WO2000058269A1

WO2000058269A1 - Dtpa esters with orthogonal removable protecting groups

Info

Publication number: WO2000058269A1
Application number: PCT/US2000/007541
Authority: WO
Inventors: Samuel I. Achilefu; Ananthachari Srinivasan
Original assignee: Mallinckrodt Inc
Current assignee: Mallinckrodt Inc
Priority date: 1999-03-26
Filing date: 2000-03-21
Publication date: 2000-10-05
Anticipated expiration: 2001-09-26
Also published as: AU4019100A

Abstract

The synthesis of compounds of formula (XXXIII) wherein each R4 is a removable protecting group; R5 is a removable protecting group different from and removable separately from R4; each R6 and R8 is a linking moiety having 1 to 10 carbon atoms; and R9 is hydrogen or a C1 to C50 alkyl moiety; is disclosed. These compounds are useful intermediates in the synthesis of compounds of formula (X). These compounds are useful in the preparation of nuclear pharmaceuticals such as those used for tumor imaging or tumor therapy.

Description

DTPA ESTERS WITH ORTHOGONAL REMOVABLE PROTECTING GROUPS

BACKGROUND OF THE INVENTION

This invention relates to the synthesis of esters of diethylenetriaminepentaacetic acid (DTPA) and of intermediates useful in the synthesis of such esters. These esters are well known, and have the general formula:

(I)

wherein R_{ and R₂ are H, and R₃, R₄, and R₅ are t-butyl (compound (II)) or a similar protecting group. Such compounds are useful in the preparation of nuclear pharmaceuticals where they serve as a metal chelate and a link between a peptide and a radionuclide. Activation of the free dicarboxylic acid rapidly forms intramolecular acid anhydride which then reacts with the amino group on a peptide to form the DTPA-peptide conjugate. Acid mediated cleavage of the esters gives free tetra-carboxylic acid which readily forms stable metal complexes with the radionuclide of choice.

Using conventional techniques, these compounds are prepared by the following sequence (t-Bu = t-butyl):

(III) (IV)

Compound (IV) is commercially available. Compound (III) was prepared by the method taught in Rapoport, J. Org. Chem. 1993, 58, 1151-1158 (incorporated herein by reference). This reaction yields two compounds in about a 1 :4 ratio. The major product is the penta ester (compound (V)), (i.e.: compound (I) in which all of R,-R₅ = t-butyl). This product is useless and must be disposed of. The minor product is:

(VI)

Compound (VI) is reacted with the compound (Bz = benzyl):

(VII) This compound is not commercially available, but may be synthesized by literature method as described by Rapoport (see the reference for compound (III) above).

The reaction product of (VI) and (VII) is:

(VIII)

This compound is subjected to catalytic hydrogenation at room temperature to yield

(II) (i.e.: compound (I) where R and R₂ are H and R₃, R₄, and R₅ are t-butyl).

Although these compounds are useful, their syntheses are often complicated and expensive. Further, it would be useful to have other compounds available with different binding affinities.

US 5,618,513 (Mallinckrodt: A. Srinivasan) teaches a general method for using DTPA compounds in the preparation of radiopharmaceuticals. This reference also teaches the preparation of such compounds as outlined above.

US 4,479,930 (Univ. of Massachusetts: D. Hnatowich) teaches the use of a dicyclic dianhydride compound to couple polypeptides. Although useful, this method results in diaddition products which are not medically useful.

S. Ram and L. Spicer, Rapid Debenzylation of N-Benzylamino Derivatives to Amino-Derivatives Using Ammonium Formate as Catalytic Hydrogen Transfer Agent, Tetrahedron Letters, Vol. 28, No. 5, pp 515-516, 1987, teaches the deprotection of various N-benzyl compounds using ammonium formate as the hydrogen source.

S. Ram and L. Spicer, Debenzylation of N-Benzylamino Derivatives by Catalytic Transfer Hydrogenation with Ammonium Formate, Synthetic Communication, 17(4), 415-418 (1987), is similar to the first Ram and Spicer reference.

C. Grote, D. Kim, and H. Rapoport, Stereocontrolled Synthesis of DTPA Analogues Branched in the Ethylene Unit, J. Org. Chem., 1995, 60, 6987-6997, teaches a synthesis similar to that outlined above, with the addition of stereo control of the reaction. M. Brechbiel and O. Gansow, Backbone-Substituted DTPA Ligands for ⁹⁰Y Radioimmunotherapy, Bioconjugate Chem. 1991, 2, 187-194, teaches the synthesis of new bifunctional DTPA ligands.

