AU4490099A - Vitamin B12 derivatives and methods for their preparation - Google Patents

Description

WO 99/65930 PCT/AU99/00462 VITAMIN B 1 2 DERIVATIVES AND METHODS FOR THEIR PREPARATION 5 TECHNICAL FIELD The present invention generally relates to novel derivatives of vitamin Bl2 carrier molecules 10 for the delivery of therapeutic substances by administration of a complex comprising these substances linked to vitamin B 1 2

(VB

1 2 ) 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 VBl 2 with electrophiles to prepare these VB 1 2 derivatives. 15 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 20 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

1 2 uptake system, mediated by the binding of VBl 2 to intrinsic factor (IF), to transport the active substance/VBl 2 complex. Once delivered into the lymphatic drainage system or 25 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 Bl2 (cobalamin, Cbl, VB12), the vitamin must first bind to the plasma protein transcolbamin II 30 (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. R., Quadros, E. B., 5 Rothenberg, S. P., Morgan, A. C., Schrader, J. W., and Ziltener, H. J., 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. C., Pinson, P. R., Munger, J. M., West, F. G., and Grissom, C. B., 1999 Cytotoxicities of two new cobalamin bioconjugates. Proceedings 10 Ninth International Symposium on Recent Advances in Drug Delivery Systems, pp 232-3; Canon, M.J., Munger, J. M., West, F. G., and Grissom, C. B., 1999 Synthesis and uptake of radiolabeled cobalamin bioconjugate, Proceedings Ninth International Symposium on Recent Advances in Drug Delivery Systems, pp 230-1; Pinson, P. R., Munger, J. M., West, F. G., and Grissom, C. B., 1999 Synthesis of two doxorubicin-cobalamin bioconjugates, Proceedings 15 Ninth International Symposium on Recent Advances in Drug Delivery Systems, pp 228-9). In order for VB 12 to co-transport pharmaceuticals across the intestinal epithelial cell layer and into the circulatory system the pharmaceuticals must first be covalently linked to the VBl 2 moleulue. Similarly, in order that VB12 can target an anti-tumrnour agent to a tumour, the agent must also be 20 covalently linked to the VB 12 molecule. For this to occur, the VB 12 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 structures (see Figure 1). This hydrolysis results in the formation of the "b", "d" and "e" monocarboxylic acids of VBl2 . 2 The isolated monocarboxylic acid derivatives can then be 25 conjugated directly to amino groups of proteins or peptides using commercial carbodiimides such as 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide (EDAC) or dicyclohexylcarbodiimide (DCC) thereby linking the peptide to VB12 via a peptide bond.1,3 A second method of conjugation of peptides to VB 1 2 is by axial substitution of functional 30 groups onto the Co atom of the corrin ring of the VBl2 molecule (see Formula 1). In this method, the axial CN ligand of VB12 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 2 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 5 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 VB 1 2 with a 54 fold excess of succinic anhydride in a large volume of DMSO (VB 12 at 5 mg/ml) plus a large excess of pyridine (128 fold w/w). These authors found that the linkage formed was not only unstable at basic pH, 10 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., 5 as well as Russell-Jones et al.

3 ' 6 and produced 15 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 co workers, 5 gm of cyanocobalamin (VB 12 - 1356 MW) was dissolved in 1,000 ml of DMSO and 200 gmn of glutaric anhydride (116 MW) was added in 160 ml of pyridine. The product yield was 20 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 VB 1 2 monoacid was prepared by treatment with 25 acid for 72 hrs and subsequent purification on Dowex 1X8 and Dowex 1X2 to afford a yield of only about 5%. 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 al 7 and Annunziato et 30 al., 5 there are other methods which could be used to form covalent linkages to the 5'OH group of

