molecule showing three sites for the possible conjugation of agents and peptides to VB 2. These sites of conjugation are as follows: a) axial conjugation through substitution onto the Co atom of the corrin ring; b) direct conjugation following acid modification of the ePropianimide side chain; and c) conjugation to the 5'OH group of the ribose moiety of the nucleotide residue.

DETAILED DESCRIPTION OF THE INVENTION The VB 12 derivatives of the present invention are suitable for conjugation or linking to polymers, nanoparticles, therapeutic agents, proteins and peptides and other such pharmaceutically active agents. The methods for the production of these VB 1 2 derivatives enable the derivatives to be obtained in generally good to high yields and are of good purity.

In general these derivatives are obtained by dissolving VB, 2 or an analogue thereof in a solvent, preferably a suitable non-aqueous solvent such as dry DMF, dry THF or dry DMSO, and activating the 5'OH group of VB 1 2 by reaction with a carbonyl electrophile, preferably 1,1'-carbonyldiimidazole at 1-5 molar excess. Quantities above 5 molar excess may be used, however this is generally not required. Preferably VB 1 2 is dissolved at high concentration in DMSO. The activated VB, 2 intermediate may then be coupled directly to peptides or proteins, or may be reacted with diamino-spacers, or amino-spacer-acids, or alternatively with amino-alkyl chains to form hydrophobic derivatives of VB 12 suitable for insertion into the hydrophobic surface of micro- or nanoparticles or into lipids or liposomes.

WO 99/65930 PCT/AU99/00462 An alternative method of this invention also utilises the 5'OH group of VB 1 2 in the production of 5'OH ester derivatives of VB 1 2 In the synthesis of the 5'OH ester derivatives an active electrophilic intermediate is first prepared from the reaction of a carboxylic acid spacer molecule with a bifunctional carbonyl electrophile to prepare the active electrophilic intermediate. VB 1 2 or analogues thereof are then subjected to reaction with the electrophilic intermediate whereby the 5'OH group of VB 12 attacks the carbonyl electrophile and displaces a leaving group to yield the VBi 2 derivative. The VBi 2 is preferably linked to an amino acid spacer or to an acid lipid in the preparation of the 5'OH ester derivative of VB 1 2 These derivatives have the added advantage that they are easy to make and produce spacers, or linkages that are readily cleaved by serum esterases to regenerate the native VB 1 2 in vivo.

The present inventors have utilised carbonyl electrophiles to enable attack of the weak nucleophile by the strongly electropositive carbonyl group in combination with good leaving groups attached to the carbonyl group. The methods overcome problems in the prior art where strong basis have been used to attach cross-linking agents to the VB 2 molecule, these strong base of which can denature the VB 12 In a preferred embodiment, the carbonyl electrophile is a bifunctional carbonyl electrophile selected from carbonyldiimidazole, phosgene, triphosgene, N,N'-disuccinimidyl carbonate, carbonyl dipiperidine, 1,1'-carbonyldi(1,2,4-triazole), di(2-pyridyl)ketone, or di(lbenzotriazolyl)carbonate, more preferably carbonyldiimidazole.

The present invention also provides a VBl2 derivatives of the formula

VB

1 2 -5'O-CO-NH-R' (I) or a salt thereof, wherein R' is Cl- 24 alkyl, C 2 24 alkenyl, C 2 24 alkynyl, C3-8cycloalkyl, (C 3 cycloalkyl)alkyl, amino, -(Ci.

2 alkyl)C(O)R 2

-(C

2 1 2 alkenyl)C(O)R 2 -NHC(O)-Ci.-alkyl-C(O)NHNH 2 or

-CH(R

3

)C(O)R

4 all of which optionally may be substituted by one or more groups selected from amino, amido, hydroxy, alkyl, halo, haloalkyl, carboxy, alkoxycarbonyl, acetoxy, sulfanyl, aryl, arylalkyl and alkylarylalkyl,

R

2 is amino, hydroxy, Ci-6alkoxy or C 2 -6alkenyloxy,

R

3 is an amino acid side chain or a derivative thereof, and

R

4 is hydroxy, Cl_.alkoxy, an amino acid or a peptide.

