WO2000058265A1 - Preparation of dtpa esters with different ester groups - Google Patents

Abstract

The synthesis of compounds of formula (XI) 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

PREPARATION OF DTPA ESTERS WITH DIFFERENT ESTER GROUPS

BACKGROUND OF THE INVENTION

This invention relates generally 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):

-COOBz

V ^■COOBz (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 Ri 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, Stereo controlled 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

(XI) and

(X) 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 R-g and R₈ are each 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 Re, is hydrogen or a 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: H

H₂N R₆ N R₈ NH₂

(XII) wherein Rg and R₈ are as defined above, with a compound Z which is any protecting group that will react with primary amines but not (under the same conditions) with secondary amines. Z is desirably trifluoroacetyl in the presence of crown ethers, 2-acetyldimedone (Dde) , or phthaloyl (Pth), preferably Pth for Fmoc procedures and preferably Dde for Boc procedures. Compound (XII) is commercially available.

The reaction yields:

H ZN R₆— N R₈ NZ

(XIII)

Compound (XIII) can be reacted with the compound:

X-Y (XX) wherein X is a group that will react with the secondary amine of compound (XIII), desirably a halide, mesylate, active ester, alkyl ester, or triflate, more desirably an allyl ester, a methyl ester, an ethyl ester, or a halide, preferably Cl or Br, and most preferably Br; and Y is a protecting group that is different from Z, preferably trifluoroacetyl, chlorotrityl, methoxybenzyl, or nitrobenzyl, and preferably benzyl.

The resultant compound is Y

ZN R₆ N R₈ NZ

(XXI) Selective removal of the Z groups yields

(XXII) This compound is then reacted with

X COOR₄

(XVII) wherein X is a group that will react with the amines of compound (XXII), desirably a halide, a mesylate, or a triflate, more desirably a halide, preferably Cl or Br, and most preferably Br; and R₄ is as defined above. The result is

(XXIII)

Y is selectively removed, for instance, by hydrogenation, to give:

(XXIII') This compound is then reacted with a compound of the formula

(XIV) wherein Rg is hydrogen or a to C₅₀ alkyl moiety, desirably such as that taught by US 5,514,810 (incorporated herein by reference), preferably hydrogen; and R₅ is a removable protecting group different from and removable separately from R₄(defined below), 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 (XXIII), desirably a halide, a mesylate, or a triflate, more desirably a halide, preferably Cl or Br, and most preferably Br. If B^ 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. The reaction product is:

(XI) Selective removal of R₅, yields:

(X)

The invention is further illustrated in the following examples.

EXAMPLE 1

Synthesis of N,N,N",N"-bis(phthaloyl)diethylenetriamine (Compound (XIII) where R^ and R₈ = ethylene and Z = phthaloyl). Phthalic anhydride (40 g, 270 mmol) and diethylenetriamine (12.7 g; 123 mmol) were added to 300 ml of glacial acetic acid and the mixture was refluxed for 2 hours. The mixture was allowed to cool to room temperature and 200 ml of heptane was added. The azeotropic mixture formed was evaporated on a rotary evaporator. The orange residue obtained was dissolved in 300 ml of dichloromethane and washed with saturated sodium bicarbonate until the solution became alkaline on a pH indicator paper. Evaporation of the solvent gave about 45 g of the crude product. Methanol (300 ml) was added to the solid residue and the mixture was heated until a fine solid dispersion was obtained. The solid was filtered, washed with methanol and dried under vacuum to give 35 g (80% yield) of the pure compound.

EXAMPLE 2

Synthesis of N'-benzyl-N,N,N",N"-bis(phthaloyl)diethylenetriamine

(Compound (XXI) where Rg and R₈ = ethylene, Z = phthaloyl and Y = benzyl).

N,N,N",N"-bis(Phthaloyl)diethylenetriamine (10 g, 27.54 mmol), benzyl bromide (5.65 g; 33.05 mmol) and diisopropylethylamine (6 g, 38.55 mmol) were added to

100 ml of acetonitrile and the mixture was refluxed for 24 hours. The solvent was evaporated and dichloromethane (100 ml) was added to the residue. The solution was first washed with 0.1 M aqueous hydrochloric acid to remove unreacted amine and the organic layer was washed with a saturated aqueous solution of sodium bicarbonate until a clear aqueous layer was observed. The dichloromethane layer was then washed with brine and water, in that order, and the solution was dried over magnesium sulfate. Evaporation of the solvent gave 12.2g of the crude product as a yellow solid. This was dissolved in 50 ml of chloroform and the solution was concentrated on a rotary evaporator until solid precipitates began to form. Diethyl ether was added to the mixture to completely precipitate the solid which was filtered and washed with diethyl ether to give a pale yellow powder (11 g, 88% yield).

