WO2001052898A1

WO2001052898A1 - Methods for incorporating metal chelators at carboxyl-terminal site of peptides

Info

Publication number: WO2001052898A1
Application number: PCT/US2001/001640
Authority: WO
Inventors: Samuel I. Achilefu; Ananthachari Srinivasan
Original assignee: Mallinckrodt Inc
Current assignee: Mallinckrodt Inc
Priority date: 2000-01-21
Filing date: 2001-01-18
Publication date: 2001-07-26
Anticipated expiration: 2002-07-21
Also published as: WO2001052898A9; AU2001232845A1

Abstract

Methods for incorporating metal chelators at carboxyl-terminal sites of peptides are presented. In a first method, a metal chelator which is a polyazacarboxylate with two free carboxyl groups is attached to a functionalized resin via one carboxyl group and to a diamine or orthogonally protected diamine via a second carboxyl group. The protecting group is removed and a sequence of amino acids is added to the diamine residue on solid support to form the desired peptide. After addition of the amino acid or peptide, the compound is cleaved from the resin and blocking groups are removed. In a second method, an orthogonally protected polyazacarboxylate with a free carboxyl and protected amine groups is reacted with a functionalized resin. The amine protecting group is removed and amino acids are added to obtain the desired peptide. The compound is then cleaved from the resin and blocking groups are removed. The cleavage from the resin and removal of blocking groups can be performed simultaneously.

Description

METHODS FOR INCORPORATING METAL CHELATORS AT CARBOXYL-

TERMINAL SITE OF PEPTIDES

FIELD OF INVENTION This invention relates to a new approach for the synthesis of metal chelators at the C- terminal site of bioactive peptides. Particularly, the invention relates to methods of incorporating polyazacarboxylic acid ligands on solid support and their use in the synthesis of biomolecules useful for diagnostic and therapeutic applications. The compounds of this invention have the general formula: H₂N-(AA)_n-LS

Formula 1 wherein (AA)_n is a bioactive molecule wherein n equals 1-50, preferably 3-25, especially peptides, and LS is a cyclic or linear polyazarcarboxylate attached to the carboxyl terminus of peptides. The formulations of this invention are useful for therapeutic and contrast agents in biomedical applications.

BACKGROUND OF THE INVENTION

The surge of interest in the use of peptides and other biocompatible markers to target tumors has led to the identification of a host of receptors that are over-expressed by certain tumors (J. C. Reubi, Neuropeptide receptors in health and disease: the molecular basis for in vivo imaging. Journal of Nuclear Medicine, 1995, 36, 1825-1835; A. J. Fischman, J. . Babich, and

H. W. Strauss, A ticket to ride: Peptide radiopharmaceuticals. Journal of Nuclear Medicine,

1993, 34, 2253-2263). The tumors can then be visualized and destroyed by agents that target the receptors which are over-expressed in the given tumor (J. E. Bugaj, J. L. Erion, M. A. Schmidt,

R. R. ilhelm, S. I. Achilefu, A. Srinivasan, Biodistribution and Radiotherapy Studies Using

Samarium-153 and Lutetium-177 DTPA Conjugates of Y³- Octreotate. Journal Nuclear

Medicine, 1999, 40(5), 223P). This site-specific delivery of contrast agents enables the differentiation of normal from diseased tissues and also preserves normal tissues from lethal therapeutic drugs.

A current method for tumor imaging involves the conjugation of radioactive metal chelates to antibodies or peptides that target the abundant receptors on a given tumor (R. Albert, E. P. Krenning, S. W. J. Lamberts, and J. Pless, Use of certain somatostatin peptides for the in vivo imaging of somatostatin receptor-positive tumors and metastasis. US 5,753,627). Careful selection of metals and peptides determines the imaging modality and therapeutic potential of the chelate-peptide conjugate. For example, gadolinium chelates are used for magnetic resonance imaging (A. D. Nunn, K. E. Linder, and M. F. Tweendle, Can receptors be imaged with MRI agent? The Quarterly Journal of Nuclear Medicine, 1997, 41(2), 155-162), radioactive metals are used for scintigraphy (e.g. technetium-99, indium- 111, see A. Srinivasan, M. M. Dyszlewski, J. E. Bugaj, and J. L. Erion, Radiolabeled peptide compositions for site-specific targeting. US 5,830,431), or therapy (e.g. lutetium, yttrium, see J. E. Bugaj, J. L. Erion, M. A. Schmidt, R. R. Wilhelm, S.I. Achilefu, A. Srinivasan, Biodistribution and Radiotherapy Studies Using Samarium-153 and Lutetium-177 DTPA Conjugates of Y³- Octreotate. Journal Nuclear Medicine, 1999, 40(5), 223P), and bioactive peptides can function as both delivery and therapeutic agents (S. W. J. Lamberts, E. P. Krenning, and J. C. Reubi, The role of somatostatin and its analogs in the diagnosis and treatment of tumors. Endocrine Reviews, 1991, 12(4), 450- 478). In general, the metal chelates are attached to free amino groups of antibodies and peptides

(R. Albert, E. P. Krenning, S. W. J. Lamberts, and J. Pless, Use of certain somatostatin peptides for the in vivo imaging of somatostatin receptor-positive tumors and metastasis. US 5,753,627).

However, some peptides require this terminal amino group for their bioactivity which would be compromised by the presence of a variety of substituents, including DTPA ligands. Further, the activity of some peptides may be enhanced by the presence of chelators at the C- terminal amino acid. An improved representative method of making carboxy-chelate peptides is described in the literature (J.C. Reubi, B. Waser, J.C. Schaer, U. Laederach, J. Erion, A. Srimvasan, M.A. Schmidt, and J.E. Bugaj, Unsulfated DTPA- and DOTA-CCK analogs as specific high affinity ligands for CCK-B receptor-expressing human and rat tissues in vitro and in vivo. European Journal of Nuclear Medicine, 1998, 25(5), 481-490). The synthesis begins with preparation of the desired peptide on solid support by standard automated Fmoc peptide synthesis using diaminoethane-trityl resin. The first amino acid is attached to a solid support and the Fmoc protecting group is removed with a solution of 20% piperidine in DMF. The carboxyl group of the next Fmoc-protected amino acid is activated and condensed on the amino terminus of the resin-bound amino acid. This sequence of deprotection of the resin-bound amino acid, activation of the carboxyl group and condensation of the next amino acid is repeated until the desired peptide is synthesized. The last amino acid in this sequence must be left protected with Boc or Fmoc. The peptide, with all side chain protecting groups, is carefully cleaved from the resin with trifluoroacetic acid (TFA). The TFA and its amine salt are neutralized with a base and the mixture is then evaporated to dryness. Care must be taken to avoid the decomposition of heat-labile peptides or amino acid residues. At this stage, the crude product is then taken up in DMF and t-butyl DTPA (which must be activated with a variety of reagents) is added. At the end ^"of the reaction, the reaction solvents must be removed under reduced pressure and the resulting crude product is subject to another TFA treatment in order to remove all protecting groups on the peptide and chelator.

This approach has several shortcomings. After synthesis, the peptide must be cleaved selectively from resin without concomitant removal of the side-chain protecting groups. This requirement limits the choice of useful resins to those that can be cleaved under very mild conditions (for example trityl resins which must be stored under inert condition). Another implication is that the side chain protecting groups of amino acids must be stable under the cleavage condition which precludes the use of some conventional protecting groups. Once the orthogonally protected peptide is obtained, another sequence of solution phase reactions is required. Worse still, purification of the final crude product is very tasking because of the many side-products that must be removed.

This approach is also cumbersome and involves several reaction steps which precludes the systematic evaluation of the effect of chelators at the C-terminal portion of bioactive peptides. There remains a need to develop dynamic methods for the incorporation of chelators at the carboxyl terminus of bioactive peptides. Such methods would be a tremendous advancement in the art and would encourage the systematic evaluation of the effect of chelators at both amino and carboxyl termini of bioactive peptides. This invention discloses such methods and ligands to accomplish the synthesis. The publications and other materials used herein to support the background of the invention or provide additional details respecting the practice, are incorporated by reference. ^'

SUMMARY OF THE INVENTION The present invention relates particularly to the method of preparing a carboxyl-terminal chelator composition comprising polyazacarboxylates of the formula 1 or la:

la wherein R_t to _t may be the same or different and are selected from the group consisting of alkyl, aryl, heterocarbocyclic, NH-k-NHR₃₀, CH₂CO₂H, hydroxyl, amino, Cl-ClO alkoxyl, Cl-ClO aryloxyl, Cl-ClO polyalkoxyalkyl, -CH₂(CH₂-O-CH₂) -CH₂-R_a, C1-C20 polyhydroxyalkyl, Cl- CIO polyhydroxyaryl, or X-Y; W is selected from the group consisting of alkyl, aryl, -CH₂(CH₂- O-CH₂)_b-CH₂-R_a, polyhydroxyalkyl, or polyhydroxyaryl; X is selected from the group consisting of NH-, CONH-, -CH₂NH-, -CH₂NR₅-, -COO-, -O-, -C(0)-, -S-, -NHCO-, or -NHC(S)-; Y is selected from H, CH₂COOH, peptide, biomolecule, alkyl amines, aryl amines, polyhydroxyalkyl amines, polyalkoxyalkyl amines or Fmoc protected amines for Fmoc peptide synthesis or Boc- protected amines for Boc peptide synthesis; b varies from 1-100; R_a may be H, OH, -O-, alkyloxy, aryl, alkyl or single bond; R₅ is as defined for Ri; R₃₀ is an amine protecting group; k is alkyl, aryl, heterocarbocyclic, CH₂CO₂H, Cl-ClO alkoxyl, Cl-ClO aryloxyl, Cl-ClO polyalkoxyalkyl, -CH₂(CH₂-O-CH₂)_b-CH₂-R_a, C1-C20 polyhydroxyalkyl, C1-C10 polyhydroxyaryl, carbocyclic, heterocyclic, or X-Y; and z varies from 1-10, preferably 1-3.

The present invention also relates to the method of preparing a carboxyl-terminal chelator composition comprising polyazacarboxylates of the formula 2 or 2a:

wherein Re to Rio are defined in the same manner as Ri to Rt; X₂ and Y₂ are defined in the same manner as X and Y respectively; W₂ and W₃ are as defined for W; W₁₅ is C=0, CH₂, or OG₂H₄; and z varies from 1-10, preferably 1-3.

The present invention also relates to the method of preparing a carboxyl-terminal chelator composition comprising polyazacarboxylates of the formula 3:

wherein Rπ to R₁₅ are defined in the same manner as Ri to Rt; X₃ and Y₃ are defined in the same manner as X and Y, respectively; W₄ and W₅ are as defined for W; and W₁₆ is as defined for ι₅. The present invention also relates to a method of preparing a carboxyl-terminal chelator composition comprising polyazacarboxylates of the formula 4:

⁴ wherein Rj₆ to R₁₉ are defined in the same manner as Rj to R_t; X₄ and Y₄ are defined in the same manner as X and Y, respectively; W₆ and W₇ are as defined for W; and W₁₇ is C=O, CH₂, or OC₂H₄.

