Multidentate bifunctional chelating agents for radionuclide complexation in diagnostics and therapy

Abstract

The invention relates to octadentate ligands of a general formula R.sup.1-D-X-D-X-D-X-D-E-R.sup.2, wherein D is C(O)N(OH) or N(OH)C(O), pyrimidinone or pyridinone, each X independently of any other X is a saturated or partially unsaturated, substituted or unsubstituted linker comprising 8-11 atoms selected from any of N, C, O; R.sup.1 is alkyl, cycloalkyl, arene, or heteroarene, E is a saturated or partially unsaturated, substituted or unsubstituted chain comprising 1-50 atoms and R.sup.2 is a moiety capable of selectively binding to a biomolecule, or a nanoparticle. The invention further relates to complexes of the ligand, particularly radionuclides and their use in radioimmunotherapy and imaging.

Claims

1. A ligand having the structure ##STR00026## wherein R.sup.1 is a C.sub.1-C.sub.5 alkyl, or a C.sub.3-C.sub.6 cycloalkyl, an arene, and/or a heteroarene, wherein the arene or heteroarene consists of between five and fifteen carbon or hetero atoms, E is a liner linker and, R.sup.2 is a) an OH, NH.sub.2, SH, COOH, CHO, N.sub.3, SCN, CH.sub.2X with X being CI, Br or I, an activated ester, an ene-one system, a diene/dienophile, an alkene, an alkyne, (CO)—(CH.sub.2).sub.2—COOH, ##STR00027## or b) a polypeptide, that specifically binds to a target site on cells and/or tissues with an association constant of lower than (<) 10.sup.−6 mol/l, <10.sup.−7 mol/l, <10.sup.−8 mol/1 or <10.sup.−9 mol/l, wherein the ligand binds to a metal atom selected from the group consisting of Zr, Ga, Lu and Y.

2. The ligand according to claim 1, wherein R.sup.1 is methyl, ethyl or propyl and E is (CH.sub.2).sub.5—NH—.

3. The ligand according to claim 1, described by the formula ##STR00028##

4. The ligand according to claim 1, wherein R.sup.2 comprises a polypeptide being a functional homologue of bombesin, somatostatin, gastrin, trans-activator of transcription peptide, prostate-specific antigen, neuropeptide Y, octreotide, gastric inhibitory polypeptide, neurokinin A, neurotensin, exendin-3, exendin-4, or substance P, said peptide comprising an amino acid sequence of at least 80%, 85%, 90%, 95%, 98%, or 99% identity to SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6, SEQ ID NO 7, SEQ ID NO 8, SEQ ID NO 9, SEQ ID NO 10, SEQ ID NO 11, SEQ ID NO 12, or SEQ ID NO 13, respectively.

5. A complex comprising a ligand according to claim 1, coordinatively bound to Zr, Ga, Lu or Y.

6. The ligand of claim 1, wherein the metal is Zr.

7. A complex comprising a ligand according to claim 1, wherein the metal is selected from the group consisting of .sup.67Ga, .sup.68Ga, .sup.89Zr, .sup.90Y, and .sup.177Lu.

8. A method of synthesis of a complex comprising a ligand according to claim 1, coordinatively bound to Zr, Ga, Lu or Y comprising steps of: (a) providing the ligand; and (b) adding a metal selected from the group consisting of Zr, Ga, Lu and Y.

9. A method of treatment of neoplastic disease comprising administering the complex according to claim 5 to a patient in need thereof.

10. A solid support comprising the ligand of claim 1 covalently attached to a solid support.

11. The solid support according to claim 10, wherein said solid support is a nanoparticle.

12. The solid support according to claim 11, wherein said nanoparticle is selected from gold, silica, lipids, polymeric, metal or metal oxide compositions comprising the metals iron, manganese, or titanium.

13. The ligand according to claim 1, wherein R.sup.1 is a methyl.

14. The ligand according to claim 1, wherein R.sup.2NH.sub.2, (CO)—(CH.sub.2).sub.2—COOH, or BBN.sub.Ago(Beta-Ala).sub.3.

15. The ligand according to claim 1, wherein E is (CH.sub.2).sub.5, (CH.sub.2).sub.5NH, or (CH.sub.2).sub.5NH—(CO)—(CH.sub.2).sub.2—(CO)N—.

