WO2001052899A1

WO2001052899A1 - Chelating agents and method for their use as tandem metal chelators and hydrophilic spacers for medical diagnosis and therapy

Info

Publication number: WO2001052899A1
Application number: PCT/US2001/001641
Authority: WO
Inventors: Samuel I. Achilefu
Original assignee: Mallinckrodt Inc.
Priority date: 2000-01-21
Filing date: 2001-01-18
Publication date: 2001-07-26
Also published as: AU2001234478A1

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. An amino acid or peptide is added at this latter carboxyl group. 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.

Claims

13

Scheme 4

(viii) Fmoc-OSu

14

Scheme 5

28

(v) FmocNHC₂H₄NH

30

15

Scheme 6

16

Scheme 7

40

Q^NHTrtorCOzBn Q₂ = C0₂HorNH₂

17 Scheme l

41 (iii) BrCH₂C0₂t-Bu 42

(viii) Fmoc-OSu

18

Scheme 9

(iii) BrCH₂C0₂Bn C0₂Bn (_{iv) NzH4} -C0₂Bn CF₃C(0)NH₂ --_^. XX*. CF₃C(0)- HN

45 C0₂Bn -C0₂Bn (v) DCC/DMAP

46 47

(vi) FmocNHC₂H₄NH₂

19

Scheme 9b

-C0₂H (i) BnOH j— C0₂Bn _{(iii) TFA} — C0₂Bn

Boc— N Boc — N ^►* HN _{co H} (ii) DCC/DMAP -C0₂Bn -C0₂Bn

45b 46b 47

20

Scheme 10

50

(iv) H₂/Pd-C 51

52

21 Scheme 11

(ii) BrCH₂C0₂t-Bu

53 54

55 56

57

22 Scheme 12

60

61

23

Scheme 1₃

H₂N-(AA)_n-CO-T-(p)

H0₂C

H0₂C- -CONH-(AA)n-CO-T-H

Mⁿ⁺

H0₂C- -CONH-CH₂CH₂NHCO-(AA)m-NH₂

64

24 Scheme 14

(i) 14, HBTU/HOBt

(ii) 20% piperidine in DMF

H₂N-(AA)_n- CO-T-(p) ^*•*••--

(iii) Synthesize Peptide 2

(iv) TFA

T = -O- or -NH-

Mⁿ⁺ (AA)m = Peptide 2 (AA)n = Peptide 1 (?) = Resin M = Metal

65

25

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 affinities 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 26 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 27 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 28 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 29 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. 30 EXAMPLE 5

Synthesis of

-C0₂t-Bu

Bn-N

-C0₂t-Bu

A mixture of benzylamine (10 g, 0.93 ol) 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 MgSO₄ , 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 i C0₂t-Bu

HN ^ C0₂t-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. 31

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 MgSO₄ 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%) ether in hexane.

EXAMPLE 8A 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. 32

EXAMPLE 8B Synthesis of

-C0₂Bn

HN

-C0₂Bn

[Scheme 9b, compound 47] Benzyl alcohol (5.1 g, 47.17 mmol) was added to a suspension of t-butoxycarbonyl (Boc) imminodiacetic acid (45b, 5 g, 21.44 mmol) in anhydrous dichloromethane (DCM). Dimethylformamide (DMF) was added dropwise until a clear solution was obtained. The solution was cooled to 0°C and a mixture of dicyclohexylcarbodiimide (DCC, 9.7 g, 47.59 mmol) and dimethylaminopyridine (DMAP, 0.4 g, 3.43 mmol) in anhydrous DCM was added slowly to the reaction medium. The resulting mixture was stirred at room temperature for 12 hours and the precipitate formed was filtered. The filtrate was washed with saturated aqueous sodium bicarbonate. The organic layer was dried over MgSO₄ and the solvent was evaporated to give the dibenzyl ester (46b) in quantitative yield. The Boc protecting group was removed with 50%> aqueous TFA to give the desired product (47) in 95%> yield.

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

33

[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 MgSO₄ , 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,N₅N-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/DCM.

