IIIa Receptor Antagonists with 99mTc

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Bioconjugate Chem. 1996, 7, 196−202

196

Labeling Cyclic Glycoprotein IIb/IIIa Receptor Antagonists with 99mTc by the Preformed Chelate Approach: Effects of Chelators on Properties of [99mTc]Chelator-Peptide Conjugates Shuang Liu,* D. Scott Edwards,* Richard J. Looby, Michael J. Poirier, Milind Rajopadhye, Jeffrey P. Bourque, and Timothy R. Carroll Radiopharmaceuticals Division, The DuPont Merck Pharmaceutical Company, 331 Treble Cove Road, North Billerica, Massachusetts 01862. Received September 18, 1995X

Several cyclic GPIIb/IIIa receptor antagonists were labeled with 99mTc by the preformed chelate approach using chelators such as H4L1 [4,5-bis(mercaptoacetamido)pentanoic acid], H4L2 [3,4-bis(mercaptoacetamido)benzoic acid], H3L3 [2-(mercapto)ethylaminoacetyl-L-cysteine], H4L4 [N-(mercaptoacetyl)glycylglycylglycine], H4L5 [N-[2-(mercapto)propionyl]glycylglycylglycine], and H4L6 [N-[2(mercapto)propionyl]glycylglycyl-γ-aminobutyric acid]. In this approach, the [99mTc]chelator complexes are formed first, followed by the activation of the carboxylic group on the complex by formation of its tetrafluorophenol (TFP) ester and the conjugation of the TFP ester with an amino group of a cyclic GPIIb/IIIa receptor antagonist. The 99mTc-labeled cyclic GPIIb/IIIa receptor antagonists were characterized by radio-HPLC (high-performance liquid chromatography); differences in lipophilicity of the [99mTc]chelator-peptide conjugate are attributable to the effects of both the cyclic peptide and the chelator.

INTRODUCTION

Venous and arterial thrombus formations are common and potentially life-threatening events. Existing diagnostic modalities are inadequate for diagnosis and determination of the morphology of the evolving thrombus (1). Thus, the development of agents that will not only detect the location but, in addition, determine the age of the thrombi is a critical unmet need in diagnostic nuclear medicine. We have been actively pursuing a research program to develop a thrombus-imaging agent by labeling a platelet GPIIb/IIIa receptor antagonist with 99mTc (2, 3). Cyclic peptides containing the RGD (Arg-Gly-Asp) sequence have been shown to be high-affinity antagonists for the GPIIb/IIIa receptor (4). Since the GPIIb/IIIa receptor is expressed only on activated platelets (5), the 99mTc-labeled cyclic GPIIb/IIIa receptor antagonists should only be bound to platelets intimately involved in the thromboembolic event. 99mTc is the preferred radionuclide for diagnostic nuclear medicine because of its ideal physical properties (e.g. 6 h half-life, 140 keV, γ-emission), easy availability, and low cost. A large number of radiolabeling techniques have been developed in the last decade (6-10). These include the direct- and indirect-labeling approaches and the preformed chelate approach. The direct-labeling approach uses a reducing agent (such as stannous chloride) to convert disulfide linkages in a protein into free thiols, which bind strongly to the Tc. This approach is easy to carry out, but very little is known about the coordination chemistry of the Tc. There is little control over the stability of the 99mTc complex or the nonspecific binding. This method applies only to proteins or their fragments because many small peptides do not have any disulfide bonds or in some cases the disulfide bond is too critical * To whom correspondence should be addressed. Telephone: 508-671-8696 (S.L.) or 508-671-8311 (D.S.E.). Fax: 508-4367500. X Abstract published in Advance ACS Abstracts, February 1, 1996.

1043-1802/96/2907-0196$12.00/0

for maintaining their biological properties to be reduced. In the indirect-labeling approach, a bifunctional chelator is first attached to the peptide or protein to form a chelator-peptide (protein) conjugate. The radiolabeling can be achieved either by direct reduction of 99mTcO4- in the presence of chelator-peptide (protein) conjugate or by ligand exchange with an intermediate 99mTc complex such as [99mTc]glucoheptonate. The preformed chelate approach involves formation of the [99mTc]chelator complex and conjugation of the [99mTc]chelator complex to a peptide or protein in a separate step on the tracer level. In this approach, the chemistry is well-defined, and the peptide or protein is not exposed to the sometimes forcing conditions used to prepare the complex. Fritzberg and co-workers have used an N2S2 diamidedithiol (H4L1) and an N3S triamidethiol (MAG2-gaba, mercaptoacetylglycylglycyl-γ-aminobutyric acid) in labeling antibodies and their fragments with 99mTc (11-13) and 186Re (14, 15) by the preformed chelate approach. The attachment of the chelator and the 99mTc or 186Re does not significantly affect the physical and biological properties of the antibodies or their fragments, since the radiolabeling occurs only on a small portion of these macromolecules. We have applied the preformed chelate approach to label several cyclic GPIIb/IIIa receptor antagonists with 99mTc using chelators (Figure 1) such as N S diamidedi2 2 thiols (H4L1 and H4L2), an N2S2 monoamide-monoaminedithiol (H3L3), and N3S triamidethiols (H4L4-H4L6). We found that chelators have significant effects on properties (such as lipophilicity) of the [99mTc]chelatorpeptide conjugates. Chelators in their protected forms were used for the 99mTc labeling in the preformed chelate approach. Synthesis and properties of several [99mTc]chelator-peptide conjugates are descibed in this report. The biological evaluation of these conjugates in two canine thrombosis models will be described in a separate communication (16). EXPERIMENTAL SECTION