16. The ligand according to claim 1, wherein the polypeptide is an antibody.

17. The ligand of claim 16, wherein in the antibody is trastuzumab or pertuzumab.

18. A complex comprising a ligand according to claim 1, wherein the metal is .sup.89Zr.

19. The ligand according to claim 1, wherein R.sup.2 is ##STR00029##

20. A solid support comprising a complex of claim 5 covalently attached to a solid support.

Description

SHORT DESCRIPTION OF THE FIGURES

(1) FIG. 1 shows a stability profile of the octa co-ordinated complex III (ligand-conjugated complex; top two lines) in comparison to the hexadentate complex known in the art (DFO-conjugated complex; bottom two curves). The graph shows results of competition experiments using 300-fold excess of free DFO in a 24 hour time (Challenging against 0.1 mM DFO-mesylate in 0.5 M HEPES (pH 7.3)). X axis: time; Y axis: percentage of intact complex.

(2) FIG. 2 shows the calculated structure of the complex formed by reaction of ligand I with zirconium chloride. The atoms are depicted in different colours comprising zirconium (center), oxygen (gray), nitrogen (white with bold line), and carbon (white).

(3) FIG. 3 shows the synthesis of complex III

GENERAL METHODS

(4) Materials:

(5) All chemicals were of reagent grade quality or better, obtained from commercial suppliers and used without further purification. Solvents were used as received or dried over molecular sieves. All preparations were carried out using standard Schlenk techniques.

(6) Instrumentation and Methods:

(7) Instrumentation and Methods. .sup.1H and .sup.13C NMR spectra were recorded in deuterated solvents on 400 (.sup.1H: 400 MHz, .sup.13C: 100.6 MHz) or 500 (.sup.1H: 500 MHz, .sup.13C: 126 MHz) MHz spectrometers at room temperature. The chemical shifts, δ, are reported in ppm (parts per million). The residual solvent peaks have been used as an internal reference. The abbreviations for the peak multiplicities are as follows: s (singlet), d (doublet), dd (doublet of doublets), t (triplet), q (quartet), m (multiplet), and br (broad). The .sup.1H and .sup.13C signals were assigned with the help of 2D NMR techniques. Elemental microanalyses were performed on a LecoCHNS-932 elemental analyser. ESI-MS were performed using a Bruker Daltonics HCT 6000 mass spectrometer. LC-MS spectra were recorded on a HPLC apparatus (Acquity Ultra Performance LC, Waters) that was connected to a mass spectrometer (Bruker Esquire 6000) operated in ESI mode. A Nucleosil 100-5 C18 (250×3 mm) reverse phase column was used with a flow rate of 0.5 mL min.sup.−1 and UV-absorption was measured at 254 nm. The runs were performed with a linear gradient of A (acetonitrile (Sigma-Aldrich HPLC-grade) and B (distilled water containing 0.02% TFA and 0.05% HCOOH): t=0-3 min, 5% A; t=17 min, 100% A; t=20 min, 100% A; t=21 min, 5% A; t=25 min, 5% A.

(8) Synthesis of the peptide precursor compound characterising a modified amino acid sequence of bombesin (SEQ ID NO. 14) was performed on an Applied Biosystem automatic peptide synthesizer using Rink Amide resin and standard Fmoc-protocoll. Analysis of peptide precursor, ligand I and ligand II were performed on a HPLC system using a Phenomenex Jupiter column, 4μ Proteo 90A, 250×4.6 mm, with a flow rate of 2 mL/min. Analysis of peptide precursor compound and ligand I were performed with a linear gradient of HPLC-grade solvent A (acetonitrile) from Sigma-Aldrich and B (distilled water containing 0.1% TFA). Peptide precursor: t=0 min, 5% A; t=12.5 min, 50% A; t=13.5 min, 95% A; t=14.5 min, 95% A; t=15 min, 5% A; t=17 min, 5% A. Ligand I: t=0 min, 10% A; t=12.5 min, 40% A; t=13.5 min, 95% A; t=14.5 min, 95% A; t=15 min, 10% A; t=17 min, 10% A. Ligand II: t=0 min, 15% A; t=12.5 min, 40% A; t=13.5 min, 95% A; t=14.5 min, 95% A; t=15 min, 15% A; t=17 min, 15% A. MS acquisitions were performed in the full scan mode in the mass range from m/z 100 to 2000 at 20′000 resolution and 1 scan per second. Masses were calibrated with a 2 mmol/l solution of sodium formate over m/z 158 to 1450 mass range with accuracy below 2 ppm.

