WO1997047329A2 - Radiopharmaceutical compositions capable of localizing at sites of thrombus - Google Patents

Radiopharmaceutical compositions capable of localizing at sites of thrombus Download PDF

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Publication number
WO1997047329A2
WO1997047329A2 PCT/US1997/009292 US9709292W WO9747329A2 WO 1997047329 A2 WO1997047329 A2 WO 1997047329A2 US 9709292 W US9709292 W US 9709292W WO 9747329 A2 WO9747329 A2 WO 9747329A2
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Prior art keywords
conh
substituted
containing ligand
protecting group
sulfur protecting
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PCT/US1997/009292
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French (fr)
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WO1997047329A3 (en
WO1997047329A9 (en
Inventor
Larry P. Feigen
Daniel Lee Flynn
Philip Needleman
Ananthachari Srinivasan
Jeffery Alan Zablocki
Michelle A. Schmidt
Original Assignee
G.D. Searle & Co.
Mallinckrodt Medical, Inc.
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Application filed by G.D. Searle & Co., Mallinckrodt Medical, Inc. filed Critical G.D. Searle & Co.
Priority to EP97931012A priority Critical patent/EP0910416A2/en
Priority to JP50164398A priority patent/JP2001504801A/en
Priority to AU34749/97A priority patent/AU3474997A/en
Publication of WO1997047329A2 publication Critical patent/WO1997047329A2/en
Publication of WO1997047329A3 publication Critical patent/WO1997047329A3/en
Publication of WO1997047329A9 publication Critical patent/WO1997047329A9/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/06—Dipeptides
    • C07K5/06104—Dipeptides with the first amino acid being acidic
    • C07K5/06113—Asp- or Asn-amino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K51/00—Preparations containing radioactive substances for use in therapy or testing in vivo
    • A61K51/02—Preparations containing radioactive substances for use in therapy or testing in vivo characterised by the carrier, i.e. characterised by the agent or material covalently linked or complexing the radioactive nucleus
    • A61K51/04—Organic compounds
    • A61K51/08—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins
    • A61K51/088—Peptides, e.g. proteins, carriers being peptides, polyamino acids, proteins conjugates with carriers being peptides, polyamino acids or proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2121/00—Preparations for use in therapy
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2123/00—Preparations for testing in vivo

Definitions

  • This invention relates generally to radiopharmaceutical compositions and, more specifically, to a radiopharmaceutical composition capable of imaging or providing radiotherapy to sites of thrombus in a warm-blooded individual.
  • platelets During the blood clotting mechanism, platelets rapidly respond to form a thrombus in cooperation with fibrinogen. At the site of injury, platelets bind to fibrinogen which initiates platelet aggregation to form the thrombus. As currently understood, a significant aspect of the aggregation process involves Glycoprotein(gp)IIb/IIIa, a platelet surface integrin which binds fibrinogen and links together activated platelets to form an aggregate.
  • DVT Deep vein thrombosis
  • PE pulmonary embolism
  • the present invention is directed to a radiopharmaceutical capable of localizing at a site of thrombus containing activated platelets within a mammalian body wherein the radiopharmaceutical comprises a linear peptidomimetic capable of specifically binding to the GPIIb/IIIa integrin receptor of activated platelets in the thrombus and a radionuclide covalently bound to the peptidomimetic.
  • a ligand composition having the following general formula is provided:
  • A is -CH- or -N-
  • B is -CH- or -N-, with the proviso that when A is -N-, B is -CH-, and when B is -N-, then A is -CH-
  • -D-E- is -CH 2 -CH 2 -, or -NHCO- with the proviso that when A is -N-, then -D-E is -CH 2 -CH 2 -, and when B is -N-, then -D-E is -NHCO-
  • Ri is hydrogen, lower alkyl, or acyloxyalkyl
  • K is hydrogen, carboxyl, lower alkyl, aralkyl, substituted or unsubstituted aromatic, or an alkylene or substituted alkylene substituted with one of the following substituents: primary amine, secondary amine, cyclic or acyclic tertiary amine, carboxyl, ester, hydroxyl, ether, thi
  • CSNH(CH 2 ) M CONH n is 0-5; n' is 0 or 1 ; m is 0-10; and Z is a metal binding group capable of covalently binding a radionuclide.
  • the ligand compositions of the invention as described above are provided complexed with a selected metal radionuclide to form a diagnostic or therapeutic radiopharmaceutical.
  • a diagnostic radionuclide the composition is capable of imaging a site of thrombus and when complexed with a therapeutic radionuclide the composition is capable of providing radiotherapy to the site of thrombus.
  • kits for preparing radioimaging or radiotherapeutic compositions that include the ligand compositions of the invention and the reagents necessary to produce a radiolabelled ligand composition.
  • Kits for labeling with the selected radionuclide are comprised of a container containing a selected amount of the ligand composition in a pharmaceutically acceptable carrier and a sufficient amount of the other reagents necessary to label the ligand composition, such as a reducing agent.
  • a radiopharmaceutical composition that is capable of selectively imaging or providing therapy to sites of thrombus and that rapidly clears from the blood; the provision of such a composition comprising a radiolabelled, linear peptidomimetic that inhibits platelet aggregation; and the provision of such compositions that are useful as diagnostic or therapeutic agents for thrombus imaging or therapy at sites of thrombus including embolism, deep vein thrombosis, cerebral vascular thrombus, coronary vascular thrombus, and peripheral arterial thrombus.
  • compositions are relatively small in size, approximately 1000-3000 daltons, and can be readily produced Moreover, the compositions are not immunogenic and clear rapidly from the circulating blood. This feature permits rapid imaging of thrombi with little background interference which could complicate a diagnosis.
  • the ligand compositions described herein comprise a linear peptidomimetic region that is capable of binding to activated platelets and a region capable of binding a metal, preferably a metal radionuclide.
  • a metal preferably a metal radionuclide.
  • the presence of a metal binding region and a metal radionuclide complexed thereto does not adversely affect the ability of the linear peptidomimetic region to localize at the activated platelets.
  • a linear peptidomimetic composition capable of localizing at or binding to activated platelets incorporating a metal binding group is provided and has the following general formula:
  • A is -CH- or -N-
  • B is -CH- or -N-, with the proviso that when A is -N-, B is -CH-, and when B is -N-, then A is -CH-
  • -D-E- is -CH 2 -CH r , or -NHCO- with the proviso that when A is -N-, then -D-E is -CH 2 -CH 2 -, and when B is -N-, then -D-E is -NHCO-
  • Ri is hydrogen, lower alkyl, or acyloxyalkyl
  • K is hydrogen, carboxyl, lower alkyl, aralkyl, substituted or unsubstituted aromatic, or an alkylene or substituted alkylene substituted with one of the following substituents: primary amine, secondary amine, cyclic or acyclic tertiary amine, carboxyl, ester, hydroxyl, ether, thi
  • lower alkyl either alone or within other terms such as phenylalkyl and alkyloxycarbonyl, embraces a linear or branched chain saturated hydrocarbon radical having 1-6 carbon atoms.
  • Illustrative of such radicals are methyl, ethyl, propyl, 1 -methylethyl, butyl, 2-methylpropyl, 1-methylpropyl, 1, 1-dimethylethyl, pentyl and hexyl.
  • alkylene either alone or within other terms, embraces linear or branched chain alkene radicals having 1-6 carbon atoms.
  • Illustrative of such radicals are emthylene, ethylene, propylene, butylene, propylene and hexalene.
  • the metal binding group Z of the ligand compositions described is capable of covalently binding a selected radionuclide thereto.
  • the metal binding group is coupled to or inco ⁇ orated in the peptidomimetic in a manner that does not interfere or adversely affect the binding properties or specificity of the peptidomimetic.
  • the use of various metal binding groups for radiolabeling compounds is well known in the art. Suitable metal binding groups generally include those which contain a tetradentate ligand for binding the metal radionuclide such as known polyaminocarboxyiate, N 3 S and N 2 S 2 ligands.
  • metal binding groups that may be used in conjunction with the peptidomimetics of the present invention include 2,3- bis(mercaptoacetamido)propanoate (U.S. Patent No. 4,444,690), S- benzoylmercaptoacetylglycylglycylglycine (U.S. Patent No. 4,861,869), dicyciic dianhydrides such as DTP A and EDTA and derivatives thereof (U S. Patent No. 4,479,930), NS chelates containing amino groups to enhance chelation kinetics (U.S. Patent No. 5,310,536), N 2 S 2 chelates as described in U.S. Patent No.
  • Z is defined by the general formula:
  • R 2 is COCH(R 5 )-S-Ro; Rs is H, -(CH 2 ) P -R 7 ; p is 1-5; R 7 is a hydrogen, primary amine, secondary amine, cyclic or acyclic tertiary amine, carboxyl, ester, hydroxyl, ether, thiol, thioether, guanido, or an imine; R « is a hydrogen, aliphatic or aromatic acyl, acetamidoalkyl, benzamidoalkyl, unsubstituted or substituted tetra-hydropyranyl, unsubstituted or substituted tetrahydrofuranyl, or alkoxyalkyl; R 3 is (CH 2 ) P -Q where p' is 0-6, Q is hydrogen, alkylene or substituted alkylene, aryl or substituted aryl group for attachment to Y; R4 is (CH 2 ),-T where s is
  • the metal binding group is coupled to or inco ⁇ orated into the peptidomimetic by standard methodology known in the field of the invention and may be added at any location on the peptidomimetic provided that the biological activity of the peptidomimetic is not adversely affected
  • Suitable peptidomimetic containing ligands within the scope of the invention include the following compositions.
  • R_ ⁇ is a suitable sulfur protecting group.
  • a suitable sulfur protecting group is a pharmaceutically acceptable compound capable of preventing potential oxidation of the sulfur or reaction of the sulfur with other reactive groups.
  • Illustrative protecting groups include hydrogen, acetals such as ethoxyethyl, methoxymethyl, substituted and unsubstituted tetrahydrofuranyl, substituted and unsubstituted tetrahydropyranyl, acetamidoalkyl such as acetamidomethyl, acyl such as alkanoyl, benzoyl and substituted benzoyl.
  • radionuclide complexes may be prepared by reacting a specified amount of the selected composition with a metal salt of the selected radionuclide in the presence of a reducing agent and a transfer agent
  • reducing agents include, but are not limited to, dithionite, stannous ion, and ferrous ion
  • Preferred transfer agents include, but are not limited to, sodium gluconate, sodium tartrate, sodium citrate, and mannitol Any radionuclide having diagnostic or therapeutic value can be used as the radiolabel for the compositions of this invention.
  • the radionuclide is a ⁇ -emitting or ⁇ -emitting radionuclide selected from the lanthanide or actinide series of the elements Positron-emitting radionuclides, e.g 68Ga, may also be used Suitable ⁇ -emitting radionuclides include those which are useful in diagnostic imaging applications.
  • the ⁇ -emitting radionuclides preferably have a half-life of from 1 hour to 40 days, preferably from 12 hours to 3 days. Examples of suitable ⁇ -emitting radionuclides include 67Ga, 1 1 lln, 99mTc, 169Yb and 186Re Most preferably, the radionuclide is 99mTc.
  • Suitable ⁇ -emitting radionuclides include those which are useful in therapeutic applications. Examples include 90Y, 67Cu, 186Re, 188Re, 169Er, 121Sn, 127Te, 143Pr, 198Au, 109Pd, 165Dy, 32P, 142Pr, and 153Sm.
  • the ⁇ -emitting radionuclide preferably has a half-life of from 2 hours to two weeks, and more preferably from about 2 hours to 100 hours
  • Suitable radiopharmaceutical complexes include the following compositions
  • M is selected from the group consisting of 67Ga, 1 1 lln, 99mTc, 169Yb, 186Re 90Y, 67Cu, 186Re, 188Re, 169Er, 121 Sn, 127Te, 143Pr, 198Au, 109Pd, 165Dy, 32P,
  • the radiolabeled compositions of the invention and their pharmaceutically acceptable salts are useful as a diagnostic imaging agent or in therapeutic applications.
  • the radiolabeled composition is prepared in a pharmaceutically acceptable carrier, e.g. saline or blood plasma, and is administered to an individual in a diagnostically or 0 therapeutically effective amount as determined using standard methods known to those in the art.
  • the carrier may also contain pharmaceutically acceptable adjunct materials such as salts, buffers, preservatives and the like.
  • the radiopharmaceutical composition of the present invention is provided in a kit whereby the radionuclide is provided in one container, e.g. a vial, and the composition capable of complexing with 5 the radionuclide is provided in a second container and the contents mixed just prior to administration.
  • the mixture may be heated if necessary to effect complete labelling.
  • the provision of such radiolabeled complexes in kit form and the preparation of the final radiolabeled product are standard and routine in the field of nuclear medicine.
  • the final radiopharmaceutical product should be of high radiochemical purity, preferably greater than 95%, and at least greater than 90%, as determined by standard protocols known in the art.
  • the radiolabeled complex is prepared to provide a radioactive dose of between about 0.05 mCi and about 40 mCi, preferably about lmCi to about 20mCi, to the individual in accordance with standard radiopharmaceutical dosing determinations.
  • a diagnostically effective amount means an amount of the radiopharmaceutical sufficient to permit its detection by scintigraphic means and "a therapeutically effective amount” means an amount sufficient to effect a therapeutic treatment at the targeted biological site.
  • the radiolabeled peptides may be administered intravenously in any conventional medium for intravenous injection.
  • Imaging of the biological site may be effected within about one hour post-injection, but may also take place several hours post-injection. Any conventional method of imaging for diagnostic pu ⁇ oses may be utilized.
  • compositions of the present invention may be synthesized either in a sequential manner or by segment condensation methodology as further described below.
  • Boc-Asp( ⁇ -OtBu)-NH(CH 2 ) 4 -CH(NH-Cbz)-COOH N- ⁇ -Cbz-L-Lysine (7.25 g, 25.9 mmol) was suspended in a mixture of 1 N sodium bicarbonate (65 mL) and dioxane (65 mL).
  • the N-hydroxysuccinimide ester of N-Boc-L-Asp( ⁇ -t-Bu) (10.0 g, 25.9 mmol) in dioxane was subsequently added, and the reaction was permitted to stir overnight at room temperature before concentrating under reduced pressure.
  • Boc-Asp( ⁇ -OtBu)-NH-(CH 2 ) 4 -CH(NH 2 )-CO-Gly-NH-(CH 2 ) 2 -N(CH 3 ) 2 N-Boc-L-Asp( ⁇ -t-Bu)- ⁇ -L-Lys( ⁇ -Cbz)-Gly-dmen (1 97 g, 2.90 mmol) was dissolved in methanol (25 mL). After flushing with nitrogen, the 10% Pd / C catalyst (0.2 g) was added. The mixture was shaken for 6 hours at ambient temperature under an atmosphere of hydrogen (40 p.s.i ).
  • N-Boc-L-Asp( ⁇ -t-Bu)- ⁇ -L-Lys-Gly-dmen 2 AcOH (1.54 g, 2.32 mmol) and the N-hydroxysuccinimide ester of S-THP-mercaptoacetic acid (0.66 g, 2 43 mmol) were dissolved in dichloromethane (40 mL) in the presence of triethylamine, and the reaction was continued overnight at ambient temperature. The reaction was subsequently diluted with dichloromethane and extracted with water (lx) and saturated sodium bicarbonate (3x) and washed with brine before drying over anhydrous magnesium sulfate.
  • N-Boc-L-Asp( ⁇ -t-Bu)- ⁇ -L-Lys( ⁇ -(S-THP-mercaptoacetyl))-Gly-dmen (40 mg, 0.057 mmol) was dissolved in 1 : 1 TFA / dichloromethane (0.5 mL each). The rea ⁇ ion was stirred for 5 hours at room temperature before removing the solvent under reduced pressure. The orange, oily residue was purified by reverse phase Cu with a 3% acetic acid / water mobile system (29 mg, 76% yield). Mass Spec.
  • Step f ABAS-L-Asp- ⁇ -L-Lys( ⁇ -(S-THP-mercaptoacetyl))-Gly-dmen
  • the hydrochloride salt of aminobenzamidinosuccinate ( 81 mg, 0.30 mmol) was added to dry DMF (4 mL) followed by N-methylmorphoiine (30 mg, 0.30 mmol) and isobutyl chloroformate (41 mg, 0.30 mmol) at 0°C under nitrogen. After stirring for 5 minutes, a solution of L-Asp-e-L-Lys(a-(S-THP-mercaptoacetyl))-Gly-dmen (200 mg 0.30 mmol) and N-methylmorpholine (91 mg, 0.90 mmol) in DMF (2 mL) was added. Stirring was continued for 2 hours afterwhich the solvent was removed under reduced pressure.
  • Example 2 This Example describes the radiolabeling of the compound of Example 1 with
  • Method B Alternatively the componets that are present in the Merck-Frosst kit (gluconate salts, SnCl 2 ) can be added individulally to form the kit. Compound in Example 1 was radiolabelled with Tc-99m according to the procedure described in Method A.
  • This Example describes the labelling of the compound of Example 1 with a non-radioactive rhenium 185,187 isotope to confirm the composition of the final composition.
  • Example 4 This Example describes Platelet Aggregation Inhibition Assays of the compound of Example 2 to illustrate that the compound binds to GPIIb/IIIa receptors when radiolabelled.
  • Platelet rich plasma (0.45 mL) was aliquoted into siliconized cuvettes and stirred (1 100 ⁇ m) at 37°C for 1 min. prior to adding 50 mL of prediluted test compound. After 1 minute of mixing, aggregation was initiated by the addition of 50 mL of 200 mM of ADP. Aggregation was recorded for 3 minutes in a Payton dual channel aggregometer (Payton Scientific, Buffalo, NY). The percent inhibition of maximal response (saline control) for a series of test compound dilutions was used to determine a dose response curve. The compounds were tested in duplicate and half- maximal inhibition (IC50) was calculated graphically from the dose response curve.
  • Example 2 The compound of Example 2 was injected (25mL, 1.5-3.5 mCi/mL) into Sprague-Dawley rats. Groups of three animals were sacrificed at the time points indicated below to determine the amount of radioactivity remained in the organs. Results: (to be filled)
  • This example describes the stepwise preparation of a composition having the structure:
  • N-Cbz-L-Glu(g-t-Bu)-dmen (7.00 g, 17.2 mmol) was dissolved in methanol (130 mL). After flushing with nitrogen, 10% Pd/C catalyst (0.7 g) was added. The mixture was shaken for 6 hours at ambient temperature under an atmosphere of hydrogen (40 p.s.i.). The reaction was filtered through a pad of Celite, and the filtrate was
  • the ethyl acetate layer was discarded, and the aqueous layer was acidified to pH 3 with 1 N hydrochloric acid.
  • the product was extracted into ethyl acetate; the combined organic layer was washed with water (2x) and brine (lx) before drying over anhydrous sodium sulfate. Filtration and evaporation of the solvent under reduced pressure revealed a white foam.
  • the material was purified on SiO 2 using a methanol / dichloromethane gradient to afford 2.60 g of product (67% yield).
  • Example 6 The compound of Example 6 was radiolabeled with Tc-99m according to method A described in Example 2.
  • This Example describes the labelling of the compound of Example 6 with a non-radioactive rhenium 185,187 isotope to confirm the composition of the final composition.
  • This Example describes Platelet Aggregation Inhibition Assays of the compound of Example 8 to illustrate that the compound binds to GPIIb/IIIa receptors when radiolabelled following the procedure of Example 4.
  • Example 7 The compound of Example 7 was injected (25mL, 1.5-3.5 mCi/mL) into Sprague-Dawley rats. Groups of three animals were sacrificed at the time points indicated below to determine the amount of radioactivity (%LD/g) remaining in the organs.
  • N- ⁇ -Boc-p-Fmoc-amino-L-Phe (10.0 g, 19.9 mmol) and N- hydroxysuccinimide (2.52 g, 21.9 mmol) were dissolved in dichloromethane (315 mL).
  • Dicyclohexylcarbodiimide (4.93 g, 23.9 mmol) was subsequently added, and the reaction was stirred overnight at ambient temperature.
  • the dicyclohexylurea was removed by filtration, and the filtrate was diluted with dichloromethane.
  • the organic layer was then extracted with saturated sodium bicarbonate (3x) and washed with brine before drying over anhydrous magnesium sulfate.
  • Gly-dmen hydrochloride (2.98 g, 16.4 mmol) was dissolved in a mixture of 1 N sodium bicarbonate (45 mL) and dioxane (45 mL).
  • the N-hydroxysuccinimide ester of N-a-Boc-p-Fmoc-amino-L-Phe (10.8 g, 18.0 mmol) in dioxane (45 mL) was subsequently added, and the reaction was stirred overnight at ambient temperature. The dioxane was then removed under reduced pressure.
  • the product was extracted into ethyl acetate, and the combined organic layer was extracted with saturated sodium bicarbonate (3x) and washed with brine before drying over anhydrous magnesium sulfate.
  • N- ⁇ -Boc-p-Fmoc-amino-L-Phe-Gly-dmen (5.00 g, 7.94 mmol) was dissolved in 1 : 1 TFA / dichloromethane (50 mL each) at 0°C. After the addition was complete, the ice bath was removed, and the reaction was stirred at ambient temperature for 3 hours before removing the solvent under reduced pressure. The residue was purified by reverse phase C ⁇ 8 chromatography utilizing a 30% acetonitrile / 3% acetic acid / water mobile phase to provide 4.1 g of a clear oil (80% yield). ⁇ NMR (DMSO-d*) ⁇
  • the diacetate salt of p-Fmoc-amino-L-Phe-Gly-dmen (4.00 g, 6.17 mmol) was dissolved in dioxane (20 mL) and 1 N sodium bicarbonate (20 mL).