VB

12 . 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 1 2 derivatives, however, the yields were either so low as to make the process non 3 WO 99/65930 PCT/AU99/00462 commercial, or the quantities of reagents employed were so high as to make them similarly non commercial. Thus it is an object of the present invention to overcome, or at least alleviate one or more of the 5 abovementioned disadvantages of the prior art. In particular, it is an object of the present invention to provide novel methods for preparing derivatives of VBl 2 carrier molecules which utilise the 5'OH group of VBl 2 for chemical bonding with spacer molecules. It is a preferred object of the present invention that the VB 12 derivatives are easy to make, obtained in good to high yields and readily purified. 10 SUMMARY OF THE INVENTION Surprisingly it has been found by the present inventors that VB 12 derivatives, which are suitable for conjugation to polymers, nanoparticles and pharmaceutically active agents, are 15 readily prepared by reaction of the 5'OH group on the ribose moiety of VB 1 2 with carbonyl electrophiles. According to an aspect of the present invention there is provided a method for preparing VB12 derivatives suitable for linking to a polymer, nanoparticle or therapeutic agent, protein 20 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 VBl 2 derivative. According to another aspect of the present invention there is provided a method for preparing 25 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. 30 There are also provided derivatives of VBl 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. 4 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 5 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: 10 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; 15 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 20 The VBl 2 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. 25 In general these derivatives are obtained by dissolving VB 12 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 VBl 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 12 is dissolved at high concentration in 30 DMSO. The activated VB 12 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 1 2 suitable for insertion into the hydrophobic surface of micro- or nanoparticles or into lipids or liposomes. 5 WO 99/65930 PCT/AU99/00462 An alternative method of this invention also utilises the 5'OH group of VB 12 in the production of 5'OH ester derivatives of VBl 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 5 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 VB 12 derivative. The VB 12 is preferably linked to an amino acid spacer or to an acid lipid in the preparation of the 5'OH ester derivative of VB 12 . These derivatives have the added advantage that they are easy to make and produce spacers, or 10 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 5'OH 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 15 where strong basis have been used to attach cross-linking agents to the VB 12 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, 20 carbonyl dipiperidine, 1,1'-carbonyldi(1,2,4-triazole), di(2-pyridyl)ketone, or di(1 benzotriazolyl)carbonate, more preferably carbonyldiimidazole. The present invention also provides a VB 12 derivatives of the formula (I):

VB

1 2 -5'O-CO-NH-R' (I) 25 or a salt thereof, wherein

R

1 is CI.

24 alkyl, C 2

-

2 4 alkenyl, C 2

.

24 alkynyl, C 3 -8.scycloalkyl, (C 3 -8cycloalkyl)alkyl, amino, -(Cl-.

1 2 alkyl)C(O)R 2 , -(C 2 -1 2 alkenyl)C(O)R 2 , -NHC(O)-C-.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, 30 sulfanyl, aryl, arylalkyl and alkylarylalkyl,

R

2 is amino, hydroxy, C 1 -6alkoxy or C 2

.-

6 alkenyloxy, R3 is an amino acid side chain or a derivative thereof, and

R

4 is hydroxy, CI- 6 alkoxy, an amino acid or a peptide. 6 WO 99/65930 PCT/AU99/00462 Preferably RI is hexyl, dodecyl, tetradecyl, hexadecyl, octadecyl, aminoethyl, aminobutyl, aminohexyl, aminododecanyl, t-butyl-Phe, succinylhydrazidyl, adipylhydrazidyl, Gly-OMe or Gly-OH. 5 The present invention also provides a VBl 2 derivative of the formula (II):

VB

12 -5'O-CO-RI (II) or a salt thereof, wherein RI is Cl- 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, 10 acetoxy, sulfanyl, aryl, arylalkyl and alkylarylalkyl, or

R

I is -CH(R 2 )-NHR3,

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. 15 Preferably R' is C8- 24 alkyl, C8-2 4 alkenyl, or -CH(R2)-NHR3 where R 2 is glycine and R 3 is Boc or hydrogen, or R 2 is phenylalanine and R 3 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 20 subjecting these reactants to the coupling reactions of the present invention. The VBl 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 25 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 30 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 VBl2 derivatives of the present invention may be formulated as a pharmaceutical composition by combining the polymers or 7 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. 5 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. 10 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 12 15 Materials: VB 12 was obtained from Rousell-Uclaf. 1. CDI / DMSO O

VB

1 z-OH ) 2. R-NH 2 VB12-O NH-R

VB

12 FW 1355.4 20 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 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 (1:1, 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 60% 30 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

70 HIolN 15 0 15 CoP 1482, found 1505 (M+23)+; UV (H 2 0) k 3 6 1 (s = 10500). 8 SUBSTITUTE SHEET (Rule 26) (RO/AU) WO 99/65930 PCT/AU99/00462 Example 2. Preparation of 5'OH-(dodecyl)-VB 1 2 Solid 1,1'-carbonyldiimidazole (CDI, 260 mg) was added to cyanocobalamin (1.0 g, 0.74 5 mmol) previously dissolved in dimethylsulfoxide (12 mL) at 30 0 C and the mixture stirred for 25 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 (1:1, 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 10 chromatography, eluting the unmodified VBl 2 with 25% ethanol and the product with 60% 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 oC (dec); MS (ESI) mass called for