This invention is also related to methods of attaching any of the composition of formulas 1 to 4 to a solid support and the subsequent synthesis of a bioactive peptide.

DETAILED DESCRIPTION OF THE INVENTION

In this disclosure, 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 novel compositions of the present invention comprising polyazacarboxylates of formulas 1 to 4 offer significant advantages over those currently described in the art. A desirable criterion for the synthesis of C-terminal peptide-chelator conjugates on a solid support is compatibility with solid phase synthesis conditions. As illustrated below in Schemes 1 to 12, two types of chelators are used. In the first method (Method A), the chelators are designed to possess two free carboxyl groups on the same molecule. One of the carboxyl groups is attached to the resin and the other carboxyl group is attached to a diamine. Functionalized resins are used. A functionalized resin is one known to those of skill in the art, e.g., resins used for solid phase synthesis of peptides which are preactivated as alkyl halides, carboxyl, amino, thiol, hydroxy or similar derivatives. Monoprotected Fmoc or Boc diamines which can be used include, but are not limited to: ethylenediamine; bis(2-aminoethyl)-ether; O-bis(aminoethyl)ethylene glycol; O- bis(aminoethyl)tetraethylene glycol; O-bis(aminoethyl)hexaethylene glycol; 1,4- bis(aminomethyl)benzene; l,3-bis(aminomethyl)benzene; 1,4-diaminobutane; 1,2- diaminocyclohexane; 4,4'-diaminodicyclohexylmethane; N-l ,3-diamino-2-propanol; homopiperazine; piperazine; histidine; and lysine. This approach is illustrated in Scheme 13. In the second method (Method B), the chelators are modified with diamines to mimic amino acids and are loaded on a solid support by standard procedure. This is illustrated in Scheme 14. In all cases, at the end of the reaction on a solid support, the product cleavage f om, the resin and removal of side chain protecting groups are carried out simultaneously, thus eliminating several reaction steps required in the conventional approach. This novel method also facilitates the selective functionalization of one of the carboxyl groups as a primary amide by loading the chelator on amide resins (for example, Rink Amide resin. See H. Rink, Tetrahedron Letters, 1987, 28, 3787.). The final chelator-peptide conjugates are purified by HPLC. Depending on the metal chelate desired, each chelating group is designed to assure in vivo stability of the final chelator-peptide conjugates. The synthetic procedures described in this invention are amenable to both solid and solution phase synthesis and are compatible with the synthesis of a combinatorial library of products. In a preferred embodiment, the carboxyl-terminal chelator peptide conjugates according to the present invention have the general formula 5:

S wherein each R₂₀ is H, t-butyl or benzyl; Wg may be selected from the group consisting of (CH₂)_h or (CH₂CH₂O)j wherein h varies from 1 to 10 and j varies from 1 to 50; L is -(CH₂)_t-₅ -CH₂- (CH₂-O-CH₂)_uCH₂- or polyhydroxyalkyl; t varies from 1 to 10; u varies from 1 to 50; (AA)_n is a bioactive peptide with an affinity for a tumor receptor wherein n is 1-50, preferably 3-25; and z varies from 1 to 10, preferably from 1 to 3.

In another preferred embodiment, the carboxyl-terminal chelator peptide conjugates according to the present invention have the general formula 6:

wherein R₂o, L, and (AA)_π are as defined in formula 5; W9 and Wι₀ are defined in the same manner as W₈; W₁₈ is defined in the same manner as Wι₅; and z varies from 1 to 10, preferably from 1 to 3.

In yet another preferred embodiment, the polyazacarboxylic acid bis-peptide conjugates according to the present invention have the general formula 7:

wherein R₂₀, L, and (AA)_n are as defined in formula 5; Wu and W]₂ are defined in the same manner as W₈; and W₁₉ is defined in the same manner as W₁₅.

In another preferred embodiment, the carboxyl-terminal chelator peptide conjugates according to the present invention have the general formula 8:

8 wherein R₂o, L, and (AA)_n are as defined in formula 5; Wι₃ and Wμ are defined in the same manner as W₈; and W₂o is defined in the same manner as W₁₅.

The invention also includes the use of the formulations disclosed herein for the synthesis of a combinatorial library of compounds .

Scheme 1

11

FmocNHCH₂CH₂NH₂

Scheme 2

19

Scheme 3

Bn-NH

20 21

Scheme 3b

C0₂t-Bu

C0₂t-Bu ^Bn°2^C \ Bn0₂C— \ I — C0₂Bn

j t-Bu0₂C ,.Bu0₂C— ' - CQjt-Bu

22b

23b

Scheme 4

(viii) Fmoc-OSu

Scheme 5

28

29

(v) FmocNHC₂ 2H' ₄N' H

30

Scheme 6

Scheme 7

(vii)

40

Q^NHTrtorCC^Bn Q₂ = C0₂HorNH₂

Scheme 8

41 (iii) BrCH ₂C0₂t-Bu 42

(viii) Fmoc-OSu

Scheme 9

Scheme 10

50 (iv) H₂Pd-C

^NHFmoc

H₂N

52

Scheme 11

(i) BrCH₂C0₂Bn (ii) BrCH₂C0₂t-Bu

S3 54

55 56

57

58 Scheme 12

Scheme 13

Scheme 14

Mⁿ⁺

T = -O-, -NH-

65 (?)= Resin M = metal

Compounds of Schemes 1, 4, 5 and 9 to 12 are particularly useful for the synthesis of bis- peptides that have the same receptor affinity where such constructs serve to augment tumor receptor binding and enhance specificity. They are also useful for the synthesis of peptides with affinities towards different receptors but do not cause detrimental intramolecular interactions between each peptide. Compounds of Schemes 2, 3 and 6 to 8 are particularly useful for the synthesis of bis-peptides with affimties for different tumor receptors and minimize intramolecular interaction.

The compositions of the invention can be formulated into diagnostic compositions for enteral or parenteral administration. These compositions contain an effective amount of the dye along with conventional pharmaceutical carriers and excipients appropriate for the type of administration contemplated. For example, parenteral formulations advantageously contain a sterile aqueous solution or suspension of dye according to this invention. Parenteral compositions may be injected directly or mixed with a large volume parenteral composition for systemic administration. Such solutions also may contain pharmaceutically acceptable buffers and, optionally, electrolytes such as sodium chloride.

Formulations for enteral administration may vary widely, as is well known in the art. In general, such formulations are liquids which include an effective amount of the dye in aqueous solution or suspension. Such enteral compositions may optionally include buffers, surfactants, thixotropic agents, and the like. Compositions for oral administration may also contain flavoring agents and other ingredients for enhancing their organoleptic qualities.

The diagnostic compositions are administered in doses effective to achieve the desired enhancement. Such doses may vary widely, depending upon the particular dye employed, the organs or tissues which are the subject of the imaging procedure, the imaging equipment being used, and the like. The diagnostic compositions of the invention are used in the conventional manner. The compositions may be administered to a patient, typically a warm-blooded animal, either systemically or locally to the organ or tissue to be imaged, and the patient is then subjected to the imaging procedure.

A combination of the above represents an important approach to the synthesis and use of novel polyazacarboxylates as chelators and linkers in the preparation of multi-bioactive molecules. The present invention is further detailed in the following Examples, which are offered by way of illustration and are not intended to limit the scope of the invention in any manner.

EXAMPLE 1 Synthesis of

[Scheme 1, lib] A solution of 50 ml of dimethylformamide and benzyl bromoacetate (16.0 g, 70 mmol) was stirred in a 100 ml three-neck flask. Solid potassium bicarbonate (7.8 g, 78 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 (1.9 g, 31 mmol) and 4 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 overnight. The reaction mixture was partitioned between 100 ml of methylene chloride and 100 ml of saturated sodium bicarbonate solution. The layers were separated and the methylene chloride layer was again washed with 100 ml of saturated sodium bicarbonate solution. The combined aqueous layers were extracted twice with 25 ml of methylene chloride. The combined methylene chloride layers were 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 with vacuum at about 45°C. The crude material was left on a vacuum line overnight at room temperature.

< The crude material from above was dissolved in 100 ml of methylene chloride at room temperature. Triphenylphosphine (8.91 g, 34 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 (6.05 g, 34 mmol) was added portionwise over 2 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 200 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 100 ml of ether. The ether solution was decanted and the oil was again triturated with a 100 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 100 ml of ether. The volume of the combined ether abstracts was reduced with vacuum until a volume of about 25 ml was obtained. This was allowed to stand overnight at 0°C. Ether (10 ml) was added to the cold mixture which was mixed to suspend the solid. The mixture was percolated through a column of 45 g of silica gel and eluted with ether and 75 ml fractions were collected. The fractions that contained product by TLC were pooled and the ether removed with vacuum. This gave 10.1 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 with vacuum. This gave 7.4 g (57% yield) of pure product.

EXAMPLE 2 Synthesis of

[Scheme 5, 27] 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 overnight 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 portion-wise 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 500 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 3 ^' Synthesis of

[Scheme 1, 10] N-Benzylethylenediamine (5g, 33.28 mmol) and potassium bicarbonate

(19.3 g, 139.7 mmol) were added to 200 ml of anhydrous acetonitrile and stirred vigorously under argon. t-Butyl bromoacetate (22.7 g, 116.5 mmol) was diluted in 30 ml of anhydrous acetonitrile and the solution was added dropwise to the reaction mixture over 90 minutes. The progress of the reaction was monitored by TLC and was essentially complete in about 4 hours but was stirred at room temperature for about 12 hours in order to assure complete alkylation of the amine. The insoluble residue was filtered and washed with acetonitrile. The filtrate was evaporated to give 20 g of a yellow liquid. Hexane (100 ml) was added to the crude mixture and stirred vigorously until white precipitate formed. The precipitate was filtered and the filtrate was evaporated to give a yellow liquid. The pure compound was obtained by washing the crude product over dry flash chromatographic column and the desired compound was eluted with 10% diethyl ether in hexane (12.5 g, 80%> yield).

EXAMPLE 4 Synthesis of

[Scheme 1, 11] N-benzyl-N,N',N'-tris(t-butyloxycarbonylmethyl) ethylenediamine (10; 6 g, 12 mmol) was added to a heterogeneous mixture of 10% palladium on carbon (6 g, 1 weight equivalent) in 100 ml of methanol. Anhydrous ammonium formate (3.8 g, 60.26 mmol) was added to the reaction mixture in one bulk. The mixture was stirred at room temperature for 2 hours. The mixture was filtered over celite and the residue was washed with chloroform. The filtrate was evaporated until white precipitates began to form. The residue was triturated in chloroform and the insoluble formate was filtered. Evaporation of the filtrate gave a pale yellow liquid (4.6 g, 96% yield) which was identified as the pure compound by NMR analysis.