Materials. Chemicals were purchased from Aldrich Chemical Co. and were used as received. 1H NMR © 1996 American Chemical Society

Labeling Cyclic GPIIb/IIIa Receptor Antagonists

Figure 1. Chelators for receptor antagonists.

99mTc

labeling of cyclic GPIIb/IIIa

spectra were collected on a Bruker 270 MHz FT-NMR spectrometer. The NMR data are reported as δ (parts per million) relative to tetramethylsilane (TMS). Infrared spectra were recorded as KBr disks in the range of 4000-400 cm-1 on a Nicolet 5DXB IR spectrophotometer. FAB-MS and elemental analyses were performed by Oneida Research Services, Inc., Whitesboro, NY. NH3DCI mass spectra were recorded on a Finnigan MAT 8230 mass spectrometer. Na99mTcO4 was obtained from a commercial DuPont 99Mo/99mTc generator, North Billerica, MA. Deionized water was obtained from a Millipore MilliQ Water System and was of >18 MΩ quality. The benzoyl-protected chelators, Bz2-H2L1, Bz2-H2L2, Bz-H3L4, and Bz-H3L6, were prepared as previously reported (17, 18). Synthesis of the cyclic GPIIb/IIIa antagonists will be reported elsewhere (19). The high-performance liquid chromatography (HPLC) method used a Hewlett-Packard Model 1050 instrument and a Vydac C18 column (4.6 mm × 25 cm) at a flow rate of 1 mL/min with a gradient mobile phase from 100% A (10 mM phosphate buffer, pH 6) to 30% B (acetonitrile) at 15 min and 75% B at 25 min. Synthesis of the Trityl-Protected Ethyl Ester of H3L3. S-Trityl-L-cysteine Ethyl Ester (2). To a solution of L-cysteine ethyl ester (1) hydrochloride (18.6 g, 100 mmol) in 200 mL of trifluoroacetic acid (TFA) was added triphenylmethanol (52 g, 200 mmol). The resulting dark brown solution was allowed to stir for 2 h at room temperature under nitrogen. The solvent was removed in vacuo. To the residue were added ethanol (100 mL) and 1.0 M sodium ethoxide solution (50 mL). The reaction mixture was stirred for 90 min while the solution turned cloudy. The mixture was filtered, and the filtrate was concentrated in vacuo to give an oily residue. Flash column chromatography using ethyl acetate/hexane (1: 3) gave the desired product. A small amount of ethyl acetate in the product is difficult to remove. The yield was 37.5 g (96%). IR (cm-1): 3500-2500 (br, νN-H), 1750, 1685 (vs, νCdO). NH3-DCI MS: m/z 409 (M + NH4+), 392 ([M + H]+). 1H NMR (CDCl3): 1.15 (t, 3H, CH3, J ) 7.1 Hz), 2.60-2.95 (m, 2H, CH2), 3.05 (m, 1H, CH), 4.08 (m, 2H, CH2), 7.15-7.45 (m, 15H, aromatic hydrogens).