US 5,514,810 (Schering: J. Platzek et al.), disclose some of the compounds which may be synthesized by this invention. However, this reference teaches a different method of synthesis.

WO 98/05626 (Bracco: P. L. Anelli, et al.) teaches compounds that are similar to the compounds made by the instant invention.

SUMMARY OF THE INVENTION

Briefly, the invention comprises a method of synthesis of compounds of the formula

(XXXIII) and

(X)

The invention also comprises compound (XXXIII) per se. DETAILED DESCRIPTION OF THE INVENTION

In this specification and claims, numerical values and ranges are not critical unless otherwise stated. That is, the numerical values and ranges may be read as if they were prefaced with the word "about" or "substantially".

The invention includes a method for synthesizing compounds of the formula:

(X) wherein each R^ is a linking moiety having 1 to 10, desirably 1 to 6, preferably 1 to 4, and most preferably 2 carbon atoms; each R₄ is a removable protecting group, generally (a) an alkyl group having 1 to 15, desirably 2 to 10, more desirably 2 to 8, preferably 3 to 6, and more preferably 4 carbon atoms, and most preferably being t-butyl or (b) benzyl or a benzyl derivative such as methoxy benzyl or nitrobenzyl, preferably benzyl. If R₄ is t-butyl or a similar group, the compound (X) is more useful in fluorenylmethoxycarbonyl (Fmoc) peptide synthesis, and if R₄ is benzyl or a similar group, the compound (X) is more useful in acid labile t-butoxycarbonyl (Boc) peptide synthesis; and R₉ is hydrogen or a C, to C₅₀ alkyl moiety such as that taught by US 5,514,810 (incorporated herein by reference), preferably hydrogen.

The synthesis begins with the reaction of:

(XXXI) (XXXII) wherein R₄ and Rg are as defined above; and R₅ is a removable protecting group different from and removable separately from R₄, generally (a) t-butyl, allyl, or chlorotrityl, preferably t-butyl or (b) allyl, benzyl, or a benzyl derivative such as methoxy benzyl or nitrobenzyl, preferably benzyl or methoxy benzyl, and most preferably benzyl; and X is a group that will react with the amine of compound (XXXI), desirably a halide, a mesylate, or a triflate, more desirably a halide, preferably Cl or Br, and most preferably Br. If R₄ is t-butyl or a similar group, R₅ is preferably benzyl or a benzyl derivative, and if R₄ is benzyl or a similar group, R₅ is preferably t-butyl or allyl. This reaction produces:

(XXXIII) When compound (XXXIII) is subjected to selective removal of the R₅ group, for instance, by hydrogenation, the result is

(X)

Compound X can be reacted with a peptide to give a conjugate in high purity. These new compounds forms stable complexes with lanthanides and other metals, and have excellent in vivo stability. Since no free carboxylic acid is present after conjugation with the peptide (or other organic molecule), these compounds can be used for orthogonal synthesis, something not possible with prior art compounds (e.g., Compound (II)).

The invention is further illustrated in the following examples. EXAMPLE 1

Synthesis of 2-[Bis-(t-butyloxycarbonylmethyl)amino] ethyl bromide

(Compound (XXXII) where X = Br, R₄ = t-butyl, and R₆ = ethyl). A solution of 370 ml of dimethylformamide and t-butyl bromoacetate (100 g, 510 mmol) was stirred in a 1000 ml three-neck flask. Solid potassium bicarbonate (57 g, 570 mmol) was added. The flask was purged with argon and cooled to 0°C with an ice bath. To the stirring mixture was added dropwise a solution of ethanolamine (13.9 g, 230 mmol) in 30 ml of dimethylformamide over 15 minutes. After the addition was complete the mixture was stirred for 1 hour at 0°C. The ice bath was removed and the mixture stirred at room temperature for 12 hours. The reaction mixture was partitioned between 700 ml of methylene chloride and 700 ml of saturated sodium bicarbonate solution. The layers were separated and the methylene chloride layer was again washed with 700 ml of saturated sodium bicarbonate solution. The combined aqueous layers were extracted twice with 200 ml of methylene chloride. The combined methylene chloride layers were washed with 500 ml of brine, and dried over magnesium sulfate. The methylene chloride was removed with aspirator vacuum at ca. 35°C, and the remaining dimethylformamide was removed with vacuum at about 45°C. The crude material was left on a vacuum line over night at room temperature.