EXAMPLE 5 Synthesis of

A mixture of benzylamine (10 g, 0.93 mol) and KHCO₃ (35 g, 3.5 mol) in acetonitrile (100 mL) was cooled to 0°C and t-butyl bromoacetate (39 g, 2.0 mol) was added dropwise. After complete addition of the bromide, the mixture was allowed to reach room temperature and stirred for 16 hours. It was filtered and the residue was washed with acetonitrile. The solvent was evaporated from the filtrate. The crude product was taken up in 100 ml of dichloromethane and washed with water (3 X 75 mL). The organic layer was dried with MgSO , filtered and the solvent was evaporated. Further purification was performed by flash chromatography using 10% ether in hexane. This gave 26.6 g (85%) of the pure compound.

EXAMPLE 6

Synthesis of C0₂t-Bu

HN ^ CO_zt-Bu

Two methods were used for the debenzylation of N,N-bis(t-butyloxycarbonylmethyl) benzylamine (from Example 5).

In Method A, a mixture of N,N-bis(t-butyloxycarbonylmethyl) benzylamine (5 g, 14.8 mmol), ammonium formate (2.4 g) and 10% Pd-C (1 g) in methanol (50 mL) was refluxed for 30 minutes. Upon cooling to ambient temperature, the catalyst was filtered over celite and the cake was washed with methanol. The solvent was evaporated and the residue extracted with chloroform. Filtration of the extract and evaporation of the solvent gave the pure secondary amine (3.4 g, 94%) as an oil.

In Method B, a mixture of N,N-bis(t-butyloxycarbonylmethyl) benzylamine (6 g, 19.5 mmol) and 10% Pd-C (0.6 g) in methanol (60 mL) was hydrogenolyzed at 45 psi for 2 hours. The catalyst was filtered over celite and the residue was washed with methanol. The filtrate was evaporated to give the pure compound (4.4 g, 92%).

EXAMPLE 7 Synthesis of

[Scheme 9, 46] Trifluoroacetamide (1 g, 9.7 mmol) in DMF (15 mL) was cooled to 0 °C and NaH (0.5 g, 20.37 mmol,) was added. After 10 minutes stirring, benzyl bromoacetate (4.6 g, 20 mmol) was added dropwise. The mixture was allowed to reach room temperature and stirred for 16 hrs. At the end of the reaction DMF was removed in vacuo at below 40°C. The residue was partitioned into dichloromethane/water. The organic layer was washed twice with water, dried with MgSO and the solvent evaporated. Starting with hexane as eluant, the crude product was purified by flash chromatography, eluting the oily pure compound (2.8 g, 72%) with 25%o ether in hexane.

EXAMPLE 8 Synthesis of

-C0₂Bn

HN C0₂Bn

[Scheme 9, 47] Dissolve the N-trifluoroacetyl-N,N-bis(benzyloxycarbonylmethyl)amine (46; 0.2 mmol) in t-butanol and add anhydrous hydrazine (2 mmol) below 0°C. Stir at this temperature for 4 hours and add dichloromethane to the reaction mixture. Wash the mixture with saturated aqueous NaHCO₃ and dry the organic phase over MgSO₄. Evaporate the solvent in vacuo and use the product immediately as prolonged storage at room temperature leads to formation of side products.

EXAMPLE 9 Synthesis of

[Scheme 2, 17a] N- Alkylation of N-benzyl-N-ethanolamine with t-butyl bromoacetate was carried out as described in Example 2. Final yield of about 90% was obtained. EXAMPLE 10 Synthesis of

[Scheme 2, 17] A mixture of N-benzyl-N-ethanolamine (15 mmol, 1 equiv.) and KHCO₃

(22.5 mmol, 1.5 equiv.) in acetonitrile (100 mL) was cooled to 0°C and t-butyl bromoacetate (19.5 mmol, 1.3 equiv.) was added dropwise. After complete addition of the bromide, the mixture was allowed to reach room temperature and stirred for 2 hours. It was filtered and the residue was washed with acetonitrile. The solvent was evaporated from the filtrate. The crude product was taken up in 100 ml of dichloromethane and washed with water (3 X 75 mL). The organic layer was dried with MgSO , filtered and the solvent was evaporated. Further purification was performed by flash chromatography using 10% ether in hexane.

Removal of the benzyl group by catalytic hydrogenation gave the secondary amine which was alkylated with benzyl bromoacetate as described in Example 1. The alcohol was converted to bromide with triphenylphosphine and N-bromosuccinimide as described in Example 2.

EXAMPLE 11 Synthesis of

N,N,N-dibenzylethanolamine was brominated with triphenylphosphine and N- bromosuccinimide as described in Example 1.

EXAMPLE 12 Synthesis of

A solution of benzyl chloride (28 g, 0.25 mol) in DMF (10 mL) was added dropwise to a mixture of potassium bicarbonate (15 g, 0.15 mol) and 2-aminoethyloxyethanol (10.5 g, 0.1 mol) in 100 mL of DMF. After stirring for 16 hours at room temperature, the mixture was filtered and the filtrate was evaporated. The crude product was partitioned into water/dichloromethane. The organic layer was washed with water, then brine and then dried over MgSO . The solvent was evaporated and the product was isolated by flash column chromatography starting with hexane and eluting the compound with 60% ethyl acetate in hexane as a pale yellow oil (20 g, 70% yield).

EXAMPLE 13 Synthesis of

A mixture of trityl chloride (10 g, 36 mmol), tetraethyleneglycol (70 g, 360 mmol) and pyridine (4.25 g, 54 mmol) was heated at 45 °C for 16 hours. An equal volume of water was added after reaction. The mixture was centrifuged in order to accelerate the separation of phases.

The aqueous phase was decanted and the sticky product was dissolved in toluene and washed thrice with water. The organic layer was dried over MgSO₄ and the solvent was evaporated. The crude intermediate product was purified by flash chromatography to give the monotrityl tetraethyleneglycol intermediate (12.7 g, 80% yield) as pale yellow oil.

The monotrityl tetraethyleneglycol (28 mmol)) was dissolved in anhydrous dichloromethane (200 mL) and cooled to -20 °C. After addition of triethyl amine (36.75 mmol), methanesulfonyl chloride (35 mmol) was introduced dropwise. The solution was stirred at this temperature for 20 minutes then allowed to warm up to room temperature. After 3 hours, the hydrochloride salt was filtered off and the filtrate was washed twice with water then brine. Drying with MgSO₄ and removal of the solvent gave the pure monotrityl tetraethyleneglycol mesylate (93%).

A heterogeneous mixture of the mesylate (5 mmol), N,N-dibenzylaminoethyloxyethanol (4.2 mmol) and KOH (17 mmol) was refluxed for 20 hours. The mixture was filtered and the solvent evaporated. The residue was partitioned into water/dichloromethane. The organic layer was separated and washed with water, then brine. After drying with magnesium sulfate, the solvent was evaporated and the residue was purified by flash chromatography, starting with hexane and eluting the monotrityl N,N-dibenzylaminohexaethyleneglycol with 40%) ether in hexane. The monotrityl N₃N-dibenzylaminohexaethyleneglycol was hydrogenated to give the α,ω- aminoalcohol of hexaethyleneglycol. The primary amine was tritylated with trityl chloride and bromination of the primary alcohol was carried out with triphenylphosphine and NBS as described in Example 1.

EXAMPLE 14 Synthesis of

Reaction of pentaethylenegl col (50 mmol) with t-butyl propiolate (5 mmol) at room temperature for 5 hours and subsequent hydrogenation with 10% Pd-C at 45 psi gives the t- butyloxycarbonylhexaethyleneglycol. Bromination of the free primary alcohol is carried out with triphenylphosphine and NBS as described in Example 1. Removal of the t-butyl ester with HCl (1 M, 30 mL, 3 hours) and esterification of the acid with benzyl alcohol in the presence of dimethylaminopyridine gives the desired compound which could be purified by dry flash chromatography.

EXAMPLE 15 Synthesis of

Til— NH

Dropwise addition of trityl chloride (15 mmol, 1 equiv.) in dichloromethane to a solution of ethanolamine (30 mmol, 2 equiv.) in DMF at 0 °C and stirring at this temperature for 6 hours gives a yellow solution. Evaporation of the solvents at below 40°C gives a solid residue which is partitioned between ether and water. Dry the ether phase over MgSO and evaporate the solvent to obtain the crude product which is readily purified by dry flash chromatography, eluting the pure compound with 30% ethyl acetate in hexane. The alcohol was converted to bromide with triphenylphosphine and N-bromosuccinimide as described in Example 2. A variation of the above procedure begins with the reaction of commercially available

2-aminoethyl bromide with trityl chloride. In this procedure, there is no need for the additional step required for bromination.

EXAMPLE 16 Synthesis of

[Scheme 7, 38] Reaction of N-benzylaminoethanol (5 mmol) with t-butyl bromoacetate (5.2 mmol) gives a tertiary amine. The benzyl group is removed by catalytic hydrogenolysis with 10%) Pd/C at 40 psi in methanol for 4 hours. After filtration of the catalyst, the solvent is evaporated and the resulting secondary amine is immediately alkylated with benzyl bromopentaethyleneglycolacetate in acetonitrile at reflux for 24 hours. Conversion of the alcohol with triphenylphosphine and NBS is carried out as described in Example 2.

EXAMPLE 17 Synthesis of

[Scheme 1, 12] A mixture of N,N',N'-tris(t-butyloxycarbonylmethyl) ethylenediamine

(4.4 g, 9.82 mmol) and 2-[Bis-(benzyloxycarbonylmethyl)amino]ethyl bromide (5.3 g, 12.76 mmol) was added to a solution of ethyldiisopropylamine (3.8 g, 29.45 mmol) in 100 ml acetonitrile. The mixture was stirred at reflux for 24 hours under nitrogen. After the reaction was complete, the solvent was evaporated and the residue was partitioned between dichloromethane (100 ml) and distilled water (100 ml). The organic layer was washed with 100 ml of water and 100 ml of brine. It was dried over magnesium sulfate and the solvent was evaporated to give about 10 g of the crude product. The product was purified by dry flash chromatography and the pure compound was eluted with 40% of diethyl ether in hexane as a pale yellow liquid (6.5 g, 90% yield).

EXAMPLE 18

Synthesis of

[Scheme 1, 13] A mixture of 10% palladium on carbon (0.21 g) and a solution of

N,N N^,-tiis(t-butyloxycarbonylmemyl)-N\N"-bis(benzyloxycarbonylmethyl) diethylene1riamine (3.3 g, 4.45 mmol) in 50 ml of methanol was hydrogenolyzed at 40 psi for 2 hours. The mixture was filtered over celite and the residue was washed with methanol. The solvent was evaporated to give an off-white powder which was shown by mass spectral analysis, HPLC and NMR to be the pure compound (2.4 g, 96% yield).