WO 99/65930 PCT/AU99/00462 Preferably R' is hexyl, dodecyl, tetradecyl, hexadecyl, octadecyl, aminoethyl, aminobutyl, aminohexyl, aminododecanyl, t-butyl-Phe, succinylhydrazidyl, adipylhydrazidyl, Gly-OMe or Gly-OH.

The present invention also provides a VB12 derivative of the formula (II):

VB

1 2 -5'O-CO-R' (II) or a salt thereof, wherein R) is C, 24 alkyl or C2 24 alkenyl optionally which may be substituted by one or more groups selected from amino, amido, hydroxy, alkyl, halo, haloalkyl, carboxy, alkoxycarbonyl, acetoxy, sulfanyl, aryl, arylalkyl and alkylarylalkyl, or R1 is -CH(R 2

)-NHR

3

R

2 is an amino acid side chain or derivative thereof, and R3 is hydrogen, an amine protecting group, an amino acid or a peptide.

Preferably RI is C 2 4 alkyl, C8 24 alkenyl, or -CH(R2)-NHR3 where R2 is glycine and R3 is Boc or hydrogen, or R 2 is phenylalanine and R3 is Boc or hydrogen. It will be apparent to one skilled in the art that other amino acids or proteins can be used to derivatise the VB 1 2 molecule or analogues thereof. Furthermore, it will be apparent that the amino acids or proteins may require protection of pendant functional groups or other such masking prior to subjecting these reactants to the coupling reactions of the present invention.

The VB, 2 derivatives of the present invention may be linked to polymers or associated with nanoparticles or the like to prepare vitamin complexes according to standard methods known to those skilled in the art and published in the patent and scientific literature. Examples of such methods may be found in, for example, European Patent No. 0 220 030, Australian Patent No. 664365 and United States Patent Nos. 5449720 and 5548064.

The vitamin complexes are used to deliver agents or active substances, in particular hormones, drugs, prodrugs, enzymes, proteins, peptides, toxins, immunogens or DNA or RNA analogues to subjects. Subjects are preferably vertebrate hosts, more preferably veterinary, domestic and agricultural animals and humans.

The polymers or nanoparticles prepared from the VBI2 derivatives of the present invention may be formulated as a pharmaceutical composition by combining the polymers or WO 99/65930 PCT/AU99/00462 nanoparticles with a pharmaceutically acceptable carrier and/or diluent in accordance with standard formulation techniques known to those skilled in the art. The pharmaceutical compositions may be formulated in any acceptable way to meet the desired mode of administration as determined by those skilled in the art.

Major advantages of the methods taught in this specification include the increase in yield of the VB 1 2 derivatives, and cost savings due to the reduction in chemicals used during the activation of the VB 1 2 or the incoming activated acid.

The present invention is further described with reference to the following examples which are in no way limiting on the scope of the invention.

Example 1. Preparation of 5'OH-(hexyl)-VB 1 2 Materials: VB1 2 was obtained from Rousell-Uclaf.

1. CDI/ DMSO VBiz-OH 2. R-NH 2 VBIz-O NH-R

VB

1 2 FW 1355.4 CDI FW 162.2

DMSO

Solid 1,1'-carbonyldiimidazole (CDI, 260 mg) was added to cyanocobalamin (1.0 g, 0.74 mmol) previously dissolved in dimethylsulfoxide (12 mL) at 30 0 C and the mixture stirred for 25 min. Hexylamine (2.7 mmol) was added in one portion and stirring continued for a further 24 h at room temperature. The mixture was extracted with phenol dichloromethane 2 x 20 mL) and back extracted with water (2 x 20 mL from 1:4 phenol dichloromethane). The mixture was purified by phenyl sepharose (50 g) column chromatography, eluting the unmodified VB 12 with 25% ethanol and the product with ethanol. The solvent was removed at reduced pressure and the residue was resuspended by sonication for 5 min into acetone (50 mL). The mixture was filtered and the solid washed with acetone and air dried: yield, 60%; mp 213-215 0 C (dec); MS (ESI) mass calcd for

C

7 0oHio1NsO15CoP 1482, found 1505 UV (H 2 0) k 361 (s 10500).