C

76

H

11 3

NI

5 Ols 15 CoP 1566, found 1589 (M+23)+; UV (H 2 0) X 361 (s = 16900). 15 Example 3. Preparation of 5'OH-(tetradecyl)-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 oC and the mixture stirred for 20 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 (1:1, 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 VBl 2 with 25% ethanol and the product with 60% 25 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 oC (dec); MS (ESI) mass calcd for

C

78 Hll 9 NI50ls 1 5 CoP 1595, found 1618 (M+23)+; UV (H120) 1361 (E = 13000). 30 Example 4. Preparation of 5'OH-(hexadecyl)-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 25 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 (1:1, 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 60% 5 ethanol. The solvent was removed at reduced pressure and the residue was sonicated for 5 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 Cso 0

H

12 1

N

1 50 1 5 CoP 1623, found 1646 (M+23)+; UV (H 2 0) X361 (c = 20000). 10 Example 5. Preparation of 5'OH-(octadecyl)-VB 1 2 Materials: VB 12 was obtained from Rousell-Uclaf. 1. CDI / DMSO O VBl 2 -OH-, 2. R-NH 2 VB12-O NH-R 15

VB

12 FW 1355.4 CDI FW 162.2 DMSO 20 VBl 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 25 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 30 DCM phase was removed and the product separated from unmodified VB 12 by flash column chromatography (isopropanol 50%, ammonia 2%, water 48%) then lyophilysed: yield, 66%; mp 220-223 0 C (dec); MS (ESI) mass calcd for C 82

HI

25

N

1 5 0 15 sCoP 1651, found 1674 (M+23)+; UV (H 2 0) k 36 1 (8 = 17500). 10 SUBSTITUTE SHEET (Rule 26) (RO/AU) WO 99/65930 PCT/AU99/00462 Example 6. Preparation of 5'OH-(aminoethyl)-VB 1 2

VB

1 2 (1.0 g, 1.0 equivalent) was dissolved in dry DMSO (20 ml) at room temperature. Solid 5 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 (1:1, 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 10 washed with acetone. The product was purified by Flash chromatography on a silica column using isopropanol 50%, ammonia 2%, water 48%. The product was then lyophilysed: yield, 63%; mp 206-210 'C (dec); TLC ('PrOH 30/n-BuOH 45/H20 25/NH 4 OH 2) Rf= 0.22; MS (ESI) mass calcd for C 6 6

H

9 4

NI

6 0 15 CoP 1441, found 1441 (M)+; UV (H 2 0) k361 (s = 19900). 15 Example 7. Preparation of 5'OH-(aminobutyl)-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 diaminobutane (3.3 equiv) was added in one portion. The 20 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 (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 50%, ammonia 2%, water 48%) then lyophilysed: : yield, 70%; mp 242-244 oC (dec); TLC (iPrOH 25 30/n-BuOH 45/H120 25/NH40H 2) Rf = 0.08; MS (ESI) mass calcd for C 68

H

98 NI60 15 CoP 1469, found 1469 (M)+; UV (H 2 0) k 3 6 1 (s = 15500). Example 8. Preparation of 5'OH-(t-butyl-Phe)-VB 2 30 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 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 (1:1, 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 50%, ammonia 2%, water 48%) then lyophilysed. 5 Example 9. Preparation of 5'OH-(aminohexyl)-VB 1 2

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 10 solid. 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 (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 50%, ammonia 2%, water 48%) then lyophilysed: yield, 98%; mp 230-233 0 C (dec); TLC 15 ('PrOH 30/n-BuOH 45/H20 25/NH 4 OH 2) Rf = 0.11; MS (ESI) mass calcd for

C

70

H

102 NI60 15 CoP 1497, found 1497 (M)+; UV (H20) ?1 361 (E = 17000). Example 10. Preparation of 5'OH-(aminododecanyl)-VB 1 2 20 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 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 25 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 2%, water 48%) then lyophilysed: yield, 68%; mp 156-158 'C (dec); TLC ('PrOH 30/n-BuOH 45/H20 25/NH40H 2) Rj= 0.27; MS (ESI) mass calcd for C 76 HI1 4