EXAMPLE 19

Synthesis of

[Scheme 1, 14] Tri-t-butyl diethylenetriaminepentaacetic acid (5.46 g, 9.72 mmol, 1 equiv.) in 20 mL DMF and dicyclohexylcarbodiimide (DCC, 2 g, 9.72 mmol, 1 equiv.) in the presence of a catalytic amount of dimethylaminopyridine (DMAP) (0.1 equiv.) were stirred at room temperature for 1 hour and mono-Fmoc ethylenediamine (2.74 g, 9.72 mmol, 1 equiv.) was added. The resulting mixture was stirred for 6 hours at room temperature and the crude product was partitioned between dichloromethane and saline. The organic phase was washed with water and dried over MgSO₄. The solvent was evaporated and the DCC urea formed was precipitated with ether. After filtration, the solvent was evaporated and the product was purified by dry flash chromatography on silica gel, eluting the compound with ethyl acetate.

EXAMPLE 20 Synthesis of

[Scheme 9, 44] A mixture of N,N\N'-tris(t-butyloxycarbonylmethyl) ethylenediamine (11; 4.4 g, 9.82 mmol), N,N-benzyloxycarbonylmethyl-t-butyloxycarbonylmethylaminoethyl bromide (17;12.76 mmol) and diisopropylethylamine (3.8 g, 29.45 mmol) in 100 ml acetonitrile was heated at reflux for 18 hours. After the reaction was complete, the solvent was evaporated and the residue was partitioned between dichloromethane (100 ml) and distilled water (100 ml). The organic layer was washed with water and brine in that order. It was dried over MgSO₄ and the solvent was evaporated. The crude product was purified by dry flash chromatography and the pure compound was eluted with 40% of diethyl ether in hexane. Hydrogenolysis of the benzyl ester was carried out as described in Example 18.

EXAMPLE 21 Synthesis of

[Scheme 9, 48] Reaction of the monocarboxylic acid tetra-t-butyl diethylenetriaminepentaacetic acid (DTPA) (44; 1 mmol) with N,N- dibenzyloxycarbonylmethylamine (47; 1.2 mmol), diisopropylethylamine (1.2 mmol) and 2-(lH- Benzotriazole-l-yl)-l,l,3,3-tetramethyluronium hexafluorophosphate (HBTU) (1.1 mmol) in 39 butanol at 0 °C and react the ensuing secondary amine with N-tritylethyl bromide as described in Example 17. Catalytic hydrogenation of the product with 10% Pd-C catalyst and subsequent protection of the free amine with Fmoc-succinimide yields the desired compound which can be purified by flash chromatography.

EXAMPLE 24 Synthesis of

The product is prepared as described in Example 23 starting with benzyl diethylenetriamine.

EXAMPLE 25 Synthesis of

[Scheme 6, 35] Add triglycine (31) to DMF and diisopropylethylamine. Slowly add benzoyl chloride at 0°C. After addition is complete, gently evaporate the solvent and purify the

• intermediate by dry flash chromatography. Redissolve the benzylamide in DMF and transfer it to a pressure bottle. Activate the free carboxyl group with HBTU for 30 minutes and cool the mixture to 0°C. Charge the pressure bottle with ammonia and seal the bottle. Stir the mixture for 4 hours, then cool it to 0°C before opening the bottle. Purify the primary amide (32) by flash chromatography and reduce the tetraamide with lithium aluminum hydride. Selectively protect the ensuing primary amine with ethyl trifluoroacetate to give the intermediate orthogonally- protected secondary amine (33). Add t-butyl bromoacetate to a mixture of 33 and potassium carbonate in acetonitrile and stir the mixture at room temperature for 16 hours. Evaporate the solvent and purify the resulting product. Selectively remove the benzyl protecting group by 40 catalytic hydrogenolysis with 10% Pd-C and alkylate the secondary amine with N- tritylaminotetraethyleneglycolethyl bromide to give 34. Remove the trifluoroacetyl group as described in Example 8 and alkylate the secondary amine with benzyl bromoacetate. Catalytic hydrogenolysis at 50 psi with 10% Pd-C in methanol removes both N-trityl and benzyl ester to give the unprotected amino acid. Reaction of the free amine with Fmoc-succinimide yields compound 35.

EXAMPLE 26 Synthesis of

[Scheme 5, 28] A mixture of 2-[Bis-(t-butyloxycarbonylmethyl)arnino]ethyl bromide (27; 6.0 g, 17.05 mmol), diisopropylethylamine (4.4 g, 34.1 mmol) and benzylamine (0.9 g, 8.41 mmol) in 100 ml of anhydrous acetonitrile was refluxed for 16 hours under argon. After reaction, the solvent was evaporated en vacuo and the residue was partitioned between dichloromethane (100 ml) and water (100 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 7 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 4.2 g (76%) of the pure compound as a pale yellow liquid.

A mixture of 10%) palladium on carbon (0.4 g) and a solution of the purified intermediate N'-berιzyl-N,N''-tetrakis(t-butyloxycarbonylmethyl)-diethylenetriamine (6.16 mmol) in 100 ml of methanol was hydrogenolyzed at 50 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 product (95%) as a viscous oil. 41

EXAMPLE 27 Synthesis of

[Scheme 5, 29] A mixture of 2-[Bis-(benzyloxycarbonylmethyl)amino]ethyl bromide (lib; 17.05 mmol), diisopropylethylamine (34.1 mmol) and N,N"-tetrakis(t- butyloxycarbonylmethyl) diethylenetriamine (28;15 mmol) in 200 ml of anhydrous acetonitrile was refluxed for 16 hours under argon. After reaction, the solvent was evaporated in vacuo and the residue was partitioned between dichloromethane (200 ml) and water (200 ml). The two layers formed were separated and the organic phase was washed with water (200 ml) and brine (200 ml) in that order. The dichloromethane layer was dried over magnesium sulfate and the solvent was removed in vacuo to give a viscous liquid residue which was dissolved in hexane and purified by dry flash chromatography with 20% diethyl ether in hexane to give the pure compound (65%) as a pale yellow liquid. The benzylester was removed by catalytic hydrogenation in methanol (200 mL) with 10% palladium on carbon (0,4 g) at 50 psi for 1 hour. The mixture was filtered over celite and the residue was washed with methanol (2 50 ml). The solvent was evaporated to give the pure product .

42

EXAMPLE 28 Synthesis of

[Scheme 5, 30] Activation of N'- [Bis-(carboxylmethyl)amino]ethyl-N,N"-tetrakis(t- butyloxycarbonylmethyl) diethylenetriamine (29; 5 mmol, 1 equiv.) with HBTU (5.1 mmol) and diisopropylethylamine (10 mmol) in 40 mL DMF for 1 hour and subsequent reaction of the intermediate with mono-Fmoc ethylenediamine (5 mmol, 1 equiv) at room temperature for 6 hours gives a heterogeneous mixture. Partition the mixture between dichloromethane and saline and wash the organic phase with water. Dry the dichloromethane solution over MgSO and evaporate the solvent to give the crude product which is readily purified by dry flash chromatography, starting with 10% ethyl acetate in hexane and eluting the pure compound with ethyl acetate.

EXAMPLE 29 Synthesis of

[Scheme 11, 55] Cyclen [1,4,7,10-tetraazacyclododecane] (53; 2.9 g, 16.8 mmol) was dissolved in chloroform (50 mL) and a solution of benzyl bromoacetate (1.92, 8.4 mmol) in acetonitrile was added dropwise. The mixture was stirred for 1.5 hours and the solvent was evaporated to give an oil which was purified by flash chromatography to give monobenzyloxycarbonylmethylcyclen (54; 2 g, 75%). 43 t-Butyl bromoacetate (3.5 g, 18 mmol) in 5 mL acetonitrile was added dropwise to a mixture of cyclen mono-benzyl ester (1.41 g, 4.4 mmol) and K₂CO₃ (2.5 g, 18 mmol) in acetonitrile (25 mL). The resulting mixture was stirred at room temperature for 2 hours and the salt was filtered. The filtrate was evaporated and the residue was purified by flash chromatography to give the N-benzyloxycarbonylmethyl-N',N"N'"-tris(t- butyloxycarbonyImethyl)cyclen (55; 3 g).

EXAMPLE 30 Synthesis of

[Scheme 11, 57] The benzyl ester of benzyloxycarbonylmethyl-N',N"N'"-tris(t- butyloxycarbonylmethyl)cyclen (55, 3 g) was removed by catalytic hydrogenation using 10% Pd- C as described in Example 18. React the cyclen monoacetic acid (56) with N,N- bis(benzyloxycarbonylmethyl)amine (47) as described in Example 19 and hydrogenolyze the dibe zyl ester as described in Example 18 to give compound 57.

EXAMPLE 31 Synthesis of

[Scheme 11, 58] Reaction of mono-Fmoc ethylenediamine with the dicarboxylic acid 57 from Example 30 follows the same procedure described in Example 19. 44

EXAMPLE 32 Synthesis of

[Scheme 12, 60] Reaction of N^,,N",N'"-tris(t-butyloxycarbonylmethyl)cyclen (59; 1 mmol) wim bis(benzyloxycarbonylmethyl)aminoethyl bromide (lib; 1.1 mmol) as described in Example 17 gives the dibenzyl ester which was hydrogenolyzed as described in Example 18.

EXAMPLE 33 Synthesis of

[Scheme 12, 61] Reaction of N-trityl-pentaethyleneglycolethyl bromide (2 .1 mmol) with N,N-benzylethanolamine (2.0 mmol) in acetonitrile at room temperature in the presence of K₂CO₃ (2 mmol) gives N'-trityl-pentaethyleneglycolethyl-N-benzylethanol and the alcohol is brominated with triphenylphosphine and NBS as described in Example 1. Conjugation of the bromide to N",N'",N""-tris(t-butyloxycarbonylmethyl)cyclen [59] and subsequent removal of the N-benzyl group gives the secondary alkylamine. Reaction of this amine with benzyl bromoacetate and removal of the benzyl group yields the desired product [61], 45

EXAMPLE 34 Synthesis of

The reaction of N",N'",N""-tris(t-butyloxycarbonylmethyl)cyclen (59) with N,N- bis(benzyloxycarbonylmethyl)-N-pentaethyleneglycolethyl bromide as described in Example 17 gives the dibenzyl ester which was hydrogenolyzed as described in Example 18 to give the dicarboxylic acid.

EXAMPLE 35 Synthesis of

The procedure for the conjugation of the mono-Fmoc ethylenediamine with the dicarboxylic acid of Example 34 is the same as in Example 22.