The crude material from above was dissolved in 600 ml of methylene chloride at room temperature. Triphenylphosphine (65.8 g, 250 mmol) was added and dissolved with stirring. An argon purge was started and the mixture cooled to 0°C with an ice bath. The N-bromosuccinimide (44.7 g, 250 mmol) was added portionwise over 5 minutes. The mixture was stirred for 1.5 hours at 0°C. The methylene chloride was removed with vacuum and gave a purple oil. This oil was triturated with 500 ml of ether with constant manual stirring. During this time the oil became very thick. The ether solution was decanted and the oil was triturated with 500 ml of ether. The ether solution was decanted and the oil was again triturated with a 500 ml portion of ether. The ether was decanted and the combined ether solutions allowed to stand for about 2 hours to allow the triphenylphosphine oxide to crystallize. The ether solution was decanted from the crystals and the solid washed with 500 ml of ether. The volume of the combined ether abstracts was reduced with vacuum until a volume of about 80 ml was obtained. This was allowed to stand over night at 0°C. Ether (100 ml) was added to the cold mixture which was mixed to suspend the solid. The mixture was filtered and washed ten times with 4 ml of ether. The solution was percolated through a column of 500g g of silica gel and eluted with 500 ml portions of ether, 500 ml fractions were collected. The fractions that contained product by TLC were pooled and the ether removed en vacuo. This gave 68.6 g of crude product. The material was flash chromatographed on silica gel with hexane, changing to 9:1 hexane: ether. The product-containing fractions were pooled and the solvents removed en vacuo. This gave 54 g (67% yield) of pure product.

EXAMPLE 2

Synthesis of N'-(benzyloxycarbonylmethyl)-N,N,N",N"-tetrakis(t- butyloxycarbonylmethyl)diethylenetriamine (Compound (XXXIII) where R₄ = t-butyl and R₅ = benzyl). A mixture of 2-[Bis-(t- butyloxycarbonylmethyl)amino]ethyl bromide (2.3 g, 6.52 mmol), diisopropylethylamine (1.5 g, 11.86 mmol) and benzyl glycinate p-toluene sulfonic acid salt (1 g, 2.96 mmol) in 50 ml of anhydrous acetonitrile was refluxed for 24 hours under argon. After reaction, the solvent was evaporated en vacuo and the residue was partitioned between dichloromethane (50 ml) and water (50 ml). The two layers formed were separated and the organic phase was washed with water (100 ml) and brine (100 ml) in that order. The dichloromethane layer was dried over magnesium sulfate and the solvent was removed en vacuo to give 2.5 g of the crude product. The crude product was dissolved in hexane and purified by dry flash chromatography with 20% diethyl ether in hexane to give 1.8 g (86%) of the pure compound as a pale yellow liquid.

EXAMPLE 3

Preparation of N'-acetic acid-N,N,N",N"-tetrakis(t-butyloxycarbonylmethyl)- N'-acetic acid)diethylenetriamine (Compound (X) where R₄ = t-butyl and R₆ = ethyl). A mixture of 10% palladium on carbon (0.1 g) and a solution of N'-

(benzyloxycarbonylmethyl)-N,N,N",N"-tetrakis(t- butyloxycarbonylmethyl)diethylenetriamine (0.6 g, 0.85 mmol) in 30 ml of methanol was hydrogenolyzed at 45 psi for 2 hours. The mixture was filtered over celite and the residue was washed with methanol (50 ml). The solvent was evaporated to give the pure mono-carboxylic acid (0.5 g, 96%) as a viscous pale yellow oil.

EXAMPLE 4 Synthesis of DTPA-Octreotate derivative. The DTPA-Octreotate conjugate was prepared by solid phase synthesis using pre-loaded fluorenemethoxycarbonyl- threonine (Fmoc-Thr) Wang resin on 0.025 mmol scale. Commercially available automated peptide synthesizer from Applied Biosystems (Model 432A SYNERGY Peptide Synthesizer) was used. Cartridges containing Fmoc-protected amino acids were used in the solid phase synthesis. Cysteines were protected with acetamidomethyl group. Coupling reaction was carried out with 0.075 mmol of the protected amino acid and 2-(lH-benzotriazole-lyl)-l,l,3,3-tetramethyluronium hexafluorophosphate (HBTU)/N-hydroxybenzotriazole (HOBT). The amino acids and tetra-t-butyl DTPA (compound X) cartridges were placed on the peptide synthesizer and the product was synthesized from the C-terminal to the N-terminal position. After the synthesis was completed, the product was cleaved from the solid support with a cleavage mixture containing trifluoroacetic acid (85%):water (5%):phenol (5%):thioanisole (5%) for 6 hours. Note that the t-butyl esters of tetra- t-butyl DTPA were also cleaved to give the free tetra-carboxylic acid. The DTPA- peptide conjugate was precipitated with t-butyl methyl ether and lyophihzed with water : acetonitrile (2/3) mixture. Mass spectral analysis indicated that only the mono peptide-DTPA conjugate was obtained in accordance with the following sequence: DTPA-D-Phe-Cys(Acm)-Tyr-D-T -Lys-The-Cys(Acm)-Thr.