NI

6 0 15 CoP 1581, found 1581 (M)+; UV (H20) 30 21 361 (E = 33000). 12 SUBSTITUTE SHEET (Rule 26) (RO/AU) WO 99/65930 PCT/AU99/00462 Example 11. Preparation of 5'OH-(succinylhydrazidyl)-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 5 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 (1:1,, 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 Flash column chromatography 10 (isopropanol 50%, ammonia 2%, water 48%) then lyophilysed: yield, 68%; mp 206-208 °C (dec); TLC ('PrOH 30/n-BuOH 45/H 2 0 25/NH 4 OH 2) Rf = 0.36; MS (ESI) mass calcd for

C

68

H

96 NI80 1 7 CoP 1581, found 1581 (M)+; UV (H20) k 3 61 (6 = 15700). Example 12. Preparation of 5'OH-(adipylhydrazidyl)-VB 1 2 15 VBl 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, 20 200 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 silica column Flash chromatography (isopropanol 50%, ammonia 2%, water 48%) then lyophilysed: yield, 73%; mp 208-210 'C (dec); TLC ('PrOH 30/n-BuOH 45/H120 25/NH 4 OH 2) Rf = 0.33; MS (ESI) mass calcd for 25 C 7 oHloo 1 00

N

18 0 17 CoP 1555, found 1555 (M)+; UV (H 2 0) k361 (F = 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 30 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 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 VBl 2 . The product was then purified by Flash 13 SUBSTITUTE SHEET (Rule 26) (RO/AU) WO 99/65930 PCT/AU99/00462 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% NH 4 OH. 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. 5 Example 14 Preparation of ester-linked VB 12 -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 10 evolution of CO 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-Gly, CDI and DIEA were removed by addition of 90 ml acetone to precipitate the VB 1 2 . The product was then purified by Flash chromatography on a silica column (2.5 X 50 cm) using a solvent mixture of 45% butanol, 15 30% propan-2-ol, 23% DW and 2% NH 4 OH. 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 VB 12 -glycine acid 20 Cyanocobalamin (1.0 g, 0.74 mmol) and 1,1l'-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 tL) and the mixture stirred for 24 h at room temperature. The mixture was poured into ethyl acetate (30 25 mL) and left to stand. The supernatant 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 oC (dec); TLC ('PrOH 30/n-BuOH 45/H120 25/NH40H 2) Rf = 0.41; MS (ESI) mass calcd for 30 C 66

H

90

N

1 50 17 CoP 1456, found 1456 (M) ; UV (H 2 0) X 36 1 (8 = 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 1 2 derivatives. Reagents 5 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. 57 CoVB 12: 57 Co stock (50 il) (Kodak) was diluted into 50ul of stock in 25ml of IF buffer to 10 give a solution of 1 ng 57 CoVB 1 2 / 25 ml. 250 ng cold VB 1 2 was added to 25 ml of hot 5 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. 15 BSA-coated charcoal: BSA (1%) 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: 20 Ten fold up dilutions of VBl 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 20 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 25 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 57CoVB12 binding when compared to unmodified VB 12 . 15 SUBSTITUTE SHEET (Rule 26) (RO/AU) WO 99/65930 PCT/AU99/00462 Compound % binding relative to vitamin B 12 hexyl-5'O-VBl 2 49 dodecyl-5'O-VB1 2 35 tetradecyl-5'O-VB 1 2 4.2 hexadecy-5'O-VB 1 2 0.78 octadecyl-5'O-VB1 2 0.57 aminoethyl-5'O-VB 12 40 aminobutyl-5'O-VB 1 2 27 t-butyl-Phe-5'O-VB12 aminohexyl-5'O-VB 1 2 25 aminododecanyl-5'O-VB 1 2 31 succinylhydrazidyl-5'O-VB 1 2 37 adipylhydrazidyl-5'O-VB 1 2 29 phenylalanyl-5'O-VB 1 2 glycyl-5'O-VB 1 l2 HO-Gly-5'O-VB 12 25 Those skilled in the art will appreciate that the invention described herein is susceptible to 5 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. 16 SUBSTITUTE SHEET (Rule 26) (RO/AU) 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 5 Controlled Release ofBioactive Materials, 1996. 2. Anton, D. L. Hogenkamp, H. P. C., Walker, T. E., and Matwiyoff, N. A. Carbon-13 nuclear magnetic resonance studies of monocarboxylic acids of cyanocobalamin. Assignments of the b-, d-, and e-monocarboxylic acids. J. Am. Chem. Soc., 102: 2215, 1980. 10 3. Russell-Jones, G.J., Westwood, S. W and Habberfield, A. D Vitamin B 12 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 B12 coenzyme and their use as affinity absorbents for a study of interactions of diol 15 dehydrase with the coenzyme. J. Biol. Chem., 255, 3520, 1980. 5. Annunziato, M. E., Patel, U. S., Ranade, M., and Palumbo, P. S. p-Maleimidophenyl isocyanate: A novel heterobifunctional linker for hydroxyl to thiol coupling. Bioconj. Chem., 4, 212, 1993. 6. Westwood, S. W., and Russell-Jones, G. J. Vitamin Bl 2 mediated delivery systems for 20 GCSF and EPO. (USP 08/064,873; 5,548,064), 1993. 7. Habberfield, A. D., Kinstler, O.B., and Pitt, C. G. Conjugates of VB 12 and proteins. (USP 5,574,018) 1996. 17 SUBSTITUTE SHEET (Rule 26) (RO/AU)