EXAMPLE 36

Synthesis of carboxyl terminal peptide-chelator conjugate, method A

X = OH or NH₂ ; Peptide = Octreotate for somatostatin receptor positive tumors 46

[Scheme 13, 64] Bromomethyl Wang resin (162 mg, 0.178 mmol) in DMF (3 mL) was placed in a plastic tube and allowed to stand at room temperature for 15 minutes in order to swell the resin. Diisopropylethylamine (46 mg, 0.356 mmol), tri-t-butyl DTPA (200 mg, 0.356 mmol) and cesium iodide (70 mg, 0.267 mmol) were added to the resin in DMF. The resin and reagents were gently mixed by shaking at room temperature for 18 hours. The crude mixture was washed with DMF (3 X 5 mL), methanol (3 X 5 mL) and tetrahydrofuran (2 X 10 mL) in that order. The resin was dried under vacuum overnight (0 mm Hg, 26°C) in the presence of KOH. A portion of the dried resin (38 mg, 0.0418 mmol assuming complete DTPA attachment) was activated with 2-(lH-benzotriazole-lyl)-l,l,3,3-tetramethyluronium hexafluorophosphate (HBTU, 0.2 M in DMSO, 0.042 mmol) and N-hydroxybenzotriazole (HOBt, 0.2 M in DMSO, 0.042 mmol) in the presence of diisopropylethylamine (0.4 M in DMSO, 0.085 mmol) for 1 hour. After activation, mono-Fmoc ethylenediamine (40 mg, 0.125 mmol) was added to the reaction mixture and mixed for 3 hours. The resin was washed and dried as described above. Fmoc loading analysis was carried out by ultraviolet light analysis and indicated a loading of about 0.2 mmol/g of resin. A fraction of the intermediate product was cleaved from the resin with 85%) TFA, 5% water, 5% thioanisole, and 5% phenol for 1 hour and analyzed by HPLC and MS in order to confirm the purity and identity of the intermediate compound. The DTPA-Octreotate conjugate was prepared by solid phase synthesis using the pre-loaded Fmoc-ethylenediamine-DTPA Wang resin on 0.025 mmol scale. A 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. A 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) in the presence of diisopropylethylamine. The amino acids and tri-t-butyl DTPA cartridges were placed on the peptide synthesizer and the product was synthesized from the C-terminal to the N-terminal position.

The product was cleaved from the solid support with a cleavage mixture containing TFA (85%):water (5%):phenol (5%):thioanisole (5%) for 6 hours. Note that the t-butyl esters of tri-t- butyl DTPA were also cleaved during this process. The DTPA-peptide conjugate was precipitated 47 with t-butyl methyl ether and lyophilized with water : acetonitrile (2:3) mixture. The crude product was purified by HPLC to give the desired product as shown by mass spectral analysis. EXAMPLE 37 Synthesis of carboxyl terminal peptide-chelator conjugate, method B

X = OH or H₂; Peptide = Octreotate for somatostatin receptor positive tumors

[Scheme 14, 64] In this method, the mono-Fmoc ethylene diamine tri-t-butyl DTPA (14, Example 19) was used in place of tri-t-butyl DTPA. This procedure permitted the automatic synthesis of the carboxyl terminal peptide-DTPA conjugate without interruption. The disulfide bond was formed and the peptide on solid support was cleaved as described in the preceding example.

EXAMPLE 38

Synthesis of carboxyl terminal peptide-chelate conjugate

X = OH or NH₂; Peptide = Octreotate for somatostatin receptor positive tumors; M = indium-115 (ⁿ⁵In)

[Scheme 14, 65] The ^{1 I5}In-DTPA-peptide complex was prepared by reacting the DTPA- peptide (64, 50 mmol) with ¹¹⁵InCl₃ (90 mmol) in 170 μL of aqueous HCl (5 nM) at room temperature for 30 minutes. The solution was purified by HPLC and lyophilized to obtain the desired compound.

Claims

48WHAT IS CLAIMED IS:

1. A method for preparing a peptide-polyazacarboxylate composition at the C-terminal carboxyl group of a peptide comprising the steps of:

(a) preparing an orthogonally protected dicarboxylic acid of a polyazacarboxylic acid ester of general formula 1:

1 wherein R_\ to R₄ may be the same or different and are selected from the group consisting of alkyl, aryl, heterocarbocyclic, NH-k-NHR ₀, CH₂CO₂H, hydroxyl, amino, Cl-ClO alkoxyl, Cl-ClO aryloxyl, Cl-ClO polyalkoxyalkyl, -CH₂(CH₂-O-CH₂)_b-CH₂-R_a, C1-C20 polyhydroxyalkyl, Cl-ClO polyhydroxyaryl, and X-Y; W is selected from the group consisting of alkyl, aryl, -CH₂(CH₂-O-CH2)b-CH₂-Ra, polyhydroxyalkyl, and polyhydroxyaryl; X is selected from the group consisting of NH-, CONH-, -CH₂NH-, - CH₂NR₅-, -COO-, -O-, -C(O)-, -S-, -NHCO-, and -NHC(S)-; Y is selected from the group consisting of H, CH₂COOH, peptide and biomolecule; b varies from 1-100; R_a may be H, OH, -O-, alkyloxy, aryl alkyl or single bond; R₅ is as defined for Ri; R₃₀ is an amine protecting group; k is alkyl, aryl, heterocarbocyclic, CH₂CO₂H, Cl-ClO alkoxyl, Cl-ClO aryloxyl, Cl-ClO polyalkoxyalkyl, -CH2(CH2-O-CH₂)b-CH2- _a, C1-C20 polyhydroxyalkyl, Cl-ClO polyhydroxyaryl, carbocyclic, heterocyclic, or X-Y; and z varies from 1-10;

(b) activating the dicarboxylic acid of step (a) to form a reactive intermediate;

(c) reacting the reactive intermediate of step (b) with a functionalized resin to form a resin-bound polyazacarboxylic acid ester of general formula 67: 49

67

(d) activating the resin-bound monocarboxylic acid of step (c) to form an active ester;

(e) reacting the active ester of step (d) with a diamine to yield a compound of general formula 68:

68

(f) removing any amine protecting group from R ₀;

(g) synthesizing a desired peptide at the diamine on said compound of formula 68 to form a peptide-polyazacarboxylate conjugate of formula

and

(h) cleaving the peptide-polyazacarboxylate conjugate from the resin and removing all protecting groups; wherein T is -O-, -NH-, -S-, alkyl, aryl, CH₂(CH₂OCH₂)_bCH₂-. 50

The method of claim 1 wherein said polyazacarboxylate is selected from the group consisting of:

The method of claim 1 wherein said diamine is selected from the group consisting of: ethylenediamine; bis(2-aminoethyl)-ether; O-bis(aminoethyl)ethylene glycol; O- bis(aminoethyl)tetraethylene glycol; O-bis(aminoethyl)hexaethylene glycol; 1,4- bis(aminomethyl)benzene; l,3-bis(aminomethyl)benzene; 1,4-diaminobutane; 1,

2- diaminocyclohexane; 4,4'-diaminodicyclohexylmethane; N-1 ,

3-diamino-2-propanol; homopiperazihe; piperazine; histidine; and lysine. 51

4. The method of claim 1 wherein said diamine is an orthogonally protected diamine.

5. The method of claim 1 wherein R30 is selected from the group consisting of H, Fmoc and Boc.

6. A method for preparing a peptide-polyazacarboxylate composition at the C-terminal carboxyl group of a peptide comprising the steps of:

(a) preparing an orthogonally protected monocarboxylic acid of a polyazacarboxylic amino acid ester of general formula 1:

1 wherein one of Ri to Rj is OH; R\ to R4 may be the same or different and are selected from the group consisting of alkyl, aryl, heterocarbocyclic, NH-k-NHR₃₀, CH₂CO₂H, hydroxyl, amino, Cl -CIO alkoxyl, Cl-ClO aryloxyl, Cl-ClO polyalkoxyalkyl, -CH₂(CH₂- O-CH₂)_b-CH₂-Ra, C1-C20 polyhydroxyalkyl, Cl-ClO polyhydroxyaryl, and X-Y; W is selected from the group consisting of alkyl, aryl, -CH₂(CH2-O-CH2)b-CH2-R_a, polyhydroxyalkyl, and polyhydroxyaryl; X is selected from the group consisting of NH-, CONH-, -CH₂NH-, -CH2NR₅-, -COO-, -O-, -C(O)-, -S-, -NHCO-, and -NHC(S)-; Y is H, CH₂COOH, peptide, biomolecule, an Fmoc protected amine or a Boc protected amine; b varies from 1-100; R_a may be H, OH, -O-, alkyloxy, aryl, alkyl or single bond; R₅ is as defined for Rj; R₃₀ is an amine protecting group; k is alkyl, aryl, heterocarbocyclic, CH₂CO₂H, Cl-ClO alkoxyl, Cl-ClO aryloxyl, Cl-ClO polyalkoxyalkyl, -CH₂(CH₂-O- CH₂)_b-CH₂-R_a, C1-C20 polyhydroxyalkyl, Cl-ClO polyhydroxyaryl, carbocyclic, heterocyclic, or X-Y; and z varies from 1-10;

(b) activating the monocarboxylic acid of step (a) to form a reactive intermediate;

(c) reacting the reactive intermediate of step (b) with a functionalized resin to form a resin-bound polyazacarboxylic amino acid ester of general formula 68: 52 ^'

68

(d) removing any amine protecting group from R₃₀;

(e) synthesizing a desired peptide at the diamine on said compound of formula 68 to form to form a peptide-polyazacarboxylate conjugate of formula

;and (f) cleaving the peptide-polyazacarboxylate conjugate from the resin and removing all protecting groups; wherein T is -O-, -NH-, -S-, alkyl, aryl, CH₂(CH₂OCH₂)_bCH₂-.

7. The method of claim 6 wherein said polyazacarboxylate is selected from the group consisting of:

53

8. The method of claim 6 wherein said diamine is selected from the group consisting of: ethylenediamine; bis(2-aminoethyl)-ether; O-bis(aminoethyl)ethylene glycol; O- bis(aminoethyl)tetraethylene glycol; O-bis(aminoethyl)hexaethylene glycol; 1,4- bis(aminomethyl)benzene; l,3-bis(aminomethyl)benzene; 1 ,4-diaminobutane; 1,2- diaminocyclohexane; 4,4'-diaminodicyclohexylmethane; N-1 ,3-diamino-2-propanol; homopiperazine; piperazine; histidine; and lysine.