EXAMPLES

a. Synthesis of compound a (4-[benzyloxy-[5-(benzyloxycarbonylamino)pentyl]amino]-4-oxo-butanoic acid) was synthesised following the procedures described in Na. Chem. Biol., 2007, 3, 652-656, and Na. Chem. Biol., 2007, 3, 652-656

(9) ##STR00016##

t-butyl N-benzyloxy-N-[5-(benzyloxycarbonylamino)pentyl]carbamate

(10) ##STR00017##

b. Synthesis of Compound b [(benzyl N-[5-[[4-[5-[[4-[5-[[4-[5-[acetyl(hydroxy)amino]pentylamino]-4-oxo-butanoyl]-hydroxy-amino]pentylamino]-4-oxo-butanoyl]-hydroxy-amino]pentylamino]-4-oxo-butanoyl]-benzyloxy-amino]pentyl]carbamate)]

(11) To a stirred solution of carboxylic acid (1.13 g, 2.6 mmol), addition of HATU (1.46 g, 3.8 mmol) in DMF (10 mL), and DIPEA (0.66 g, 5.1 mmol) was carried out under N.sub.2 atmosphere. After stirring the mixture for 40 min at room temperature, deferoxamine mesylate salt (1.68 g, 2.6 mmol) followed by DIPEA (0.66 g, 5.1 mmol) and 4-methyl morpholine (2 mL) were added. The mixture was then stirred for 48 h at room temperature. The solvent was removed using a high vacuum pump. Addition of 50 mL ice cold acetone to the resulting paste resulted in a white solid, which was isolated by centrifugation, followed by decantation of the acetone. This procedure was repeated twice. Then a similar washing procedure was followed using double distilled water instead of acetone (3×30 mL). The wet white solid was lyophilized to give result to a white powder.

(12) Data: 1H NMR (400 MHz, DMSO-d6): δ (ppm) 1.16-1.27 (m, 8H), 1.32-1.43 (m, 8H), 1.41-1.56 (m, 8H), 1.97 (s, 3H), 2.24-2.33 (m, 6H), 2.54-2.65 (m, 6H), 2.94-3.03 (m, 8H), 3.43-3.48 (m, 6H), 3.53-3.58 (m, 2H), 4.87 (s, 2H), 4.99 (s, 2H), 7.17-7.47 (m, 11H), 7.76 (s, br, 3H), 9.56-9.67 (m, 3H). 13C{1H} NMR (100 MHz, DMSO-d6): δ (ppm) 20.8, 23.8, 23.9, 26.5, 26.6, 27.7, 28.1, 29.3, 29.4, 30.1, 30.4, 38.9, 45.1, 47.3, 47.6, 65.5, 75.9, 128.2, 128.7, 128.9, 129.1, 129.7, 135.4, 137.8, 156.8, 170.6, 171.4, 171.7, 172.4. ESI-MS (positive detection mode): m/z (%) 1007.3 (100) [M+Na]+. Anal. Calcd for C.sub.49H.sub.76N.sub.8O.sub.13: C, 59.74, H, 7.78, N, 11.37. Found: C, 59.99, H, 7.67, N, 11.73.

(13) ##STR00018##

c. Synthesis of Ligand I

(14) A mixture of compound b (125 mg, 0.13 mmol) and 100 mL of MeOH was sonicated for 10 min in an ultrasonic bath. To the resulting suspension, 10% Pd/C (38 mg) was added and hydrogenation was carried out for 6 h under H.sub.2 (1 bar) atmosphere. The reaction mixture was then filtered by cotton plug followed by a filter paper. The solution was evaporated to give a white solid that was washed with 10 mL of acetonitrile and 20 mL of Et.sub.2O and dried.