Claims (28)

1. A method for preparing VBl 2 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 1 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.

2. A method of claim 1, 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.

3. A method of claim 2, wherein the bifunctional carbonyl electrophile is carbonyldiimidazole.

4. A method of claim 1, wherein the nucleophilic spacer molecule is an amino- or hydrazidyl-substituted spacer molecule.

5. A method of claim 4, wherein the spacer molecule is octadecylamine.

6. A method of claim 4, wherein the spacer molecule is diaminoethane.

7. A method of claim 4, wherein the spacer molecule is diaminobutane.

8. A method of claim 4, wherein the spacer molecule is diaminohexane.

9. A method of claim 4, wherein the spacer molecule is diaminododecane.

10. A method of claim 4, wherein the spacer molecule is diaminooctadeccane.

11. A method of claim 4, wherein the spacer molecule is an amino acid or a peptide.

12. A method of claim 4, wherein the spacer molecule is a dihydrazide. 18 WO 99/65930 PCT/AU99/00462

13. A method of claim 12, wherein the dihydrazide is succinic acid dihydrazide.

14. A method of claim 12, wherein the dihydrazide is adipic acid dihydrazide.

15. 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 VB 1 2 with the active intermediate to yield said VB 12 derivative.

16. A method of claim 15, 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.

17. A method of claim 16, wherein the bifunctional carbonyl electrophile is carbonyldiimidazole.

18. A method of claim 15, wherein the carboxylic acid spacer molecule is N-Boc-Phe.

19. A method of claim 15 wherein the carboxylic acid spacer molecule is N-Boc-Gly.

20. A VB 12 derivative prepared by a method of any preceding claim.

21. A VB 12 derivative of the formula (I): VB 12 -5'O-CO-NH-R' (I) or a salt thereof, wherein R' is Ci. 24 alkyl, C 2 - 24 alkenyl, C 2 . 24 alkynyl, C 3 .scycloalkyl, (C 3 .scycloalkyl)alkyl, amino, -(CI. 12 alkyl)C(O)R 2 , -(C2-1 2 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, R2 is amino, hydroxy, Ci.6alkoxy or C2.- 6 alkenyloxy, 19 WO 99/65930 PCT/AU99/00462 R 3 is an amino acid side chain or a derivative thereof, and R 4 is hydroxy, C I. 6 alkoxy, an amino acid or a peptide.

22. A VB12 derivative of claim 21, wherein R 1 is hexyl, dodecyl, tetradecyl, hexadecyl, octadecyl, aminoethyl, aminobutyl, aminohexyl, aminododecanyl, t-butyl-Phe, succinylhydrazidyl, adipylhydrazidyl, Gly-OMe or Gly-OH.

23. A VB 1 2 derivative of the formula (II): VB 12 -5'O-CO-R' (II) or a salt thereof, wherein R' is Ci.- 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(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.

24. A VB 12 derivative of claim 23, wherein R' is C8- 24 alkyl or Cs. 24 alkenyl.

25. A VB 12 derivative of claim 23, wherein R' is -CH(R 2 )-NHR 3 , R 2 is Gly and R 3 is Boc or hydrogen.

26. A VB 1 2 derivative of claim 23, wherein R' is -CH(R 2 )-NHR 3 , R 2 is Phe and R 3 is Boc or hydrogen.

27. A polymer or nanoparticle suitable for therapeutic administration to a subject, said polymer of nanoparticle comprising a VB 12 derivative of any one of claims 20-26.

28. A pharmaceutical composition comprising a polymer or nanoparticle of claim 27 in association with a pharmaceutically acceptable carrier and/or diluent. 20