9. The method of claim 6 wherein said diamine is an orthogonally protected diamine.

10. The method of claim 6 wherein R₃₀ is selected from the group consisting Fmoc and Boc.

11. A method for preparing a peptide-polyazacarboxylate composition at the C-terminal carboxyl group of a peptide comprising the steps of:

(a) preparing ah orthogonally protected dicarboxylic acid of a polyazacarboxylic acid ester of general formula la: 54

la wherein R_t to R₄ may be the same or different and are selected from the group consisting of alkyl, aryl, heterocarbocyclic, NH-k-NHR₃₀, CH₂CO2H, hydroxyl, amino, Cl-ClO alkoxyl, Cl-ClO aryloxyl, Cl-ClO polyalkoxyalkyl, -CH₂(CH2-O-CH₂)b-CH2-Ra, C1-C20 polyhydroxyalkyl, Cl-ClO polyhydroxyaryl, and X-Y; W is selected from the group consisting of alkyl, aryl, -CH₂(CH2-O-CH₂)_b-CH₂-R_a, polyhydroxyalkyl, and polyhydroxyaryl; X is selected from the group consisting of NH-, CONH-, -CH₂NH-, - CH₂NR₅-, -COO-, -O-, -C(O)-, -S-, -NHCO-, and -NHC(S)-; Y is selected from the group consisting of H, CH₂COOH, peptide and biomolecule; b varies from 1-100; R_a may be H, OH, -O-, alkyloxy, aryl, alkyl or single bond; R5 is as defined for Ri; R₃₀ is an amine protecting group; k is alkyl, aryl, heterocarbocyclic, CH₂CO2H, Cl-ClO alkoxyl, Cl-ClO aryloxyl, Cl-ClO polyalkoxyalkyl, -CH2(CH₂-O-CH₂)b-CH2-Ra, C1-C20 polyhydroxyalkyl, Cl-ClO polyhydroxyaryl, carbocyclic, heterocyclic, or X-Y; and z varies from 1-10;

(c) reacting the reactive intermediate of step (b) with a functionalized resin to form a resin-bound polyazacarboxylic acid ester of general formula 69:

55

69

•>

(e) reacting the active ester of step (d) with a di-Uiήne to yield a compound of general formula 70:

70

(f) removing any amine protecting group from R₃₀;

(g) synthesizing a desired peptide at the diamine on said compound of formula 70 to form a peptide-polyazacarboxylate conjugate of formula

56

and

(h) cleaving the peptide-polyazacarboxylate conjugate from the resin and removing all protecting groups; wherein T is -O-, -NH-, -S-, alkyl, aryl, CH₂(CH₂OCH₂)_bCH₂-.

12. The method of claim 11 wherein said polyazacarboxylic acid is

29

13. The method of claim 11 wherein said diamine is selected from the group consisting of: ethylenediamine; bis(2-aminoethyl)-ether; O-bis(aminoethyl)ethylene glycol; O- bis(aminoethyl)tetraethylene glycol; O-bis(aminoethyl)hexaethylene glycol; 1,4- bis(aminomethyl)benzene; l,3-bis(aminomethyl)benzene; 1,4-diaminobutane; 1,2- diaminocyclohexane; 4,4'-diaminodicyclohexylmethane; N-1 ,3-diamino-2-propanol; homopiperazine; piperazine; histidine; and lysine. 57

14. The method of claim 11 wherein said diamine is an orthogonally protected diamine.

15. The method of claim 11 wherein R₃₀ is selected from the group consisting of H, Fmoc and Boc.

16. A method for preparing a peptide-polyazacarboxylate composition at the C-terminal carboxyl group of a peptide comprising the steps of:

(a) preparing an orthogonally protected monocarboxylic acid of a polyazacarboxylic acid ester of general formula la:

la wherein one of Ri to R4 is OH; Rj to Rt may be the same or different and are selected from the group consisting of alkyl, aryl, heterocarbocyclic, NH-k-NHR₃o, CH₂CO₂H, hydroxyl, amino, Cl-ClO alkoxyl, Cl-ClO aryloxyl, Cl -CIO polyalkoxyalkyl, -CH₂(CH₂- O-CH₂)_b-CH₂-R_a, C1-C20 polyhydroxyalkyl, Cl-ClO polyhydroxyaryl, and X-Y; W is selected from the group consisting of alkyl, aryl, -CH₂(CH2-O-CH₂)b-CH₂-Ra, polyhydroxyalkyl, and polyhydroxyaryl; X is selected from the group consisting of NH-, CONH-, -CH2NH-, -CH₂NR5-, -COO-, -O-, -C(O)-, -S-, -NHCO-, and -NHC(S)-; Y is H, CH₂COOH, peptide, biomolecule, an Fmoc protected amine or a Boc protected amine; b varies from 1-100; R_a may be H, OH, -O-, alkyloxy, aryl, alkyl or single bond; R₅ is as defined for Ri; R₃₀ is an amine protecting group; k is alkyl, aryl, heterocarbocyclic, CH₂CO₂H, Cl-ClO alkoxyl, Cl-ClO aryloxyl, Cl-ClO polyalkoxyalkyl, -CH₂(CH₂-O- CH₂)_b-CH₂-R_a, C1-C20 polyhydroxyalkyl, Cl-ClO polyhydroxyaryl, carbocyclic, heterocyclic, or X-Y; and z varies from 1-10;

(b) activating the monocarboxylic acid of step (a) to form a reactive intermediate; 58

(c) reacting the reactive intermediate of step (b) with a functionalized resin to form a resin-bound polyazacarboxylic acid ester of general formula 70:

70

(d) removing any amine protecting group from R₃₀;

(e) synthesizing a desired peptide at the diamine on said compound of formula 70 to form a peptide-polyazacarboxylate conjugate of formula

and

(f) cleaving the peptide-polyazacarboxylate conjugate from the resin and removing all protecting groups; wherein T is -O-, -NH-, -S-, alkyl, aryl, CH₂(CH₂OCH₂)_bCrk..

17. The method of claim 16 wherein said polyazacarboxylic acid is 59

18. The method of claim 16 wherein said diamine is selected from the group consisting of: ethylenediamine; bis(2-aminoethyl)-ether; O-bis(aminoethyl)ethylene glycol; O- bis(aminoethyl)tetraethylene glycol; O-bis(aminoethyl)hexaethylene glycol; 1,4- bis(aminomethyl)benzenej l,3-bis(aminomethyl)benzene; 1,4-diaminobutane; 1,2- diaminocyclohexane; 4,4'-diaminodicyclohexylmethane; N-l,3-diamino-2-propanol; homopiperazine; piperazine; histidine; and lysine.

19. The method of claim 16 wherein said diamine is an orthogonally protected diamine.

20. The method of claim 16 wherein R₃₀ is selected from the group consisting of Fmoc and Boc.

21. A method for preparing a peptide-polyazacarboxylate composition at the C-terminal carboxyl group of a peptide comprising the steps of:

(a) preparing an orthogonally protected dicarboxylic acid of a polyazacarboxylic acid ester of general formula 2:

60 wherein Re to Rio may be the same or different and are selected from the group consisting of alkyl, aryl, heterocarbocyclic, NH-k-NHR₃₀, CH₂CO₂H, hydroxyl, amino, C1-G10 alkoxyl, Cl-ClO aryloxyl, Cl-ClO polyalkoxyalkyl, -CH^CH O-CH^_b-CH R_a, C1-C20 polyhydroxyalkyl, Cl-ClO polyhydroxyaryl, and X2-Y2; W₂ and W₃ are selected from the group consisting of alkyl, aryl, -CH2(CH2-O-CH2)b-CH2-R_a, polyhydroxyalkyl, and polyhydroxyaryl; X₂ is selected from the group consisting of NH-, CONH-, -CH₂NH-, - CH₂NR₅-, -COO-, -O-, -C(O)-, -S-, -NHCO-, and -NHC(S)-; Y is selected from the group consisting of H, CH₂COOH, peptide and biomolecule; W1₅ is C=O, CH₂, or OC₂H₄; b varies from 1-100; Ra may be H, OH, -O-, alkyloxy, aryl, alkyl or single bond; R₅ is as defined for Re; R₃o is an amine protecting group; k is alkyl, aryl, heterocarbocyclic, CH₂CO₂H, Cl-ClO alkoxyl, Cl-ClO aryloxyl, Cl-ClO polyalkoxyalkyl, -CH₂(CH₂-O- CH₂)_b-CH₂-R_a, C1-C20 polyhydroxyalkyl, Cl-ClO polyhydroxyaryl, carbocyclic, heterocyclic, or X-Y; and z varies from 1-10;

(c) reacting the reactive intermediate of step (b) with a functionalized resin to form a resin-bound polyazacarboxylic acid ester of general formula 71:

(d) activating the resin-bound monocarboxylic acid of step (c);

(e) reacting the activated acid of step (d) with a diamine to yield a compound of general formula 72: 61

(f) removing any amine protecting group from R₃₀;

(g) synthesizing a desired peptide at the diamine on said compound 72 to form a peptide- polyazacarboxylate conjugate of formula

and

22. The method of claim 21 wherein said polyazacarboxylate is

62

23. The method of claim 21 wherein said diamine is selected from the group consisting of: ethylenediamine; bis(2-aminoethyl)-ether; O-bis(aminoethyl)ethylene glycol; O- bis(aminoethyl)tetraethylene glycol; O-bis(aminoethyl)hexaethylene glycol; 1,4- bis(aminomethyl)benzene; l,3-bis(aminomethyl)benzene; 1,4-diaminobutane; 1,2- diaminocyclohexane; 4,4'-diaminodicyclohexylmethane; N-1 ,3-diamino-2-propanol; homopiperazine; piperazine; histidine; and lysine.

24. The method of claim 21 wherein said diamine is an orthogonally protected diamine.

25. The method of claim 21 wherein R₃₀ is selected from the group consisting of H, Fmoc and Boc.

26. A method for preparing a peptide-polyazacarboxylate composition at the C-terminal carboxyl group of a peptide comprising the steps of:

(a) preparing an orthogonally protected monocarboxylic acid of a polyazacarboxylic acid ester of general formula 2:

wherein one of Rg to R₁₀ is OH; Rg to Rio may be the same or different and are selected from the group consisting of alkyl, aryl, heterocarbocyclic, NH-k-NHR₃o, CH₂CO₂H, hydroxyl, amino, Cl-ClO alkoxyl, Cl-ClO aryloxyl, Cl-ClO polyalkoxyalkyl, -CH₂(CH₂- O-CH₂)_b-CH₂-R_a, C1-C20 polyhydroxyalkyl, Cl -CIO polyhydroxyaryl, and X₂-Y₂; W₂ and W₃ are selected from the group consisting of alkyl, aryl, -CH₂(CH₂-O-CH₂)b-CH₂-R_a, polyhydroxyalkyl, and polyhydroxyaryl; X₂ is selected from the group consisting of NH-, CONH-, -CH₂NH-, -CH₂NR₅-, -COO-, -O-, -C(O)-, -S-, -NHCO-, and -NHC(S)-; Y₂ is 63 H, CH₂COOH, peptide, biomolecule, an Fmoc protected amine or a Boc protected amine; W15 is C=O, CH₂, or OC₂H ; b varies from 1-100; R_a may be H, OH, -O-, alkyloxy, aryl, alkyl or single bond; R₅ is as defined for Re; R30 is an amine protecting group; k is alkyl, aryl, heterocarbocyclic, CH₂CO₂H, Cl-ClO alkoxyl, Cl-ClO aryloxyl, Cl-ClO polyalkoxyalkyl, -CH₂(CH₂-O-CH2)b-CH2-R_a, C1-C20 polyhydroxyalkyl, Cl-ClO polyhydroxyaryl, carbocyclic, heterocyclic, or X-Y; and z varies from 1-10, comprising steps of:

(c) reacting the reactive intermediate of step (b)^' with a functionalized resin to form a resin-bound polyazacarboxylic acid ester of general formula 72 :

(d) removing any amine protecting group from R₃₀;

(e) synthesizing a desired peptide at the diamine on said compound of formula 72 to form a peptide-polyazacarboxylate conjugate of formula

and

(f) cleaving the peptide-polyazacarboxylate conjugate from the resin and removing all protecting groups; wherein T is -O-, -NH-, -S-, alkyl, aryl, CH₂(CH₂OCH₂)_bCH₂-. 64

27. The method of claim 26 wherein said polyazacarboxylate is

28. The method of claim 26 wherein said diamine is selected from the group consisting of: ethylenediamine; bis(2-aminoethyl)-ether; O-bis(aminoethyl)ethylene glycol; O- bis(aminoethyl)tetraethylene glycol; O-bis(aminoethyl)hexaethyIene glycol; 1,4- bis(aminomethyl)benzene; l,3-bis(aminomethyl)benzene; ^; 1 ,4-diaminobutane; 1,2- diaminocyclohexane; 4,4'-diaminodicyclohexylmethane; N-1 ,3-diamino-2-propanol; homopiperazine; piperazine; histidine; and lysine.