  • the N- hydroxysuccinimide ester of S-tetrahydropyranyl-mercaptoacetic acid (1.85 g, 6.78 mmol) in dioxane (20 mL) was subsequently added, and the reaction was stirred
  • ABAS hydrochloride (0.64 g, 2.4 mmol) was dissolved in DMF(30 mL). N- methylmo ⁇ holine (0.24 g, 2.4 mmol) and isobutyl chloroformate (0.32 g, 2.4 mmol) were added after cooling to 0°C. After stirring for 0.5 hour, L-Asp( ⁇ -t-Bu)-L-Lys( ⁇ - Boc)-OtBu AcOH (1.2 g, 2.3 mmol) and N-methylmo ⁇ holine (0.23 g, 2.3 mmol) were added in DMF (15 mL). The reaction was then stirred overnight at ambient temperature before removing the solvent under reduced pressure.
  • the reaction was stirred overnight at ambient temperature before removing the solvent under reduced pressure.
  • the target compound was finally cleanly isolated by HPLC using a Nova-Pak Cu 30x300 mm column to provide 40 mg of a glassy solid (10% yield).
  • Example 1 1 The compound of Example 1 1 was radiolabeled with Tc-99m according to method A described in Example 2.
  • This Example describes the labelling of the compound of Example 11 with a non-radioactive rhenium 185,187 isotope to confirm the composition of the final composition.
  • the composition of the Re-complexes were confirmed by mass spectra (m/e 1 102 (M+l).
  • This Example describes Platelet Aggregation Inhibition Assays of the compound of Example 13 to illustrate that the compound binds to GPIIb/IIIa receptors when radiolabelled following the procedure of Example 4.
  • IC 5 o 4.6 X 10- 8 M
  • Example 15 This study was designed to evaluate the biodistribution pattern of the compound of Example 12 to determine the clearance profile from blood, route of excretion and in vivo stability of the complex by HPLC analysis of urine samples, in a rat model.
  • Example 12 The compound of Example 12 was injected into Sprague-Dawley rats according to the procedure outlined in Example 5. The amount of radioactivity present in the major excretionary organs at different time points (1, 4 and 24 hours) are given below. All values are %ID/g
  • the dog is first anesthetized with an intravenous injection of sodium pentobarbital (30mg/kg).
  • An IV catheter is placed into the jugular vein and advanced to the pulmonary artery.
  • Visualization of the catheter placement is facilitated using fluoroscopy.
  • Embolization coils of various sizes (3-8mm) are next released via 0 the catheter using the appropriate guide wire, and localization of the coils is followed by fluoroscopy.
  • 3-5 a satisfactory number of coils (3-5) have been placed and visualized, the catheter is removed and the vein ligated.
  • the formation of a deep vein thrombus (DVT) in the saphenous vein is facilitated by the placement of a 16 gauge needle into the vein followed by the passage of a 5 mm emboilzation coil through the 5 needle directly into the vein.
  • DVD deep vein thrombus
  • Example 17 This Example describes the stepwise preparation of the composition having the structure
  • ABAS-L-Asp-L-Lys-p-amino-Phe( ⁇ -S-THP-mercaptoacetyl)-Glu-dmen Thiourea (MP-2068) was prepared in a manner similar to ABAS-L-Asp-L-Lys-p- amino-Phe(a-S-THP-mercaptoacetyl)-Glu-dmen Thiourea (see example 3) except that Glu(g-t-Bu)-dmen was substituted for Gly-dmen.
  • This Example describes the stepwise preparation of the composition having the structure 2) 2 -N(CH 3 ) 2
  • the material (2.25 g, 3.19 mmol) was dissolved in methanol (25 mL). After flushing with nitrogen, the catalyst was added, and the hydrogenolysis was performed in the usual manner. After five hours, the reaction was filtered through Celite, and the filtrate was concentrated to reveal a quantitative yield of a white foam.
  • the ABAS-Asp( ⁇ -t-Bu) (1 30 g, 2 79 mmol) was dissolved in DMF (70 mL) at 0°C N-methylmo ⁇ holine (0 28 g, 0 31 mL, 2 79 mmol) and isobutyl chloroformate (0 38 g, 0 36 mL, 2.79 mmol) were subsequently added After stirring for 0 5 hour at 0°C, a solution of Ala-e-L-Lys(a-Boc)-Glu(g-t-Bu)-dmen (1 6 g, 2.79 mmol) in DMF (30 mL) was added.
  • Example 18 was radiolabeled with Tc-99m according to method A and B described in Example 2.
  • This Example describes the labelling of the compound of Example 11 with a non-radioactive rhenium 185,187 isotope to confirm the composition of the final composition
  • the composition of the Re-complexes were confirmed by mass spectra (m/e 1013 (M+l)).
  • Example 21 This Example describes Platelet Aggregation Inhibition Assays of the compound of Examples 18 and 20 to illustrate that the compound binds to GPirb/TIIa receptors when radiolabelled following the procedure of Example 4.
  • This study was designed to evaluate the biodistribution pattern of the compound of Example 19 to determine the clearance profile from blood, route of excretion and in vivo stability of the complex by HPLC analysis of urine samples, in a rat model.
  • the compound of Example 19 was injected into Sprageue-Dawley rats according to the procedure outlined earlier.
  • the amount of radioactivity present in the major excretionary organs at different time points (1 , 4 and 24 hours) are given below.
  • the compound of claim 19 was administered to a canine according to the general protocol described in Example 16 The following results were obtained.
  • This Example describes the stepwise preparation of a compound having the structure
  • ABAS-L-Asp-L-Asp- ⁇ , ⁇ -Lys( ⁇ -S-THP-mercaptoacetyl)-Glu-dmen was prepared in a manner similar to Example 18 (as previously desribed) except that Cbz-L-Asp( ⁇ -OSu)- OBn was substituted for Cbz- ⁇ -Ala-OSu Mass Spec (ESI) 950 (M+l, 10%), 476.5 ((M+2)/2, 100%), Retention Time 22 min (0 46 x 25 cm Vydac Cu, 1 mL / min flow rate), Gradient 95% A - 70% A over 35 minutes (Solvent A: 0 1% TFA / water, Solvent B 0 1% TFA / 10% water / acetonitrile)
  • This Example describes the stepwise preparation of a compound having the structure
  • ABAS-L-Asp-L-Lys2TFA (25.6 mg, .034 mmol) and S-THP-mercaptoacetyl- AAA( ⁇ -OSu)-Glu( ⁇ -t-Bu)-dmen (19.5 mg, .041 mmol) were dissolved in DMF (0.5 mL) in the presence of triethylamine (.012 mL). The reaction was continued overnight before diluting with 1 : 1 acetonitrile / water (2 mL) and acidifying to pH 4 with 0.2 N hydrochloric acid. The solvent was subsequently removed under reduced pressure.
  • Example 25 was radiolabeled according to the method A described Example 2.
  • This Example describes the labelling of the compound of Example 25 with non radioactive rhenium 185, 187 isotope to confirm the composition of the final composition.
  • Example 28 This Example describes Platelet Aggregation Inhibition Assays of the compound of Example 26 and 27 to illustrate that the compound binds to GPIIb/IIIa receptors when radiolabelled following the procedure of Example 4
  • This study was designed to evaluate the biodistribution pattern of the compound of Example 26 to determine the clearance profile from blood, route of excretion and in vivo stability of the complex by HPLC analysis of urine samples, in a rat model.
  • the compound of Example 26 was injected (25mL, 1.5-3.5 mCi/mL) into Sprague-Dawley rats. Groups of three animals were sacrificed at the time points indicated below to determine the amount of radioactivity remaining in the organs. All values are %LD/g
  • N-Boc-L-Asp( ⁇ -OtBu)-L-Lys( ⁇ -Cbz)-OtBu L-Lys( ⁇ -Cbz)-OtBu hydrochloride (7.93 g, 21.2 mmol) was dissolved in dichloromethane (140 mL) in the presence of the triethylamine (2.15 g, 21.2 mmol, 3.0 mL).
  • N-Boc-L-Asp( ⁇ -OtBu)-OSu (10.0 g, 21.2 mmol) was subsequently added, and the reaction was continued overnight at ambient temperature before diluting with dichloromethane.
  • Step c N-Boc-L-Asp( ⁇ -OtBu)-L-Lys( ⁇ - ⁇ -L-Glu(N- ⁇ -Cbz)-OBn)-OtBu
  • N-Boc-L-Asp( ⁇ -OtBu)-L-Lys( ⁇ - ⁇ -L-Glu(N- ⁇ -Cbz)-OBn)-OtBu 5.8 g, 7.01 mmol was dissolved in methanol (75 mL). After flushing with nitrogen, 10% P ⁇ VC catalyst (0.5 g) was added. The mixture was shaken for 6 hours at ambient temperature under an atmosphere of hydrogen (40 p.s.i.). The reaction was filtered through a pad of Celite, and the filtrate was concentrated under reduced pressure to reveal a white foam in quantitative yield.
  • Step f N-Boc-L-Asp( ⁇ -OtBu)-L-Lys( ⁇ - ⁇ -L-Glu(N- ⁇ -S-THP-mercaptoacetyl)-Gly-dmen)-
  • N-Boc-L-Asp( ⁇ -OtBu)-L-Lys( ⁇ - ⁇ -L-Glu(N- ⁇ -S-THP-mercaptoacetyl))-OtBu (1.91 g, 2.51 mmol) and N-hydroxysuccinimide (0.32 g, 2.76 mmol) were dissolved in dichloromethane (40 mL). Dicyclohexylcarbodiimide (0.62 g, 3.01 mmol) was subsequently added, and the reaction was continued overnight at ambient temperature. The reaction was then filtered to remove the dicyclohexylurea, and the filtrate was diluted with dichloromethane.
  • the organic layer was extracted with saturated sodium bicarbonate (3x) and washed with brine (lx) before drying over anhydrous magnesium sulfate. Filtration and evaporation of the solvent under reduced pressure revealed a foam which was immediately used without further purification.
  • the active ester (1.05 g, 1.27 mmol) was added to a suspension of Gly-dmen hydrochloride (0.23 g, 1.27 mmol) in dichloromethane (25 mL) in the presence of triethylamine (0.13 g, 1.27 mmol). After adding DMF (2 mL) to improve solubility, the reaction was continued overnight at room temperature before diluting with dichloromethane.
  • N-Boc-L-Asp( ⁇ -OtBu)-L-Lys( ⁇ - ⁇ -L-Glu(N- ⁇ -S-THP-mercaptoacetyl)-Gly- dmen)-OtBu (0.99, 1.11 mmol) was dissolved in 1 : 1 TFA / dichloromethane (4 mL each). After two hours, the solvent was removed under reduced pressure, and the residue was purified by reverse phase Cu chromatography using 3% acetic acid / water as the mobile phase to afford 300 mg of product (34% yield).
  • ABAS HCl (104 mg, 0.38 mmol) was dissolved in DMF (5 mL). N- methylmo ⁇ holine (39 mg, 0.38 mmol, 0.042 mL) and isobutyl chloroformate (52 mg, 0.38 mmol) were added after cooling to 0°C. After stirring for one half hour, Asp-L- Lys(e-g-L-Glu(N-a-S-THP-mercaptoacetyl)-Gly-dmen) (290, 0.36 mmol) and N- methylmo ⁇ holine (73 mg, 0.72 mmol) were added, and the reaction was continued overnight at ambient temperature.
  • This Example describes the stepwise preparation of a compound having the structure
  • Step b ABAS-L-Asp-L-Lys phenylpropionyl- ⁇ -L-Lys(N- ⁇ -S-THP-mercaptoacetyl)-Gly-dmen
  • a 99m-Tc labeled molecule consisting of an N3S-chelate chemically linked to a peptidominmatic moiety which has high affinity for the GpIIb/IIIa receptor expressed on activated platelets was prepared from an instant kit and 99m-pertechnatate with radiochemical purity >95% and is stable for up to 6 hours. Studies in normal volunteers showed rapid blood clearance 11 ⁇ 4 min) and extensive liver uptake (31.7 ⁇ 1.6% i.d. 30 min p.i.). The pu ⁇ ose of this procedure was to assess the labeled compound for imaging fresh thrombi in humans.

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Abstract

A radiopharmaceutical capable of localizing at a site of thrombus containing activated platelets within a mammalian body is provided wherein the radiopharmaceutical comprises a linear peptidomimetic containing ligand capable of specifically binding to the GPIIb/IIIa integrin receptor of platelets in the thrombus and a radionuclide covalently bound to the peptidomimetic containing ligand. The ligand compositions of the invention are provided complexed with a selected metal radionuclide to form a diagnostic or therapeutic radiopharmaceutical to image a site of thrombus or provide radiotherapy to the site of thrombus, respectively. Methods of imaging a site of thrombus in a mammalian body by administering a diagnostically effective amount of the radiopharmaceutical composition of this invention complexed with a selected diagnostic metal radionuclide and methods of providing therapy to a site of thrombus in a mammalian body by administering a therapeutically effective amount of the radiopharmaceutical composition of this invention complexed with a selected therapeutic metal radionuclide are also provided as well as kits for preparing such radiopharmaceutical compositions.

Description

RADIOPHARMACEUTICAL COMPOSITIONS CAPABLE OF LOCALIZING AT
SITES OF THROMBUS
The present application claims priority under 35 USC §1 10 (c) of United States provisional application Serial No. 60/019,513, filed June 10, 1996.
Field of the Invention
This invention relates generally to radiopharmaceutical compositions and, more specifically, to a radiopharmaceutical composition capable of imaging or providing radiotherapy to sites of thrombus in a warm-blooded individual.
Background of the Invention
During the blood clotting mechanism, platelets rapidly respond to form a thrombus in cooperation with fibrinogen. At the site of injury, platelets bind to fibrinogen which initiates platelet aggregation to form the thrombus. As currently understood, a significant aspect of the aggregation process involves Glycoprotein(gp)IIb/IIIa, a platelet surface integrin which binds fibrinogen and links together activated platelets to form an aggregate. Deep vein thrombosis (DVT) and pulmonary embolism (PE) are common clinical observations resulting from thrombus formation and are associated with nearly 5 million patients in the United States alone. PE results in over 100,000 deaths per year and patients with DVT formation have a high probablity of PE formation. Several interventional methods using an anticoagulant or fibrinolytic have been utilized to treat DVT and PE, including treatment with heparin, streptoicinase or recombinant tissue plasminogen activators. Before these treatments can begin, however, the diagnosis of the condition must be made. Diagnostic radionuclides have been successfully used to detect various pathological conditions. The method is effective when the radionuclide can be effectively attached to a moiety that can localize to a selected biological site without interfering with the localization or binding of the localizing moiety. In this manner, the rapid identification of these sites becomes possible.
Various approaches for the detection of thrombi have been proposed. Existing non-radionuclide based methods of detecting and diagnosing thrombus formation include contrast venography and ultrasound. A prior knowledge of thrombus formation is highly desirable for either of these methods to be used advantageously and the venography technique is invasive. Radiolabeled thrombin inhibitors, plasmin, plasminogen activators such as TPA, heparin, fibronectin and anti-platelet monoclonal antibodies have also variously been proposed and used to radioimage thrombi. These radiolabelled approaches, however, present numerous disadvantages which limit their desirability as a diagnostic radiopharmaceutical. Most significantly, a long waiting period between injection and imaging is typically associated with these radiolabelled compositions which is considered undesirable in terms of patient comfort and ease of use.
Hence, a need exists for a diagnostic composition that rapidly locates sites of thrombus in vivo to facilitate a determination as to whether further diagnostic and/or therapeutic treatment is necessary or to provide a quick and reliable means to follow the course of prior treatment of a thrombus.
Summary of the Invention
The present invention is directed to a radiopharmaceutical capable of localizing at a site of thrombus containing activated platelets within a mammalian body wherein the radiopharmaceutical comprises a linear peptidomimetic capable of specifically binding to the GPIIb/IIIa integrin receptor of activated platelets in the thrombus and a radionuclide covalently bound to the peptidomimetic. In one significant aspect of the invention, a ligand composition having the following general formula is provided:
Figure imgf000005_0001
where A is -CH- or -N-, and B is -CH- or -N-, with the proviso that when A is -N-, B is -CH-, and when B is -N-, then A is -CH-; -D-E- is -CH2-CH2-, or -NHCO- with the proviso that when A is -N-, then -D-E is -CH2-CH2-, and when B is -N-, then -D-E is -NHCO-; Ri is hydrogen, lower alkyl, or acyloxyalkyl; K is hydrogen, carboxyl, lower alkyl, aralkyl, substituted or unsubstituted aromatic, or an alkylene or substituted alkylene substituted with one of the following substituents: primary amine, secondary amine, cyclic or acyclic tertiary amine, carboxyl, ester, hydroxyl, ether, thiol, thioether, guanido, imine, aminoalkyl, monoaminoalkyl, diaminoalkyl, hydroxyalkyl, alkoxylalkyl, hydroxycarbonylalkyl, alkoxycarbonylalkyl, mercaptoalkyl, or alkylthioalkyl; X is NH, CO, S, or O; Y is NH, CO, CS, CONH, CSNH, NHCO, NHCS, CONH0(CH2),. 4CONH, CONH(CH2)MCONH, CSNH0(CH2)1-4CONH, or CSNH(CH2)MCONH, with the proviso that X and Y are different except when X is S, and when X is O, Y is CO, and when X is S, Y is S, CO or CS, and when X is NH, Y is CO, CONH, CSNH, CONH0(CH2),_,CONH, CONH(CH2)MCONH, CSNH0(CH2)MCONH, or CSNH(CH2)MCONH, and when X is NH, Y is not NH, NHCO or NHCS, and when X is CO, Y is NH, O or S, and when X is CO, Y is not CO, CS, CONH, CSNH, CONH0(CH2)MCONH, CONH(CH2)1-4CONH, CSNH0(CH2)MCONH, or
CSNH(CH2)MCONH; n is 0-5; n' is 0 or 1 ; m is 0-10; and Z is a metal binding group capable of covalently binding a radionuclide.
In a further significant aspect of the invention, the ligand compositions of the invention as described above are provided complexed with a selected metal radionuclide to form a diagnostic or therapeutic radiopharmaceutical. When complexed with a diagnostic radionuclide the composition is capable of imaging a site of thrombus and when complexed with a therapeutic radionuclide the composition is capable of providing radiotherapy to the site of thrombus.
Also provided are methods of imaging a site of thrombus in a mammalian body by administering a diagnostically effective amount of the radiopharmaceutical composition of this invention complexed with a selected diagnostic metal radionuclide in a pharmaceutically acceptable carrier and methods of providing therapy to a site of thrombus in a mammalian body by administering a therapeutically effective amount of the radiopharmaceutical composition of this invention complexed with a selected therapeutic metal radionuclide in a pharmaceutically acceptable carrier.
The invention also provides kits for preparing radioimaging or radiotherapeutic compositions that include the ligand compositions of the invention and the reagents necessary to produce a radiolabelled ligand composition. Kits for labeling with the selected radionuclide are comprised of a container containing a selected amount of the ligand composition in a pharmaceutically acceptable carrier and a sufficient amount of the other reagents necessary to label the ligand composition, such as a reducing agent.
Among the many objects and advantages of the present invention include the provision of a radiopharmaceutical composition that is capable of selectively imaging or providing therapy to sites of thrombus and that rapidly clears from the blood; the provision of such a composition comprising a radiolabelled, linear peptidomimetic that inhibits platelet aggregation; and the provision of such compositions that are useful as diagnostic or therapeutic agents for thrombus imaging or therapy at sites of thrombus including embolism, deep vein thrombosis, cerebral vascular thrombus, coronary vascular thrombus, and peripheral arterial thrombus.
Detailed Description of the Invention
According to the present invention, it has been discovered that certain linear, peptidomimetic antagonists of GPIIb/IIIa can be labeled with a radionuclide without compromising the ability of the peptidomimetic to localize at the site of the thrombus. The compositions are relatively small in size, approximately 1000-3000 daltons, and can be readily produced Moreover, the compositions are not immunogenic and clear rapidly from the circulating blood. This feature permits rapid imaging of thrombi with little background interference which could complicate a diagnosis.
The ligand compositions described herein comprise a linear peptidomimetic region that is capable of binding to activated platelets and a region capable of binding a metal, preferably a metal radionuclide. Surprisingly, the presence of a metal binding region and a metal radionuclide complexed thereto does not adversely affect the ability of the linear peptidomimetic region to localize at the activated platelets.