8 SUBSTITUTE SHEET (Rule 26) (RO/AU)

I_

WO 99/65930 PCT/AU99/00462 Example 2. Preparation of 5'OH-(dodecyl)-VB 12 Solid 1,1'-carbonyldiimidazole (CDI, 260 mg) was added to cyanocobalamin (1.0 g, 0.74 mmol) previously dissolved in dimethylsulfoxide (12 mL) at 30 0 C and the mixture stirred for min. Dodecylamine (2.7 mmol) was added in one portion and stirring continued for a further 24 h at room temperature. The mixture was extracted with phenol dichloromethane 2 x 20 mL) and back extracted with water (2 x 20 mL from 1:4 phenol dichloromethane). The mixture was purified by phenyl sepharose (50 g) column chromatography, eluting the unmodified VB 1 2 with 25% ethanol and the product with ethanol. The solvent was removed at reduced pressure and the residue was resuspended by sonicated for 5 min into acetone (50 mL). The mixture was filtered and the solid washed with acetone and air dried: yield, 52%; mp 215-218 °C (dec); MS (ESI) mass calcd for

C

76

H

1 3 Ni50 15 CoP 1566, found 1589 UV (H 2 0) X 36 1 (s 16900).

Example 3. Preparation of 5'OH-(tetradecyl)-VBi 2 Solid 1,1'-carbonyldiimidazole (CDI, 260 mg) was added to cyanocobalamin (1.0 g, 0.74 mmol) previously dissolved in dimethylsulfoxide (12 mL) at 30 °C and the mixture stirred for 25 min. Tetradecylamine (2.7 mmol) was added in one portion and stirring continued for a further 24 h at room temperature. The mixture was extracted with phenol dichloromethane 2 x 20 mL) and back extracted with water (2 x 20 mL from 1:4 phenol dichloromethane). The mixture was purified by phenyl sepharose (50 g) column chromatography, eluting the unmodified VB 1 2 with 25% ethanol and the product with ethanol. The solvent was removed at reduced pressure and the residue resuspended by sonication for 5 min into acetone (50 mL). The mixture was filtered and the solid washed with acetone and air dried: yield, 46%; mp 228-233 °C (dec); MS (ESI) mass calcd for

C

78

H

1 1 9 NI50iCoP 1595, found 1618 UV (H 2 0) k 36 1 (E 13000).

Example 4. Preparation of 5'OH-(hexadecyl)-VB 2 Solid 1,1'-carbonyldiimidazole (CDI, 260 mg) was added to cyanocobalamin (1.0 g, 0.74 mmol) previously dissolved in dimethylsulfoxide (12 mL) at 30 0 C and the mixture stirred for min. Hexadecylamine (2.7 mmol) was added in one portion and stirring continued for a 9 SUBSTITUTE SHEET (Rule 26) (RO/AU) WO 99/65930 PCT/AU99/00462 further 24 h at room temperature. The mixture was extracted with phenol dichloromethane 2 x 20 mL) and back extracted with water (2 x 20 mL from 1:4 phenol dichloromethane). The mixture was purified by phenyl sepharose (50 g) column chromatography, eluting the unmodified VB 1 2 with 25% ethanol and the product with ethanol. The solvent was removed at reduced pressure and the residue was sonicated for min into acetone (50 mL). The mixture was filtered and the solid washed with acetone and air dried: yield, 48%; mp 223-227 0 C (dec); MS (ESI) mass calcd for C 8 0

HI

21

N

15 0 15 CoP 1623, found 1646 UV (H 2 0) X 361 (e 20000).

Example 5. Preparation of 5'OH-(octadecyl)-VBi2 Materials: VB 12 was obtained from Rousell-Uclaf.

1. CDI DMSO

O

VBi 2 -OH 2. R-NH 2 VBi2-O NH-R VBi 2 FW 1355.4 CDI FW 162.2

DMSO

VB

1 2 (1.0 g, 1.0 equivalent) was dissolved in dry DMSO (20 ml) at room temperature. Solid carbonyldiimidazole (CDI; 400 mg, 3.3 equivalents) was added and the mixture stirred at room temperature for 1 hr. The reaction mix was split into 4 equal parts and added to 500 mg of octadecylamine (Aldrich) dissolved in acetone, ethanol, dichloromethane or chloroform.