Bioconjugate Chem., Vol. 7, No. 2, 1996 197

N-(Bromoacetyl)-S-trityl-L-cysteine Ethyl Ester (3). A solution of S-trityl-L-cysteine ethyl ester (18 g, 46 mmol) and triethylamine (6.4 mL, 46 mmol) in dry tetrahydrofuran (THF) (250 mL) under nitrogen was cooled to 0 °C. A solution of bromoacetyl bromide (9.28 g, 46 mmol) in dry THF (60 mL) was added dropwise, during which time the solution turned cloudy. After it was stirred at 0 °C for 1 h and room temperature for an additional 1 h, the reaction mixture was filtered. The filtrate was concentrated in vacuo to give an oil, which was partitioned between methylene chloride and water (60 mL each). The organic layer was washed with 5% HCl and saturated sodium bicarbonate solution and dried over anhydrous magnesium sulfate. The solution was filtered, and volatiles were removed under reduced pressure to give the desired product. The yield was 16.3 g (69%). IR (cm-1): 3310 (s, νN-H), 2900-3100 (m, νC-H), 1750, 1715, 1660 (vs, νCdO). NH3-DCI MS: m/z 531 (M + NH4+), 514 ([M + H]+). 1H NMR (CDCl3): 1.26 (t, 3H, CH3, J ) 7.0 Hz), 2.60-2.95 (m, 2H, CH2), 3.82 (s, 2H, CH2), 4.20 (q, 2H, CH2, J ) 7.0 Hz), 4.50 (m, 1H, CH), 6.95 (d, 1H, NH, J ) 6.0 Hz), 7.20-7.45 (m, 15H, aromatic hydrogens). 2-[[[(S-Tritylmercapto)ethyl]amino]acetyl]-S-trityl-L-cysteine Ethyl Ester (4). To a solution of N(bromoacetyl)-S-trityl-L-cysteine ethyl ester (1.0 g, 1.98 mmol) and triethylamine (0.4 mL, 2.9 mmol) in methylene chloride (20 mL) was added S-trityl-2-aminoethanethiol (0.64 g, 2.0 mmol). The reaction mixture was stirred at room temperature for 7 days. To the mixture was added water (10 mL). The organic layer was separated, washed with saturated sodium bicarbonate solution (2 × 10 mL), water (2 × 10 mL), and brine (10 mL), and dried over anhydrous magnesium sulfate. After filtration, the filtrate was concentrated in vacuo to give a foamy product. Flash chromatography using ethyl acetate/hexane (3:1) gave the expected product. The yield was 0.56 g (38%). IR (cm-1): 3500-2500 (br, νN-H), 1745, 1615 (vs, νCdO). FAB-MS: m/z 751 ([M + H]+). 1H NMR (CDCl3): 1.25 (m, 3H, CH3), 2.30-2.80 (m, 6H, CH2), 3.10 (s, 2H, CH2CO), 4.15 (m, 2H, CH2), 4.54 (m, 1H, CH), 7.20-7.50 (m, 30H, aromatic hydrogens), 7.75 (d, 1H, NH, J ) 6.0 Hz). Anal. Calcd (found) for C47H46N2O3S2: C, 75.17 (74.15), H, 6.17 (6.07), N, 3.73 (3.68), S, 8.54 (8.57). Synthesis of Benzoyl-Protected H4L5. N-[2-(Benzoylmercapto)propionyl]glycine (6). Sodium hydroxide (4.5 g, 109 mmol) and N-(2-mercaptopropionyl)glycine (5) (8.20 g, 50 mmol) were dissolved in a mixture of water (40 mL) and toluene (30 mL). The temperature was lowered to 5-15 °C using an ice bath. Benzoyl chloride (4.6 mL, 51 mmol) in toluene (10 mL) was added dropwise with vigorous stirring. After addition, the mixture was stirred at 5-15 °C for another 30 min and then at room temperature for 2 h. The organic layer was separated, washed with H2O (2 × 20 mL), and discarded. Aqueous fractions were combined and acidified to pH ∼1.5 using concentrated HCl while a white precipitate formed. The solid was collected by filtration, washed with H2O and a small amount of ethanol, and dried under vacuum. The yield was 13.0 g (97%). IR (cm-1): 3375 (s, νN-H), 32002500 (br, νO-H), 1745 (vs, thio ester νCdO), 1663, 1625 (vs, amide and carboxylic νCdO). FAB-MS: m/z 268 ([M + H]+). 1H NMR (DMSO-d6): 1.47 (d, 3H, CH3, J ) 7.0 Hz), 3.79 (d, 2H, CH2, J ) 5.9 Hz), 4.40 (q, 1H, CH, J ) 7.0 Hz), 7.53 (m, 2H, dCH), 7.69 (m, 1H, dCH), 7.90 (dd, 2H, dCH, J ) 7.0 Hz), 8.59 (t, 1H, NH, J ) 5.8 Hz), 12.6 (bs, 1H, COOH). Anal. Calcd (found) for C12H13NO4S: C, 53.90 (53.89), H, 4.90 (4.81), N, 5.24 (5.22). N-[2-(Benzoylmercapto)propionyl]glycine Succinimide Ester (7). To a suspention of N-[2-(benzoylmercapto)propionyl]glycine (13.5 g, 50 mmol) and N-hydroxysuc-