In one aspect of the invention, a linear peptidomimetic composition capable of localizing at or binding to activated platelets incorporating a metal binding group is provided and has the following general formula:
Figure imgf000007_0001
where A is -CH- or -N-, and B is -CH- or -N-, with the proviso that when A is -N-, B is -CH-, and when B is -N-, then A is -CH-; -D-E- is -CH2-CHr, or -NHCO- with the proviso that when A is -N-, then -D-E is -CH2-CH2-, and when B is -N-, then -D-E is -NHCO-; Ri is hydrogen, lower alkyl, or acyloxyalkyl; K is hydrogen, carboxyl, lower alkyl, aralkyl, substituted or unsubstituted aromatic, or an alkylene or substituted alkylene substituted with one of the following substituents: primary amine, secondary amine, cyclic or acyclic tertiary amine, carboxyl, ester, hydroxyl, ether, thiol, thioether, guanido, imine, aminoalkyl, monoaminoalkyl, diaminoalkyl, hydroxyalkyl, alkoxylalkyl, hydroxycarbonylalkyl, alkoxycarbonylalkyl, mercaptoalkyl, or alkylthioalkyl; X is NH, CO, S, or O; Y is NH, CO, CS, CONH, CSNH, NHCO, NHCS, CONH0(CH2),. 4CONH, CONH(CH2)MCONH, CSNH0(CH2)1-4CONH, or CSNH(CH2)1-4CONH, with the proviso that X and Y are different except when X is S, and when X is O, Y is CO, and when X is S, Y is S, CO or CS, and when X is NH, Y is CO, CONH, CSNH, CONH0(CH2)1-4CONH, CONH(CH2),.4CONH, CSNH0(CH2)1.4CONH, or CSNH(CH2)MCONH, and when X is NH, Y is not NH, NHCO or NHCS, and when X is CO, Y is NH, O or S, and when X is CO, Y is not CO, CS, CONH, CSNH, CONH0(CH_),-4CONH, CONH(CH2),.4CONH, CSNH0(CH2)1-4CONH, or CSNH(CH2)MCONH; n is 0-5; n' is 0 or 1 ; m is 0-10; and Z is a metal binding group capable of covalently binding a radionuclide.
As used herein, the term "lower alkyl", either alone or within other terms such as phenylalkyl and alkyloxycarbonyl, embraces a linear or branched chain saturated hydrocarbon radical having 1-6 carbon atoms. Illustrative of such radicals are methyl, ethyl, propyl, 1 -methylethyl, butyl, 2-methylpropyl, 1-methylpropyl, 1, 1-dimethylethyl, pentyl and hexyl. The term "alkylene", either alone or within other terms, embraces linear or branched chain alkene radicals having 1-6 carbon atoms. Illustrative of such radicals are emthylene, ethylene, propylene, butylene, propylene and hexalene.
The metal binding group Z of the ligand compositions described is capable of covalently binding a selected radionuclide thereto. The metal binding group is coupled to or incoφorated in the peptidomimetic in a manner that does not interfere or adversely affect the binding properties or specificity of the peptidomimetic. The use of various metal binding groups for radiolabeling compounds is well known in the art. Suitable metal binding groups generally include those which contain a tetradentate ligand for binding the metal radionuclide such as known polyaminocarboxyiate, N3S and N2S2 ligands. More particularly, metal binding groups that may be used in conjunction with the peptidomimetics of the present invention include 2,3- bis(mercaptoacetamido)propanoate (U.S. Patent No. 4,444,690), S- benzoylmercaptoacetylglycylglycylglycine (U.S. Patent No. 4,861,869), dicyciic dianhydrides such as DTP A and EDTA and derivatives thereof (U S. Patent No. 4,479,930), NS chelates containing amino groups to enhance chelation kinetics (U.S. Patent No. 5,310,536), N2S2 chelates as described in U.S. Patent No. 4,965,392, the N3S chelates as described in U.S. Patent No. 5,120,526, and the N2S2 chelates containing cleavable linkers as described in U.S. Patent No. 5,175,257. All of the patents referred to above and the teachings therein are hereby incorporated by reference hereto.
In a preferred embodiment, Z is defined by the general formula:
R2-NH-CH(R3)-[CH(R4)]p"-CO-NH-AA,-NH(CH-L)p".-(CH-L')P....-L"
where R2 is COCH(R5)-S-Ro; Rs is H, -(CH2)P-R7 ; p is 1-5; R7 is a hydrogen, primary amine, secondary amine, cyclic or acyclic tertiary amine, carboxyl, ester, hydroxyl, ether, thiol, thioether, guanido, or an imine; R« is a hydrogen, aliphatic or aromatic acyl, acetamidoalkyl, benzamidoalkyl, unsubstituted or substituted tetra-hydropyranyl, unsubstituted or substituted tetrahydrofuranyl, or alkoxyalkyl; R3 is (CH2)P-Q where p' is 0-6, Q is hydrogen, alkylene or substituted alkylene, aryl or substituted aryl group for attachment to Y; R4 is (CH2),-T where s is 0-6, T is hydrogen, alkylene or substituted alkylene,, aryl or substituted aryl group for attachment to Y; p" is 0,1 if p" is 1 only one of the groups defined under Q or T is attached to Y; AAi is any natural or unnatural α-amino acid or β-amino acid; L, L' = is a hydrogen, carboxyl, lower alkyl, aralkyl, substituted or unsubstituted aromatic group, or an alkylene bearing one of the following substituents: a primary amine, secondary amine, cyclic or acyclic tertiary amine, carboxyl, ester, hydroxyl, ether, thiol, thioether, guanido, imine, aminoalkyl, monoaminoalkyl, diaminoaikyl, hydroxyalkyl, alkoxylalkyl, hydroxycarbonylalkyl, alkoxycarbonylalkyl, mercaptoalkyl, or alkylthioalkyl; p'" is 0- 3, p"" is 0-3, L" is a hydrogen, lower alkyl, aralkyl, substituted or unsubstituted aromatic group, or an alkylene bearing one of the following substituents: a primary amine, secondary amine, cyclic or acyclic tertiary amine, carboxyl, ester, hydroxyl, ether, thiol, thioether, guanido, imine, aminoalkyl, monoaminoalkyl, diaminoaikyl, hydroxyalkyl, alkoxylalkyl, hydroxycarbonylalkyl, alkoxycarbonylalkyl, mercaptoalkyl, or alkylthioalkyl.
The metal binding group is coupled to or incoφorated into the peptidomimetic by standard methodology known in the field of the invention and may be added at any location on the peptidomimetic provided that the biological activity of the peptidomimetic is not adversely affected
Illustrative of suitable peptidomimetic containing ligands within the scope of the invention include the following compositions.
Figure imgf000010_0001
Figure imgf000010_0002
Figure imgf000010_0003
Asp-NH-(CH2)2-CO-NH.(CH2)4-CH-CO-Glu-NH-(CH2)2-N(CH3)2
NHCO-CHrS-R8
Figure imgf000010_0004
Figure imgf000011_0001
Figure imgf000011_0002
where R_§ is a suitable sulfur protecting group. As used herein, a suitable sulfur protecting group is a pharmaceutically acceptable compound capable of preventing potential oxidation of the sulfur or reaction of the sulfur with other reactive groups. Illustrative protecting groups include hydrogen, acetals such as ethoxyethyl, methoxymethyl, substituted and unsubstituted tetrahydrofuranyl, substituted and unsubstituted tetrahydropyranyl, acetamidoalkyl such as acetamidomethyl, acyl such as alkanoyl, benzoyl and substituted benzoyl.
The radiopharmaceutical compositions of the present invention may be complexed with a radionuclide (radiolabeled) by methods known in the art. Briefly, radionuclide complexes may be prepared by reacting a specified amount of the selected composition with a metal salt of the selected radionuclide in the presence of a reducing agent and a transfer agent Preferred reducing agents include, but are not limited to, dithionite, stannous ion, and ferrous ion Preferred transfer agents include, but are not limited to, sodium gluconate, sodium tartrate, sodium citrate, and mannitol Any radionuclide having diagnostic or therapeutic value can be used as the radiolabel for the compositions of this invention. In a preferred embodiment, the radionuclide is a γ-emitting or β-emitting radionuclide selected from the lanthanide or actinide series of the elements Positron-emitting radionuclides, e.g 68Ga, may also be used Suitable γ-emitting radionuclides include those which are useful in diagnostic imaging applications. The γ-emitting radionuclides preferably have a half-life of from 1 hour to 40 days, preferably from 12 hours to 3 days. Examples of suitable γ-emitting radionuclides include 67Ga, 1 1 lln, 99mTc, 169Yb and 186Re Most preferably, the radionuclide is 99mTc. Suitable β-emitting radionuclides include those which are useful in therapeutic applications. Examples include 90Y, 67Cu, 186Re, 188Re, 169Er, 121Sn, 127Te, 143Pr, 198Au, 109Pd, 165Dy, 32P, 142Pr, and 153Sm. The β-emitting radionuclide preferably has a half-life of from 2 hours to two weeks, and more preferably from about 2 hours to 100 hours Suitable radiopharmaceutical complexes include the following compositions
Figure imgf000012_0001
Figure imgf000013_0001
Figure imgf000013_0002
Figure imgf000013_0003
Figure imgf000014_0001
Figure imgf000014_0002
Figure imgf000014_0003
Figure imgf000015_0001
where M is selected from the group consisting of 67Ga, 1 1 lln, 99mTc, 169Yb, 186Re 90Y, 67Cu, 186Re, 188Re, 169Er, 121 Sn, 127Te, 143Pr, 198Au, 109Pd, 165Dy, 32P,
5 142Pr, and 153Sm.
The radiolabeled compositions of the invention and their pharmaceutically acceptable salts are useful as a diagnostic imaging agent or in therapeutic applications. The radiolabeled composition is prepared in a pharmaceutically acceptable carrier, e.g. saline or blood plasma, and is administered to an individual in a diagnostically or 0 therapeutically effective amount as determined using standard methods known to those in the art. The carrier may also contain pharmaceutically acceptable adjunct materials such as salts, buffers, preservatives and the like. Preferably, the radiopharmaceutical composition of the present invention is provided in a kit whereby the radionuclide is provided in one container, e.g. a vial, and the composition capable of complexing with 5 the radionuclide is provided in a second container and the contents mixed just prior to administration. The mixture may be heated if necessary to effect complete labelling. The provision of such radiolabeled complexes in kit form and the preparation of the final radiolabeled product are standard and routine in the field of nuclear medicine. The final radiopharmaceutical product should be of high radiochemical purity, preferably greater than 95%, and at least greater than 90%, as determined by standard protocols known in the art.
The radiolabeled complex is prepared to provide a radioactive dose of between about 0.05 mCi and about 40 mCi, preferably about lmCi to about 20mCi, to the individual in accordance with standard radiopharmaceutical dosing determinations. As used herein, "a diagnostically effective amount" means an amount of the radiopharmaceutical sufficient to permit its detection by scintigraphic means and "a therapeutically effective amount" means an amount sufficient to effect a therapeutic treatment at the targeted biological site. The radiolabeled peptides may be administered intravenously in any conventional medium for intravenous injection.
Imaging of the biological site may be effected within about one hour post-injection, but may also take place several hours post-injection. Any conventional method of imaging for diagnostic puφoses may be utilized.
It is well known in the art that technetium and rhenium form complexes with chemical structures that are identical, but because 99mTc is produced at tracer levels, one cannot readily perform chemical characterization or in vitro studies on such complexes. The corresponding rhenium (185,187 isotopes) complexes are preferred for structure determination and in vitro assays because they can be obtained in sufficient amount and are non-radioactive. To confirm the structure of the specific embodiments of this invention, each was reacted with perrhenate-185, 187 in the presence of stannous ion and an exchange agent, sodium tartarate. The Re-complexes were isolated by reverse phase liquid chromatography and identified by mass spectrometry. Retention times of the rhenium complexes closely match those of the Tc-99m complexes. This ensures the chemical composition of the Tc-99m complexes. The following examples describe preferred embodiments of the invention.
Other embodiments within the scope of the claims herein will be apparent to one skilled in the art from consideration of the specification or practice of the invention as disclosed herein. It is intended that the specification, taken together with the examples, be considered exemplary only, with the scope and spirit of the invention being indicated by the claims. In the Examples that follow, all solvents and reagents used were as supplied from the vendors without further purification Ammo acid derivatives were purchased commercially Thin layer chromatography (TLC) was performed with 1x3 in Whatman SiO2 and Cu plates with fluorescent indicators TLC visualization was accomplished using UV light, iodine, and/or vanillin stain HPLC analyses and purification were performed on Waters systems using either Nova-Pak or Vydac Cu columns. NMR spectra were recorded on a 300 MHZ Varian Gemini spectrometer while all mass spectra were recorded on a Finnigan TSQ 500 instrument using electrospray ionization. The standard three letter abbvreviations for amino acids and the following abbreviations have been used in the Examples-
Figure imgf000017_0001
AAA = 2- ammoadipic acid dmen = NH-(CH2)2-N(CH3)3
The compositions of the present invention may be synthesized either in a sequential manner or by segment condensation methodology as further described below.
Example 1
This Example describes the stepwise preparation of the compound having the formula
Figure imgf000018_0001
Step a N-α-Boc-L-Asp(β-t-Bu)-ε-L-Lys(α-Cbz)-OH
Boc-Asp(β-OtBu)-NH(CH2)4-CH(NH-Cbz)-COOH N-α-Cbz-L-Lysine (7.25 g, 25.9 mmol) was suspended in a mixture of 1 N sodium bicarbonate (65 mL) and dioxane (65 mL). The N-hydroxysuccinimide ester of N-Boc-L-Asp(β-t-Bu) (10.0 g, 25.9 mmol) in dioxane was subsequently added, and the reaction was permitted to stir overnight at room temperature before concentrating under reduced pressure. The remaining aqueous layer was acidified to pH 3 with 1 N HCl, and the product was extracted into ethyl acetate. The combined organic layer was washed with water (2x) and brine before drying over anhydrous magnesium sulfate. Filtration and evaporation of the solvent under reduced pressure revealed a white foam (12.6 g, 88% yield). TLC (10% MeOH / CH2C12) showed only one spot, so no further purification was required. Η NMR (CDC13) δ 8.05 (br m, 1H, amide NH), 7.27 (m, 5H, aromatic), 5.74 (d, 1H, carbamate NH), 5.06 (dd, 2H, benzylic CH2), 4.41 (m, 1H, α-H), 4.39 (m, 1H, α-H), 3.12 - 3.67 (m, 2H, CH2N), 2.58 - 2.75 (m, 2H, CH2CO2-), 1.25 - 1.83 (m, 6H, 3 CH2's), 1.39 (s, 9H, C(CH3)3), 1.37 (s, 9H, C(CH3)3); l3C NMR (CDC13) δ 175, 172, 171, 156, 155, 136, 129, 128, 81.7, 80.5, 66.9, 53.5, 38.8, 37.3, 31.4, 28.5, 28.0, 27.8, 21.8, 13.9; Mass Spec. (ESI) 552 (M+l, 100%).
Step b N-Boc-L-Asp(β-t-Bu)-ε-L-Lys)α-Cbz)-Gly-dmen
Boc-Asp(β-OtBu)-NH-(CH2)4-CH-(NH-Cbz)-CO-Gly-NH-(CH2)2-N(CH3)2
N-Boc-L-Asp(β-t-Bu)-ε-L-Lys(α-Cbz) (12.2 g, 22.1 mmol) and N- hydroxysuccinimide (2.80 g, 24.3 mmol) were dissolved in dichloromethane (250 mL) afterwhich dicyclohexylcarbodiimide (5 47 g, 26 5 mmol) was added The reaction was permitted to stir overnight at ambient temperature before filtering to remove the dicyclohexylurea After diluting with dichloromethane, it was extracted with saturated sodium bicarbonate (3x) and washed with water (lx) and brine (lx) before drying over anhydrous magnesium sulfate Filtration and evaporation of the solvent under reduced pressure revealed a white foam (12.2 g, 85% yield) This intermediate (4 00 g, 6.17 mmol) was added to a suspension of glycyl-dmen hydrochloride (1.02 g, 5 61 mmol) in acetonitrile (42 mL) in the presence of triethylamine (0 57 g, 5 61 mmol) The reaction was continued overnight at ambient temperature before diluting with ethyl acetate. The organic layer was extracted with saturated sodium bicarbonate (3x) and washed with brine before drying over anhydrous magnesium sulfate. Filtration and evaporation of the solvent under reduced pressure revealed an off-white foam. It was redissolved in 30% acetonitrile / 3% acetic acid / water with a small amount of methanol and loaded onto a reverse phase Cu column packed with the same solvent system. Isocratic elution with 30% acetonitrile / 3% acetic acid / water gave a white foam in 58% isolated yield. Rf = 0.57 (60% acetonitrile / 3% acetic acid / 37% water); 1H NMR (DMSO-d6) δ 8.23 (t, 1H, amide NH), 7 76 (m, 2H, 2 amide NH), 7 49 (d, 1H, carbamate NH), 7 32 (m, 5H, aromatic), 6.93 (d, 1H, carbamate NH), 5 02 (s, 2H, benzylic CH2), 4 23 (m, 1H, α-H), 3 91 (m, 1H, α-H), 3 66 (m, 2H, Gly CH2), 3.20 (m, 2H, CH2N), 3 17 (m, 2H, CH2N), 2.24 (s, 6H, N(CH3)2), 2 04 - 2.56 (m, 4H,
CH2CO2-t-Bu + CH2N), 1.35 (2 s's, 18H, 2 C(CH3)3), 1.13 - 1 84 (m, 6H, 3 CH2's),
1.87 (s, 3H, CH3CO2H), ,3C NMR (DMSO-d6) δ 173, 171, 170, 169, 157, 156, 137,
129, 128.3, 128 1, 80 3, 78 6, 65.8, 57.5, 55.1, 44 5, 42.3, 36 1, 31.2, 28.7, 28.2,
27.7, 22.8, 21 3
Step c
N-Boc-L-Asp(β-t-Bu)-ε-L-Lys-Gly-dmen
Boc-Asp(β-OtBu)-NH-(CH2)4-CH(NH2)-CO-Gly-NH-(CH2)2-N(CH3)2 N-Boc-L-Asp(β-t-Bu)-ε-L-Lys(α-Cbz)-Gly-dmen (1 97 g, 2.90 mmol) was dissolved in methanol (25 mL). After flushing with nitrogen, the 10% Pd / C catalyst (0.2 g) was added. The mixture was shaken for 6 hours at ambient temperature under an atmosphere of hydrogen (40 p.s.i ). The reaction was filtered through a pad of Celite, and the filtrate was concentrated under reduced pressure to reveal a white foam. By Η and 13C NMR, it appeared that a quantitative yield of the diacetate salt was obtained due to the presence of excess acetic acid from the prior purification. Η NMR (CDC13) δ 8.53 (t, IH, amide NH), 8.36 (t, IH, amide NH), 6.97 (t, IH, amide NH), 5.89 (d, IH, J = 8.2 Hzm, carbamate NH), 4.41 (m, IH, α-H), 3.86 (m, 2H, Gly CH2), 3.64 (m, IH, a-H), 3.52 (m, 2H, CH2N), 3.12 - 3.41 (m, 2H, CH2N), 2.97 (t, 2H, CH2N), 2.61 (s, 6H, N(CH3)2), 2.55 - 2.58 (m, 2H, CH2CO2tBu), 1.94 (s, 6H, 2 CH3CO2H), 1.39 (s, 9H, C(CH3)3), 1.38 - 1.81 (m, 6H, 3 CH2's); 13C NMR (CDC13) δ 177.4, 177.3, 172, 171, 170, 158, 81.4, 80.2, 57.0, 54.1, 43.7, 42.6, 38.7, 37.6, 35.1, 32.4, 28.6, 28.1, 27.8, 22.2, 22.0.
Step d N-Boc-L-Asp(β-t-Bu)-ε-L-Lys(α-(S-THP-mercaptoacetyl))-Gly-dmen
Boc-Asp(β-OtBu)-NH-(CH2)4-CH-CO-Gly-NH-(CH2)2-N(CH3)2
NHCO-CH2-S-THP
N-Boc-L-Asp(β-t-Bu)-ε-L-Lys-Gly-dmen 2 AcOH (1.54 g, 2.32 mmol) and the N-hydroxysuccinimide ester of S-THP-mercaptoacetic acid (0.66 g, 2 43 mmol) were dissolved in dichloromethane (40 mL) in the presence of triethylamine, and the reaction was continued overnight at ambient temperature. The reaction was subsequently diluted with dichloromethane and extracted with water (lx) and saturated sodium bicarbonate (3x) and washed with brine before drying over anhydrous magnesium sulfate. Filtration and evaporation of the solvent under reduced pressure revealed a glassy solid (1.1 g, 69% yield) which was deemed of sufficient purity; no further purification was necessary. lH NMR (CDC13) δ 7.60 (t, IH, amide NH), 7.49 (m, IH, amide NH), 6.94 (m, IH, amide NH), 5.74 (d, IH, carbamate NH), 4.84 (rn, IH, S-CH-O), 4.39 (m, IH, a-H), 4.21 (m, IH, a-H), 3.10 - 4.01 (m's, 10H, Gly CH2 + 2 CH2N + OCH2 +SCH2CO), 2.78 (m, 2H, CH2CO2tBu), 2.59 (m, 2H, CH2N), 2.34 (s, 3H, NCH3), 2.32 (s, 3H, NCH3), 1.41 (s, 9H, OC(CH3)3), 1.40 (s, 9H, OC(CH3)3), 1.19 - 2.01 (m, 12H, 6 CH2's); Mass Spec. (ESI) 545 (M+l , 100%), 273 ((M+2)/2, 20%).
Step e L-Asp-ε-L-Lys(α-(S-THP-mercaptoacetyl))-Gly-dmen
Asp-NH-(CH2)4-CH-CO-Gly-NH-(CH2)2-N(CH3)2
NHCO-CH2-S-THP
N-Boc-L-Asp(β-t-Bu)-ε-L-Lys(α-(S-THP-mercaptoacetyl))-Gly-dmen (40 mg, 0.057 mmol) was dissolved in 1 : 1 TFA / dichloromethane (0.5 mL each). The reaαion was stirred for 5 hours at room temperature before removing the solvent under reduced pressure. The orange, oily residue was purified by reverse phase Cu with a 3% acetic acid / water mobile system (29 mg, 76% yield). Mass Spec. (ESI) 547 (M+l, 100%), 274 ((M+2)/2, 20%); 'H NMR (D2O) δ 4.21 (m, IH, S-CH-O), 3.90 (m, IH, a-H), 3.55 (m, IH, a-H), 2.86 (s, 6H, N(CH3)2), 2.71 - 3.33 (m's, 14H, Gly CH2 + CH2CO2H + 2 CH2N + CH2N(CH3)2 + S-CH2-CO + CH2O), 2.01 (s, 6H, 2 CH3CO2H), 1.30 -2.00 (m's, 12H, 6 CH2's).