29. The method of claim 26 wherein said diamine is an orthogonally protected diamine.

30. The method of claim 26 wherein R₃o is selected from the group consisting of Fmoc and Boc.

31. A method for preparing a peptide-polyazacarboxylate composition at the C-terminal carboxyl group of a peptide comprising the steps of:

(a) preparing an orthogonally protected dicarboxylic acid of a polyazacarboxylic acid ester of general formula 2a:

65

wherein Re to Rio may be the same or different and are selected from the group consisting of alkyl, aryl, heterocarbocyclic, NH-k-NHR₃o, CH2CO₂H, hydroxyl, amino, Cl-ClO alkoxyl, Cl-ClO aryloxyl, Cl-ClO polyalkoxyalkyl, -CH₂(CH2-O-CH2)b-CH2-R_a> C1-C20 polyhydroxyalkyl, Cl -CIO polyhydroxyaryl, and X₂-Y₂; ₂ and W₃ are selected from the group consisting of alkyl, aryl, -CH₂(CH2-O-CH2)b-CH2-R_a, polyhydroxyalkyl, and polyhydroxyaryl; X₂ is selected from the group consisting of NH-, CONH-, -CH₂NH-, - CH₂NR₅-, -COO-, -O-, -C(O)-, -S-, -NHCO-, and -NHC(S)-; Y₂ is selected from the group consisting of H, CH₂COOH, peptide and biomolecule; W₁₅ is C=O, CH₂, or OC₂H ; b varies from 1-100; R_a may be H, OH, -O-, alkyloxy, aryl, alkyl or single bond; R₅ is as defined for R^; R₃₀ is an amine protecting group; k is alkyl, aryl, heterocarbocyclic, CH₂CO₂H, Cl-ClO alkoxyl, Cl-ClO aryloxyl, Cl-ClO polyalkoxyalkyl, -CH₂(CH₂-O-CH₂)_b-CH₂-R_a, C1-C20 polyhydroxyalkyl, Cl-ClO polyhydroxyaryl, carbocyclic, heterocyclic, or X-Y; and z varies from 1-10;

(c) reacting the reactive intermediate of step (b) with a functionalized resin to form a resin-bound polyazacarboxylic acid ester of general formula 73:

66

(d) activating the resin-bound monocarboxylic acid of step (c);

(e) reacting the active ester of step (d) with a diamine to yield a compound of general formula 74:

(f) removing any amine protecting group from R₃₀;

(g) synthesizing a desired peptide at the diamine on said compound of formula 74 to form a peptide-polyazacarboxylate conjugate of formula 67

and

32. The method of claim 31 wherein said polyazacarboxylate is

33. The method of claim 31 wherein said diamine is selected from the group consisting of: ethylenediamine; bis(2-aminoethyl)-ether; O-bis(aminoethyI)ethylene glycol; O- bis(aminoethyl)tetraethylene glycol; O-bis(aminoethyl)hexaethylene glycol; 1,4- bis(aminomethyl)benzene; l,3-bis(aminomethyl)benzene; 1,4-diaminobutane; 1,2- diaminocyclohexane; 4,4'-diaminodicyclohexylmethane; N-1 ,3-diamino-2-propanol; homopiperazine; piperazine; histidine; and lysine.

34. The method of claim 31 wherein said diamine is an orthogonally protected diamine. 68

35. The method of claim 31 wherein R30 is selected from the group consisting of H, Fmoc and Boc.

36. A method for preparing, a peptide-polyazacarboxylate composition at the C-terminal carboxyl group of a peptide comprising the steps of:

(a) preparing an orthogonally protected monocarboxylic acid of a polyazacarboxylic acid ester of general formula 2a:

wherein one of Rg to Rio is OH; Re to Rio may be the same or different and are selected from the group consisting of alkyl, aryl, heterocarbocyclic, NH-k-NHR₃o, CH₂CO₂H, hydroxyl, amino, Cl-ClO alkoxyl, Cl-ClO aryloxyl, Cl-ClO polyalkoxyalkyl, -CH₂(CH₂- O-CH₂)_b-CH₂-R_a, C1-C20 polyhydroxyalkyl, Cl -CIO polyhydroxyaryl, and X₂-Y₂; W₂ and W₃ are selected from the group consisting of alkyl, aryl, -CH₂(CH2-O-CH2)b-CH₂-Ra₉ polyhydroxyalkyl, and polyhydroxyaryl; X₂ is selected from the group consisting of NH-, CONH-, -CH₂NH-, -CH2NR5-, -COO-, -O-, -C(O)-, -S-, -NHCO-, and -NHC(S)-; Y₂ is H, CH₂COOH, peptide, biomolecule, an Fmoc protected amine or a Boc protected amine; W₁₅ is OO, CH₂, or OC2H₄; b varies from 1-100; R_a may be H, OH, -O-, alkyloxy, aryl, alkyl or single bond; R₅ is as defined for R^; R₃₀ is an amine protecting group; k is alkyl, aryl, heterocarbocyclic, CH₂CO₂H, Cl-ClO alkoxyl, Cl-ClO aryloxyl, Cl-ClO polyalkoxyalkyl, -CH₂(CH₂-O-CH₂)_b-CH2-Ra_s C1-C20 polyhydroxyalkyl, Cl-ClO polyhydroxyaryl, carbocyclic, heterocyclic, or X-Y; and z varies from 1-10; (b) activating the monocarboxylic acid of step (a) to form a reactive intermediate; 69 (c) reacting the reactive intermediate of step (b) with a functionalized resin to form a resin-bound polyazacarboxylic acid ester of general formula 74:

(d) removing any amine protecting group from R₃₀;

(e) synthesizing a desired peptide at the diamine on said compound of formula 74 to form a peptide-polyazacarboxylate conjugate of formula

and

(f) cleaving the peptide-polyazacarboxylate conjugate from the resin and removing all protecting groups; wherein T is -O-, -NH-, -S-, alkyl, aryl, CH2(CH₂OCH₂)_bCH₂-.

37. The method of claim 36 wherein said polyazacarboxylate is 70

38. The method of claim 36 wherein said diamine is selected from the group consisting of: ethylenediamine; bis(2-aminoethyl)-ether; O-bis(aminoethyl)ethylene glycol; O- bis(aminoethyl)tetraethylene glycol; O-bis(aminoethyl)hexaethylene glycol; 1,4- bis(aminomethyl)benzene; l,3-bis(aminomethyl)benzene; 1,4-diaminobutane; 1,2- diaminocyclohexane; 4,4^l-diaminodicyclohexylmethane; N- 1 ,3 -diamino-2-propanol; homopiperazine; piperazine; histidine; and lysine.

39. The method of claim 36 wherein said diamine is an orthogonally protected diamine.

40. The method of claim 36 wherein R₃o is selected from the group consisting of Fmoc and Boc.

41. A method for preparing a peptide-polyazacarboxylate composition at the C-terminal carboxyl group of a peptide comprising the steps of:

(a) preparing an orthogonally protected dicarboxylic acid of a polyazacarboxylic acid ester of general formula 3 :

71

wherein Rπ to R₁₅ may be the same or different and are selected from the group consisting of alkyl, aryl, heterocarbocyclic, NH-k-NHR₃o, CH₂CO₂H, hydroxyl, amino, Cl-ClO alkoxyl, Cl-ClO aryloxyl, Cl-ClO polyalkoxyalkyl, -CH₂(CH₂-O-CH₂)_b-CH₂-R_a, C1-C20 polyhydroxyalkyl, Cl-ClO polyhydroxyaryl, and X3-Y3; W₄ and W₅ are selected from the group consisting of alkyl, aryl, -CH₂(CH₂-O-CH₂)b-CH₂-R_a, polyhydroxyalkyl, and polyhydroxyaryl; X₃ is selected from the group consisting of NH-, CONH-, -CH₂NH-, -CH₂NR₅-, -COO-, -O-, -C(O , -S-, -NHCO-, and -NHC(S)-; Y₃ is selected from the group consisting of H, CH2COOH, peptide and biomolecule; iβ is C=O, CH₂, or OC₂H ; b varies from 1-100; R_a may be H, OH, -O-, alkyloxy, aryl, alkyl or single bond; R₅ is as defined for Rπ; R3₀ is an amine protecting group; k is alkyl, aryl, heterocarbocyclic, CH₂CO₂H, CT-C10 alkoxyl, Cl-ClO aryloxyl, Cl-ClO polyalkoxyalkyl, -CH₂(CH₂-O-CH₂)_b-CH₂-R_a, C1-C20 polyhydroxyalkyl, Cl-ClO polyhydroxyaryl, carbocyclic, heterocyclic, or X-Y; and z varies from 1-10;

(c) reacting the reactive intermediate of step (b) with a functionalized resin to form a resin-bound polyazacarboxylic acid ester of general formula 75:

72

75

(e) reacting the active ester of step (d) with a diamine to yield a compound of general formula 76:

76

(f) removing any amine protecting group from R₃o;

(g) synthesizing a desired peptide at the diamine on said compound of formula 76 to form a peptide-polyazacarboxylate conjugate of formula

73

and

(h) cleaving the peptide-polyazacarboxylate conjugate from the resin and removing all protecting groups; > wherein T is -O-, -NH-, -S-, alkyl, aryl, CH2(CH2OCH₂)_bCH₂-.