198 Bioconjugate Chem., Vol. 7, No. 2, 1996

cinimide (5.75 g, 50 mmol) in THF (200 mL) was added 1-cyclohexyl-3-(2-morpholineethyl)carbodiimide metho-ptoluenesulfonate (25 g, 59 mmol), in a mixture of THF (100 mL) and water (100 mL). The reaction mixture was stirred for 24 h at room temperature. Upon addition of acetic acid, the mixture was stirred for another 2 h. The solid was separated by filtration and discarded. The filtrate was evaporated to dryness to give a white solid, which was collected, washed with diethyl ether, and dried in air. The yield was 10.5 g (57.5%). IR (cm-1): 3290 (s, νN-H), 1820 (m, succinimide νCdO), 1785 (m, ester νCdO), 1735 (vs, thio ester νCdO), 1600 (vs, amide νCdO). FABMS: m/z 365 ([M + H]+). 1H NMR (CDCl3): 1.57 (d, 3H, CH3, J ) 7.0 Hz), 2.79 (s, 4H, CH2), 4.33 (q, 1H, CH, J ) 7.0 Hz), 4.39 (m, 2H, CH2), 7.00 (t, 1H, NH, J ) 5.8 Hz), 7.44 (m, 2H, dCH), 7.59 (m, 1H, dCH), 7.93 (dd, 2H, dCH, J ) 7.0 Hz). Anal. Calcd (found) for C16H16N2O6S: C, 52.72 (52.70), H, 4.43 (4.21), N, 7.69 (7.69). N-[2-(Benzoylmercapto)propionyl]glycylglycylglycine (BzH3L5). N-[2-(Benzoylmercapto)propionyl]glycine succinimide ester (1.82 g, 5 mmol) and glycylglycine (0.66 g, 5 mmol) were suspended in a mixture of ethanol (150 mL) and water (30 mL). The mixture was heated to reflux for 5 h, during which time the cloudy mixture became a clear solution. The solution was then cooled to room temperature and was kept stirring overnight. Evaporation of solvents under reduced pressure gave the expected product. The crude product was washed with water. Recrystallization from 50% aqueous ethanol solution produced the pure product. The yield was 1.58 g (63%). IR (cm-1): 3380, 3300 (s, νN-H), 3100-2500 (br, νO-H), 1738 (vs, thio ester νCdO), 1680, 1660 (vs, amide νCdO). FAB-MS: m/z 382 ([M + H]+)]. 1H NMR (DMSO-d6): 1.48 (d, 3H, CH3, J ) 7.0 Hz), 3.78 (m, 4H, CH2), 3.85 (d, 2H, CH2, J ) 6.0 Hz), 4.41 (m, 1H, CH), 7.52 (m, 2H, dCH), 7.70 (m, 1H, dCH), 7.90 (m, 2H, dCH), 8.15 (t, 1H, NH, J ) 3.0 Hz), 8.51 (t, 1H, NH, J ) 3.0 Hz), 8.80 (t, 1H, NH, J ) 3.0 Hz). Anal. Calcd (found) for C16H19N3O6S: C, 50.39 (50.59), H, 5.02 (5.78), N, 11.02 (10.70). Deprotection of 2-[[[(S-Tritylmercapto)ethyl]amino]acetyl]-S-trityl-L-cysteine Ethyl Ester. 2-[[[(STritylmercapto)ethyl]amino]acetyl]-S-trityl-L-cysteine ethyl ester (10 mg) was dissolved in 2 mL of TFA to give a bright yellow solution. The mixture was stirred at room temperature for 5 min. Upon addition of triethylsilane (1 mL), the reaction mixture became clear. TFA and excess triethylsilane were removed under vacuum to give a white solid residue. To the residue were added 0.5 mL of 5 N NaOH solution and 1.5 mL of THF. The mixture was heated on a boiling water bath for 5 min. To the mixture was added 0.5 M phosphate buffer (1.5 mL, pH 12), and the pH was adjusted to 10-11 using 5 N HCl. The solution was filtered, and the filtrate was diluted to 5 mL using 0.5 M phosphate buffer (pH 12). The freshly prepared solution was used immediately for 99mTc labeling. General Procedure for 99mTc Labeling of Cyclic GPIIb/IIIa Receptor Antagonists. Chelation. To a 10 mL vial was added 0.5 mL of 99mTcO4- solution (200400 mCi/mL in saline), 0.5 mL of ligand solution (2 mg/ mL in 0.5 M phosphate buffer, pH 12), 0.15-0.20 mL of sodium dithionite solution (5 mg/mL in 0.5 M phosphate buffer, pH 12) for H4L1-H3L3, or SnCl2‚2H2O solution (5 mg/mL in 1.0 N HCl) for H4L4-H4L6. The pH was adjusted to 11 (8-10 for H3L3), and the mixture was heated at 100 °C for 30 min. The reaction mixture was analyzed by radio-HPLC. Activation. To the reaction mixture were added 0.2 mL

Liu et al. Table 1. HPLC Chromatographic Data for Complexes

99mTc

complexa

retention time (min)a

labeling yield (%)

[99mTcO(L1)]2[99mTcO(L1-TFP)][99mTcO(L1-III)][99mTcO(L1-IV)][99mTcO(L1-V)][99mTcO(L2)]2[99mTcO(L2-TFP)][99mTcO(L2-III)][99mTcO(L2-IV)][99mTcO(L2-V)][99mTcO(L3)][99mTcO(L3-TFP)] [99mTcO(L3-III)] [99mTcO(L3-IV)] [99mTcO(L4)]2[99mTcO(L4-TFP)][99mTcO(L4-III)][99mTcO(L5)]2[99mTcO(L5-TFP)][99mTcO(L5-III)][99mTcO(L6)]2[99mTcO(L6-TFP)][99mTcO(L6-III)][99mTcO(L6-IV)][99mTcO(L6-V)]-