The reaction was allowed to proceed for 2 hours after which the reaction was monitored by TLC and RP-HPLC to determine the quantity of product (5'OH-(octadecyl)-VB 1 2 which was formed.

The product was then separated from the unreacted VB 1 2 by addition of an equal volume of water and DCM, followed by centrifugation in a Beckman high speed (5K, 10 min). The DCM phase was removed and the product separated from unmodified VB 1 2 by flash column chromatography (isopropanol 50%, ammonia water 48%) then lyophilysed: yield, 66%; mp 220-223°C (dec); MS (ESI) mass calcd for Cs 2

HI

25

N

1 5 0 1 5 CoP 1651, found 1674 UV (H 2 0) 36 1 (e 17500).

SUBSTITUTE SHEET (Rule 26) (RO/AU) WO 99/65930 PCT/AU99/00462 Example 6. Preparation of 5'OH-(aminoethyl)-VB 12

VB

12 (1.0 g, 1.0 equivalent) was dissolved in dry DMSO (20 ml) at room temperature. Solid carbonyldiimidazole (CDI; 400 mg, 3.3 equivalents) was added and the mixture stirred at room temperature for 1 hr. Diaminoethane (3.3 equiv) was added to the reaction mix. The mixture was stirred for 12 h and then poured into acetone ethyl acetate 200 mL) and left to stand. The supernatant was poured off and the residue resuspended in acetone (50 mL) by sonicationed for 5 min. The mixture was filtered onto a sintered glass funnel and the solid washed with acetone. The product was purified by Flash chromatography on a silica column using isopropanol 50%, ammonia water 48%. The product was then lyophilysed: yield, 63%; mp 206-210 °C (dec); TLC ('PrOH 30/n-BuOH 45/H20 25/NH40H 2) 0.22; MS (ESI) mass calcd for C 66

H

94 NI60 1 5 CoP 1441, found 1441 UV (H 2 0) 1 361 (s 19900).

Example 7. Preparation of 5'OH-(aminobutyl)-VB 12

VB

12 (1.0 g, 1.0 equivalent) was dissolved in DMSO (35 mL) at room temperature. Solid carbonyldiimidazole (CDI; 400 mg, 3.3 equivalents) was added and the mixture stirred at room temperature for 1 hr. Solid diaminobutane (3.3 equiv) was added in one portion. The mixture was stirred for 12 h and then poured into acetone ethyl acetate 200 mL) and left to stand. The supernatant was poured off and the residue in acetone (50 mL) sonicated for 5 min. The mixture was filtered onto a sintered glass funnel and the solid washed with acetone. The product was purified by column chromatography (silica, isopropanol ammonia water 48%) then lyophilysed: yield, 70%; mp 242-244 °C (dec); TLC ('PrOH 30/n-BuOH 45/H20 25/NH 4 0H 2) Rf 0.08; MS (ESI) mass calcd for C 68

H

98 NI60 15 CoP 1469, found 1469 UV (H 2 0) 3 6 1 (e 15500).

Example 8. Preparation of 5'OH-(t-butyl-Phe)-VB, 2

VB

1 2 (1.0 g, 1.0 equivalent) was dissolved in DMSO (35 mL) at room temperature. Solid carbonyldiimidazole (CDI; 400 mg, 3.3 equivalents) was added and the mixture stirred at room temperature for 1 hr. Solid t-butyl-Phe (3.3 equiv) was added in one portion. The mixture was stirred for 12 h and then poured into acetone ethyl acetate 200 mL) and left to stand. The supernatant was poured off and the residue in acetone (50 mL) sonicated 11 SUBSTITUTE SHEET (Rule 26) (RO/AU) WO 99/65930 PCT/AU99/00462 for 5 min. The mixture was filtered onto a sintered glass funnel and the solid washed with acetone. The product was purified by Flash column chromatography (silica, isopropanol ammonia water 48%) then lyophilysed.