Step f ABAS-L-Asp-ε-L-Lys(α-(S-THP-mercaptoacetyl))-Gly-dmen
Figure imgf000022_0001
The hydrochloride salt of aminobenzamidinosuccinate ( 81 mg, 0.30 mmol) was added to dry DMF (4 mL) followed by N-methylmorphoiine (30 mg, 0.30 mmol) and isobutyl chloroformate (41 mg, 0.30 mmol) at 0°C under nitrogen. After stirring for 5 minutes, a solution of L-Asp-e-L-Lys(a-(S-THP-mercaptoacetyl))-Gly-dmen (200 mg 0.30 mmol) and N-methylmorpholine (91 mg, 0.90 mmol) in DMF (2 mL) was added. Stirring was continued for 2 hours afterwhich the solvent was removed under reduced pressure. The residue was purified by reverse phase Cu flash chromatography using 3% acetic acid / water as an isocratic eluent. Mass Spec. (ESI) 764 (M+l, 5%), 382 ((M+2)/2, 100%); 1H NMR (D2O) δ 7.69 (dd, 4H, p-substituted aromatic), 4.58 (m, IH, S-CH-O), 4.19 (m, IH, a-H), 3.10 - 4.00 (m's 13H's, 1 a-H + Gly CH2 + 2 CH2N + CH2NMe2 + OC-CH2-S + OCH2), 2.86 (s, 6H, N(CH3)2), 2.70 (m's 6H,
OC(CH2)2CO + CH2CO2H), 2.01 (s, 6H, 2 CH,CO2H), 1.20 - 1.97 (m's, 12H, 6 CH2's).
Example 2 This Example describes the radiolabeling of the compound of Example 1 with
Tc-99m and a kit formulation.
Method A: Tc-99m labeling was performed using a commercially available
Merck-Frosst kit. The kit components were dissolved in 1 mL of water. In a separate vial containing 1 mL pertechnetate (10-100 mCi) from commercially available generator, 100 mL of the solution from Merck-Frosst kit was added and the solution was allowed to stand for 15 minutes. To this solution 30-100 mg of the above derivatives were added and the solution was heated at 75-100°C for 15-20 minutes. The solution was ready for administration after filtration through a sterile filter.
Method B: Alternatively the componets that are present in the Merck-Frosst kit (gluconate salts, SnCl2) can be added individulally to form the kit. Compound in Example 1 was radiolabelled with Tc-99m according to the procedure described in Method A.
Example 3
This Example describes the labelling of the compound of Example 1 with a non-radioactive rhenium 185,187 isotope to confirm the composition of the final composition.
The composition of the Re-complexes were confirmed by mass spectra (m/e 880 (M+l). Example 4 This Example describes Platelet Aggregation Inhibition Assays of the compound of Example 2 to illustrate that the compound binds to GPIIb/IIIa receptors when radiolabelled.
Healthy aspirin free donors were randomly selected from a pool of volunteers. The harvesting of platelet rich plasma and subsequent ADP induced platelet aggregation assays were performed as described in Zucker, M. B. " Platelet
Aggregation Measured by the Photometric Method", Methods in Enzymology 169 (1989): 1 17-133. Standard venipuncture techniques using butterfly allowed the withdrawal of 45 mL of whole blood into a 60 mL syringe containing 5 mL 3.8% trisodium citrate. Following thorough mixing in the syringe, the anti-coagulated whole blood was transferred to a 50 mL conical polyethylene tube. The blood was centrifuged at room temperature for 12 minutes at 200 xg to sediment non-platelet cells. Platelet poor plasma was obtained from a second centrifugation of the remaining blood at 2000 xg for 15 minutes. Platelet counts are typically 300,000 to 500,000 per microliter. Platelet rich plasma (0.45 mL) was aliquoted into siliconized cuvettes and stirred (1 100 φm) at 37°C for 1 min. prior to adding 50 mL of prediluted test compound. After 1 minute of mixing, aggregation was initiated by the addition of 50 mL of 200 mM of ADP. Aggregation was recorded for 3 minutes in a Payton dual channel aggregometer (Payton Scientific, Buffalo, NY). The percent inhibition of maximal response (saline control) for a series of test compound dilutions was used to determine a dose response curve. The compounds were tested in duplicate and half- maximal inhibition (IC50) was calculated graphically from the dose response curve.
During these studies, IC50 values were obtained for both the parent molecules as well as the corresponding Re- 185, 187 complexes.
Example 5
This study was designed to evaluate the biodistribution pattern of the compound of Example 2 to determine the clearance profile from blood, route of excretion and in vivo stability of the complex by HPLC analysis of urine samples, in a rat model.
The compound of Example 2 was injected (25mL, 1.5-3.5 mCi/mL) into Sprague-Dawley rats. Groups of three animals were sacrificed at the time points indicated below to determine the amount of radioactivity remained in the organs. Results: (to be filled)
Example 6
This example describes the stepwise preparation of a composition having the structure:
Figure imgf000024_0001
Step a ABAS-Asp(β-OtBu)-OH
Figure imgf000025_0001
Aminobenzamidinosuccinic acid hydrochloride (500 mg, 1.84 mmol) was
5 suspended in dry DMF (25 mL) under an atmosphere of nitrogen. N- methylmorpholine (186 mg, 1.84 mmol, 0.20 mL) and isobutyl chloroformate (251 mg, 1.84 mmol, 0.24 mL) were added after cooling the reaction to 0°C. Stirring was continued at 0°C for one half hour before adding a suspension of L-Asp(β-t-Bu) (324 mg, 1.84 mmol) and N-methylmorpholine (0.20 mL) in DMF (12 mL). The reaction
10 was subsequently stirred at ambient temperature overnight. The solvent was removed under reduced pressure, and the residue was dissolved in 3% acetic acid / water. The solution was loaded onto a reverse phase Cu column packed with the same solvent system. Initially, 3% acetic acid / water was used to elute unreacted ABAS followed by 10%o acetonitrile / 3% acetic acid / water to elute the desired product. Evaporation
15 of the appropriate fractions revealed an off-white solid (650 mg, 76% yield). Mass Spec. (ESI) 407 (M+l , 100%); Η NMR (DMSO-d6) δ 1 1.3 (br s, I H), 10.4 (s, IH, C=NH), 8.81 (br s, IH), 7.75 (m, 4H, aromatic), 4.32 (m, IH, Asp-α-H), 2.42 - 2.62 (m, 6H, 3 CH2's), 1.87 (s, 3H, CH3CO2H), 1.35 (s, 9H, OC(CH3)3); 13C NMR (DMSO-d6) δ 175, 173, 172, 170.8, 170.7, 166, 144, 129, 123, 1 19, 79.5, 50.8, 38.8,
20 31.9, 30.2, 27.7, 21.5.
Step b Cbz-L-Glu(γ-t-Bu)-dmen
Cbz-L-Glu-(γ-OtBu)-NH-(CB)2-N(CH3)2 25 The N-hydroxysuccinimide ester of N-Cbz-L-Glu(γ-t-Bu) (7.70 g, 17.7 mmol) and N.N-dimethylethylenediamine (dmen) (1.56 g, 17.7 mmol) were dissolved in dichloromethane ( 170 mL), and the reaction was stirred overnight at ambient temperature. The reaction was diluted with dichloromethane, extracted with saturated sodium bicarbonate (3x), and washed with brine before drying over anhydrous magnesium sulfate. Filtration and evaporation of the solvent under reduced pressure
5 revealed a white foam (7.08 g, 98% yield). No purification was necessary. Η NMR (CDC13) δ 7.28 (m, 5H, aromatic), 6.67 (br t, IH, amide NH), 5.78 (d, J = 7.7 Hz, IH, ZNH), 5.05 (s, 2H, benzylic CH2), 4.17 (m, IH, Glu α-H), 3.28 (q, 2H, amide, N- CH2), 2.28 (m, 4 H, CH2-NMe2 + CH2CO2), 2.17 (s, 6H, N(CH3)2), 2.04 (m, IH, 1 b- H of Glu), 1.88 (m, IH, 1 b-H of Glu), 1.39 (s, 9H, OC(CH3)3); l3C NMR (CDC13) d
10 173, 171, 156, 136, 128.6, 128.2, 128.1, 80.7, 66.8, 57.5, 54.2, 44.9, 36.6, 31.4, 28.0, 27.8.
Step c L-Glu(γ-t-Bu)-dmen
, 5 L-Glu-(γ-OtBu)-NH-(CH2)2-N(CH3)2
N-Cbz-L-Glu(g-t-Bu)-dmen (7.00 g, 17.2 mmol) was dissolved in methanol (130 mL). After flushing with nitrogen, 10% Pd/C catalyst (0.7 g) was added. The mixture was shaken for 6 hours at ambient temperature under an atmosphere of hydrogen (40 p.s.i.). The reaction was filtered through a pad of Celite, and the filtrate was
20 concentrated under reduced pressure to reveal a yellow oil in quantitative yield. Η NMR (CDC13) δ 7.37 (br s, IH, amide NH), 3.32 (m, 3H, Glu α-H + CONHCHj), 2.40 (t, 2H, CH2NMe2), 2.03 (m, 3H, NH2 + 1 Glu β-H), 1.76 (m, IH, 1 Glu β-H), 1.39 (s, 9H, OC(CH3)3); ,3C NMR (CDC13) δ 175, 173, 80.4, 58.0, 54.6, 45.0, 36.3, 31.8, 30.2, 27.8.
25
Step d S-Tetrahydropyranyl-mercaptoacetyl-L-Lys(ε-Fmoc)
Fmoc-NH-(CH2)4-C 1-COOSu.
NHCO-CB,-S-THP L-Lys(e-Fmoc) (2.70 g, 7.32 mmol) was suspended in a mixture of dioxane (15 mL) and 1 N sodium bicarbonate (15 mL). The N-hydroxysuccinimide ester of S- THP-mercaptoacetic acid (2.00 g, 7.32 mmol) in dioxane (20 mL) was subsequently added, and the reaction was stirred overnight at ambient temperature. The reaction was then concentrated under reduced pressure to remove the dioxane, and the remaining aqueous solution was extracted with ethyl acetate. The ethyl acetate layer was discarded, and the aqueous layer was acidified to pH 3 with 1 N hydrochloric acid. The product was extracted into ethyl acetate; the combined organic layer was washed with water (2x) and brine (lx) before drying over anhydrous sodium sulfate. Filtration and evaporation of the solvent under reduced pressure revealed a white foam. The material was purified on SiO2 using a methanol / dichloromethane gradient to afford 2.60 g of product (67% yield). Η NMR (CDC13) δ 7.72 (d, 2H, aromatic), 7.54 (d, 2H, aromatic), 7.33 (t, 2H, aromatic), 7.22 (t, 2H, aromatic), 6.09 (br s, IH, amide NH), 5.31 (br m, IH, carbamate NH), 4.81 (m, IH, SCHO), 4.29 - 4.51 (m, 3H), 4.18 (m, IH), 3.99 (m, IH), 2.99 - 3.48 (m, 5H), 1.28 - 2.05 (m, 12H, 6 CH2's). Step e
S-Tetrahydropyranyl-mercaptoacetyl-L-Lys(ε-Fmoc)-OSu.
Fmoc-NH-(CH2)4-CH-COOSu. NHCO-CH2-S-THP
S-Tetrahydropyanyl-mercaptoacetyl-L-Lys(ε-Fmoc) (2.56 g, 4.86 mmol) and N-hydroxysuccinimide (0.62 g, 5.35 mmol) were dissolved in dichloromethane (75 mL). Dicyciohexylcarbodiimide (1.20 g, 5.83 mmol) was subsequently added, and the reaction was stirred at ambient temperature overnight. The reaction was filtered to remove dicyclohexylurea, and the filtrate was diluted with dichloromethane. The organic layer was extracted with saturated sodium bicarbonate (3x) and washed with brine before drying over anhydrous sodium sulfate. Filtration and evaporation of the solvent under reduced pressure revealed a white foam (2.50 g, 87% yield) which was used immediately in the next step. Η NMR (CDC13) δ 7.83 (d, IH, amide NH), 7.72 (d, 2H, aromatic), 7 68 (d, IH, amide NH), 7 59 (d, 2H, aromatic), 7 38 (t, 2H, aromatic), 7 29 (t, 2H, aromatic), 5 18 (m, IH), 4 98 (m, IH), 4 71 (m, IH), 4 37 (m, 2H), 4 19 (m, IH), 3 99 (m, IH), 3 12 - 3 52 (m, 4H), 2 79 (s, 4H, COCH2CH2CO), 1.20 - 2.02 (m, 12H)
Step f
S-Tetrahydropyranyl-mercaptoacetyl-L-Lys(ε-Fmoc)-L-Glu(γ-t-Bu)-dmen Fmoc-NH-(CH2)4-CH-CO-L-Glu(γ-OtBu)-NH-(CH2)2-N(CH3)2
NHCO-CH2-S-THP S-Tetrahydropyranyl-mercaptoacetyΙ-L-Lys(ε-Fmoc) (520 mg, 0 88 mmol) and L-Glu(γ-t-Bu)-dmen (240 mg, 0 88 mmol) were dissolved in dichloromethane (8 mL), and the reaction was stirred overnight at ambient temperature before diluting with dichloromethane. The organic layer was extracted with saturated sodium bicarbonate(3x) and washed with brine (lx) before drying over anhydrous sodium sulfate. Filtration and evaporation of the solvent under reduced pressure revealed a white solid It was redissolved in 10% methanol / dichloromethane and loaded onto a SiO2 column packed with the same solvent system. Elution was isocratic with 10% methanol / dichloromethane (Rf = 0.51, 10% methanol / dichloromethane) The product-containing fractions were combined and concentrated under reduced pressure to reveal 350 mg of a white solid (51 % yield) Η NMR (CDC13) δ 7 73 (d, 2H, aromatic), 7 58 (d, 2H, aromatic) , 7 50 (m, IH, amide NH), 7 39 (t, 2H, aromatic), 7 29 (t, 2H aromatic), 7 12 (m, IH, amide NH), 6.71 (m, IH, amide NH), 5 19 (m, IH), 4 79 (m, IH), 4 37 (m, 4H), 4 19 (m, IH), 3.99 (m, IH), 3 00 - 3 60 (m's, 7H), 2.41 (m, 2H), 2.29 (t, 2H, CH2NMe2), 2.20 (s, 3H, NCH3), 2.10 (m, 2H), 1 48 - 2.00 (m, 12H), 1 41 (s, 9H, OC(CH3)3)
Sjep_g S-Tetrahydropyranyl-mercaptoacetyl-L-Lys-L-Glu(γ-t-Bu)-dmen NH2-(CH2)4-CH-CO-L-Glu(γ-OtBu)-NH-(CH2)2-N(CH3)2
NHCO-CHrS-THP
S-Tetrahydropyranyl-mercaptoacetyl-L-Lys(ε-Fmoc)-L-Glu(γ-t-Bu)-dmen (320 mg, 0.41 mmol) was dissolved in DMF (4.1 mL) afterwhich moφholine (0.82 mL) was added. The reaction was stirred for two hours at ambient temperature before removing the solvent under reduced pressure. The residue was redissolved in methanol and filtered to remove the moφholine adduct of dibenzofulvene. The solvent was removed one more time under reduced pressure. The remaining material was purified by dissolving in 30% acetonitrile / water and loading onto a reverse phase Cu column packed with the same solvent system. Elution was isocratic with 30% acetonitrile / water (Rf = 0.14, 30% acetonitrile / 5 % acetic acid / water). The product-containing fractions were combined, and the solvent was removed under reduced pressure to reveal a glassy solid (200 mg, 87% yield). Η NMR (CD3OD) δ 4.75 (m, IH), 4.08 (m, 2H), 3.81 (m, IH), 3.3 l(m, IH), 3.09 (m, 4H), 2.48 (t, 2H), 2.22 (t, 2H), 2.09 (m, 2H), 2.01 (s, 6H), 1.88 (m, IH), 1.12 - 1.75 (m, 13H), 1.20 (2, 9H); 13C NMR (CD3OD) δ 175, 174.5, 174, 173, 84.2, 84.0, 82.0, 65.9, 59.0, 56.8, 56.0, 45.9, 41.9, 38.0, 32.5, 32.2, 32.1, 32.0, 28.1, 28.0, 26.3, 24.0, 22.9. Step h
ABAS-L-Asp(β-t-Bu)-ε-L-Lys(α-(S-THP-mercaptoacetyl))-Glu(γ-t-Bu)-dmen
Figure imgf000029_0001
ABAS-L-Asp(β-t-Bu) AcOH (360 mg, 0.77 mmol) was dissolved in DMF (20 mL). N-methylmoφholine (78 mg, 0.77 mmol) and isobutyl chloroformate (105 mg, 0.77 mmol) were added after cooling to 0°C. After stirring for one half hour, the S- THP-mercaptoacetyl-L-Lys-L-Glu(g-t-Bu)-dmen (431 mg, 0.77 mmol) was added in DMF (5 mL). The reaction was subsequently continued at 0°C for 4 hours before removing the solvent under reduced presssure. The material was purified by reverse phase Cu flash chromatography using 30% acetonitrile / 3% acetic acid / water as the eluent to afford 220 mg of product (29% yield). Mass Spec. (ESI) 948 (M+H, 10%), 5 475 ((M+2)/2, 100%); Η NMR (CDC13) d_10.9 (s, IH, amidino H), 7.80 - 8.70 (m's, 6H, amide NH's), 7.61 (m, 4H, aromatic), 4.91 (m, 2H), 4.78 (m, IH), 3.89 - 4.23 (m, 3H), 3.05 - 3.80 (m, 7H), 2.78 (s, 6H), 1.20 - 2.70 (m, 22H), 1.39 (2 s's, 18H, 2 OC(CH3)3).
0 Step i
ABAS-L-Asp-ε-L-Lys(α-(S-THP-mercaptoacetyl))-Glu-dmen
Figure imgf000030_0001
ABAS-L-Asp(β-t-Bu)-ε-L-Lys(a-(S-THP-mercaptoacetyl))-Glu(γ-t-Bu)-dmen 5 (209 mg, 0.21 mmol) was dissolved in formic acid (5 mL). The reaction was stirred overnight at ambient temperature before removing the solvent under reduced pressure. The residue was purified by reverse phase Cu chromatography using initially 3% acetic acid / water and finally 10% acetonitrile / 3% acetic acid / water as the eluent to give 35 mg of a white powder (20% yield). Mass Spec. (ESI) 836 (M+l, 5%), 418 0 ((M+2)/2, 100%); lH NMR (D2O) δ 7.66 (dd, J = 25, 7.1 Hz, 4H), 4.52 (t, IH), 4.12 - 4.25 (m, 3H), 3.90 (m, 2H), 3.07 - 3.56 (m's, 9H), 2.86 (2 s's, 6H), 2.62 (m, 5H), 2.34 (m, 3H), 2, 17 (m, 4H), 1.99 (s, CH3CO2H), 1.20 - 1.90 (m, 9H). Example 7
The compound of Example 6 was radiolabeled with Tc-99m according to method A described in Example 2.
Example 8
This Example describes the labelling of the compound of Example 6 with a non-radioactive rhenium 185,187 isotope to confirm the composition of the final composition.
The composition of the Re-complexes were confirmed by mass spectra (m/e 952 (M+l)).
Example 9
This Example describes Platelet Aggregation Inhibition Assays of the compound of Example 8 to illustrate that the compound binds to GPIIb/IIIa receptors when radiolabelled following the procedure of Example 4.
Results: IC50: 1.8 X 10'7 M
Example 10
This study was designed to evaluate the biodistribution pattern of the compound of Example 7 to determine the clearance profile from blood, route of excretion and in vivo stability of the complex by HPLC analysis of urine samples, in a rat model.
The compound of Example 7 was injected (25mL, 1.5-3.5 mCi/mL) into Sprague-Dawley rats. Groups of three animals were sacrificed at the time points indicated below to determine the amount of radioactivity (%LD/g) remaining in the organs.