9.5/10.0 23.0 15.0 15.7 16.2 14.5 25.0 18.1 17.8 19.1 7.4/8.7 24.6 14.8 16.2 7.2 20.8 13.2 8.1/8.4 21.9 13.5 10.8/11.0 22.8 14.5 14.9 15.5

g90 g90 60 45 50 85 80 33 21 30 g75 g90 35 34 g90 g80 50 90 g80 55 98 g90 47 35 70

aUnder the experimental conditions described in the Experimental Section, the carboxylate groups in complexes [99mTcO(L)]n(L ) L1-L6) are deprotonated.

of 1.0 N HCl, 0.5 mL of 2,3,5,6-tetrafluorophenol (TFP) solution (100 mg/mL in 90% aqueous CH3CN), and 0.5 mL of 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride (WS-CDI) solution (100 mg/mL in 90% aqueous CH3CN). The pH was adjusted to 6, and the reaction mixture was heated at 45-50 °C for 30 min. Radio-HPLC showed g85% of the activated ester. Conjugation. To the mixture was added 2-3 mg of peptide in 0.5 mL of 0.5 M phosphate buffer (pH 9). After the pH was adjusted to 9.0-10.0, the reaction mixture was incubated at 40 °C for 30 min and analyzed by radioHPLC. The conjugation yields ranged from 30 to 60%, depending on the identity and the amount of the peptide. The yields and retention times for the [99mTc]chelator complexes, the TFP-ester complexes, and [99mTc]chelator-peptide conjugates are listed in Table 1. The products were purified and separated from the unlabeled cyclic peptide before biological evaluation. RESULTS AND DISCUSSION

Selection of Target Molecules. Platelet GPIIb/IIIa is a membrane protein that mediates the aggregation of platelets. In response to stimulation by a variety of agonists, including thromboxane, thrombin, or epinephrine, this protein undergoes a conformational change, allowing it to bind to fibrinogen (4). Multiple GPIIb/IIIa molecules appear to bind to a single molecule of fibrinogen, which leads to platelet aggregation and formation of a platelet-rich thrombus. Thus, inhibition of fibrinogen binding to the GPIIb/IIIa receptor blocks platelet aggregation and thrombus formation. As with many members of the integrin superfamily of adhesion molecules, the GPIIb/IIIa receptor recognizes proteins and peptides bearing the tripeptide sequence Arg-Gly-Asp (RGD). A number of RGD-containing small molecules, including peptides (4, 20-22) and peptidomimetics (23, 24), have been synthesized and studied for their antithrombotic activities. These studies are directed toward the development of antithrombotic agents

Labeling Cyclic GPIIb/IIIa Receptor Antagonists

Bioconjugate Chem., Vol. 7, No. 2, 1996 199 Scheme 1. Synthesis of Trityl-Protected Ethyl Ester of H3L3

Scheme 2. Synthesis of Benzoyl-Protected H4L5 Figure 2. Cyclic GPIIb/IIIa receptor antagonists.

for the treatment of thrombosis. It seems that cyclization of a peptide can give rise to impressive gains in affinity and specificity due to restriction of conformational mobility. Recently, DeGrado (4) and co-workers reported a series of very potent cyclic GPIIb/IIIa receptor antagonists, two (I and II) of which are shown in Figure 2. Both peptides contain a rigid m-(aminomethyl)benzoic acid (Mamb) moiety and show very high affinity for the GPIIb/ IIIa receptor with IC50 values in the nanomolar range for inhibition of both platelet aggregation and fibrinogen binding (4). This makes them the candidates of choice as target molecules to carry the radionuclide (99mTc) to thrombi. If the attachment of a chelator and 99mTc labeling do not block RGD sequence binding, the 99mTclabeled cyclic peptides should be able to bind strongly to the GPIIb/IIIa receptor and thus incorporate into rapidly growing thrombi under both arterial and venous conditions. Since peptides are expected to exhibit more rapid blood clearance than the larger proteins, the 99mTc-labeled cyclic GPIIb/IIIa receptor antagonists should have the potential to permit earlier imaging of thrombi. In order to label cyclic GPIIb/IIIa receptor antagonists with 99mTc, a linker is required to connect the cyclic peptide framework with a Tc-binding group. Since the RGD sequence is vital for maintaining biological activity, we prepared linker-modified cyclic peptides by incorporation of a lysine residue between the arginine residue and the Mamb ring or by attachment of a 6-aminocaproic acid group on the Mamb ring. The use of a 6-aminocaproic acid linker keeps the Tc center far from the RGD tripeptide sequence to minimize the effect of 99mTc labeling on the binding affinity for the GPIIb/IIIa receptor (19). The amino group can be used for the conjugation of either a chelator by the indirect-labeling approach or a 99mTc complex by the preformed chelate approach. Selection of a Chelator. A bifunctional chelator can be divided into three parts: a binding unit, a conjugation group, and a spacer, which connects the binding unit and the conjugation group. There are several requirements for an ideal chelator. First, the binding unit can selectively stabilize an intermediate or lower oxidation state of Tc so that the 99mTc complex is not subject to redox reactions; oxidation changes are often accompanied by transchelation of 99mTc from a [99mTc]chelator-peptide conjugate to the native chelating ligands in biological systems. Second, the chelator forms a 99mTc complex which is kinetically inert with respect to dissociation. Finally, the conjugation group can be easily attached to the target peptide.