(15) Data: 1H NMR (500 MHz, DMSO-d6): δ (ppm) 1.19-1.28 (m, 8H), 1.33-1.39 (m, 7H), 1.46-1.53 (m, 9H), 1.97 (s, 3H), 2.24-2.33 (m, 8H), 2.55-2.60 (m, 6H), 2.73-2.76 (m, 2H), 2.97-3.03 (m, 6H), 3.41-3.49 (m, 8H), 7.77 (s, br, 4H), 9.60 (s, br, 3H). 13C{1H} NMR (125 MHz, DMSO-d6): δ (ppm) 20.8, 23.3, 23.9, 26.2, 26.5, 27.1, 27.9, 28.1, 29.2, 30.2, 30.3, 38.9, 39.2, 47.2, 47.5, 170.6, 171.7, 172.4, 172.5. ESI-MS (positive detection mode): m/z (%) 761.5 (100) [M+H]+. Anal. Calcd for C.sub.34H.sub.64N.sub.8O.sub.11: C, 53.67, H, 8.48, N, 14.73. Found: C, 53.60, H, 8.25, N, 14.66.

(16) ##STR00019##

d. Synthesis of Complex I

(17) 600 uL (0.0072 mM) of a solution of the ligand I in 0.1 M HCl was added to 200 uL of the solution of ZrCl.sub.4 in 0.1 M HCl. The pH of the mixture was then slowly adjusted to ca. 8 by slow addition of 0.1 M K.sub.2CO.sub.3 solution and stirred for addition 40 min. Then the mixture was lyophilized to give white solid. Formation of the desired product was confirmed by the single peak observed in the LC-MS analysis that has the expected mass.

(18) For scaling up, to a suspension of the ligand (21.8 mg) in 667 uL of 0.1 mM HCl, a solution (667 uL, 10 mg/mL) of ZrCl.sub.4 in 0.1M HCl was added and stirred for 10 min. The suspension slowly disappears during this time. The pH of the mixture was then slowly adjusted to ca. 8 by slow addition of 0.1 M K.sub.2CO.sub.3 solution, stirred for additional 1.5 h and then lyophilized to give a white solid powder.

(19) Data: t.sub.R (LC-MS) 9.3 min. Mass 847.3 [M+H]+.

(20) ##STR00020##

e. Synthesis of Ligand II

(21) To a stirred mixture of ligand I (20 mg, 0.03 mmol) and succinic anhydride (3.9 mg, 0.04 mmol) in 2 mL DMF, NEt.sub.3 (7.6 mg, 0.07 mmol) was added under N.sub.2 atmosphere. After 48 h, DMF was removed in vacuum and the white solid obtained was washed with small portions acetone and Et.sub.2O.

(22) Data: 1H NMR (500 MHz, DMSO-d6): δ (ppm) 1.18-1.26 (m, 8H), 1.34-1.42 (m, 8H), 1.46-1.54 (m, 8H), 1.97 (s, 3H), 2.24-2.33 (m, 8H), 2.39-2.43 (m, 2H), 2.56-2.61 (m, 6H), 2.97-3.03 (m, 8H), 3.41-3.49 (m, 8H), 7.77 (s, br, 4H), 9.64 (s, br, 3H), 12.04 (s, br, 1H). 13C{1H} NMR (125 MHz, DMSO-d6): δ (ppm) 20.8, 23.9, 26.4, 28.1, 29.2, 29.6, 30.3, 30.4, 38.9, 47.2, 47.4, 170.6, 171.2, 171.7, 172.4, 174.3. ESI-MS (positive detection mode): m/z (%) 883.4 (100) [M+Na]+. Anal. Calcd for C.sub.38H.sub.68N.sub.8O.sub.14: C, 53.01, H, 7.96, N, 13.01. Found: C, 52.89, H, 7.73, N, 12.88.

(23) 1H NMR (500 MHz, DMSO-d6)

(24) ##STR00021##

f. Synthesis of Complex II

(25) Ligand II is not soluble either in 0.1 M HCl or in H.sub.2O. The complexation was carried out in MeOH using zirconium (IV) acetylacetonate [Zr(acac).sub.4]. A mixture of ligand II (50 mg, 0.058 mmol) and Zr(acac).sub.4 (28.3 mg, 0.058 mmol) in 15 mL MeOH was heated at reflux for 15 h with continuous stirring under N.sub.2 atmosphere. The solvent was then removed and the residue was then washed with Et.sub.2O (20 mL) and acetone (10 mL). The compound was obtained as off white solid (48 mg, 87%). The compound is it not soluble in common organic solvents including DMSO. When trying to dissolve in 0.5% NaOD in D.sub.2O, decomposition of the complex was observed. Therefore due to the limited solubility NMR spectra of the compound could not be obtained. However, the formation of the complex can be confirmed using LC-MS. Elemental analysis calculated for C.sub.38H.sub.64N.sub.8O.sub.14Zr.6H.sub.2O.(CH.sub.3).sub.2CO.sub.3C, 44.19, H, 7.42, N, 10.06. Found C, 44.11, H, 7.04, N, 10.16.