The organic layer was washed with water, then brine and then dried over MgSO₄. The solvent 34 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. 35

The monotrityl N,N-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 pentaethyleneglycol (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 HC1 (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

^\ _/Br Trt— NH x^ 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 MgSO₄ 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. 36

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)ammo]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 37

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'-tris(t-butyloxycarbonylmemyl)-N' N"-bis(berrzyloxycarbonylmethyl)diethylenetriamine (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

[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 38 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

39

[Scheme 9, 48] Reaction of the monocarboxylic acid tetra-t-butyl diethylenetriaminepentaacetic acid (DTP A) (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 DMF for 5 hours at room temperature gives the dibenzyl ester conjugate which is hydrogenolyzed as described in Example 18 to give the dicarboxylic acid, 48.

EXAMPLE 22

[Scheme 9, 49] Conjugation of mono-Fmoc ethylenediamine to the dicarboxylic acid of Example 21 was carried out as described in Example 19.

EXAMPLE 23 Synthesis of

Dissolve benzylethylenediamine (9; 10.3 mmol, 1 equiv.) in dry dichloromethane (10 mL) and cool the solution to -5 °C. Add ethyl trifluoroacetate (10.3 mmol, 1 equiv.) dropwise while maintaining the temperature below 0 °C. After addition, stir at this temperature for 2 hours and allow the reaction mixture to warm up to room temperature and stir for additional 2 hours. Evaporate the solvent and any unreacted trifluoroacetate and purify the crude product by dry flash chromatography if desired. Dissolve the crude product in anhydrous DMF (20 mL) and cool the 40 solution to 0 °C. Add sodium hydride (20.6 mmol, 2 equiv.) and stir the mixture for 10 minutes before adding t-butyl bromoacetate (20.6 mmol, 2 equiv.) dropwise. Allow the mixture to warm up to room temperature and stir for 12 hours. Remove the solvent in vacuo and partition the residue between dichloromethane and water. Wash the organic layer with water and dry it over MgSO₄. After evaporating the solvent, redissolve the residue in acetonitrile and react it with N,N-benzyloxycarbonylmethyl-t-butyloxycarbonylmethylaminoethyl bromide (17; 22 mmol) as described in Example 17. Deprotect the trifluoroacetyl group with hydrazine in t-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

41

[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 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)amino]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 42 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 1 % palladium on carbon (0.4 g) and a solution of the purified intermediate N'-benzyl-N,N''-teljakis(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.

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 43 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 x 50 ml). The solvent was evaporated to give the pure product .

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 MgSO₄ 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. 44

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%>) 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- butyloxycarbonylmethyl)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- 45

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 dibenzyl 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.

EXAMPLE 32

Synthesis of

[Scheme 12, 60] Reaction of N',N",N'"-tris(t-butyloxycarbonylmethyl)cyclen (59; 1 mmol) with 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. 46

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].

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. 47

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 bis-peptide-chelate conjugate, method A

X = COOH; (AA)m = Octreotate for somatostatin receptor positive tumors (AA)n = Bombesin (7-14) for bombesin receptor positive tumors

[Scheme 13, 64] 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- (1 H-benzotriazole- 1 yl)- 1 , 1 ,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. 48

After the synthesis of the first peptide and conjugation of the chelator was complete, the free acid from tri-t-butyl DTPA was activated on solid support with HBTU/HOB (1.5 equiv.) for 30 minutes and mono-Fmoc ethylenediamine (3 equiv.) was added in the presence of diisopropylethylamine (3 equiv.). The mixture was shaken for 2 hours and the resin was washed with DMF and THF. After drying the resin, it was placed on the resin cartridge and the second peptide (bombesin (7-14) was synthesized automatically. At the end of the reaction, the disulfide bond was formed between the two Cysteines of the octreotate peptide with thallium trifluoroacetate. 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 tri-t-butyl DTPA were also cleaved to give the free tetra-carboxylic acid. The DTPA-bispeptide conjugate was precipitated 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 bis-peptide-chelator conjugate, method B

[Scheme 14, 64] In this method, the mono-Fmoc ethylene diamine tri-t-butyl DTPA (compound of Example 19) was used in place of tri-t-butyl DTPA. This procedure permitted the automatic synthesis of the bis-peptide without interruption. The disulfide bond was formed and the bis-peptide on solid support was cleaved as described in the preceding example. 49

EXAMPLE 38

Synthesis of bis-peptide-chelate conjugate

X = COOH; (AA)m = Octreotate for somatostatin receptor positive tumors (AA)n = Bombesin (7-14) for bombesin receptor positive tumors M = indium-115 (¹¹⁵In)