Various chelators have been used in labeling proteins and peptides with 99mTc and 186Re. These include DTPA (23), N2S2 diamidedithiols (11-13, 17), N3S triamidethiols (14, 15, 17), BATOs (26, 27), N2S2 diaminedithiols (28), and hydrazino nicotinamide (HYNIC) (29, 30). The chelators used in this study are chosen because they all form stable anionic or neutral technetium complexes (neglecting charges on the carboxylate group) with the TcdO core. The coordination chemistry of these three types of chelators with technetium is well-documented (31-35). The use of these chelators allows us to study their effect on the physical and biological properties of the [99mTc]chelator-peptide conjugates. Synthesis of Chelators. H3L3. The S-trityl-protected ethyl ester of H3L3 was prepared (Scheme 1) in three steps from L-cysteine ethyl ester (1). The reaction of L-cysteine ethyl ester hydrochloride with triphenylmethanol in TFA afforded compound 2, which was then allowed to react with bromoacetyl bromide in THF in presence of triethylamine to give compound 3. The reaction of compound 3 with S-trityl-2-aminoethanethiol (33) in the presence of Et3N in dichloromethane produced the S-trityl-protected ethyl ester of H3L3 (4). It was characterized by spectroscopic (IR, NMR, and FAB-MS) methods and elemental analysis. Deprotection of the trityl groups in compound 4 was achieved by the radical cleavage of the C-S bonds. Hydrolysis of the ethyl ester of H3L3 under basic conditions afforded unprotected H3L3. The freshly prepared H3L3 solution was used immediately for 99mTc labeling. Benzoyl-Protected H4L5. The benzoyl-protected H4L5 was readily prepared (Scheme 2) from commercially available N-(2-mercaptopropionyl)glycine (5) in three steps. The thiol group was protected by reaction of compound 5 with benzoyl chloride under basic conditions. Activation of the carboxylic group of compound 6 was achieved by formation of its succinimide ester (7). Con-

200 Bioconjugate Chem., Vol. 7, No. 2, 1996

Liu et al.

Scheme 3. Labeling Cyclic GPIIb/IIIa Peptides by the Preformed Chelate Approach Using H4L1

jugation of compound 7 with glycylglycine afforded the benzoyl-protected H4L5, which has been characterized by IR, 1H NMR, and FAB-MS spectroscopy and elemental analysis. Labeling of Cyclic GPIIb/IIIa Antagonists by the Preformed Chelate Approach. The preformed chelate approach used in this study involves three steps of tracer level (99mTc) synthesis: chelation of the radionuclide (99mTc), activation of the pendant carboxylate group on the chelate by formation of its TFP ester, and conjugation of the TFP ester with the amino group of a peptide. The reaction product in each step is characterized by radioHPLC. The final products, the [99mTc]chelator-peptide conjugates, are purified by HPLC before biological evaluation. N2S2 Diamidedithiols. Since the coordination chemistry of diamidedithiols with technetium is well-understood, we used chelators, H4L1 and H4L2, to label the cyclic IIb/IIIa antagonists with 99mTc. We prepared the complex [99mTcO(L1)]2- by reducing pertechnetate with sodium dithionite in the presence of H4L1 under basic conditions (pH 10-12). Heating at 100 °C for 30 min is necessary for successful radiolabeling. As expected, the coordination of H4L1 to the Tc center results in two diastereomers (syn and anti) due to the relative orientation of the functional group (CH2CH2COOH) to the TcdO core. The carboxylic group was activated by formation of its tetrafluorophenol (TFP) ester, [99mTcO(L1-TFP)]-. Conjugation of [99mTcO(L1-TFP)]- with a cyclic GPIIb/ IIIa peptide was carried out at pH 9.5-10.0 to form the complex [99mTcO(L1-peptide)]- (Scheme 3). The conjugation yields varied from 50 to 80%, depending upon the amount of cyclic peptide used for conjugation. Total time for radiolabeling was about 3 h. As an example, Figure 3 shows radio-HPLC chromatograms for complexes [99mTcO(L1)]2- (A), [99mTcO(L1-TFP)]- (B), [99mTcO(L1V)] (C), and the HPLC-purified [99mTcO(L1-V)] (D). The HPLC-purified [99mTcO(L1-V)] was found to be stable for at least 12 h in saline solution. H4L2 is of interest because the carboxylic group is wellseparated from the metal center by a rigid aromatic benzene ring so that intramolecular interaction between the carboxylate O and the Tc(V) center is minimized. Unlike chelators functionalized on a saturated carbon, it does not form diastereomers upon coordination to the TcdO core. We have used H4L2 in labeling the cyclic GPIIb/IIIa peptides by a procedure similar to that described for H4L1. It is noted that the conjugation of [99mTcO(L2-TFP)]- with the cyclic IIb/IIIa peptides requires higher pH (10-10.5) than that of [99mTcO(L1TFP)]-. The conjugation yields are lower (20-30%) than those obtained using H4L1.