(26) Data: t.sub.R (LC-MS) 8.7 and 8.8 min. Mass 947.3 [M+H]+

(27) ##STR00022##

g. Synthesis of a Modified Amino Acid Sequence of Bombesin BBN.SUB.Ago.(Beta-Ala).SUB.3., Identical to SEQ ID NO 14: (R-Ala).SUB.3.-Gln-Trp-Ala-Val-Gly-Hi-Leu-14Nle-CONH.SUB.2

(28) Synthesis was performed on a solid phase synthesis using an Applied Biosystem automatic peptide synthesizer with low-loaded Rink Amid resin (on a Phenomenex Jupiter 250×4.6 mm column) and standard Fmoc-protocoll, followed by HPLC analysis to test for chemical purity. Molecular Weight: 1135.32 g/mol; Chemical Formula: C.sub.53H.sub.82N.sub.16O.sub.12

(29) ##STR00023##

h. Synthesis of Ligand III

(30) Coupling of ligand II to BBN.sub.Ago(beta-Ala).sub.3 was done by manual synthesis. 50-60 mg (9-12 μmol) of peptide resin was Fmoc-deprotected with 20% Piperidine/DMF (5×2 mL, 3-5 min each). MP-Zr-19 (2 eq.) was dissolved in 1 mL DMF (peptide grade) and shortly warmed up in a heating block at 60-70° C., then vortexed and sonicated. The solution was transferred to an eppendorff tube containing HATU (2 eq), and DIPEA (4 eq.) was added. The mixture was transferred to a syringe reactor containing the swollen peptide resin. After 3 h the reaction was stopped and the coupling procedure repeated over night. The peptide conjugate was cleaved from the resin with ˜0.6 mL TFA/phenol/TIS/water (87.5/5/2.5/5) for 3 h and afterwards precipitated in ice cold Et.sub.2O. The pellet was broken and washed three times with Et.sub.2O. The remaining pellet was purified by preparative HPLC. Combined HPLC fractions were concentrated and lyophilized. ESI-MS spectra were recorded at positive electrospray ionization mode on a Bruker Esquire 3000 plus (Bruker Daltonics GmbH, Bremen, Germany) at the University of Basel. MALDI-MS analysis was performed on an Applied Biosystem 4800 TOF-TOF.

(31) Characterization HPLC: t.sub.R=9.6 min (85_60, analyt.); t.sub.R=8.9 min (75_68, analyt.), t.sub.R=14.8 min (75_65, prep.)

(32) Characterization ESI-MS: m/z=990.1 [M+2H].sup.2+, 1979.8 [M+H]+

(33) Characterization MALDI-MS: m/z=1978.1 [M+H].sup.+, additional signals due to degradation of chelator during measurement (e.g. 1333.7, 1533.8 break of Hydroxamate-bonds).

(34) Molecular Weight: 1978.29 g/mol; Chemical Formula: C.sub.91H.sub.148N.sub.24O.sub.25