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

A. CLASSIFICATION OF SUBJECT MATTER

IPC 7 A61K47/48 A61P39/04

According to International Patent Classification (IPC) orto both national classification and IPC

B. FIELDS SEARCHED

Minimum documentation searched (classification system followed by classification symbols)

IPC 7 A61K

Documentation searched other than minimum documentation to the extent that such documents are included in the fields searched

Electronic data base consulted during the international search (name of data base and, where practical, search terms used)

EPO-Internal , BIOSIS , CHEM ABS Data, WPI Data , PAJ

C. DOCUMENTS CONSIDERED TO BE RELEVANT

Category " Citation of document, with indication, where appropnate, of the relevant passages Relevant to claim No

EP 0345723 A (NIHON MEDIPHYSICS CO LTD) 1-5 , 13 December 1989 (1989-12-13) 13-60 exampl es

EP 0649857 A (NIHON MEDIPHYSICS CO LTD) 1-5,

26 April 1995 (1995-04-26) 13-60 page 3 , l i ne 7 - l i ne 34 ; exampl e 8

D Further documents are listed in the continuation of box C Patent family members are listed in annex

° Special categories of cited documents

"T" later document published after the international filing date or priority date and not in conflict with the application but

"A" document defining the general state of the art which is not cited to understand the principle or theory underlying the considered to be of particular relevance invention

"E" earlier document but published on or after the international "X" document of particular relevance, the claimed invention filing date cannot be considered novel or cannot be considered to

^■L" document which may throw doubts on pπonty claιm(s) or involve an inventive step when the document is taken alone which is cited to establish the publication date of another "Y" document of particular relevance, the claimed invention citation or other special reason (as specified) cannot be considered to involve an inventive step when the

"O" document referring to an oral disclosure, use, exhibition or document is combined with one or more other such docuother means ments, such combination being obvious to a person skilled

"P" document published prior to the international filing date but in the art later than the priority date claimed "&" document member of the same patent family

Date of the actual completion of the international search Date of mailing of the international search report

31 May 2001 12/06/2001

Name and mailing address of the ISA Authorized officer

European Patent Office, P B 5818 Patentlaan 2 NL - 2280 HV Rljswijk Tel (+31-70) 340-20₄0, Tx 31 651 epo nl, Fax (+31-70) 3 0-3016 Dδpfer, K-P

Form PCT/ISA 210 (second sheet) (July 1992) International Application No. PCT/US 01 Λ1641

FURTHER INFORMATION CONTINUED FROM PCT/ISA/ 210

Continuation of Box 1.2 Claims Nos.: 6-12, 61 ff.

Claims 6 (partially), 7-11, 12 (partially), 61 ff. are missing in the application documents. The subject matter of claims 6 and 12 is so uncleat that a meaningful search is not possible.

The applicant's attention is drawn to the fact that claims, or parts of claims, relating to inventions in respect of which no international search report has been established need not be the subject of an international preliminary examination (Rule 66.1(e) PCT). The applicant is advised that the EPO policy when acting as an International Preliminary Examining Authority is normally not to carry out a preliminary examination on matter which has not been searched. This is the case irrespective of whether or not the claims are amended following receipt of the search report or during any Chapter II procedure.

Patent document Publication Patent family Publication cited in search report date member(s) date

EP 0345723 13-12-1989 AT 106075 T 15-06- 1994 AU 617816 B 05-12-^■1991 AU 3603989 A 14-12-^■1989 CA 1331450 A 16-08-^■1994 DE 68915476 D 30-06-^■1994 DE 68915476 T 29-09- 1994 DK 276789 A 08-12-^■1989 ES 2056150 T 01-10-^•1994 JP 2085239 A 26-03-^•1990 JP 2815615 B 27-10- 1998 KR 126238 B 26-12-^•1997 US 5094950 A 10-03-^•1992 US 5250702 A 05-10-^■1993

EP 0649857 A 26-04-1995 AT 175976 T 15-02- 1999 AU 675166 B 23-01- 1997 AU 7597994 A 11-05-^•1995 CA 2134051 A 23-04-^■1995 DE 69416078 D 04-03-^■1999 DE 69416078 T 27-05-^•1999 DK 649857 T 13-09-^■1999 ES 2126695 T 01-04-^■1999 P 7206895 A 08-08-^■1995 US 5821330 A 13-10-^•1998

Foim PCT/ISA/210 (patent family annex) (July 1992)