Example 9. Preparation of 5'OH-(aminohexyl)-VBI2

VB

1 2 (1.0 g, 1.0 equivalent) was dissolved in dry DMSO (20 ml) at room temperature. Solid carbonyldiimidazole (CDI; 400 mg, 3.3 equivalents) was added and the mixture stirred at room temperature for 1 hr. Diaminohexane (3.3 equiv) was added to the reaction mix as a solid. The mixture was stirred for 12 h and then poured into acetone ethyl acetate 200 mL) and left to stand. The supernatant was poured off and the residue in acetone (50 mL) sonicated for 5 min. The mixture was filtered onto a sintered glass funnel and the solid washed with acetone. The product was purified by column chromatography (isopropanol ammonia water 48%) then lyophilysed: yield, 98%; mp 230-233°C (dec); TLC ('PrOH 30/n-BuOH 45/H 2 0 25/NH 4 0H 2) Rf 0.11; MS (ESI) mass calcd for

C

70

H

102

NI

6 0 15 CoP 1497, found 1497 UV (H20) .361 (E 17000).

Example 10. Preparation of 5'OH-(aminododecanyl)-VB 1 2

VB

12 (1.0 g, 1.0 equivalent) was dissolved in DMSO (35 mL) at room temperature. Solid carbonyldiimidazole (CDI; 400 mg, 3.3 equivalents) was added and the mixture stirred at room temperature for 1 byhr. followed by addition of diaminododecane (3.3 equiv) in one portion. The mixture was stirred for 12 h and then poured into acetone ethyl acetate (1:1, 200 mL) and left to stand. The supernatant was poured off and the residue resuspended in acetone (50 mL) and sonicated for 5 min. The mixture was filtered onto a sintered glass funnel and the solid washed with acetone. The product was purified by Flash column chromatography (silica resin using isopropanol 50%, ammonia water 48%) then lyophilysed: yield, 68%; mp 156-158 °C (dec); TLC ('PrOH 30/n-BuOH 45/H20 25/NH 4 0H 2) Rf= 0.27; MS (ESI) mass calcd for C 76

H

1 14

NI

6 0 15 CoP 1581, found 1581 UV (H 2 0)

X

36 1 (s 33000).

12 SUBSTITUTE SHEET (Rule 26) (RO/AU) WO 99/65930 PCT/AU99/00462 Example 11. Preparation of5'OH-(succinylhydrazidyl)-VBi 2

VB

1 2 (1.0 g, 1.0 equivalent) was dissolved in DMSO (35 mL) at room temperature. Solid carbonyldiimidazole (CDI; 400 mg, 3.3 equivalents) was added and the mixture stirred at room temperature for 1 hr followed by solid succinyldihydrazide (3.3 equiv) added in one portion. The mixture was stirred for 12 h and then poured into acetone ethyl acetate 200 mL) and left to stand. The supematant was poured off and the residue in acetone mL) sonicated for 5 min. The mixture was filtered onto a sintered glass funnel and the solid washed with acetone. The product was purified by Flash column chromatography (isopropanol 50%, ammonia water 48%) then lyophilysed: yield, 68%; mp 206-208 °C (dec); TLC ('PrOH 30/n-BuOH 45/H20 25/NH 4 0H 2) Rf 0.36; MS (ESI) mass calcd for

C

68

H

96 NI80 1 7CoP 1581, found 1581 UV (H 2 0) X 36 1 (E 15700).

Example 12. Preparation of 5'OH-(adipylhydrazidyl)-VB 2 VB1 2 (1.0 g, 1.0 equivalent) was dissolved in DMSO (35 mL) at room temperature. Solid carbonyldiimidazole (CDI; 400 mg, 3.3 equivalents) was added and the mixture stirred at room temperature for 1 hr followed by solid adipyldihydrazide (3.3 equiv) added in one portion. The mixture was stirred for 12 h and then poured into acetone ethyl acetate (1:1, 200 mL) and left to stand. The supernatant was poured off and the residue in acetone mL) sonicated for 5 min. The mixture was filtered onto a sintered glass funnel and the solid washed with acetone. The product was purified by silica column Flash chromatography (isopropanol 50%, ammonia water 48%) then lyophilysed: yield, 73%; mp 208-210 °C (dec); TLC ('PrOH 30/n-BuOH 45/H 2 0 25/NI40H 2) Rf 0.33; MS (ESI) mass calcd for

C

70

H

100

NI

8 0 7 CoP 1555, found 1555 UV (H 2 0) 36 1 (E 21100).