Claims

What is claimed is:

1. The compound

(XXXIII) wherein each R₄ is a removable protecting group; R₅ is a removable protecting group different from and removable separately from R₄; each R₆ is a linking moiety having 1 to 10 carbon atoms; and R₉ is hydrogen or a C, to C₅₀ alkyl moiety.

2. The compound of claim 1 wherein each R₄ is (a) an alkyl group having 1 to 15 carbon atoms or (b) benzyl or a benzyl derivative; if R₄ is an alkyl group then R₅ is allyl, benzyl, or a benzyl derivative, and if R₄ is benzyl or a benzyl derivative, then R₅ is t-butyl, chlorotrityl, or allyl; each Rg has 1 to 6 carbon atoms; and R₉ is hydrogen.

3. The compound of claim 2 wherein each R₄ is (a) an alkyl group having 3 to 6 carbon atoms or (b) benzyl or a benzyl derivative; if R₄ is an alkyl group then R₅ is allyl, benzyl, or a benzyl derivative, and if R₄ is benzyl or a benzyl derivative, then R₅ is t-butyl, chlorotrityl, or allyl; each Rg has 1 to 4 carbon atoms.

4. The compound of claim 3 wherein each R₄ is t-butyl; R₅ is benzyl, and Re is ethyl.

5. The compound of claim 3 wherein each R₄ is benzyl; R₅ is t-butyl, and Rg is ethyl.

6. A method of synthesizing a compound of the formula

(XXXIII) wherein each R₄ is a removable protecting group; R₅ is a removable protecting group different from and removable separately from R₄; each Rg is a linking moiety having 1 to 10 carbon atoms; and R₉ is hydrogen or a C] to C₅₀ alkyl moiety; comprising reacting together compounds of the formulas

(XXXI) (XXXII) wherein X is a group that will react with the amine of compound (XXXI); to form compound (XXXIII).

7. The method of claim 6 wherein each R₄ is (a) an alkyl group having 1 to 15 carbon atoms or (b) benzyl or a benzyl derivative; if R₄ is an alkyl group then R₅ is allyl, benzyl, or a benzyl derivative, and if R₄ is benzyl or a benzyl derivative, then R₅ is t-butyl, chlorotrityl, or allyl; each Re has 1 to 6 carbon atoms; R₉ is hydrogen; and X is a halide, a mesylate, or a triflate.

8. The method of claim 7 wherein each R₄ is (a) an alkyl group having 3 to 6 carbon atoms or (b) benzyl or a benzyl derivative; if R₄ is an alkyl group then R₅ is allyl, benzyl, or a benzyl derivative, and if R₄ is benzyl or a benzyl derivative, then R₅ is t-butyl, chlorotrityl, or allyl; each Rg has 1 to 4 carbon atoms; and X is a halogen.

9. The method of claim 8 wherein each R₄ is t-butyl; R₅ is benzyl, R^ is ethyl; and X is Br.

10. The method of claim 8 wherein each R₄ is benzyl; R₅ is t-butyl, Rg is ethyl; and X is Br.

11. A method of synthesizing a compound of the formula

(X) wherein each R₄ is a removable protecting group; each Rg is a linking moiety having 1 to 10 carbon atoms; and R₉ is hydrogen or a to C₅₀ alkyl moiety comprising, reacting a compound of the formula

(XXXIII) wherein R₅ is a removable protecting group different from and removable separately from R₄; under conditions such that R₅ is replaced by a hydrogen.

12. The method of claim 11 wherein the conditions such that R₅ is replaced by a hydrogen comprise a hydrogenation reaction.

13. The method of claim 11 wherein the compound of the formula

(XXXIII) is prepared by reacting together compounds of the formulas