WO1999040882A2 - STEREOSELECTIVE Tc-99m LIGANDS - Google Patents

Abstract

The present invention concerns novel ligands, represented by Formulae (I), (II), (I') and (II'), that form complexes with a radioactive metal through a chelate bond. The complexes are useful in radiodiagnostic compositions employed for imaging. In said formulae, X, R?1 and R2, R3 and R4, R5 and R6¿, m and n are defined herein. The compounds of the invention avoid the formation of diastereomers based on incorporating [TcvO]+3N2S2 as a chelating moiety since these compounds form only one isomer when complexed with [?99mTc][TcvO]+¿.

Description

Stereoselective Tc-99m Ligands

This patent claims the benefit of filing of U.S. Provisional Patent Application Nos. 60/073,957, filed February 6, 1998, and 60/078,052, filed March 16, 1998, which Applications are herein incorporated by reference in their entirety.

Background of the Invention

Statement as to Rights to Inventions Made Under

Federally-Sponsored Research and Development

Part of the work performed during development of this invention utilized U.S. Government funds. The U.S. Government has certain rights in this invention pursuant to Grant No. NS 18509 from the National Institutes of Health.

Field of the Invention

The present invention relates to novel stereoselective diaminedithiol ligands. The ligands chelate a radioactive metal to form complexes that are useful as radioactive diagnostic and therapeutic agents.

Related Art

Technetium-99m (t_1/2 = 6 hours, 140 KeV) is the most commonly used radionuclide in diagnostic nuclear medicine (Jurisson, S.S., et al., Chem. Rev. 93:1137-1156 (1993); Steigman, J. and Eckelman, W.C, The Chemistry of Technetium In Medicine, National Academy, Washington, D.C. (1992); Tisato, F., et al., Coord. Chem. Rev. 135/136:325-397 (1994)). Its popularity is due to several unique characteristics: the radionuclide can be readily produced by a "Mo/"^mTc generator, the medium gamma-ray energy emitted by Tc-99m (140 KeV) is suitable for gamma camera detection and the physical half-life is compatible with the biological localization and residence time required for in vivo imaging. Currently about 85% of routine nuclear medicine procedures are performed with radiopharmaceuticals based on Tc-99m. In the past ten years, significant progress has been made in the chemistry of technetium using

= 2.1 x 10⁵ years) as well as the non-radioactive rhenium (Tisato, F., et al., Coord. Chem. Rev. 135/136:325-397 (1994)). Improvements in the understanding of technetium chemistry have significantly enhanced the development of a new generation of technetium radiopharmaceuticals for clinical use and potentially will benefit millions of patients who receive Tc-99m agents for routine nuclear medicine diagnosis.

Despite its attractive physical properties, technetium is a difficult metallic element for designing small molecule-based (non-peptide) ligands for receptor or site- specific imaging. Technetium is a transition metal and requires a complexing agent to stabilize it at different valence states (Steigman, J. and Eckelman, W.C, The Chemistry of Technetium In Medicine, National Academy, Washington, D.C (1992); Tisato, V., et al, Coord. Chem. Rev. 135/136:325-397 (1994)). Valence states achieved after reduction can vary from +7 (as pertechnetate) to zero (0), depending on the reaction conditions and chelating agents used during preparation. After complexation, the molecules invariably become big and bulky, which can be a limiting factor in designing a molecule targeted to a specific receptor or biological process.

It is well established that when [^99mTc]pertechnetate (TcO⁴ ) is reduced in the presence of a reducing agent, such as stannous chloride, and a " soft" chelating ligand, such as N₂S₂ or NS₃, a neutral and lipophilic [Tc^vO]⁺³N₂S₂ or [TcO]⁺³NS₃ center core is formed (Davison, A., et al., Inorg. Chem. 20:1629-1632 (1981); Lever, S.Z., etal, J. Nucl. Med. 26: 1287-1294 (1985); Mahmood, A., etal, Ada. Crystallogr., SectC: Cryst. Struct. Commun. 47:25A-257 (1991); Steigman, J. and Eckelman, W.C, The Chemistry ofTechnetium In Medicine, National Academy, Washington, D.C. (1992)). When an N-alkyl substitution group is added to the ligand, syn- and αntz^'-isomers are formed (Lever, S.Z., et al., Inorg. Chim. Ada 77(5:183-184 (1990)) (Scheme 1).