Organ 5 min 30 min 60 min 120 min
Blood 0.397 0.099 0.057 0.022
Liver 1.668 0.902 0.734 0.450 Kidney 4.613 1.713 0.978 0.983 Example 1 1
This Example describes the stepwise preparation of the composition having the structure
Figure imgf000032_0001
Step a
N-Hydroxysuccinimide ester of N-α-Boc-p-Fmoc-amino-L-Phe
Figure imgf000032_0002
N-α-Boc-p-Fmoc-amino-L-Phe (10.0 g, 19.9 mmol) and N- hydroxysuccinimide (2.52 g, 21.9 mmol) were dissolved in dichloromethane (315 mL). Dicyclohexylcarbodiimide (4.93 g, 23.9 mmol) was subsequently added, and the reaction was stirred overnight at ambient temperature. The dicyclohexylurea was removed by filtration, and the filtrate was diluted with dichloromethane. The organic layer was then extracted with saturated sodium bicarbonate (3x) and washed with brine before drying over anhydrous magnesium sulfate. Filtration and evaporation of the solvent under reduced pressure revealed a white foam (quantitative yield) which was used immediately without further purification. 1H NMR (CDCI3) δ 7.75 (d, J = 7.6 Hz, 2H, aromatic), 7.59 (d, J = 7.3 Hz, 2H, aromatic), 7.39 (t, 2H, aromatic), 7.29 (t, 2H, aromatic), 7.19 (d, J = 8 Hz, IH, carbamate NH), 6.85 (s, IH carbamate NH), 4.91 (m, IH, α-H), 4.49 (d, J = 6.7 Hz, 2H, Fmoc CH2), 4.24 (t, IH, Fmoc CH), 3.17 (m, 2H, CH2-Ar), 2.78 (s, 4H, succinimide CH2's), 1.39 (s, 9H, OC(CH3)3); 13C NMR (CDCl3) δ 169, 168, 155, 154, 144, 142, 137, 131, 130, 128, 127, 125, 120, 1 19, 80.5, 66.8, 46.9, 37.2, 34,7, 28.0, 25.3. Step b
N-α-Boc-p-Fmoc-amino-L-Phe-Gly-dmen
Figure imgf000033_0001
Gly-dmen hydrochloride (2.98 g, 16.4 mmol) was dissolved in a mixture of 1 N sodium bicarbonate (45 mL) and dioxane (45 mL). The N-hydroxysuccinimide ester of N-a-Boc-p-Fmoc-amino-L-Phe (10.8 g, 18.0 mmol) in dioxane (45 mL) was subsequently added, and the reaction was stirred overnight at ambient temperature. The dioxane was then removed under reduced pressure. The product was extracted into ethyl acetate, and the combined organic layer was extracted with saturated sodium bicarbonate (3x) and washed with brine before drying over anhydrous magnesium sulfate. Filtration and evaporation of the solvent under reduced pressure revealed a white solid (7.1 g, 69% yield) which was used without further purification. !H NMR (CDC13) δ 7.85 (br m, IH, amide NH), 7.73 (d, 2H, aromatic), 7.58 (d, 2H aromatic), 7.43 (br m, IH, amide NH), 7.02 - 7.39 (m, 8H, aromatic), 5.62 (br s, IH, carbamate NH), 2.44 (m, 2H, Gly CH2), 4.34 (m, IH, α-H), 4.21 (t, IH, Fmoc CH), 3.99 (d, 2H, Fmoc CH2), 3.57 (m, 2H, CH2N), 2.94 (m, 2H, CH2-Ar), 2.58 - 2.65 (m, 2H CH2N), 2.40 (s, 6H, N(CH3)2), 1.35 (s, 9H, OC(CH3)3).
Step c p-Fmoc-amino-L-Phe-Gly-dmen
Figure imgf000033_0002
N-α-Boc-p-Fmoc-amino-L-Phe-Gly-dmen (5.00 g, 7.94 mmol) was dissolved in 1 : 1 TFA / dichloromethane (50 mL each) at 0°C. After the addition was complete, the ice bath was removed, and the reaction was stirred at ambient temperature for 3 hours before removing the solvent under reduced pressure. The residue was purified by reverse phase Cι8 chromatography utilizing a 30% acetonitrile / 3% acetic acid / water mobile phase to provide 4.1 g of a clear oil (80% yield). Η NMR (DMSO-d*) δ
5 9.77 (s, IH, carbamate NH), 8.83 (t, IH, amide NH), 8.31 (t, IH, amide NH), 8.19 (br s, 2H, NH2), 7.90 (d, 2H, aromatic), 7.78 (d, 2H, aromatic), 7.39 (m, 6H, aromatic), 7.19 (d, 2H, aromatic), 4.45 (d, 2H, Fmoc CH2), 4.32 (t, IH, Fmoc CH2), 4.03 (rn, IH, α-H), 3.79 (d, 2H, Gly CH2), 3.42 (q, 2H, NCH2), 3.18 (t, 2H, CH2N), 2.99 (dd, 2H, b-CH2), 2.81 (s, 6H, N(CH3)2), 1.98 (s, 6H, 2 AcOH); I3C NMR (DMSO-d*) δ
10 172, 170, 169, 154, 144, 141, 138, 132, 130, 128, 127, 125, 120, 118, 81.2, 65.6, 63.6, 58.0, 46.6, 45.0, 36.6, 30.6, 25.0, 20.9.
Step d
S-Tetrahydropyranyl-mercaptoacetyl-p-Fmoc-amino-L-Phe-Gly-dmen
15
Figure imgf000034_0001
The diacetate salt of p-Fmoc-amino-L-Phe-Gly-dmen (4.00 g, 6.17 mmol) was dissolved in dioxane (20 mL) and 1 N sodium bicarbonate (20 mL). The N- hydroxysuccinimide ester of S-tetrahydropyranyl-mercaptoacetic acid (1.85 g, 6.78 mmol) in dioxane (20 mL) was subsequently added, and the reaction was stirred
20 overnight at ambient temperature before concentrating under reduced pressure. The product was extracted from the remaining aqueous mixture with ethyl acetate (3x). The combined organic layer was washed with saturated sodium bicarbonate (3x), water (lx), and brine (lx) before drying over anhydrous magnesium sulfate. Filtration and evaporation of the solvent under reduced pressure revealed a yellow solid. The
25 crude material (2.20 g, 52% yield) was used immediately without further purification. 'HNMR (DMSO-dβ) δ 9.64 (s, IH, carbamate NH), 8.38 (d, IH, amide NH), 8.19 (m, IH, amide NH), 7.91 (d, 2H, aromatic), 7.68 (d, 2H, aromatic), 7.62 (t, IH, amide NH), 7.39 (m, 6H, aromatic), 7 18 (d, 2H, aromatic), 4.59 (m, IH, SCHO), 443, d, 2H, Fmoc CH2), 4,36 (t, IH, Fmoc CH), 3.82 (m, IH, a-H), 3.18 - 3.68 (m, 8H, Gly CH2 + CONHCHj + OCH2 + COCH2S), 2.99 (dd, IH, b-H of Phe), 2.68 (m, IH, b-H of Phe), 2.32 (t, 2H, CH2NMe2), 2.18 (s, 6H, N(CH3)2), 1.40 - 1.80 (m, 6H, 3 CH2's).
Step e S-Tetrahydropyranyl-mercaptoacetyl-p-amino-L-Phe-Gly-dmen
Figure imgf000035_0001
S-Tetrahydropyranyl-mercaptoacetyl-p-Fmoc-amino-L-Phe-Gly-dmen (2.09 g, 10 3.04 mmol) was dissolved in DMF (30 mL) afterwhich moφholine (6 mL) was added. Due to lack of time, the reaction was stirred overnight at ambient temperature before removing the solvent under reduced pressure. The residue was purified by reverse phase Cu column using 30% acetonitrile / 2% acetic acid / water to provide 1.1 g of a clear oil (62% yield). 1H NMR (CDC13) δ 8.02 (s, IH, OH), , 7.86 (m, IH, amide 15 NH), 7.72 (m, IH, amide NH), 7.61 (m, IH, amide NH), 6.96 (d, J = 8.1 Hz, 2H, aromatic), 6.57 (d, J = 8.1 Hz, 2H, aromatic), 6.12 (br s, 2H, NH2), 4.71 (m, IH, SCHO), 4.53 (m, IH, a-H), 4.40 (m, 2H, Gly CH2), 2.82 - 3.89 (m's, 10H SCH2CO, OCH2, CH2NHCO, CH2N, CH2Ar), 2.64 (s, 6H, N(CH3)2), 1.96 (s, 6H, 2 AcOH), 1.50 - 1.77 (m, 6H, 3 CH2's); 13C NMR (CDC13) δ 177, 172, 171, 170, 161, 146, 130, 20 126, 1 16, 82.8, 67.1, 66.3, 65.2, 56.7, 55.8, 45.7, 43.2, 40.5, 36.5, 34.6, 30.8, 25.0, 21.7.
Step f
Fmoc-L-Asp(β-t-Bu)-L-Lys(ε-Boc)-OtBu 25 Fmoc-NH-CH(CH2-COOtBu)-CO-NH-CH(COOtBu)(CH2)4-NH-tBoc
Fmoc-L-Asp(β-t-Bu) (10.0 g, 24.3 mmol) and N-hydroxybenzotriazole (4.93 g, 36.4 mmol) were dissolved in DMF (190 mL). The solution was cooled in an ice bath at 0°C afterwhich EDCHC1 (5.12 g, 26.7 mmol) was added. Stirring was continued at 0°C for one hour before adding a mixture of L-Lys(ε-Boc)-OtBu hydrochloride (8.23 g, 24.3 mmol) and N-methylmoφholine (4.67 g, 46.2 mmol) in DMF (50 mL) dropwise. After the addition was complete, the reaction was stirred
5 overnight at ambient temperature. The reaction mixture was poured into saturated sodium bicarbonate, and the product was extracted into ethyl acetate. The combined organic layer was extracted with saturated sodium bicarbonate (2x) , 1 N HCl (2x), and brine (lx) before drying over anhydrous magnesium sulfate. Filtration and evaporation of the solvent under reduced pressure revealed a yellow foam. The 0 material was purified on SiO2 using a methanol / dichloromethane mobile phase to provide 7 grams of a white foam (44% yield). Η NMR (CDC13) δ 7.78 (d, 2H, aromatic), 7.59 (d, 2H, aromatic), 7.38 (t, 2H, aromatic), 7.29 (t, 2H, aromatic), 7.10 (m, 2H, 2 NH), 5.98 (m, IH, carbamate NH), 4.63 (m, IH, a-H), 4.57 (m, IH, α-H), 4.41 (d, 2H, Fmoc CH2), 4.22 (t, IH, Fmoc CH), 3.33 (m, 2H, CH2N), 2.93 (dd, IH, 5 β-H of Asp), 2.61 (dd, IH, b-H of Asp), 1.20 - 1.84 (m, 6H, 3 CH2's), 1.44 (3 s's, 27H, 3 OC(CH3)3).
Step g
L-Asp(b-t-Bu)-L-Lys(e-Boc)-OtBu 0 NH2-CH(CH2-COOtBu)-CO-NH-CH(COOtBu)(CH2)4-NH-tBoc
Fmoc-L-Asp(β-t-Bu)-L-Lys(ε-Boc)-OtBu (2.69 g, 3.87 mmol) was dissolved in DMF (38.7 mL) afterwhich moφholine (7.74 mLO was added. Due to lack of time, the reaction was stirred overnight before removing the solvent under reduced pressure. The residue was purified by reverse phase Cu chromatography using 30% ethanol / 5 3% acetic acid / water as the eluent to provide 1.3 g of a clear oil (63 % yield). Η NMR (CDC13) δ 7.80 (d, IH, amide NH), 5.58 (br s, 2H, NH2), 4.81 (m, IH, carbamate NH), 4.39 (m, IH, a-H), 3.79 (m, IH, a-H), 3.05 (m, 2H, CH2N), 2.78 (dd, IH, b-H of Asp), 2.59 (dd, IH, b-H of Asp), 1.42 (3 s's, 27H, 3 OC(CH3)3), 1.25 - 1.82 (m, 6H, 3 CH2's); I3C NMR (CDC13) δ 177, 173, 172, 171, 82.0, 81.6, 79.1, 30 52.3, 51.4, 40.0, 39.4, 31.8, 29.0, 28.2, 27.8, 27.7, 22.0, 20.8. Step h ABAS-L-Asp(β-t-Bu)-L-Lys(ε-Boc)-OtBu
Figure imgf000037_0001
ABAS hydrochloride (0.64 g, 2.4 mmol) was dissolved in DMF(30 mL). N- methylmoφholine (0.24 g, 2.4 mmol) and isobutyl chloroformate (0.32 g, 2.4 mmol) were added after cooling to 0°C. After stirring for 0.5 hour, L-Asp(β-t-Bu)-L-Lys(ε- Boc)-OtBu AcOH (1.2 g, 2.3 mmol) and N-methylmoφholine (0.23 g, 2.3 mmol) were added in DMF (15 mL). The reaction was then stirred overnight at ambient temperature before removing the solvent under reduced pressure. The residue was purified by reverse phase Cu chromatography using 30% acetonitrile / 2% acetic acid / water as the eluent to provide a white foam (1.3 g, 76% yield). lH NMR (CDC13) δ 10.3 (br s, IH, amidine NH), 8.72 (br s, IH, Ar-NH), 7.98 (d, IH, amide NH), 7.66 (m, 4H, aromatic), 7.57 (br d, IH, amide NH), 5.04 (t, IH, carbamate NH), 4.81 (m, IH, a-H), 4.28 (m, IH, a-H), 2.99 (m, 2H, CH2NH), 2.79 (m, 4H, COCH2CH2CO), 2.59 (m, 2H, b-H's of Asp), 2.01 (s, 3H, AcOH), 1.10 - 1.80 (m, 6H, 3 CH2's), 1.39 (3 s's, 27H, 3 OC(CH3)3).
Step i ABAS-L-Asp-L-Lys2TFA
Figure imgf000037_0002
ABAS-L-Asp(β-t-Bu)-L-Lys(ε-Boc)-OtBu (1.25 g, 1.66 mmol) was dissolved in 1 : 1 TFA / dichloromethane ( 10 mL each). The reaction was stirred overnight at ambient temperature before removing the solvent under reduced pressure to reveal a yellow powder (0.96 g, 82 %) which was used without further purification. lH NMR (D2O) δ 7.78 (d, 2H, aromatic), 7.62 (d, 2H, aromatic), 4.72 (m, IH, a-H), 4.38 (m, IH, a-H), 2.89 (m, 4H, COCH2CH2CO), 2.69 (m, 4H, CH2N + 2 b-H's of Asp), 1.25 - 1.89 (m, 6H, 3 CH2's).
Step i ABAS-L-Asp-L-Lys p-amino-Phe(α-S-THP-mercaptoacetyl)-Gly-dmen Thiourea
Figure imgf000038_0001
The diacetate salt of S-tetrahydropyranyl-mercaptoacetyl-p-amino-L-Phe-Giy- dmen (260 mg, 0.44 mmol) was dissolved in DMF (4 mL) in the presence of N- methylmoφholine (90 mg, 0.89 mmol, 98 ml). Thiocarbonyldiimidazole (83 mg, 0.47 mmol) was subsequently added. After stirring for 3 hours, ABAS-L-Asp-L-Lys2TFA (310 mg, 0.44 mmol) was added in DMF (4 mL) followed by additional N- methylmoφholine (222 mg, 2.20 mmol, 242 ml). The reaction was stirred overnight at ambient temperature before removing the solvent under reduced pressure. The material was initially flash chromatographed on Cu using an isocratic 30% acetonitrile / 2% acetic acid / water mobile phase. This rapid elution resulted in the separation of unreacted starting materials from the desired product (Rf = 0.23, 30% acetonitrile / 3% acetic acid / water) and the symmetrical thiourea side product. The target compound was finally cleanly isolated by HPLC using a Nova-Pak Cu 30x300 mm column to provide 40 mg of a glassy solid (10% yield). Mobile Phase A: 0.1% TFA / water; Mobile Phase B: 0.1% TFA / 10% water / acetonitrile; Gradient: 90% A / 10%B to 60% A / 40% B over 15 minutes holding at 60% A / 40% B for 15 minutes; Retention Time: 19 minutes; Mass Spec. (ESI) 986 (M+l, 10%), 493 ((M+2)/2, 100%). Example 12
The compound of Example 1 1 was radiolabeled with Tc-99m according to method A described in Example 2.
Example 13
This Example describes the labelling of the compound of Example 11 with a non-radioactive rhenium 185,187 isotope to confirm the composition of the final composition. The composition of the Re-complexes were confirmed by mass spectra (m/e 1 102 (M+l).
Example 14
This Example describes Platelet Aggregation Inhibition Assays of the compound of Example 13 to illustrate that the compound binds to GPIIb/IIIa receptors when radiolabelled following the procedure of Example 4. IC5o: 4.6 X 10-8M
Example 15 This study was designed to evaluate the biodistribution pattern of the compound of Example 12 to determine the clearance profile from blood, route of excretion and in vivo stability of the complex by HPLC analysis of urine samples, in a rat model.
The compound of Example 12 was injected into Sprague-Dawley rats according to the procedure outlined in Example 5. The amount of radioactivity present in the major excretionary organs at different time points (1, 4 and 24 hours) are given below. All values are %ID/g
Organ 1 hour 4 hours 24 hours
Blood 0.1 18 0.062 0.010
Liver 0.187 0.096 0.044
5 Kidney 1.620 1.434 1.066
Sml.Int &
Contents 0.448 0.087 0.008
%TD excreted in the urine at 24 hr. 49.95
%LD excreted in the feces at 24 hr. 43.80 n υ
Example 16
Canine Model for Pulmonary Embolism
These studies are designed to serve as a preliminary screen of various radiolabeled pharmaceuticals for the puφose of imaging pulmonary emboli (PE) and/or 5 deep vein thrombus (DVT).
Procedure: The dog is first anesthetized with an intravenous injection of sodium pentobarbital (30mg/kg). An IV catheter is placed into the jugular vein and advanced to the pulmonary artery. Visualization of the catheter placement is facilitated using fluoroscopy. Embolization coils of various sizes (3-8mm) are next released via 0 the catheter using the appropriate guide wire, and localization of the coils is followed by fluoroscopy. After a satisfactory number of coils (3-5) have been placed and visualized, the catheter is removed and the vein ligated. The formation of a deep vein thrombus (DVT) in the saphenous vein is facilitated by the placement of a 16 gauge needle into the vein followed by the passage of a 5 mm emboilzation coil through the 5 needle directly into the vein.
IV injection (cephalic vein) of the radiolabeled test article occurs approximately 30-45 minutes post placement of the embolization coils. Approximate activity injected is 2.5-3.5mCi. Beginning at 30 minutes post injection and continuing for several hours at 30 minute intervals, the animal is scintigraphed in both the lateral and supine 0 positions. At the conclusion of the imaging portion of the study (3 hours) the animal is sacrificed, and selected tissues removed for scintillation counting. The coils are located, removed and quantitated for uptake of the radiotracer. From these values is determined the target: non-target ratios and blood clearance of the test article. Sample Clot/blood %LD/g
Blood - 0.001
Clot 1 (DVT) 320 0.29
Clot 2 (PE) 38 0.03
Clot 3 (PE) 33 0.03 Clot 4 (PE) 30 0.03
Clot 5 (PE) 57 0.05
Example 17 This Example describes the stepwise preparation of the composition having the structure
Figure imgf000041_0001
ABAS-L-Asp-L-Lys-p-amino-Phe(α-S-THP-mercaptoacetyl)-Glu-dmen Thiourea Step a
ABAS-L-Asp-L-Lys-p-amino-Phe(α-S-THP-mercaptoacetyl)-Glu-dmen Thiourea (MP-2068) was prepared in a manner similar to ABAS-L-Asp-L-Lys-p- amino-Phe(a-S-THP-mercaptoacetyl)-Glu-dmen Thiourea (see example 3) except that Glu(g-t-Bu)-dmen was substituted for Gly-dmen.
Example 18
This Example describes the stepwise preparation of the composition having the structure 2)2-N(CH3)2
Figure imgf000042_0001
Step a
N-Hydroxysuccinimide Ester of Cbz-β-Ala
Cbz-NH-(CH2)2-COOSu. N-Carbobenzyloxy-β-Ala (15.0 g, 67.2 mmol), N-hydroxysuccinimide (8.5 g, 73.9 mmol), and dicyclohexylcarbodiimide (16.6 g, 80.6 mmol) were dissolved in dichloromethane (600 mL). The reaction was continued overnight before filtering to remove the dicylohexylurea. The filtrate was then diluted with dichloromethane and extracted with saturated sodium bicarbonate (3x) and washed with brine before drying over anhydrous magnesium sulfate. Filtration and evaporation of the solvent under reduced pressure revealed a clear oil (20.9 g, 97% yield). The material was used immediately without further purification.
Step b
N-Carbobenzoxy-β-Alanyl-(α-Boc)-lysyl-succinimidate ester
Cbz-β-Ala-ε-L-Lys(α-Boc)-OSu. The α-Boc-L-Lys (10.0 g, 31.2 mmol) was dissolved in water (85 mL) in the presence of sodium bicarbonate (2.62 g, 31.2 mmol). The N-hydroxysuccinimide ester of Z-β-Ala (7.68 g, 31.2 mmol) in acetonitrile (85 mL) was subsequently added, and the reaction was continued overnight before concentrating under reduced pressure. The remaining aqueous layer was acidified with 1 N HCl, and the product was extracted into ethyl acetate (3x). The combined organic layer was washed with brine and dried over anhydrous magnesium sulfate. Filtration and evaporation of the solvent under reduced pressure revealed an off-white foam (5.68 g, 40 % yield). The crude foam (5.62 g, 12.4 mmol), N-hydroxysuccinimide (1.58 g, 13 7 mmol), and dicyclohexylcarbodiimide (3.0 g, 14.9 mmol) were dissolved in dichloromethane (110 mL). The reaction was continued overnight before filtering to remove the dicyclohexylurea. The filtrate was diluted with dichloromethane and extracted with saturated sodium bicarbonate (3x) before washing with brine. The solution was dried over anhydrous magnesium sulfate, filtered, and evaporated to reveal an off-white foam (6.2 g, 91% yield). This material was used without further purification.