Example 13. Preparation of ester-linked VB12-phenylalanine Boc-phenylalanine (1.57 g, 0.0059 mol) and carbonyl diimidazole (1.01 g, 0.0062 mol) were dissolved in DMF (6 ml) and the solution stirred at room temperature for 1 h with vigorous evolution of C0 2 A solution of VB 1 2 (1.0 g) in DMSO (10 ml) was added dropwise to the active ester solution followed by DIEA (1.2 ml, 0.89 g, 0.0069 mol) and stirring was continued at room temperature overnight. Unreacted Boc-Phe, CDI and DIEA were removed by addition of 90 ml acetone to precipitate the VB 12 The product was then purified by Flash 13 SUBSTITUTE SHEET (Rule 26) (RO/AU)

I_

WO 99/65930 PCT/AU99/00462 chromatography on a silica column (2.5 X 50 cm) using a solvent mixture of 45% butanol, propan-2-ol, 23% DW and 2% NH 4 0H. The purified product was lyophilized and the dry powder deprotected by the addition of neat TFA (1 ml /100 mg) for 10 minutes. The product was then precipitated by the addition of ethyl acetate, and dried.

Example 14 Preparation of ester-linked VB 1 2 -glycine Boc-glycine (1.57 g, 0.0059 mol) and carbonyl diimidazole (1.01 g, 0.0062 mol) were dissolved in DMF (6 ml) and the solution stirred at room temperature for 1 h with vigorous evolution of CO 2 A solution of VB 12 (1.0 g) in DMSO (10 ml) was added dropwise to the active ester solution followed by DIEA (1.2 ml, 0.89 g, 0.0069 mol) and stirring was continued at room temperature overnight. Unreacted Boc-Gly, CDI and DIEA were removed by addition of 90 ml acetone to precipitate the VB 12 The product was then purified by Flash chromatography on a silica column (2.5 X 50 cm) using a solvent mixture of 45% butanol, 30% propan-2-ol, 23% DW and 2% NH40H. The purified product was lyophilized and the dry powder deprotected by the addition of neat TFA (1 ml /100 mg) for 10 minutes. The product was then precipitated by the addition of ethyl acetate, and dried.

Example 15. Preparation of VB12-glycine acid Cyanocobalamin (1.0 g, 0.74 mmol) and 1,1'-carbonyldiimidazole (CDI, 260 mg) were added sequentially to dimethylsulfoxide (12 mL) at 30 0 C and the mixture stirred for 25 min. OMe- Gly (2.7 mmol) was added in one portion followed by triethylamine (200 PL) and the mixture stirred for 24 h at room temperature. The mixture was poured into ethyl acetate mL) and left to stand. The supematant was poured off and the residue sonicated for 5 min in acetone (50 mL). The mixture was filtered and the solid washed with acetone. The residue was then dissolved in 0.1 M HCI solution and stirred for 30 min. The crude acid was then purified on Dowex 1X4 resin eluting with 2% acetic acid: yield, 95%; mp 239-242 °C (dec); TLC ('PrOH 30/n-BuOH 45/H20 25/NH 4 0H 2) Ry 0.41; MS (ESI) mass calcd for

C

66

H

90 Ni 5 0 7 CoP 1456, found 1456 UV (H 2 0) X 361 19800).

14 SUBSTITUTE SHEET (Rule 26) (RO/AU) WO 99/65930 PCT/AU99/00462 Example 16. Determination of the relative IF affinity of various 5'O-VB, 2 derivatives.

Reagents IF Buffer: BSA (VB 1 2 and IF deficient) BSA (Sigma A-3902) was dissolved at 1 mg/ml in 0.1M potassium phosphate buffer pH 7.5 7 CoVB2: 57 Co stock (50 il) (Kodak) was diluted into 50ul of stock in 25ml of IF buffer to give a solution of 1 ng 57 CoVB 12 25 ml. 250 ng cold VB 1 2 was added to 25 ml of hot 7 CoVB 1 2 solution to give a 10 ng/ml solution.