Figure 3. Radio-HPLC chromatograms for [99mTcO(L1)]2- (A), [99mTcO(L1-TFP)]- (B), [99mTcO(L1-V)]- (C), and the HPLCpurified [99mTcO(L1-V)]- (D).

Figure 4. Syn and anti isomers of [99mTcO(L3)]-.

Monoamide-Monoaminedithiol. H3L3 contains a secondary amine nitrogen, an amide nitrogen, and two thiolate sulfur donors. It acts as a tribasic ligand in binding to the TcdO core to form a neutral complex, [99mTcO(L3)], providing that the carboxylate group is protonated. We prepared [99mTcO(L3)] by reducing pertechnetate with sodium dithionite in the presence of free ligand H3L3 at pH 8-10. Two isomers were observed because of the anti and syn orientations of the carboxylate group relative to the TcdO moiety (Figure 4). The carboxylate group in [99mTcO(L3)] was activated by formation of its TFP ester, [99mTcO(L3-TFP)], which was then conjugated with a peptide at pH 9.5-10 to afford [99mTcO(L3-peptide)] conjugates. The conjugation yields were 30-50%. The HPLC-purified [99mTcO(L3V)] was found to be stable for 24 h in saline solution. N3S Triamidethiols. Like N2S2 diamidedithiols, N3S triamidethiols also form stable Tc(V) complexes with the TcdO core. For H4L5 and H4L6, the presence of a methyl group on the ligand backbone results in two diastereomers because of its relative orientation to the TcdO bond (Figure 5). Stannous chloride was used instead of sodium dithionite as the reducing agent for pertechnetate. The chelation reaction was carried out

Labeling Cyclic GPIIb/IIIa Receptor Antagonists

Figure 5. Syn and anti isomers of [99mTcO(L)]2- (L ) L5 and L6).

at pH ∼11 with heating for 30 min at 100 °C. The activation of the carboxylic group and conjugation of the TFP ester with a peptide were performed in a fashion similar to that described for H4L1. The conjugation yields ranged from 30 to 70%, depending on the amount of cyclic peptide used for conjugation. The 99mTc-labeled cyclic GPIIb/IIIa antagonists [[99mTcO(L-peptide)] (L ) L6, peptide ) III and V)] were stable for 12 h in saline solution. HPLC Data for 99mTc Complexes. Table 1 summarizes radio-HPLC data for all the 99mTc complexes, which include [99mTcO(L)]n-, [99mTcO(L-TFP)]n-, and [99mTcO(L-peptide)]n-. For the 99mTc-labeled cyclic GPIIb/ IIIa receptor antagonists, [99mTcO(L-peptide)]n- (L ) L1, L3, L5, and L6), the two isomeric forms are not wellseparated using the HPLC method described in the Experimental Section. They are isolated as a mixture of two diastereomers for the in vivo animal studies in a canine AV model (16). In general, the conversion of the anionic carboxylate group into its TFP ester results in a dramatic increase in lipophilicity. Conjugation of the TFP ester with a cyclic peptide results in a sharp decrease in lipophilicity. These changes are reflected by the differences in the HPLC retention times and are caused by the change in the molecular charge and the identity of the molecule attached to the carboxylic group. For complexes [99mTcO(L-V)]- (L ) L1-L6), the rank order in retention time is the same as that for complexes [99mTcO(L)]n- (L ) L1-L6). For complexes [99mTcO(L1peptide)]- and [99mTcO(L6-peptide)]- (peptide ) III-V), the retention time follows the rank order of the peptide. Therefore, both cyclic peptide and the chelator have significant effects on physical properties (such as lipophilicity) of the [99mTc]chelator-peptide conjugates. These effects are also seen on biological properties (such as thrombus uptake and the target-to-background ratio) of the conjugates, [99mTcO(L-peptide)]- (L ) L1 and L6, peptide ) III and V), in two canine thrombosis models (16). CONCLUSIONS