42. The method of claim 41 wherein said polyazacarboxylate is selected from the group consisting of

51 and

74

43. The method of claim 41 wherein said diamine is selected from the group consisting of: ethylenediamine; bis(2-aminoethyl)-ether; O-bis(aminoethyl)ethylene glycol; O- bis(aminoethyl)tetraethylene glycol; O-bis(aminoethyl)hexaethylene glycol; 1,4- bis(aminomethyl)benzene; l,3-bis(aminomethyl)benzene; 1,4-diaminobutane; 1,2- diaminocyclohexane; 4,4'-diaminodicyclohexylmethane; N-1 ,3-diamino-2-propanol; homopiperazine; piperazine; histidine; and lysine.

44. The method of claim 41 wherein said diamine is an orthogonally protected diamine.

45. The method of claim 41 wherein R₃0 is selected from the group consisting of H, Fmoc and Boc.

46. A method for preparing a peptide-polyazacarboxylate composition at the C-terminal carboxyl group of a peptide comprising the steps of:

(a) preparing an orthogonally protected monocarboxylic acid of a polyazacarboxylic acid ester of general formula 3:

wherein one of Rπ to R1₅ is OH; Rn to R15 may be the same or different and are selected from the group consisting of alkyl, aryl, heterocarbocyclic, NH-k-NHR₃₀, CH₂CO₂H, hydroxyl, amino, Cl-ClO alkoxyl, Cl-ClO aryloxyl, Cl-ClO polyalkoxyalkyl, -CH₂(CH₂- O-CH₂)_b-CH₂-R_a, C1-C20 polyhydroxyalkyl, Cl-ClO polyhydroxyaryl, and X₃-Y₃; W₄ and W₅ are selected from the group consisting of alkyl, aryl, -CH₂(CH₂-O-CH₂)_b-CH₂-R_a, polyhydroxyalkyl, and polyhydroxyaryl; X₃ is selected from the group consisting of NH-, CONH-, -CH₂NH-, -CH₂NR₅-, -COO-, -O-, -C(O)-, -S-, -NHCO-, and -NHC(S)-; Y₃ is 75

H, CH₂COOH, peptide, biomolecule, an Fmoc protected amine or a Boc protected amine; Wi₆ is 0=0, CH₂, or OC₂H4; b varies from 1-100; R_a may be H, OH, -O-, alkyloxy, aryl, alkyl or single bond; R₅ is as defined for Ri; R₃o is an amine protecting group; k is alkyl, aryl, heterocarbocyclic, CH₂CO₂H, Cl-ClO alkoxyl, Cl-ClO aryloxyl, Cl-ClO polyalkoxyalkyl, -CH₂(CH₂-O-CH₂)b-CH2-Ra₅ C1-C20 polyhydroxyalkyl, Cl-ClO polyhydroxyaryl, carbocyclic, heterocyclic, or X-Y; and z varies from 1-10;

(c) reacting the reactive intermediate of step (b) with a functionalized resin to form a resin-bound polyazacarboxylic acid ester of general formula 76:

76

(d) removing any amine protecting group from R₃0;

(e) synthesizing a desired peptide at the diamine on said compound of formula 76 to form a peptide-polyazacarboxylate conjugate of formula

and 76

(f) cleaving the peptide-polyazacarboxylate conjugate from the resin and removing all protecting groups; wherein T is -O-, -NH-, -S-, alkyl, aryl, CH₂(CH₂OCH₂)_bCH₂-.

47. The method of claim 46 wherein said polyazacarboxylate is selected from the group consisting of

and

48. The method of claim 46 wherein said diamine is selected from the group consisting of: ethylenediamine; bis(2-aminoethyl)-ether; O-bis(aminoethyl)ethylene glycol; O- bis(aminoethyl)tetraethylene glycol; O-bis(aminoethyl)hexaethylene glycol; 1,4- bis(aminomethyl)benzene; l,3-bis(aminomethyl)benzene; 1,4-diaminobutane; 1,2- diaminocyclohexane; 4,4'-diammodicyclohexylmethane; N-1 ,3-diamino-2-propanol; homopiperazine; piperazine; histidine; and lysine. ι

49. The method of claim 46 wherein said diamine is an orthogonally protected diamine.

50. The method of claim 46 wherein R₃o is selected from the group consisting of Fmoc and Boc. 77

51. A method for preparing a peptide-polyazacarboxylate composition at the C-terminal carboxyl group of a peptide comprising the steps of:

(a) preparing an orthogonally protected dicarboxylic acid of a polyazacarboxylic acid ester of general formula 4:

wherein Rι₆ to R₁₉ may be the same or different and are selected from the group consisting of alkyl, aryl, heterocarbocyclic, NH-k-NHR₃₀, CH₂CO₂H, hydroxyl, amino, Cl-ClO alkoxyl, Cl-ClO aryloxyl, Cl-ClO polyalkoxyalkyl, -CH₂(CH₂-O-CH₂)b-CH₂-R_a, C1-C20 polyhydroxyalkyl, Cl -CIO polyhydroxyaryl, and X-Y; W₆ and W₇ are selected from the group consisting of alkyl, aryl, -CH2(CH2-O-CH₂)b-CH2-R_a, polyhydroxyalkyl, and polyhydroxyaryl; X4 is selected from the group consisting of NH-, CONH-, -CH₂NH-, -CH2NR₅-, -COO-, -O-, -C(O)-, -S-, -NHCO-, and -NHC(S)-; Y₄ is selected from the group consisting of H, CH2COOH, peptide and biomolecule; Wj is C=O, CH₂, or OC₂H ; b varies from 1-100; R_a may be H, OH, -O-, alkyloxy, aryl, alkyl or single bond; R₅ is as defined for R^ R₃₀ is an amine protecting group; k is alkyl, aryl, heterocarbocyclic, CH₂CO₂H, Cl-ClO alkoxyl, Cl-ClO aryloxyl, Cl-ClO polyalkoxyalkyl, -CH₂(CH2-O-CH₂)_b-CH2-R_a, C1-C20 polyhydroxyalkyl, Cl-ClO polyhydroxyaryl, carbocyclic, heterocyclic, or X-Y; and z varies from 1-10;

(c) reacting the reactive intermediate of step (b) with a functionalized resin to form a resin-bound polyazacarboxylic acid ester of general formula 77: 78

77

(e) reacting the active ester of step (d) with a diamine to yield a compound of general formula 78:

78

(f) removing any amine protecting group from R₃o;

(g) synthesizing a desired peptide at the diamine on said compound of formula 78 to form a peptide-polyazacarboxylate conjugate of formula

79

and

(h) cleaving the peptide-polyazacarboxylate conjugate from the resin and removing all protecting groups; wherein T is -O-, -NH-, -S-, alkyl, aryl, CH (CH₂OCH )bCH2-.

52. The method of claim 51 wherein said polyazacarboxylate is selected from the group consisting of:

57 and

60 80

53. The method of claim 51 wherein said diamine is selected from the group consisting of: ethylenediamine; bis(2-aminoethyl)-ether; O-bis(aminoethyl)ethylene glycol; O- bis(aminoethyl)tetraethylene glycol; O-bis(aminoethyl)hexaethylene glycol; 1,4- bis(aminomethyl)benzene; l,3-bis(aminomethyl)benzene; 1,4-diaminobutane; 1,2- diaminocyclohexane; 4,4'-diaminodicyclohexylmethane; N- 1 ,3-diamino-2-propanol; homopiperazine; piperazine; histidine; and lysine.

54. The method of claim 51 wherein said diamine is^' an orthogonally protected diamine.

55. The method of claim 51 wherein R30 is selected from the group consisting of H, Fmoc and Boc. ;

56. A method for preparing a peptide-polyazacarboxylate composition at the C-terminal carboxyl group of a peptide comprising the steps of:

(a) preparing an orthogonally protected monocarboxylic acid of a polyazacarboxylic acid ester of general formula 4:

4 wherein one of R₁₅ to R₁₉ is OH; R]₆ to R^ may be the same or different and are selected from the group consisting of alkyl, aryl, heterocarbocyclic, NH-k-NHR₃o, CH₂CO₂H, hydroxyl, amino, Cl-ClO alkoxyl, Cl-ClO aryloxyl, Cl-C 10 polyalkoxyalkyl, -CH₂(CH₂- O-CH₂)_b-CH₂-Ra, C1-C20 polyhydroxyalkyl, Cl-ClO polyhydroxyaryl, and X₄-Y₄; W₆ and W₇ are selected from the group consisting of alkyl, aryl, -CH₂(CH₂-O-CH₂)_b-CH₂-R_a, 81 polyhydroxyalkyl, and polyhydroxyaryl; X4 is selected from the group consisting of NH-, CONH-, -CH2NH-, -CH₂NR₅-, -COO-, -O-, -C(O)-, -S-, -NHCO-, and -NHC(S)-; Y₄ is H, CH₂COOH, peptide, biomolecule, an Fmoc protected amine or a Boc protected amine; W17 is CO, CH₂, or OC2FU; b varies from 1-100; Ra may be H, OH, -O-, alkyloxy, aryl, alkyl or single bond; R₅ is as defined for R₁₆; R₃₀ is an amine protecting group; k is alkyl, aryl, heterocarbocyclic, CH₂CO₂H, Cl-ClO alkoxyl, Cl-ClO aryloxyl, Cl-ClO polyalkoxyalkyl, -CH2(CH2-O-CH₂)b-CH₂-R_a, C1-C20 polyhydroxyalkyl, Cl-ClO polyhydroxyaryl, carbocyclic, heterocyclic, or X-Y; and z varies from 1-10;

(c) reacting the reactive intermediate of step (b) with a functionalized resin to form a resin-bound polyazacarboxylic acid ester of general formula 78:

78

(d) removing any amine protecting group from R₃0;

(e) synthesizing a desired peptide at the diamine on said compound of formula 78 to form a peptide-polyazacarboxylate conjugate of formula

82

and

(f) cleaving the peptide-polyazacarboxylate conjugate from the resin and removing all protecting groups; wherein T is -O-, -NH-, -S-, alkyl, aryl, CH₂(CH₂OCH2)bCH2-.

57. The method of claim 56 wherein said polyazacarboxylate is selected from the group consisting of:

and

83

58. The method of claim 56 wherein said diamine is selected from the group consisting of: ethylenediamine; bis(2-aminoethyl)-ether; O-bis(aminoethyl)ethylene glycol; O- bis(aminoethyl)tetraethylene glycol; O-bis(aminoethyl)hexaethylene glycol; 1,4- bis(aminomethyl)benzene; l,3-bis(aminomethyl)benzene; 1 ,4-diaminobutane; 1,2- diaminocyclohexane; 4,4'-diaminodicyclohexylmethane; N-1 ,3-diamino-2-propanol; homopiperazine; piperazine; histidine; and lysine.

59. The method of claim 56 wherein said diamine is an orthogonally protected diamine.

60. The method of claim 56 wherein R₃₀ is selected from the group consisting of Fmoc and Boc.