Porcine Intrinsic Factor: Porcine IF (Sigma) was dissolved in IF buffer at 200 Units/ml, and frozen in 500 ul lots (100 IU aliquots) until required.

BSA-coated charcoal: BSA was added to an equal volume of 5% charcoal solution of 0.1 M potassium phosphate buffer pH 7.5 and stirred gently for 30 minutes.

Procedure: Ten fold up dilutions of VB 1 2 or VB 1 2 derivatives were prepared down to 1 ng/ml in IF buffer. An equal volume of diluted IF was added to each sample and incubated for minutes at room temperature. An equal volume of the BSA-coated charcoal was added to each sample, which was mixed prior to centrifugation. Following centrifugation the supernatant and pellet of each sample were separated and 57 CoVB 1 2 determined by counting in a gamma counter. Data is represented as the inhibition of 5CoVB12 binding when compared to unmodified VBt 2 SUBSTITUTE SHEET (Rule 26) (RO/AU) P:\WPDOCS\Hjw\Specs 2\7111348div.doc-23/0503 Compound binding relative to vitamin B12 12 49 1 2 1 2 4.2 12 0.78 1 2 0.57 1 2 1 2 27 t-butyl-Phe-5'O-VB12 aminohexyl-5'O-VB12 2 31 succinylhydrazidyl-5'O-VB12 37 adipylhydrazidyl-5'0-VBl2 29 phenylalanyl-5'O-VB12 glycyl-5'O-VB12 1 2 Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

The reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that that prior art forms part of the common general knowledge in Australia.

WO 99/65930 PCT/AU99/00462

REFERENCES

1. Russell-Jones G. J. The use of the vitamin B 1 2 transport system as a carrier for the oral delivery of peptides, proteins and nanoparticles. Proc. 23 rd International Symposium on Controlled Release of Bioactive Materials, 1996.

2. Anton, D. L. Hogenkamp, H. P. Walker, T. E.,-and Matwiyoff, N. A. Carbon-13 nuclear magnetic resonance studies of monocarboxylic acids of cyanocobalamin.

Assignments of the and e-monocarboxylic acids. J. Am. Chem. Soc., 102: 2215, 1980.

3. Russell-Jones, Westwood, S. W and Habberfield, A. D Vitamin B 1 2 mediated oral delivery systems for Granulocyte-Colony Stimulating Factor and erythropoietin.

Bioconj Chem, 6, 459-465, 1995.

4. Toraya, T. and Fukui, S. The synthesis of several immobilized derivatives of vitamin Bi 2 coenzyme and their use as affinity absorbents for a study of interactions of diol dehydrase with the coenzyme. J. Biol. Chem., 255, 3520, 1980.

Annunziato, M. Patel, U. Ranade, and Palumbo, P. S. p-Maleimidophenyl isocyanate: A novel heterobifunctional linker for hydroxyl to thiol coupling. Bioconj.

Chem., 4, 212, 1993.

6. Westwood, S. and Russell-Jones, G. J. Vitamin B 1 2 mediated delivery systems for GCSF and EPO. (USP 08/064,873; 5,548,064), 1993.

7. Habberfield, A. Kinstler, and Pitt, C. G. Conjugates of VB 1 2 and proteins.

(USP 5,574,018) 1996.