In conclusion, three cyclic GPIIb/IIIa receptor antagonists were labeled with 99mTc by the preformed chelate approach using chelators such as N2S2 diamidedithiols (H4L1 and H4L2), a monoamine-monoamidedithiol (H3L3), and N3S triamidethiols (H4L4-H4L6). The 99m Tc-labeled cyclic GPIIb/IIIa receptor antagonists were formed in moderate to good yields, depending on the amount of the peptide used in the conjugation reaction. They were purified and separated from the unlabeled cyclic peptide before biological evaluation. Although the preformed chelate approach has been widely used in labeling proteins and their fragments with 99m Tc (11-13, 17, 36) and 186Re (14, 15), there is little information available on the use of this approach in labeling biologically active small molecules such as peptides. The advantage of the approach is that the chemical and biological properties of a series of conjugates with a range of lipophilicities and differing charges

Bioconjugate Chem., Vol. 7, No. 2, 1996 201

can be determined before extensive efforts are directed at synthesizing chelator-modified peptides. We have synthesized and evaluated conjugates using six different chelators and three different peptides using milligram quantities of the peptides. For proteins and their fragments, the attachment of the chelator and 99mTc does not significantly affect their physical and biological properties because these properties are determined mainly by the much larger polypeptide structure. For small molecules such as cyclic GPIIb/ IIIa peptides, the 99mTc complexes constitute a significant portion of the [99mTc]chelator-peptide conjugates. The physical and biological properties should be a function of both the peptide and the 99mTc complex, as seen in this and other (16) studies. However, the use of the appropriate combination of chelator, linker, and peptide can still result in 99mTc-labeled GPIIb/IIIa receptor antagonists with good biological activity. ACKNOWLEDGMENT

Acknowledgment is made to J. Pietryka for the 1H NMR spectra, to N. E. Williams for analytical HPLC, and to Dr. T. D. Harris, D. Glowacka, P. R. Damphousse, and K. Yu for the synthesis of the cyclic IIb/IIIa receptor antagonists (III-V). We also thank Mr. Carl Schwartz in the Mass Spectrometry Laboratory, Central Research and Development, DuPont Merck Pharmaceutical Co., Wilmington, DE, for the mass spectra of compounds 3 and 4. LITERATURE CITED (1) Knight, L. (1990) Radiopharmaceuticals for thrombus detection. Semin. Nucl. Med. 20, 52-67. (2) Barrett, J. A., Heminway, S., Damphousse, D. J., Thomas, J., Looby, R. J., Edwards, D. S., Harris, T. D., Rajopadhye, M., Liu, S., and Carroll, T. R. (1994) Platelet GPIIb/IIIa antagonists in the canine arteriovenous shunt: potential thrombus imaging agents. J. Nucl. Med. 35, 52P (abstract 202). (3) Harris, T. D., Barrett, J. A., Bourque, J. P., Carroll, T. R., Damphousse, P. R., Edwards, D. S., Glowacka, D., Liu, S., Looby, R. J., Poirier, M. J., Rajopadhye, M., and Yu, K. Design and synthesis of radiolabeled GPIIb/IIIa receptor antagonists as potential thrombus imaging agents. J. Nucl. Med. 35, 245P (abstract 1005). (4) Jackson, S., DeGrado, W., Dwivedi, A., Parthasarathy, A., Higley, A., Krywko, J., Rockwell, A., Markwalder, J., Wells, G., Wexler, R., Mousa, S., and Harlow, R. (1994) Templateconstrained cyclic peptides: design of high-affinity ligands for GPIIb/IIIa. J. Am. Chem. Soc. 116, 3220-3230. (5) Haskel, E., Adams, S., Feigen, L., Saffitz, J., Gorczynski, R., Sobel, D., and Abendschein, D. (1989) Prevention of reoccluding platelet-rich thrombi in canine femoral arteries with a novel peptide antagonist of platelet glycoprotein IIb/ IIIa receptors. Circulation 80, 1775-1782. (6) Fritzberg, A. R., Berninger, R. W., Hadley, S. W., and Wester, D. W. (1988) Approaches to radiolabeling of antibodies for diagnosis and therapy of cancer. Pharm. Res. 5, 325334. (7) Otsuka, F. L., and Welch, M. J. (1987) Methods to label monoclonal antibodies for use in tumor imaging. Nucl. Med. Biol. 14, 243-249. (8) Hnatowich, D. J. (1990) Antibody radiolabeling, problems and promises. Nucl. Med. Biol. 17, 49-55. (9) Griffiths, G. L., Goldenberg, D. M., Jones, A. L., and Hansen, H. J. (1992) Radiolabeling of monoclonal antibodies and fragments with technetium and rhenium. Bioconjugate Chem. 3, 91-99. (10) Eckelman, W. C., Paik, C. H., and Steigman, J. (1989) Three approaches to radiolabeling antibodies with 99mTc. Nucl. Med. Biol. 18, 589-603. (11) Fritzberg, A. R., Abrams, P. G., Beaumier, P. L., Kasina, S., Morgan, A. C., Rao, T. N., Reno, J. M., Sanderson, J. A.,

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