(35) Formula of ligand III and reaction: see FIG. 3

(36) Radiolabelling and Quality Control of .sup.89Zr-ABP-27 and .sup.89Zr-ABP-28

(37) i) Synthesis of Complex III

(38) Zr-89 was obtained in 1 M oxalic acid from Perkin Elmer. ABP-27 (M.sub.w=1978.29 g/mol) was prepared in lyophilized aliquots of 50 μg/50 μL and dissolved with deionized water upon starting the labeling. 10-30 μL (11.2-28.1 MBq) was taken from the Zr-89 stock solution and filled up to 200 μL with 1.0 M oxalic acid. 90 μL of 2.0 M Na.sub.2CO.sub.3 was added and incubated for 3 min at RT. 300 μL 0.5 M HEPES (pH 7.3), 710 μL of peptide solution (10 μg/10 μL (5 nmol)+700 μL H.sub.2O) and 300 μL 0.5 M HEPES (pH 7.3) were added. The pH was checked with pH-strips and ranged from pH 7.0-7.3. The reaction solution was incubated for 240 min at ambient temperature. Quality control of radiolabelling reactions were performed by means of HPLC and ITLC. Reversed phase HPLC was done on a Bischof system equipped with HPLC pumps 2250, a λ-1010 UV-detector, a Berthold LB509 radioflow detector and a Jupiter, 4μ Proteo 90A, 250×4.6 mm column from Phenomenex. The column was eluted with mixtures of acetonitrile (solvent A) and water with 0.1% trifluoroacetic acid (TFA) (solvent B) at a flow rate of 2 mL/min and a linear gradient: 0 min 80% B, 12.5 min 60% B, 13.5 min 5% B, 14.5 min 5% B, 15 min 80% B, 17 min 80% B. 20 μL of labeling solution were diluted in 50 μL 0.1 mM Desferrioxamine in 0.5 M HEPES solution (pH 7.3) and 10 μL were injected into HPLC. Radiochemical purity was determined by manual integration and determined to be between 94-97%. ITLC was done using Biodex green ITLC-Strips. 2 μL of labelling solution were spotted directly on the strip and developed with citric acid solution (20 mM, pH 4.8) as eluent. The strip was read out with a Cyclone Plus Phosphorimager and a MultiSensitive storage phosphor screen from Perkin Elmer. Radiochemical yield was determined by manual integration and determined to be >95%

(39) j) Synthesis and Characterisation of DFO*-NCS:

(40) Method:

(41) DFO*(50 mg, 0.066 mmol) was dissolved in a mixture isopropanol/water (4:1, 8 mL) by means of sonication and heating. The solution was then added at room temperature drop wise to a solution of phenylenediisothiocyanate (126 mg, 0.66 mmol) in chloroform (8 mL) containing triethylamine (0.079 mmol, 0.011 mL). The reaction was stirred at room temperature. After 24 h UPLC-MS indicated complete conversion of DFO*. The solvents were concentrated under reduced pressure and the resulting white solid was washed with diethylether (4×10 mL). The product was further purified by preparative HPLC (Nucleodur Preparative column (ISIS-C18 16×250 mm 5 μm)) at a flow rate of 8 mL min.sup.−1 with a linear gradient of A (acetonitrile (Sigma-Aldrich HPLC-grade) and B (distilled water containing 0.1% TFA): t=0 min A 36% B 64%, t=15 min A 44% B 56%, t=17 min A 36% B 64%. After lyophilisation, the product was obtained as a white powder (6 mg, 10%).

(42) .sup.1H NMR (600 MHz, DMSO-d.sub.6): δ (ppm) 9.57-9.71 (m, br, 5H), 7.90 (s, br, 1H), 7.77 (s, br, 3H), 7.52-7.56 (d, 2H), 7.34-7.38 (d, 2H), 3.41-3.49 (m, 10H), 2.97-3.03 (m, 6H), 2.55-2.60 (m, 6H), 2.23-2.3 (m, 6H), 1.97 (s, 3H), 1.48-1.51 (m, 10H), 1.36-1.39 (m, 6H), 1.19-1.21 (m, 8H). .sup.13C{.sup.1H} NMR (125 MHz, DMSO-d6): δ (ppm) 180.1, 172.0, 171.3, 170.2, 139.3, 132.6, 126.2, 124.6, 123.1, 47.1, 46.8, 43.7, 38.4, 29.9, 28.8, 28.0, 27.6, 27.1, 26.0, 23.6, 23.5, 20.4. Some carbon signals were not observed due to overlapping signals. HR-ESI-MS: calcd for C.sub.42H.sub.68N.sub.10O.sub.11S.sub.2/z [M+H.sup.+].sup.+ 953.45887. found 953.45832, calcd for C42H68N10O11NaS2/z [M+Na.sup.+].sup.+ 975.44027. found 975.44027.

(43) .sup.1H and .sup.13C NMR measurements were carried out on Bruker AV-600 (CP-TCl (CryoPorbe)) spectrometer and referenced to residual solvent peaks. High resolution ESI-MS spectra were recorded using a Bruker ESQUIRE-LC quadrupole ion trap instrument.

(44) ##STR00024##

(45) k) Synthesis of PEG Complex:

(46) ##STR00025##
R1=Methyl
D=Hydroxymate
X═[CH.sub.2].sub.5—N—[C═O]—[CH.sub.2].sub.2