Step c Ala-ε-L-Lys(α-Boc)-Glu(γ-t-Bu)-dmen
H2N-(CH2)2-CO-NH-(CH2)4-CH-CO-Glu(γ-OtBu)-NH(CH2)2-N(CH3)2
NH-Boc
Cbz-β-Ala-ε-L-Lys(α-Boc)-OSu (3.0 g, 5.47 mmol) and Glu(γ-t-Bu)-dmen (1.42 g, 5.21 mmol) were dissolved in dichloromethane (60 mL), and the reaction was continued overnight. It was diluted with dichloromethane and extracted with saturated sodium bicarbonate(3x) and washed with brine before drying over anhydrous magnesium sulfate. Filtration and evaporation of the solvent under reduced pressure revealed a white foam which was purified by reverse phase Cu chromatography using an acetonitrile / acetic acid / H2O gradient (2.50 g, 68% yield). The material (2.25 g, 3.19 mmol) was dissolved in methanol (25 mL). After flushing with nitrogen, the catalyst was added, and the hydrogenolysis was performed in the usual manner. After five hours, the reaction was filtered through Celite, and the filtrate was concentrated to reveal a quantitative yield of a white foam.
Step d ABAS-L-Asp-β-Ala-ε-L-Lys(α-S-THP-mercaptoacetyl)-Glu-dmen
Figure imgf000043_0001
The ABAS-Asp(β-t-Bu) (1 30 g, 2 79 mmol) was dissolved in DMF (70 mL) at 0°C N-methylmoφholine (0 28 g, 0 31 mL, 2 79 mmol) and isobutyl chloroformate (0 38 g, 0 36 mL, 2.79 mmol) were subsequently added After stirring for 0 5 hour at 0°C, a solution of Ala-e-L-Lys(a-Boc)-Glu(g-t-Bu)-dmen (1 6 g, 2.79 mmol) in DMF (30 mL) was added. The reaction was gradually permitted to warm to room temperature for overnight stirring The solvent was then removed under reduced pressure The crude material was purified by reverse phase Cu chromatography using an acetonitrile / acetic acid / water gradient to afford a white solid (500 mg, 17 % yield, not accounting for the unreacted ABAS-Asp(β-tBu)) This material was then dissolved in 1.1 TFA / dichloromethane (10 mL), and the deprotection was continued overnight before removing the solvent under reduced pressure The residue was dissolved in water and filtered through CI8 to give a clear oil (390 mg, 86 % yield). This deprotected peptide was finally dissolved in water (5 mL) in the presence of sodium bicarbonate (194 mg, 2.31 mmol) The N-hydroxysuccinimide ester of S- tetrahydropyranyl-mercaptoacetic acid (115 mg, 042 mmol) in acetonitrile (5 mL) was subsequently added, and the reaction was continued overnight. The reaction was concentrated under reduced pressure, and the remaining aqueous layer was acidified with 1 N HCl to pH 3 The material was then loaded onto a flash Cu column for removal of the bulk impurities followed by HPLC purification using an acetonitrile / water / 0.1% TFA mobile phase system After lyophilization, approximately 60 mg were obtained
Example 19
The compound of Example 18 was radiolabeled with Tc-99m according to method A and B described in Example 2.
Example 20
This Example describes the labelling of the compound of Example 11 with a non-radioactive rhenium 185,187 isotope to confirm the composition of the final composition The composition of the Re-complexes were confirmed by mass spectra (m/e 1013 (M+l)).
Example 21 This Example describes Platelet Aggregation Inhibition Assays of the compound of Examples 18 and 20 to illustrate that the compound binds to GPirb/TIIa receptors when radiolabelled following the procedure of Example 4.
Results: IC50: 4 9 X 10-7M (Example 18) and 4.1 X 10"7M (Example 20)
Example 22
This study was designed to evaluate the biodistribution pattern of the compound of Example 19 to determine the clearance profile from blood, route of excretion and in vivo stability of the complex by HPLC analysis of urine samples, in a rat model. The compound of Example 19 was injected into Sprageue-Dawley rats according to the procedure outlined earlier. The amount of radioactivity present in the major excretionary organs at different time points (1 , 4 and 24 hours) are given below.
All values are %LD/g
Organ 1 hour 4 hours 24 hours
Blood 0.067 0.01 1 0.004
Liver 0.158 0.024 0.009
Kidney 0.864 0.520 0.241 Sml.Int &
Contents 0.645 0.015 0.002
%LD excreted in the urine at 24 hr. 41.217 %ED excreted in the feces at 24 hr. 32.758 Example 23
The compound of claim 19 was administered to a canine according to the general protocol described in Example 16 The following results were obtained.
Sample Clot/blood %LD/g Blood - 0.0018
Clot 1 (DVT) 43 0.0828
Clot 2 (PE) 6 0.01 14
Clot 3 (PE) 65 0.125
Clot 4 (PE) 74 0.142 Clot 5 (PE) 64 0.1232
Example 24
This Example describes the stepwise preparation of a compound having the structure
Figure imgf000046_0001
Step a
Figure imgf000046_0002
ABAS-L-Asp-L-Asp-β,ε-Lys(α-S-THP-mercaptoacetyl)-Glu-dmen was prepared in a manner similar to Example 18 (as previously desribed) except that Cbz-L-Asp(β-OSu)- OBn was substituted for Cbz-β-Ala-OSu Mass Spec (ESI) 950 (M+l, 10%), 476.5 ((M+2)/2, 100%), Retention Time 22 min (0 46 x 25 cm Vydac Cu, 1 mL / min flow rate), Gradient 95% A - 70% A over 35 minutes (Solvent A: 0 1% TFA / water, Solvent B 0 1% TFA / 10% water / acetonitrile)
Example 25
This Example describes the stepwise preparation of a compound having the structure
Figure imgf000047_0001
S-THP-mercaptoacetyl-AAA(δ-OBn)-Glu(γ-t-Bu)-dmen
BnOOC-(CH2)3-CH-CO-Glu(γ-OtBu)-NH-(CH2)2-N(CH3)2
NHCO-CH2-S-THP S-THP-mercaptoacetyt-aminoadipic acid (S-THP-MA-AAA, 3 16 g, 7 72 mmol) and N-hydroxysuccinimide (0 98 g, 8 49 mmol) were dissolved in anhydrous dichloromethane (118 mL) Dicyclohexylcarbodiimide (1 75 g, 8 49 mmol) was subsequently added, and the reaction was continued overnight at ambient temperature before removing the dicyclohexylurea by filtration The filtrate was evaporated to dryness, and the residue was immediately dissolved in 1 1 acetonitrile / water (20 mL each) along with Glu(γ-t-Bu)-dmen (2.53 g, 9 26 mmol) in the presence of sodium bicarbonate (0 78 g, 9 26 mmol) The reaction was continued overnight at room temperature The solvent was finally removed under reduced pressure, and the crude material was dissolved in 30% acetonitrile / 2% acetic acid / water in the presence of 0.4 mL of acetic acid. It was loaded onto a reverse phase Cu column packed with the same solvent system. Isocratic elution with 30% acetonitrile / 2% acetic acid / water afforded (2 70 g, 48% yield). Rf = 0 12 (30% acetonitrile / 2% acetic acid / water). Step b S-THP-mercaptoacetyl-AAA-Glu(γ-t-Bu)-dmen
HOOC-(CH2)3-CH-CO-Glu(γ--OtBu)-NH-(CB)2-N(CH3)2
NHCO-CH2-S-THP
S-THP-mercaptoacetyl-AAA(δ-OBn)-Glu(g-t-Bu)-dmen AcOH (1.93 g, 2.66 mmol) was dissolved in acetonitrile (95 mL) and water (50 mL). One equivalent of 1 N sodium hydroxide (2.6 mL) was subsequently added dropwise. After 10 minutes, another equivalent of 1 N sodium hydroxide was added. The reaction was then continued overnight at room temperature before adjusting the pH to 7 with 1 N hydrochloric acid (2 mL). The solvent was removed under reduced pressure, and the residue was dissolved in 20% acetonitrile / water and loaded onto a reverse phase Cu column packed with 10% acetonitrile / water. Isocratic elution with 20% acetonitrile / water ultimately gave 920 mg of a white powder (60% yield). Rf = 0.57 (60% acetonitrile / water).
Step c S-THP-mercaptoacetyl-AAA(δ-OSu)-Glu(γ-t-Bu)-dmen
SuOOC-(CH2)3-CH-CO-Glu(γ-OtBu)-NH-(CH2)2-N(CH3)2
NHCO-CH2-S-THP S-THP-mercaptoacetyl-AAA-Glu(γ-t-Bu)-dmen (400 mg, 0.70 mmol) and N- hydroxysuccinimide (88 mg, 0.77 mmol) were dissolved in anhydrous dichloromethane (11 mL). Dicyclohexylcarbodiimide (170 mg, 0.84 mmol) was subsequently added, and the reaction was continued overnight at ambient temperature. The dicyclohexylurea was removed by filtration, and the filtrate was diluted with dichloromethane. The organic layer was extracted with saturated sodium bicarbonate (3x) and washed with brine (lx) before drying over anhydrous magnesium sulfate. Filtration and evaporation of the solvent under reduced pressure revealed a white foam which was used without purification.
Step d ABAS-L-Asp-L-Lys(ε-AAA(N-α-S-THP-mercaptoacetyl)-Glu(γ-t-Bu)-dmen)
Figure imgf000049_0001
ABAS-L-Asp-L-Lys2TFA (25.6 mg, .034 mmol) and S-THP-mercaptoacetyl- AAA(δ-OSu)-Glu(γ-t-Bu)-dmen (19.5 mg, .041 mmol) were dissolved in DMF (0.5 mL) in the presence of triethylamine (.012 mL). The reaction was continued overnight before diluting with 1 : 1 acetonitrile / water (2 mL) and acidifying to pH 4 with 0.2 N hydrochloric acid. The solvent was subsequently removed under reduced pressure. The residue was purified by reverse phase Cu chromatography using an acetonitrile / water gradient system to afford 16 mg of product (46% yield). Rf = 0.54 (33% acetonitrile / 0.5 N sodium chloride); Η NMR (1 : 1 CD3CN / D2O, unreferenced) δ 8..12 (dd, 4H, aromatic), 5.31 (m, 2H), 5.09 (m, IH), 4.60 (m, IH), 4.36 (m, 2H), 3.93 (m, 4H), 3.74 (m, 3H), 3.61 (m, 3H), 3.45 (t, 2H), 3.18 (s + m, 10H), 2.99 (m, 2H), 2.69 (m, 4H), 2.56 (m, 2H), 2.44 (m, IH), 1.88 - 2.34 (m, 14H), 1.79 (s, 9H), 1.57 - 1.83 (m, 4H).
Step e
ABAS-L-Asp-L-Lys(ε-AAA(α-S-THP-mercaptoacetyl)-Glu-dmen)
Figure imgf000049_0002
ABAS-L-Asp-L-Lys(ε-AAA(α-S-THP-mercaptoacetyl)-Glu(γ-t-Bu)-dmen) (16 mg, .016 mmol) was dissolved in formic acid (1 mL). The reaction was continued overnight before evaporating the solvent under reduced pressure The residue was evaporated several times from 1 1 acetonitrile / water to generate 14 mg of sufficiently pure product (91% yield) Η NMR ( 1 1 CD3CN / D2O, unreferenced) δ 8.74 (d, IH, 3.4 Hz), 8 54 (s, IH), 8 19 (dd, 8 2, 1.8 Hz, 4H, aromatic), 5 34 (m, IH), 5.07 (m, IH), 4.59 (m, 2H), 4 41 (m, 2H), 3.99 (m, 4H), 3.79 (m, 2H), 3.68 (m, 4H), 3.49 (m, 2H), 3.29 (s + m, 8H), 3.1 1 (m, 6H), 2.78 (m, 4H), 1 62 - 2.62 (m, 18H)
Example 26
The compound of Example 25 was radiolabeled according to the method A described Example 2.
Example 27
This Example describes the labelling of the compound of Example 25 with non radioactive rhenium 185, 187 isotope to confirm the composition of the final composition.
The composition of the Re-complexes were confirmed by mass spectra (m/e 1096(M+1)).
Example 28 This Example describes Platelet Aggregation Inhibition Assays of the compound of Example 26 and 27 to illustrate that the compound binds to GPIIb/IIIa receptors when radiolabelled following the procedure of Example 4
Results: 2.9 X 10'7 M (Example 26) and 2.7 X 10"7 (Example 27)
Example 29
This study was designed to evaluate the biodistribution pattern of the compound of Example 26 to determine the clearance profile from blood, route of excretion and in vivo stability of the complex by HPLC analysis of urine samples, in a rat model. The compound of Example 26 was injected (25mL, 1.5-3.5 mCi/mL) into Sprague-Dawley rats. Groups of three animals were sacrificed at the time points indicated below to determine the amount of radioactivity remaining in the organs. All values are %LD/g
Organ 1 hour 4 hours 24 hours
Blood 0.067 0.011 0.007
Liver 0.043 0.015 0.014
Kidney 0.628 0.536 0.227 Sml.Int &
Contents 0.206 0.031 0.009
%ID excreted in the urine at 24 hr. 54.708 %LD excreted in the feces at 24 hr. 16.277
Example 30
This Example describes the stepwise preparation of
Figure imgf000051_0001
ABAS-L-Asp-β-Ala-ε-Lys(α-S-THP-mercaptoacetyl)-Glu-Thr(ol)
Step a
Figure imgf000051_0002
ABAS-L-Asp-β-Ala-ε-Lys(α-S-THP-mercaptoacetyi)-Glu-Thr(ol) was prepared in a manner similar to MP-2065 (as previously desribed) except that threoninol was substituted for dmen. Mass Spec. (ESI) 924 (M+l, 20%), 462.9 ((M+2)/2, 100%); Retention Time: 21 min. (0.46 x 25 cm Vydac Cu, 1 mL / min flow rate); Gradient: 100% A - 60% A over 30 minutes (Solvent A: 0 1% TFA / water, Solvent B: 0.1% TFA / 10% water / acetonitrile).
Example 31
This Example describes the stepwise preparation of the compound having the structure
Figure imgf000052_0001
Step a
N-Boc-L-Asp(β-OtBu)-L-Lys(ε-Cbz)-OtBu L-Lys(ε-Cbz)-OtBu hydrochloride (7.93 g, 21.2 mmol) was dissolved in dichloromethane (140 mL) in the presence of the triethylamine (2.15 g, 21.2 mmol, 3.0 mL). N-Boc-L-Asp(β-OtBu)-OSu (10.0 g, 21.2 mmol) was subsequently added, and the reaction was continued overnight at ambient temperature before diluting with dichloromethane. The organic layer was extracted with saturated sodium bicarbonate (3x), 1 N hydrochloric acid (2x), and washed with brine (lx) before drying over anhydrous magnesium sulfate. Filtration and evaporation of the solvent under reduced pressure revealed a yellow foam which was purified on SiO2 using dichloromethane as the eluent to provide 7.2 grams of a white foam (56% yield). Η NMR (CDC13) δ 7.28 (m, 5H, aromatic), 7.05 (m, IH, carbamate NH), 5.64 (d, IH, amide NH), 5.05 (dd, 2H, benzylic CH2), 5.00 (m, IH, carbamate NH), 4.41 (m, 2H, 2 a-H's), 3.18 (m, 2H, CH2N), 2.84 (dd, IH, Asp b-H), 2.58 (dd, IH, Asp b-H), 1.44 (3 s's, 27H, 3 OC(CH3)3), 1.20 - 1.85 (m, 6H, 3 CH2's). Step b
N-Boc-L-Asp(β-OtBu)-L-Lys-OtBu N-Boc-L-Asp(β-OtBu)-L-Lys(ε-Cbz)-OtBu (4 97 g, 8 18 mmol) was dissolved in methanol (85 mL) After flushing with nitrogen, 10% Pd/C catalyst (0 5 g) was added The mixture was shaken for 6 hours at ambient temperature under an atmosphere of hydrogen (40 p s i ) The reaction was filtered through a pad of Celite, and the filtrate was concentrated under reduced pressure to reveal a mauve oil in quantitative yield Η NMR (CDC13) δ 7 03 (d, IH, amide NH), 5 74 (d, 2H, amide NH), 4 42 (m, 2H, 2 a-H's), 2 84 (dd, IH, Asp b-H), 2 62 (m, CH2N + Asp b-H), 1 44 (3 s's, 27H, 3 OC(CH3)3), 1 20 - 1 85 (m, 6H, 3 CH2's), ,3C NMR (CDC13) δ 172, 171, 170, 156, 81 9, 81 6, 80 2, 52 7, 50 6, 41 6, 37 2, 33 0, 32 1, 28 1, 27 8, 27 7, 21 9
Step c N-Boc-L-Asp(β-OtBu)-L-Lys(ε-γ-L-Glu(N-α-Cbz)-OBn)-OtBu
N-Boc-Asp-Lys(OtBu)-NH-CO-(CH2)2-CH-COOBn
NH-Cbz N-Cbz-L-Glu-OBn (2 77 g, 7 45 mmol) and N-hydroxybenzotπazole (1 51 g, 11 2 mmol) were dissolved in DMF (55 mL) The solution was cooled in an ice bath afterwhich EDCHC1 (1 57 g, 8 20 mmol) was added Stirring was continued at O°C for one hour before adding a mixture of N-Boc-L-Asp(b-OtBu)-L-Lys-OtBu (3 78 g, 7 45 mmol) and N-methylmoφholine (0 68 g, 6 70 mmol, 1 0 mL) in DMF (15 mL) dropwise After the addition was complete, the reaction was permitted to gradually warm to room temperature for overnight stirring The reaction mixture was poured into saturated sodium bicarbonate, and the aqueous mixture was extracted into ethyl acetate (3x) The combined ethyl acetate layer was washed with saturated sodium bicarbonate (2x), water (2x), 1 N hydrochloπc acid (2x), and bπne (lx) before drying over anhydrous magnesium sulfate Filtration and evaporation of the solvent under reduced pressure revealed a yellowish foam (5 8 g, 94% yield) This mateπal appeared sufficiently pure by proton NMR, so no further purification was necessary. Η NMR (CDC13) δ 7.29 (m, 5H, aromatic), 7.04 (d, IH, carbamate NH), 6.10 (t, IH, amide NH), 5.91 (d, IH, amide NH), 5.70 (d, IH, carbamate NH), 5.07 (s, 2H, benzylic CH2), 4.38 (m, 3H, 3 a-H's), 3.13 (m, 2H, CH2N), 2.82 (dd, IH, Asp b-H), 2.56 (dd, IH, Asp b-H), 2.16 (m, 2H, Glu g CH2), 1.39 (3 s's, 27H's, 3 OC(CH3)3), 1.22 - 1.79 (m, 8H, 4 CH2's).
Step d N-Boc-L-Asp(β-OtBu)-L-Lys(ε-γ-L-Glu)
N-Boc-Asp-Lys(OtBu)-NH-CO-(CH2)2-CH-COOBn
NH2
N-Boc-L-Asp(β-OtBu)-L-Lys(ε-γ-L-Glu(N-α-Cbz)-OBn)-OtBu (5.8 g, 7.01 mmol) was dissolved in methanol (75 mL). After flushing with nitrogen, 10% PάVC catalyst (0.5 g) was added. The mixture was shaken for 6 hours at ambient temperature under an atmosphere of hydrogen (40 p.s.i.). The reaction was filtered through a pad of Celite, and the filtrate was concentrated under reduced pressure to reveal a white foam in quantitative yield. Η NMR (CDC13) δ 7.75 (br d, IH, amide NH), 7.41 (br d, IH, amide NH), 6.08 (br d, IH, carbamate NH), 4.54 (m, IH, a-H), 4.28 (m, IH, a-H), 3.61 (m, IH, a-H), 3 07 (m, 2H, CH2N), 2.67 (m, 2H, NH2), 2.37 (m, 2H, Asp b-H's), 2.31 (m, 2H, Glu g-H's), 1.39 (3 s's, 27H's, 3 OC(CH3)3), 1.19 - 1.72 (m, 8H, 4 CH2's); 13C NMR (CDC13) d 174, 173, 171.4, 171.3, 171.0, 156, 81.8, 81.4, 79.9, 54.2, 53.5, 50.6, 50.7, 38.8, 38.1, 32.0, 31.9, 28.0, 27.9, 27.8, 22.2.
Step e
N-Boc-L-Asp(β-OtBu)-L-Lys(ε-γ-L-Glu(N-α-S-THP-mercaptoacetyl))-OtBu ε- N-Boc-Asp-Lys(OtBu)-NH-CO-(CH2)2-CH-COOH
NHCO-CH2-S-THP N-Boc-L-Asp(β-OtBu)-L-Lys(ε-γ-L-Glu(N-α-Cbz)-OBn)-OtBu (4.12 g, 6.84 mmol) was suspended in a mixture of 1 N sodium bicarbonate (20 mL) and dioxane (20 mL). The NHS ester of S-THP-mercaptoacetic acid (1.78 g, 6.51 mmol) was subsequently added, and the reaction was continued overnight at room temperature. The dioxane was removed under reduced pressure, and the remaining aqueous layer was acidified with 1 N hydrochloric acid. The product was then extracted into ethyl acetate (3x), and the combined ethyl acetate layer was washed with 1 N hdrochloric acid (2x), water (2x), and brine (lx) before drying over anhydrous magnesium sulfate. Filtration and evaporation of the solvent under reduced pressure revealed a white foam which was purified on SiO2 using 5% methanol / 2% acetic acid / dichloromethane as an isocratic eluent. Mixed fractions were discarded (2.1 g, 42% isolated yield). Rf = 0.62 (20% methanol / dichloromethane).