17 SUBSTITUTE SHEET (Rule 26) (RO/AU)

Claims

2. A method of claim 1, wherein the bifunctional carbonyl electrophile is carbonyldiimidazole.
3. A method of claim 1, wherein the nucleophilic spacer molecule is an amino- or hydrazidyl-substituted spacer molecule.
4. A method of claim 3, wherein the spacer molecule is octadecylamine, diaminoethane, diaminobutane, diaminohexane, diaminododecane, diaminooctadeccane, an amino acid or a peptide or a dihydrazide. A method of claim 4, wherein the dihydrazide is succinic acid dihydrazide.
6. A method of claim 4, wherein the dihydrazide is adipic acid dihydrazide.
7. A method for preparing a VB 1 2 derivative suitable for linking to a polymer, nanoparticle or therapeutic agent, protein or peptide comprising the steps of reacting a carboxylic acid spacer molecule with a bifunctional carbonyl electrophile to form an active intermediate, and subsequently reacting the 5'OH group of VB 12 with the active intermediate to yield said VB 1 2 derivative, wherein the bifunctional carbonyl electrophile is selected from the group consisting of carbonyldiimidazole, phosgene, triphosgene, N,N'-disuccinimidyl carbonate, carbonyl dipiperidine, 1,1'- carbonyldi(1,2,4-triazole), di(2-pyridyl)ketone or di(1-benzotriazolyl)carbonate. P:WPDOCSjw\Spcc\VBl2 deriv AU claimsdoc 22/2/07
8. A method of claim 7, wherein the bifunctional carbonyl electrophile is carbonyldiimidazole. 00 9. A method of claim 7, wherein the carboxylic acid spacer molecule is N-Boc-Phe. A method of claim 7, wherein the carboxylic acid spacer molecule is N-Boc-Gly. O 11. A VB 1 2 derivative prepared by a method of any preceding claim. O 12. A VB 1 2 derivative of the formula VB 1 2 -5'0-CO-NH-R' (I) or a salt thereof, wherein R' is hexyl, dodecyl, tetradecyl, hexadecyl, octadecyl, aminoethyl, aminobutyl, aminohexyl, aminododecanyl, t-butyl-Phe, succinylhydrazidyl, adipylhydrazidyl, Gly-OMe or Gly-OH.
13. A VB 12 derivative of the formula (II): VB12-5'O-CO-R' (II) or a salt thereof, wherein R' is -CH(R 2 )-NHR, and R 2 is Gly and R 3 is Boc or hydrogen, or R 2 is Phe and R 3 is Boc or hydrogen.
14. A polymer or nanoparticle suitable for therapeutic administration to a subject, said polymer or nanoparticle comprising a VB 1 2 derivative of any one of claims 11-13 A polymer or nanoparticle suitable for therapeutic administration to a subject, said polymer or nanoparticle comprising a VB 12 derivative of the formula VB 1 2 -5'O-CO-NH-R' (1) or a salt thereof, wherein R' is Cl.-2 4 alkyl, C 2 24 alkenyl, C 2 24 alkynyl, C 3 .8cycloalkyl, (C 3 8 cycloalkyl)alkyl, amino, -(C 1 1 2 alkyl)C(O)R 2 -(C 2 -I 2 alkenyl)C(O)R2, -NHC(O)-C 8 alkyl- C(O)NHNH 2 or -CH(R 3 )C(O)R 4 all of which optionally may be substituted by one or P:AWPDOCS\Hjw\Sps\VB12 dcriv AU laim.doc 22/07 more groups selected from amino, amido, hydroxy, alkyl, halo, haloalkyl, carboxy, 0 alkoxycarbonyl, acetoxy, sulfanyl, aryl, arylalkyl and alkylarylalkyl, R is amino, hydroxy, Ci.6alkoxy or C2-6alkenyloxy, [R 3 is an amino acid side chain or a derivative thereof, and 00 R 4 is hydroxy, Ci-6alkoxy, an amino acid or a peptide. M^ 16. A polymer or nanoparticle suitable for therapeutic administration to a subject, said c polymer or nanoparticle comprising a VB 1 2 derivative of the formula (II): O VB 1 2 -5'O-CO-R' (II) mC or a salt thereof, wherein 0 SR' is Ci- 24 alkyl or C2- 2 4alkenyl optionally which may be substituted by one or more groups selected from amino, amido, hydroxy, alkyl, halo, haloalkyl, carboxy, alkoxycarbonyl, acetoxy, sulfanyl, aryl, arylalkyl and alkylarylalkyl, or R' is -CH(R 2 )-NHR 3 R 2 is an amino acid side chain or derivative thereof, and R 3 is hydrogen, an amine protecting group, an amino acid or a peptide.
17. A pharmaceutical composition comprising a polymer or nanoparticle of any one of claim 14-16 in association with a pharmaceutically acceptable carrier and/or diluent.
18. VB 1 2 derivatives, polymers or nanoparticles comprising same and/or pharmaceutical compositions comprising said polymers or nanoparticles substantially as herein described with reference to the Examples or accompanying drawing. PA\WPDOCS\HjwMSpecs\VBI2 deiv AU ciaims.doc 22/2107 c