EXAMPLE 3

Synthesis of N'-benzyl-diethylenetriamine (Compound (XXII) where Rg and

R₈ = ethylene and Y = benzyl). N'-benzyl-N,N,N",N"-bis(ρhthaloyl) diethylenetriamine (7.6 g, 16.77 mmol) and hydrazine (1.1 g; 34.38 mmol) were added to 100 ml of ethanol and the mixture was heated to a gentle reflux for 20 minutes. Hydrochloric acid (1.0 M, 10 ml) was added to the hot ethanol mixture and this was followed by a dropwise addition of a 6.0 M HCl solution until the pH became acidic on a pH indicator paper. The mixture was heated at reflux for 30 minutes , the precipitate was filtered and washed with 10% aqueous HCl (3 x 20 ml). Ethanol was evaporated and the residue was neutralized with 10% aqueous NaOH. The pH of the solution was adjusted to about 8 and the product was extracted with diethyl ether (4 x 50 ml). The aqueous phase was concentrated to about 10 ml and a mixture of diethyl ether/acetonitrile/ethanol (1 :1:2) was added to it. The white precipitate that formed was filtered and washed with ethanol (2 x 15 ml). The filtrate was combined and the solvent mixture was evaporated to give the pure compound as a yellow viscous liquid (3 g, 92% yield). EXAMPLE 4

Synthesis of N'-benzyl-N,N,N",N"-tetrakis(t-butyloxycarbonyImethyl) diethylenetriamine (Compound (XXIII) where Rή and R₈ = ethylene, R₄ = t- Butyl and Y = benzyl). A solution of t-butyl bromoacetate (13.9 g, 71.18 mmol) in 80 ml of anhydrous dimethylformamide was stirred in a 250 ml three-neck flask. Solid potassium bicarbonate (7.8 g, 78.30 mmol) was added. The flask was purged with argon and cooled to 0°C in an ice bath. To the stirring mixture was added dropwise a solution of N'-benzyl-diethylenetriamine (2.8 g, 14.24 mmol) in 5 ml of dimethylformamide over 5 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 over night. The reaction mixture was partitioned between 50 ml of methylene chloride and 50 ml of saturated sodium bicarbonate solution. The layers were separated and the methylene chloride layer was again washed with 50 ml of saturated sodium bicarbonate solution. The aqueous layer was extracted twice with 25 ml of methylene chloride. The combined methylene chloride layers was washed with 100 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 under vacuum at about 45°C to give 13 g of the crude product. This product was purified by dry flash chromatography on silica gel column and the pure compound was eluted with 30% ethyl acetate in hexane.

EXAMPLE 5 Preparation of N,N,N",N"-tetrakis(t-butyloxycarbonylmethyl) diethylenetriamine (Compound (XXIII') where Rg and R₈ = ethylene, R₄ = t- Butyl and Y = H). A mixture of N'-benzyl-N,N,N",N"-tetrakis(t- butyloxycarbonylmethyl)diethylenetriamine (4.7 g, 7.23 mmol), ammonium formate (2.3 g, 36.16 mmol) and 10% palladium on activated carbon (2 g) in 100 ml of anhydrous methanol was stirred at room temperature under argon for 4 hours. The catalyst was filtered over celite and the residue was washed with chloroform (30 ml). The combined filtrate was concentrated to dryness. Chloroform (10 ml) was added to precipitate the excess ammonium formate. After filtration, the filtrate was evaporated to give the pure compound as the formate salt (4.5 g, quantitative).

EXAMPLE 6

Preparation of N'-benzyloxycarbonylmethyl-N,N,N",N"-tetrakis(t- butyloxycarbonylmethyl)diethylenetriamine (Compound (XI) where R_<s and R₈ = ethylene, R₄ = t-Butyl, R, = H and Rj = benzyl). A mixture of N,N,N",N"- tetrakis(t-butyloxycarbonylmethyl)diethylenetriamine (2 g, 3.57 mmol), benzyl bromoacetate (1.2 g, 5.36 mmol) and diisopropylethylamine (1 g, 7.86 mmol) in 50 ml of acetonitrile was refluxed for 16 hours. The solvent was evaporated and the residue was purified on a silica gel column by dry flash chromatography. The pure compound was eluted with 30% of diethyl ether in hexane (2 g, 80%).

EXAMPLE 7

Preparation of N'-acetic acid- N,N,N",N"-tetrakis(t-butyloxycarbonylmethyl) diethylenetriamine (Compound (X) where Rg and R₈ = ethylene, R₄ = t-Butyl, R₉ and Rg = H). A mixture of 10% palladium on carbon (0.1 g) and N'- (benzy loxycarbonylmethyl)-N,N,N " ,N " -tetrakis(t-buty loxycarbony lmethyl) 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 8

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 lyophilized 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-Trp-Lys-The-Cys(Acm)-Thr.

Claims

What is claimed is:

1. A method of synthesizing a compound of the formula

(XI) wherein each R₄ is a removable protecting group; R₅ is a removable protecting group different from and removable separately from R₄; each Rg and R₈ 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

(XXIIF) and

(XIV) wherein X is a group that will react with the amine of compound (XXIII).

2. The method 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; R₉ is hydrogen; and X is a halide, a mesylate, or a triflate.

3. The method 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 -R-, is benzyl or a benzyl derivative, then R₅ is t-butyl, chlorotrityl, or allyl; each R^ has 1 to 4 carbon atoms; and X is a halogen.

4. The method of claim 3 wherein each R^ is t-butyl; R₅ is benzyl, Rg is ethyl; and X is Br.

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

6. A method of synthesizing a compound of the formula

(XXIIF) wherein each -R-j is a removable protecting group; each Rg and R₈ is a linking moiety having 1 to 10 carbon atoms; and Y is a removable protecting group; comprising reacting together compounds of the formulas Y

H₂N R₆ N R₈ NH₂ (XXII)

This compound is then reacted with

X COOR₄

(XVII) wherein X is a group that will react with the amines of compound (XXII) to form the compound

(XXIII) and selectively removing Y to form Compound (XXIIF).