Step f N-Boc-L-Asp(β-OtBu)-L-Lys(ε-γ-L-Glu(N-α-S-THP-mercaptoacetyl)-Gly-dmen)-
OtBu ε- N-Boc-Asp-Lys(OtBu)-NH-CO-(CH2)2-CH-CO-Gly-dmen
NHCO-CH2-S-THP
N-Boc-L-Asp(β-OtBu)-L-Lys(ε-γ-L-Glu(N-α-S-THP-mercaptoacetyl))-OtBu (1.91 g, 2.51 mmol) and N-hydroxysuccinimide (0.32 g, 2.76 mmol) were dissolved in dichloromethane (40 mL). Dicyclohexylcarbodiimide (0.62 g, 3.01 mmol) was subsequently added, and the reaction was continued overnight at ambient temperature. The reaction was then filtered to remove the dicyclohexylurea, and the filtrate was diluted with dichloromethane. The organic layer was extracted with saturated sodium bicarbonate (3x) and washed with brine (lx) before drying over anhydrous magnesium sulfate. Filtration and evaporation of the solvent under reduced pressure revealed a foam which was immediately used without further purification. The active ester (1.05 g, 1.27 mmol) was added to a suspension of Gly-dmen hydrochloride (0.23 g, 1.27 mmol) in dichloromethane (25 mL) in the presence of triethylamine (0.13 g, 1.27 mmol). After adding DMF (2 mL) to improve solubility, the reaction was continued overnight at room temperature before diluting with dichloromethane. The organic layer was extracted with saturated sodium bicarbonate (3x), water (lx), and brine (lx) before drying over anhydrous magnesium sulfate. Filtration and evaporation of the solvent under reduced pressure revealed 0.99 g (88% yield) of a clear oil of sufficient purity by NMR; no further purification was performed.
Step g
Asp-L-Lys(ε-γ-L-Glu(N-α-S-THP-mercaptoacetyl)-Gly-dmen) ε- Asp-Lys-NH-CO-(CH2)2-CH-CO-Gly-dmen
NHCO-CH2-S-THP
N-Boc-L-Asp(β-OtBu)-L-Lys(ε-γ-L-Glu(N-α-S-THP-mercaptoacetyl)-Gly- dmen)-OtBu (0.99, 1.11 mmol) was dissolved in 1 : 1 TFA / dichloromethane (4 mL each). After two hours, the solvent was removed under reduced pressure, and the residue was purified by reverse phase Cu chromatography using 3% acetic acid / water as the mobile phase to afford 300 mg of product (34% yield).
Step h
Figure imgf000056_0001
ABAS-L-Asp-Lys(ε-γ-L-Glu(N-α-S-THP-mercaptoacetyl)-Gly-dmen)
ABAS HCl (104 mg, 0.38 mmol) was dissolved in DMF (5 mL). N- methylmoφholine (39 mg, 0.38 mmol, 0.042 mL) and isobutyl chloroformate (52 mg, 0.38 mmol) were added after cooling to 0°C. After stirring for one half hour, Asp-L- Lys(e-g-L-Glu(N-a-S-THP-mercaptoacetyl)-Gly-dmen) (290, 0.36 mmol) and N- methylmoφholine (73 mg, 0.72 mmol) were added, and the reaction was continued overnight at ambient temperature. The solvent was removed under reduced pressure, and the residue was partially purified by reverse phase Cu flash chromatography using 30% acetonitrile / 3% acetic acid / water as an isocratic eluent (Rr= 0.23, 60% acetonitrile / 3% acetic acid / water). A final HPLC purification was performed to remove the remaining trace impurities.
5 Example 32
This Example describes the stepwise preparation of a compound having the structure
Figure imgf000057_0001
0 Step a
S-THP-mercaptoacetyl-L-Lys(ε-(4-propionylphenyl isothiocyanate)-Gly-dmen SCN-0(CH2)2-CO-NH-(CH2)4-CH-CO-Gly-NH-(CH2)2-N(CH3)2
NHCO-CH2-S-THP S-THP-mercaptoacetyl-L-Lys-Gly-dmen 2TFA (1.32 g, 2.00 mmol) was dissolved in acetonitrile (25 mL). A solution of 3-(/?-isothiocyanato-phenyl)propionyl chloride
15 (0.677 g, 3.00 mmol) in chloroform (25 mL) was subsequently added, and the reaction was stirred for one hour before adding potassium carbonate (1.38 g, 10.0 mmol). After stirring for another hour, water (10 mL) was added. Finally, after a total reaction time of four hours, the solvent was removed under reduced pressure. The residue was redissolved in ethyl acetate, and the organic solution was extracted with
20 10% sodium bicarbonate. The organic layer was set aside, and the aqueous layer was extracted with ethyl acetate (2x). The combined ethyl acetate layer was then extracted with 10%» sodium bicarbonate (2x) and washed with brine (lx) before drying over anhydrous magnesium sulfate. Filtration and evaporation of the solvent under reduced pressure revealed an oily semi-solid. The crude material was purified by preadsorbing
25 onto reverse phase Cu and dry loading this mixture onto a reverse phase Cu column packed with 25% acetonitrile / water. Elution was accomplished with a gradient from 25% acetonitrile to 50% acetonitrile to provide 200 mg of product (16% yield). Mass Spec. (ESI) 621 (M+l).
Step b ABAS-L-Asp-L-Lys phenylpropionyl-ε-L-Lys(N-α-S-THP-mercaptoacetyl)-Gly-dmen
Thiourea
Figure imgf000058_0001
S-THP-mercaptoacetyl-L-Lys(e-(4-propionylphenyl isothiocyanate)-Gly-dmen (42 mg, 68 mmol) was dissolved in DMF (1.5 mL). ABAS-L-Asp-L-Lys2TFA (96 mg, 140 mmol) and triethylamine (50 ml) were subsequently added, and the reaction was continued overnight at ambient temperature. The solvent was removed under reduced pressure, and the residue was dissolved 10% acetonitrile / water and loaded onto a reverse phase Cu column packed with the same solvent system. A gradient elution from 10% acetonitrile to 20% acetonitrile resulted in the isolation of 28 mg of product (38% yield). Mass Spec. (ESI) 1099 (M+l).
Example 33
A 99m-Tc labeled molecule consisting of an N3S-chelate chemically linked to a peptidominmatic moiety which has high affinity for the GpIIb/IIIa receptor expressed on activated platelets was prepared from an instant kit and 99m-pertechnatate with radiochemical purity >95% and is stable for up to 6 hours. Studies in normal volunteers showed rapid blood clearance
Figure imgf000058_0002
11±4 min) and extensive liver uptake (31.7 ± 1.6% i.d. 30 min p.i.). The puφose of this procedure was to assess the labeled compound for imaging fresh thrombi in humans. We studied seven patients (3 males, 4 females, 58 ± 12 yr) with diagnosis of deep vein thrombosis based on pain of one lower limb, fibrin degradation products (D-Dimer) >2.3 μg/ml, and positive compression ultra sonography examination. The labeled compound (15-20 mCi Tc- 99m) was administered to each patient within 2 hours of enrollment. Whole body images were acquired at 30 and 60 minutes p i. Activity ratios between the affected and contralateral limbs were calculated. Tracer accumulation was in all cases consistent with the diagnosis. Affected/contralateral limb activity ratios were 1.82 ± 0 58 at 30 minutes and 1.96 ± 0.73 at 60 minutes (in normals = 0.98 ± 0.004 and 0.99 ± 0.005 respectively). These results indicate that members of this class of Tc-99m compounds have potential for the imaging of fresh thrombi.

Claims

What is claimed is:
1. A radiopharmaceutical capable of localizing at a site of thrombus containing activated platelets within a mammalian body, the radiopharmaceutical comprising a linear peptidomimetic containing ligand capable of specifically binding to platelets in the thrombus and a radionuclide complexed with the peptidomimetic containing ligand.
2. The radiopharmaceutical of claim 1 wherein the radionuclide is selected from the group consisting of 67Ga, 1 1 lln, 99mTc, 169Yb, 186Re, 90Y, 67Cu, 186Re, 188Re, 169Er, 121Sn, 127Te, 143Pr, 198Au, 109Pd, 165Dy, 32P, 142Pr, and 153Sm. 3. The radiopharmaceutical of claim 2 wherein the radionuclide is 99mTc.
4. The radiopharmaceutical of claim 1 wherein the peptidomimetic containing ligand has the structure:
Figure imgf000060_0001
where A is -CH- or -N-, and B is -CH- or -N-, with the proviso that when A is -N-, B is -CH-, and when B is -N-, then A is -CH-; -D-E- is -CH2-CH2-, or -NHCO- with the proviso that when A is -N-, then -D-E is -CH2-CH2-, and when B is -N-, then -D-E is -NHCO-; Ri is hydrogen, lower alkyl, or acyloxyalkyl; K is hydrogen, carboxyl, lower alkyl, aralkyl, substituted or unsubstituted aromatic, or an alkylene or substituted alkylene substituted with one of the following substituents: primary amine, secondary amine, cyclic or acyclic tertiary amine, carboxyl, ester, hydroxyl, ether, thiol, thioether, guanido, imine, aminoalkyl, monoaminoalkyl, diaminoaikyl, hydroxyalkyl, alkoxylalkyl, hydroxycarbonylalkyl, alkoxycarbonylalkyl, mercaptoalkyl, or alkylthioalkyl; X is NH, CO, S, or O; Y is NH, CO, CS, CONH, CSNH, NHCO, NHCS, CONH0(CH2),. 4CONH, CONH(CH2)MCONH, CSNH0(CH2)MCONH, or CSNH(CH2)MCONH, with the proviso that X and Y are different except when X is S, and when X is O, Y is CO, and when X is S, Y is S, CO or CS, and when X is NH, Y is CO, CONH, CSNH, CONH0(CH2)1-4CONH, CONH(CH2)MCONH, CSNH0(CH2)1-4CONH, or CSNH(CH2)ι-4CONH, and when X is NH, Y is not NH, NHCO or NHCS, and when X is CO, Y is NH, O or S, and when X is CO, Y is not CO, CS, CONH, CSNH, CONH0(CH2)MCONH, CONH(CH2)MCONH, CSNH0(CH2)MCONH, or
CSNH(CH2)ι-4CONH; n is 0-5; n' is 0 or 1; m is 0-10; and Z is a metal binding group capable of covalently binding a radionuclide.
5. The radiopharmaceutical of claim 4 wherein Z is R2-NH-CH(R3)-[CH(R4)]p-CO-NH-AArNH(CH-L)P -(CH-L')p. .-L" where R2 is COCH(R5)-S-Ri; Ri is H, -(CH2)P-R7 ; p is 1 -5; R7 is a hydrogen, primary amine, secondary amine, cyclic or acyclic tertiary amine, carboxyl, ester, hydroxyl, ether, thiol, thioether, guanido, or an imine; R« is a hydrogen, aliphatic or aromatic acyl, acetamidoalkyl, benzamidoalkyi, unsubstituted or substituted tetra-hydropyranyl, unsubstituted or substituted tetrahydrofuranyl, or alkoxyalkyl; R3 is (CH2)P-Q where p' is 0-6, Q is hydrogen, alkylene or substituted alkylene, aryl or substituted aryl group for attachment to Y; R* is (CH2),-T where s is 0-6, T is hydrogen, alkylene or substituted alkylene,, aryl or substituted aryl group for attachment to Y; p" is 0, 1 if p" is 1 only one of the groups defined under Q or T is attached to Y; AAi is any natural or unnatural α-amino acid or β-amino acid; L, L' = is a hydrogen, carboxyl, lower alkyl, aralkyl, substituted or unsubstituted aromatic group, or an alkylene bearing one of the following substituents: a primary amine, secondary amine, cyclic or acyclic tertiary amine, carboxyl, ester, hydroxyl, ether, thiol, thioether, guanido, imine, aminoalkyl, monoaminoalkyl, diaminoaikyl, hydroxyalkyl, alkoxylalkyl, hydroxycarbonylalkyl, alkoxycarbonylalkyl, mercaptoalkyl, or alkylthioalkyl; p'" is 0- 3; p"" is 0-3; L" is a hydrogen, lower alkyl, aralkyl, substituted or unsubstituted aromatic group, or an alkylene bearing one of the following substituents: a primary amine, secondary amine, cyclic or acyclic tertiary amine, carboxyl, ester, hydroxyl, ether, thiol, thioether, guanido, imine, aminoalkyl, monoaminoalkyl, diaminoaikyl, hydroxyalkyl, alkoxylalkyl, hydroxycarbonylalkyl, alkoxycarbonylalkyl, mercaptoalkyl, or alkylthioalkyl. 6. The radiopharmaceutical of claim 5 wherein A and B are CH.
7. The radiopharmaceutical of claim 6 wherein D-E is NHCO.
8. The radiopharmaceutical of claim 7 wherein the peptidomimetic containing ligand has the structure:
Figure imgf000062_0001
where R« is a suitable sulfur protecting group.
9. The radiopharmaceutical of claim 7 wherein the peptidomimetic containing ligand has the structure:
Figure imgf000062_0002
where R« is a suitable sulfur protecting group.
10. The radiopharmaceutical of claim 7 wherein the peptidomimetic containing ligand has the structure:
Figure imgf000062_0003
where Rg is a suitable sulfur protecting group. 11. The radiopharmaceutical of claim 7 wherein the peptidomimetic containing ligand has the structure:
Figure imgf000062_0004
where R« is a suitable sulfur protecting group. 12. The radiopharmaceutical of claim 7 wherein the peptidomimetic containing ligand has the structure:
Figure imgf000063_0001
where R* is a suitable sulfur protecting group.
13. The radiopharmaceutical of claim 7 wherein the peptidomimetic containing ligand has the structure:
Figure imgf000063_0002
where R« is a suitable sulfur protecting group.
14. The radiopharmaceutical of claim 7 wherein the peptidomimetic containing ligand has the structure:
Figure imgf000063_0003
where Rs is a suitable sulfur protecting group.
15. The radiopharmaceutical of claim 7 wherein the peptidomimetic containing ligand has the structure:
Figure imgf000063_0004
where R» is a suitable sulfur protecting group. 16 The radiopharmaceutical of claim 7 wherein the peptidomimetic containing ligand has the structure
Figure imgf000064_0001
where R« is a suitable sulfur protecting group. 5 17. The radiopharmaceutical of claim 7 wherein the complex of the radionuclide and the peptidomimetic containing ligand has the structure.
Figure imgf000064_0002
where M is selected from the group consisting of 67Ga, 1 1 lln, 99mTc, 169Yb, 186Re, 90Y, 67Cu, 186Re, 188Re, 169Er, 121Sn, 127Te, 143Pr, 198Au, 109Pd, 165Dy, 32P, 10 142Pr, and 153Sm.
18. The radiopharmaceutical of claim 7 wherein the complex of the radionuclide and the peptidomimetic containing ligand has the structure:
Figure imgf000064_0003
where M is selected from the group consisting of 67Ga, 1 1 lln, 99mTc, 169Yb, 186Re, 90Y, 67Cu, 186Re, 188Re, 169Er, 121 Sn, 127Te, 143Pr, 198Au, 109Pd, 165Dy, 32P, 142Pr, and 153Sm.
5 19. The radiopharmaceutical of claim 7 wherein the complex of the radionuclide and the peptidomimetic containing ligand has the structure:
Figure imgf000065_0001
where M is selected from the group consisting of 67Ga, 1 1 lln, 99mTc, 169Yb, 186Re,
90Y, 67Cu, 186Re, 188Re, 169Er, 121 Sn, 127Te, 143Pr, 198Au, 109Pd, 165Dy, 32P,
10 142Pr, and 153Sm.
20. The radiopharmaceutical of claim 7 wherein the complex of the radionuclide and the peptidomimetic containing ligand has the structure:
Figure imgf000065_0002
where M is selected from the group consisting of 67Ga, 1 1 lln, 99mTc, 169Yb, 186Re,
15 90Y, 67Cu, 186Re, 188Re, 169Er, 121 Sn, 127Te, I43Pr, 198Au, 109Pd, 165Dy, 32P,
142Pr, and 153Sm. 21. The radiopharmaceutical of claim 7 wherein the complex of the radionuclide and the peptidomimetic containing ligand has the structure:
Figure imgf000066_0001
where M is selected from the group consisting of 67Ga, 11 lln, 99mTc, 169Yb, 186Re, 90Y, 67Cu, 186Re, 188Re, 169Er, 121 Sn, 127Te, 143Pr, 198Au, 109Pd, 165Dy, 32P, 142Pr, and 153Sm.
22. The radiopharmaceutical of claim 7 wherein the complex of the radionuclide and the peptidomimetic containing ligand has the structure:
Figure imgf000066_0002
where M is selected from the group consisting of 67Ga, 1 1 lln, 99mTc, 169Yb, 186Re, 90Y, 67Cu, 186Re, 188Re, 169Er, 121Sn, 127Te, 143Pr, 198Au, 109Pd, 165Dy, 32P, 142Pr, and l53Sm. 23 The radiopharmaceutical of claim 7 wherein the complex of the radionuclide and the peptidomimetic containing ligand has the structure
Figure imgf000067_0001
where M is selected from the group consisting of 67Ga, 1 1 lln, 99mTc, 169Yb, 186Re, 90Y, 67Cu, 186Re, 188Re, 169Er, 121Sn, 127Te, 143Pr, 198Au, 109Pd, 165Dy, 32P,
142Pr, and 153Sm.
24. The radiopharmaceutical of claim 7 wherein the complex of the radionuclide and the peptidomimetic containing ligand has the structure:
Figure imgf000067_0002
where M is selected from the group consisting of 67Ga, 11 lln, 99mTc, 169Yb, 186Re, 90Y, 67Cu, 186Re, 188Re, 169Er, 121Sn, 127Te, 143Pr, 198Au, 109Pd, 165Dy, 32P, 142Pr, and 153Sm. 25. The radiopharmaceutical of claim 7 wherein the complex of the radionuclide and the peptidomimetic containing ligand has the structure:
Figure imgf000068_0001
where M is selected from the group consisting of 67Ga, 1 1 lln, 99mTc, 169Yb, 186Re, 90Y, 67Cu, 186Re, 188Re, 169Er, 121 Sn, 127Te, 143Pr, 198Au, 109Pd, 165Dy, 32P,
142Pr, and 153Sm.
26. A ligand composition for preparing a radiopharmaceutical having the structure:
Figure imgf000068_0002
where A is -CH- or -N-, and B is -CH- or -N-, with the proviso that when A is -N-, B is -CH-, and when B is -N-, then A is -CH-; -D-E- is -CH2-CH2-, or -NHCO- with the proviso that when A is -N-, then -D-E is -CH2-CH2-, and when B is -N-, then -D-E is -NHCO-; Ri is hydrogen, lower alkyl, or acyloxyalkyl; K is hydrogen, carboxyl, lower alkyl, aralkyl, substituted or unsubstituted aromatic, or an alkylene or substituted alkylene substituted with one of the following substituents: primary amine, secondary amine, cyclic or acyclic tertiary amine, carboxyl, ester, hydroxyl, ether, thiol, thioether, guanido, imine, aminoalkyl, monoaminoalkyl, diaminoaikyl, hydroxyalkyl, alkoxylalkyl, hydroxycarbonylalkyl, alkoxycarbonylalkyl, mercaptoalkyl, or alkylthioalkyl; X is NH, CO, S, or O; Y is NH, CO, CS, CONH, CSNH, NHCO, NHCS, CONH0(CH2),. 4CONH, CONH(CH2)MCONH, CSNH0(CH2)MCONH, or CSNH(CH2)1-4CONH, with the proviso that X and Y are different except when X is S, and when X is O, Y is CO, and when X is S, Y is S, CO or CS, and when X is NH, Y is CO, CONH, CSNH, CONH0(CH2)MCONH, CONH(CH2),.4CONH, CSNH0(CH2)MCONH, or
CSNH(CH2)MCONH, and when X is NH, Y is not NH, NHCO or NHCS, and when X is CO, Y is NH, O or S, and when X is CO, Y is not CO, CS, CONH, CSNH, CONH0(CH2)MCONH, CONH(CH2)MCONH, CSNH0(CH2)MCONH, or CSNH(CH2)MCONH; n is 0-5; n' is 0 or 1; m is 0-10; and Z is a metal binding group capable of covalently binding a radionuclide. 27. The ligand of claim 26 wherein Z is R2-NH-CH(R3)-[CH(R4)]p«-CO-NH-AArNH(CH-L)n....-(CH-L)n..-L" where R2 is COCH(Rs)-S-R<;; R5 is H, -(CH2)P-R7 ; p is 1-5; R7 is a hydrogen, primary amine, secondary amine, cyclic or acyclic tertiary amine, carboxyl, ester, hydroxyl, ether, thiol, thioether, guanido, or an imine; Re is a hydrogen, aliphatic or aromatic acyl, acetamidoalkyl, benzamidoalkyl, unsubstituted or substituted tetra-hydropyranyl, unsubstituted or substituted tetrahydrofuranyl, or alkoxyalkyl; R3 is (CH2)P-Q where p' is 0-6, Q is hydrogen, alkylene or substituted alkylene, aryl or substituted aryl group for attachment to Y; R» is (CH2)S-T where s is 0-6, T is hydrogen, alkylene or substituted alkylene,, aryl or substituted aryl group for attachment to Y; p" is 0,1 if p" is 1 only one of the groups defined under Q or T is attached to Y; AAi is any natural or unnatural α-amino acid or β-amino acid; L, L' = is a hydrogen, carboxyl, lower alkyl, aralkyl, substituted or unsubstituted aromatic group, or an alkylene bearing one of the following substituents: a primary amine, secondary amine, cyclic or acyclic tertiary amine, carboxyl, ester, hydroxyl, ether, thiol, thioether, guanido, imine, aminoalkyl, monoaminoalkyl, diaminoaikyl, hydroxyalkyl, alkoxylalkyl, hydroxycarbonylalkyl, alkoxycarbonylalkyl, mercaptoalkyl, or alkylthioalkyl; p'" is 0- 3; p"" is 0-3; L" is a hydrogen, lower alkyl, aralkyl, substituted or unsubstituted aromatic group, or an alkylene bearing one of the following substituents: a primary amine, secondary amine, cyclic or acyclic tertiary amine, carboxyl, ester, hydroxyl, ether, thiol, thioether, guanido, imine, aminoalkyl, monoaminoalkyl, diaminoaikyl, hydroxyalkyl, alkoxylalkyl, hydroxycarbonylalkyl, alkoxycarbonylalkyl, mercaptoalkyl, or alkylthioalkyl
28 The ligand of claim 27 wherein A and B are CH
29 The ligand of claim 28 wherein D-E is NHCO.
30 The ligand of claim 29 having the structure:
Figure imgf000070_0001
where R« is a suitable sulfur protecting group. 31 The ligand of claim 29 having the structure.
Figure imgf000070_0002
where R« is a suitable sulfur protecting group.
32 The ligand of claim 29 having the structure"
Figure imgf000070_0003
where R» is a suitable sulfur protecting group.
33 The ligand of claim 29 having the structure
Figure imgf000070_0004
where R« is a suitable sulfur protecting group. 34 The ligand of claim 29 having the structure:
Figure imgf000071_0001
where Re is a suitable sulfur protecting group. 35. The ligand of claim 29 having the structure:
Figure imgf000071_0002
where R« is a suitable sulfur protecting group.
36. The ligand of claim 29 having the structure:
Figure imgf000071_0003
where R« is a suitable sulfur protecting group.
37 The ligand of claim 29 having the structure
Figure imgf000071_0004
where R« is a suitable sulfur protecting group.
38. The ligand of claim 29 having the structure:
2)2-N(CHj)2
Figure imgf000071_0005
where R8 is a suitable sulfur protecting group.
39. A kit for preparing a radiopharmaceutical composition comprising a container containing a selected amount of a linear peptidomimetic containing ligand in a pharmaceutically acceptable vehicle and a sufficient amount of a reducing reagent to permit labeling of the composition with a selected radionuclide.
40. The kit of claim 39 wherein the radionuclide is selected from the group consisting of 67Ga, 1 11 In, 99mTc, 169Yb, 186Re, 90 Y, 67Cu, 186Re, 188Re, 169Er, 121Sn, 127Te, 143Pr, 198Au, 109Pd, 165Dy, 32P, 142Pr, and 153Sm.
41. The kit of claim 40 wherein the radionuclide is 99mTc.
42. The kit of claim 39 wherein the peptidomimetic containing ligand has the structure:
Figure imgf000072_0001
where A is -CH- or -N-, and B is -CH- or -N-, with the proviso that when A is -N-, B is -CH-, and when B is -N-, then A is -CH-; -D-E- is -CH2-CH2-, or -NHCO- with the proviso that when A is -N-, then -D-E is -CH2-CH2-, and when B is -N-, then -D-E is -NHCO-; Ri is hydrogen, lower alkyl, or acyloxyalkyl; K is hydrogen, carboxyl, lower alkyl, aralkyl, substituted or unsubstituted aromatic, or an alkylene or substituted alkylene substituted with one of the following substituents: primary amine, secondary amine, cyclic or acyclic tertiary amine, carboxyl, ester, hydroxyl, ether, thiol, thioether, guanido, imine, aminoalkyl, monoaminoalkyl, diaminoaikyl, hydroxyalkyl, alkoxylalkyl, hydroxycarbonylalkyl, alkoxycarbonylalkyl, mercaptoalkyl, or alkylthioalkyl; X is NH, CO, S, or O; Y is NH, CO, CS, CONH, CSNH, NHCO, NHCS, CONH0(CH2),. 4CONH, CONH(CH2)1-4CONH, CSNH0(CH2),-4CONH, or CSNH(CH2)MCONH, with the proviso that X and Y are different except when X is S, and when X is O, Y is CO, and when X is S, Y is S, CO or CS, and when X is NH, Y is CO, CONH, CSNH, CONH0(CH2)MCONH, CONH(CH2)MCONH, CSNH0(CH2)MCONH, or CSNH(CH2)MCONH, and when X is NH, Y is not NH, NHCO or NHCS, and when X is CO, Y is NH, O or S, and when X is CO, Y is not CO, CS, CONH, CSNH, CONH0(CH2)MCONH, CONH(CH2),.4CONH, CSNH0(CH2)MCONH, or CSNH(CH2)ι-4CONH; n is 0-5; n' is 0 or 1 ; m is 0-10; and Z is a metal binding group capable of covalently binding a radionuclide.
43. The kit of claim 42 wherein Z is R2-NH-CH(R3)-[CH(R4)]p.l-CO-NH-AArNH(CH-L)n--(CH-L)n».»-L" where R2 is COCH(Rs)-S-R<s; R5 is H, -(CH2)P-R7 , p is 1-5; R7 is a hydrogen, primary amine, secondary amine, cyclic or acyclic tertiary amine, carboxyl, ester, hydroxyl, ether, thiol, thioether, guanido, or an imine; Re is a hydrogen, aliphatic or aromatic acyl, acetamidoalkyl, benzamidoalkyl, unsubstituted or substituted tetra-hydropyranyl, unsubstituted or substituted tetrahydrofuranyl, or alkoxyalkyl; R3 is (CH2)P-Q where p' is 0-6, Q is hydrogen, alkylene or substituted alkylene, aryl or substituted aryl group for attachment to Y; R» is (CH2),-T where s is 0-6, T is hydrogen, alkylene or substituted alkylene,, aryl or substituted aryl group for attachment to Y; p" is 0,1 if p" is 1 only one of the groups defined under Q or T is attached to Y; AAi is any natural or unnatural α-amino acid or β-amino acid; L, L' = is a hydrogen, carboxyl, lower alkyl, aralkyl, substituted or unsubstituted aromatic group, or an alkylene bearing one of the following substituents: a primary amine, secondary amine, cyclic or acyclic tertiary amine, carboxyl, ester, hydroxyl, ether, thiol, thioether, guanido, imine, aminoalkyl, monoaminoalkyl, diaminoaikyl, hydroxyalkyl, alkoxylalkyl, hydroxycarbonylalkyl, alkoxycarbonylalkyl, mercaptoalkyl, or alkylthioalkyl; p'" is 0- 3; p"" is 0-3; L" is a hydrogen, lower alkyl, aralkyl, substituted or unsubstituted aromatic group, or an alkylene bearing one of the following substituents: a primary amine, secondary amine, cyclic or acyclic tertiary amine, carboxyl, ester, hydroxyl, ether, thiol, thioether, guanido, imine, aminoalkyl, monoaminoalkyl, diaminoaikyl, hydroxyalkyl, alkoxylalkyl, hydroxycarbonylalkyl, alkoxycarbonylalkyl, mercaptoalkyl, or alkylthioalkyl. 44. The kit of claim 43 wherein A and B are CH.
45. The kit of claim 44 wherein D-E is NHCO.
46. The kit of claim 45 wherein the peptidomimetic containing ligand has the structure:
Figure imgf000074_0001
where R« is a suitable sulfur protecting group.
47. The kit of claim 45 wherein the peptidomimetic containing ligand has the structure:
Figure imgf000074_0002
where Rg is a suitable sulfur protecting group.
48. The kit of claim 45 wherein the peptidomimetic containing ligand has the structure:
Figure imgf000074_0003
where R« is a suitable sulfur protecting group. 49. The kit of claim 45 wherein the peptidomimetic containing ligand has the structure:
Figure imgf000074_0004
where R« is a suitable sulfur protecting group. 50 The kit of claim 45 wherein the peptidomimetic containing ligand has the structure
CH 2)2-N(CH3)2
Figure imgf000075_0001
where R« is a suitable sulfur protecting group 51 The kit of claim 45 wherein the peptidomimetic containing ligand has the structure-
Figure imgf000075_0002
where Rs is a suitable sulfur protecting group
52. The kit of claim 45 wherein the peptidomimetic containing ligand has the structure
Figure imgf000075_0003
where R% is a suitable sulfur protecting group
53 The kit of claim 45 wherein the peptidomimetic containing ligand has the structure
Figure imgf000075_0004
where R* is a suitable sulfur protecting group 54. The kit of claim 45 wherein the peptidomimetic containing ligand has the structure:
Figure imgf000076_0001
where Rg is a suitable sulfur protecting group.
55. A method for imaging a site of thrombus comprising administering a diagnostically effective amount of a linear peptidomimetic containing ligand complexed with a diagnostic radionuclide and detecting the radionuclide localized at the site of thrombus.
56. The method of claim 55 wherein the radionuclide is selected from the group consisting of 67Ga, 1 1 lln, 99mTc, 169Yb, and 186Re.
57. The method of claim 56 wherein the radionuclide is 99mTc.
58. The method of claim 55 wherein the peptidomimetic containing ligand prior to complexation with the radionuclide has the structure:
Figure imgf000076_0002
where A is -CH- or -N-, and B is -CH- or -N-, with the proviso that when A is -N-, B is -CH-, and when B is -N-, then A is -CH-; -D-E- is -CH2-CH2-, or -NHCO- with the proviso that when A is -N-, then -D-E is -CH2-CH2-, and when B is -N-, then -D-E is -NHCO-; Ri is hydrogen, lower alkyl, or acyloxyalkyl; K is hydrogen, carboxyl, lower alkyl, aralkyl, substituted or unsubstituted aromatic, or an alkylene or substituted alkylene substituted with one of the following substituents: primary amine, secondary amine, cyclic or acyclic tertiary amine, carboxyl, ester, hydroxyl, ether, thiol, thioether, guanido, imine, aminoalkyl, monoaminoalkyl, diaminoaikyl, hydroxyalkyl, alkoxylalkyl, hydroxycarbonylalkyl, alkoxycarbonylalkyl, mercaptoalkyl, or alkylthioalkyl; X is NH, CO, S, or O, Y is NH, CO, CS, CONH, CSNH, NHCO, NHCS, CONH0(CH2),. 4CONH, CONH(CH2)wCONH, CSNH0(CH2)1-4CONH, or CSNH(CH2)MCONH, with the proviso that X and Y are different except when X is S, and when X is O, Y is CO, and when X is S, Y is S, CO or CS, and when X is NH, Y is CO, CONH, CSNH, CONH0(CH2)MCONH, CONH(CH2)MCONH, CSNH0(CH2)MCONH, or CSNH(CH2),^CONH, and when X is NH, Y is not NH, NHCO or NHCS, and when X is CO, Y is NH, O or S, and when X is CO, Y is not CO, CS, CONH, CSNH, CONH0(CH2)MCONH, CONH(CH2)MCONH, CSNH0(CH2)MCONH, or
CSNH(CH2)MCONH; n is 0-5; n' is 0 or 1; m is 0-10; and Z is a metal binding group capable of covalently binding a radionuclide.
59. The method of claim 58 wherein Z is
R2-NH-CH(R3)-[CH(R4)]P-CO-NH-AA1-NH(CH-L)P".-(CH-L')P-.-L" where R2 is COCH(R5)-S-R«; R5 is H, -(CH2)P-R7 , p is 1-5; R7 is a hydrogen, primary amine, secondary amine, cyclic or acyclic tertiary amine, carboxyl, ester, hydroxyl, ether, thiol, thioether, guanido, or an imine; Rt, is a hydrogen, aliphatic or aromatic acyl, acetamidoalkyl, benzamidoalkyl, unsubstituted or substituted tetra-hydropyranyl, unsubstituted or substituted tetrahydrofuranyl, or alkoxyalkyl; R3 is (CH2)P-Q where p' is 0-6, Q is hydrogen, alkylene or substituted alkylene, aryl or substituted aryl group for attachment to Y; R4 is (CH2),-T where s is 0-6, T is hydrogen, alkylene or substituted alkylene,, aryl or substituted aryl group for attachment to Y; p" is 0,1 if p" is 1 only one of the groups defined under Q or T is attached to Y; AAi is any natural or unnatural α-amino acid or β-amino acid; L, L' = is a hydrogen, carboxyl, lower alkyl, aralkyl, substituted or unsubstituted aromatic group, or an alkylene bearing one of the following substituents: a primary amine, secondary amine, cyclic or acyclic tertiary amine, carboxyl, ester, hydroxyl, ether, thiol, thioether, guanido, imine, aminoalkyl, monoaminoalkyl, diaminoaikyl, hydroxyalkyl, alkoxylalkyl, hydroxycarbonylalkyl, alkoxycarbonylalkyl, mercaptoalkyl, or alkylthioalkyl; p'" is 0- 3; p"" is 0-3, L" is a hydrogen, lower alkyl, aralkyl, substituted or unsubstituted aromatic group, or an alkylene bearing one of the following substituents: a primary amine, secondary amine, cyclic or acyclic tertiary amine, carboxyl, ester, hydroxyl, ether, thiol, thioether, guanido, imine, aminoalkyl, monoaminoalkyl, diaminoaikyl, hydroxyalkyl, alkoxylalkyl, hydroxycarbonylalkyl, alkoxycarbonylalkyl, mercaptoalkyl, or alkylthioalkyl.
59. The method of claim 58 wherein A and B are CH. 60 The method of claim 59 wherein D-E is NHCO. 61. The method of claim 60 wherein the peptidomimetic containing ligand has the structure:
Figure imgf000078_0001
where R« is a suitable sulfur protecting group.
62. The method of claim 60 wherein the peptidomimetic containing ligand has the structure:
Figure imgf000078_0002
where R« is a suitable sulfur protecting group.
63. The method of claim 60 wherein the peptidomimetic containing ligand has the structure:
Figure imgf000078_0003
where R* is a suitable sulfur protecting group. 64. The method of claim 60 wherein the peptidomimetic containing ligand has the structure:
Figure imgf000079_0001
where R* is a suitable sulfur protecting group.
65. The method of claim 60 wherein the peptidomimetic containing ligand has the structure:
Figure imgf000079_0002
where R$ is a suitable sulfur protecting group. 66. The method of claim 60 wherein the peptidomimetic containing ligand has the structure:
Figure imgf000079_0003
where Rg is a suitable sulfur protecting group.
67. The method of claim 60 wherein the peptidomimetic containing ligand has the structure.
Figure imgf000079_0004
where R« is a suitable sulfur protecting group. 68. The method of claim 60 wherein the peptidomimetic containing ligand has the structure:
Figure imgf000080_0001
where Re is a suitable sulfur protecting group. 5 69. The method of claim 60 wherein the peptidomimetic containing ligand has the structure:
Figure imgf000080_0002
where Rg is a suitable sulfur protecting group.
70. A method for providing radiotherapy to a site of thrombus comprising 0 administering a therapeutically effective amount of a linear peptidomimetic containing ligand complexed with a therapeutic radionuclide that localizes at the site of thrombus for a period of time sufficient to ablate the thrombus.
71. The method of claim 70 wherein the radionuclide is selected from the group consisting of 90Y, 67Cu, 186Re, 188Re, 169Er, 121Sn, 127Te, 143Pr, 198Au,
15 109Pd, 165Dy, 32P, 142Pr, and 153Sm.
72. The method of claim 70 wherein the peptidomimetic containing ligand has the structure:
Figure imgf000080_0003
where A is -CH- or -N-, and B is -CH- or -N-, with the proviso that when A is -N-, B is -CH-, and when B is -N-, then A is -CH-; -D-E- is -CH2-CH2-, or -NHCO- with the proviso that when A is -N-, then -D-E is -CH2-CH2-, and when B is -N-, then -D-E is -NHCO-; Ri is hydrogen, lower alkyl, or acyloxyalkyl; K is hydrogen, carboxyl, lower alkyl, aralkyl, substituted or unsubstituted aromatic, or an alkylene or substituted alkylene substituted with one of the following substituents: primary amine, secondary amine, cyclic or acyclic tertiary amine, carboxyl, ester, hydroxyl, ether, thiol, thioether, guanido, imine, aminoalkyl, monoaminoalkyl, diaminoaikyl, hydroxyalkyl, alkoxylalkyl, hydroxycarbonylalkyl, alkoxycarbonylalkyl, mercaptoalkyl, or alkylthioalkyl; X is NH, CO, S, or O; Y is NH, CO, CS, CONH, CSNH, NHCO, NHCS, CONH0(CH2),. 4CONH, CONH(CH2)MCONH, CSNH0(CH2)MCONH, or CSNH(CH2)MCONH, with the proviso that X and Y are different except when X is S, and when X is O, Y is CO, and when X is S, Y is S, CO or CS, and when X is NH, Y is CO, CONH, CSNH, CONH0(CH2)MCONH, CONH(CH2)MCONH, CSNH0(CH2)MCONH, or CSNH(CH2)MCONH, and when X is NH, Y is not NH, NHCO or NHCS, and when X is CO, Y is NH, O or S, and when X is CO, Y is not CO, CS, CONH, CSNH, CONH0(CH2)MCONH, CONH(CH2)MCONH, CSNH0(CH2)MCONH, or CSNH(CH2)MCONH; n is 0-5; n' is 0 or 1; m is 0-10; and Z is a metal binding group capable of covalently binding a radionuclide. 73. The method of claim 72 wherein Z is
R2-NH-CH(R3)-[CH(R4)]P -CO-NH-AA,-NH(CH-L)p--(CH-L')p L" where R2 is COCH(Rs)-S-R6; Rs is H, -(CH2)P-R7 ; p is 1-5; R7 is a hydrogen, primary amine, secondary amine, cyclic or acyclic tertiary amine, carboxyl, ester, hydroxyl, ether, thiol, thioether, guanido, or an imine; R« is a hydrogen, aliphatic or aromatic acyl, acetamidoalkyl, benzamidoalkyl, unsubstituted or substituted tetra-hydropyranyl, unsubstituted or substituted tetrahydrofuranyl, or alkoxyalkyl; R3 is (CH2)P-Q where p' is 0-6, Q is hydrogen, alkylene or substituted alkylene, aryl or substituted aryl group for attachment to Y; R4 is (CH2),-T where s is 0-6, T is hydrogen, alkylene or substituted alkylene,, aryl or substituted aryl group for attachment to Y; p" is 0,1 if p" is 1 only one of the groups defined under Q or T is attached to Y; AAi is any natural or unnatural α-amino acid or β-amino acid; L, L' = is a hydrogen, carboxyl, lower alkyl, aralkyl, substituted or unsubstituted aromatic group, or an alkylene bearing one of the following substituents: a primary amine, secondary amine, cyclic or acyclic tertiary amine, carboxyl, ester, hydroxyl, ether, thiol, thioether, guanido, imine, aminoalkyl, monoaminoalkyl, diaminoaikyl, hydroxyalkyl, alkoxylalkyl, hydroxycarbonylalkyl, alkoxycarbonylalkyl, mercaptoalkyl, or alkylthioalkyl; p'" is 0- 3; p"" is 0-3; L" is a hydrogen, lower alkyl, aralkyl, substituted or unsubstituted aromatic group, or an alkylene bearing one of the following substituents: a primary amine, secondary amine, cyclic or acyclic tertiary amine, carboxyl, ester, hydroxyl, ether, thiol, thioether, guanido, imine, aminoalkyl, monoaminoalkyl, diaminoaikyl, hydroxyalkyl, alkoxylalkyl, hydroxycarbonylalkyl, alkoxycarbonylalkyl, mercaptoalkyl, or alkylthioalkyl.
74. The method of claim 73 wherein A and B are CH. 75. The method of claim 74 wherein D-E is NHCO.
76. The method of claim 75 wherein the peptidomimetic containing ligand has the structure:
Figure imgf000082_0001
where R« is a suitable sulfur protecting group. 77. The method of claim 75 wherein the peptidomimetic containing ligand has the structure:
Figure imgf000082_0002
where Rg is a suitable sulfur protecting group. 78. The method of claim 75 wherein the peptidomimetic containing ligand has the structure-
Figure imgf000083_0001
where R« is a suitable sulfur protecting group. 79. The method of claim 75 wherein the peptidomimetic containing ligand has the structure:
Figure imgf000083_0002
where Rg is a suitable sulfur protecting group.
80. The method of claim 75 wherein the peptidomimetic containing ligand has the structure:
Figure imgf000083_0003
where Rg is a suitable sulfur protecting group. 81. The method of claim 75 wherein the peptidomimetic containing ligand has the structure:
Figure imgf000083_0004
where Rg is a suitable sulfur protecting group. 82 The method of claim 75 wherein the peptidomimetic containing ligand has the structure
Figure imgf000084_0001
where R« is a suitable sulfur protecting group 83 The method of claim 75 wherein the peptidomimettc containing ligand has the structure
Figure imgf000084_0002
where Rg is a suitable sulfur protecting group
84 The method of claim 75 wherein the peptidomimetic containing ligand has the structure"
Figure imgf000084_0003
where R« is a suitable sulfur protecting group
85 The radiopharmaceutical of claim 7 wherein the peptidomimetic containing ligand has the structure
Figure imgf000084_0004
where Rg is a suitable sulfur protecting group
86. The radiopharmaceutical of claim 7 wherein the complex of the radionuclide and the peptidomimetic containing ligand has the structure.
Figure imgf000085_0001
where Rg is a suitable sulfur protecting group.
87. The ligand of claim 29 having the structure:
Figure imgf000085_0002
where Rg is a suitable sulfur protecting group.
88. The kit of claim 45 wherein the peptidomimetic containing ligand has the structure:
Figure imgf000085_0003
where R« is a suitable sulfur protecting group. 89. The method of claim 60 wherein the peptidomimetic containing ligand has the structure.
Figure imgf000085_0004
where R« is a suitable sulfur protecting group.
90. The method of claim 75 wherein the peptidomimetic containing ligand has the structure:
Figure imgf000085_0005
where Rg is a suitable sulfur protecting group.
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