Scheme 1

Several recent reports demonstrate that it is possible to incorporate [TcO]⁺³N₂S₂ into potential receptor-selective imaging agents for muscarinic receptors (Eckelman, W.C, J. Nucl. Med. 3f5:S5-S7 (1995); Lever, S.Z., Nucl Med. Biol. 27:157-164 (1994)) (or using boronic acid adducts of technetium dioxime-B ATO type of ligand (Jurisson, S.S., et al, Nucl. Med. Biol. 22:269-281 (1995))); vesamicol sites (del Rosario, R.B., et al., Nucl. Med. Biol. 27:197-203 (1994)); serotonin receptors (Baidoo, K.E., et al., J. Nucl. Med. 38:100? (1997); Johannsen, B., et al, Nucl. Med. Biol. 23:429-438 (1996); Samnick, S., et al, Nucl. Med. Biol. 22:573-583 (1995)); dopamine Dl (Chumpradit, S., et al, "Synthesis and characterization of a [^99mTc]SCH23390 derivative as a selective dopamine Dl receptor imaging agent" [abstract VI- 14], Xllth International Symposium on Radiopharmaceutical Chemistry, Uppsala, Sweden, (1997), p. 437) and D2 receptors (Lever, S.Z., et al, J. Nucl. Med. 38:99? (1997)); and steroid hormone receptors (Chi, O.Y. and Katzenellenbogen, J.A., J. Am. Chem. Soc. 775:7045-7046 (1993); Chi, D,Y., etal, J. Med. Chem. 57:928-937 (1994); DiZio, J.P., et al, J. Nucl. Med. 55:558-569 (1992); DiZio, J.P., et al, Bioconj. Chem. 2:353-366 (1991); O'Neil, J.P., et al, Bioconj. Chem. 5:182-193 (1994); O'Neil, J.P., etal, Inorg. Chem. 55:319-323 (1994)). Generally, design of these imaging agents can be classified into two categories: pendent approach, in which the Tc-99m complexing moiety hangs from the main body of the molecule responsible for binding to the pocket of the receptor-ligand binding site; or integrated approach, in which the Tc-99m complex is integrated as part of the receptor specific ligand (for example, as steroid analogs (Chi, D,Y., etal, J. Med. Chem. 57:928-937 (1994); Horn, R.K., et al, J. Org. Chem. 67:2624-2631

(1996); Sugano, Y. and Katzenellenbogen, J.A., Bioorg. & Med. Chem. Lett. (5:361-366 (1996)). However, many Tc-99m imaging agents have demonstrated limited in vivo success to date. There are various factors which may contribute to the failure of Tc-99m receptor imaging agents. It appears that designing a high-binding affinity ligand containing a Tc-99m core either by the pendent or integrated approach can be accomplished; however, achieving the goal of designing a Tc-99m ligand with a high uptake and low non-specific binding in vivo is much more elusive.

The [TcO]⁺³N₂S₂ core system has been successfully applied in the development of a brain perfusion imaging agent ~ [^99mTc]ECD (ethylene cysteine dimer) (Leveille, J., et al, J. Nucl. Med. 50:1902-1910 (1989); Walovitch, R.C., et al, Neuropharmacology 50:283-292 (1991); Walovitch, R.C, et al, J. Nucl. Med. 30: 1892- 1901 (1989)). This agent is currently being used for the diagnosis of epilepsy and strokes (Devous, M.D., et al, J. Nucl. Med. 54:754-61 (1993); Lancman, M.E., et al, Epilepsia 55:466-471 (1997); Lassen, N.A., Clin.

Neuropharmacol. 75:S1-S8 (1990)). ["^mTc]ECD is only active when the ligand is in a L,L configuration. Formation of the complex with a [TcO]⁺³N₂S₂ core produces only one isomer. Summary of the Invention

A first aspect of the present invention concerns novel stereoselective compounds. These compounds are represented by Formula I and II, below.

A second aspect of the present invention concerns novel stereoselective bifunctional conjugates. These compounds are represented by Formula F and IF below.

A third aspect of the present invention concerns complexes of a compound of Formula I, II, /', or IF combined with a radioactive metal through a chelate bond. A fourth aspect of the present invention concerns radiodiagnostic compositions useful for imaging, comprising a pharmaceutically acceptable carrier or diluent, and a complex of a compound of Formula I, F, II or 77' and a radioactive metal.

A fifth aspect of the present invention provides for imaging tissue in a mammal comprising injecting an effective amount of a complex of a compound of Formula I, F, II or IF and a radioactive metal, and radioimaging the mammal after allowing sufficient time for the composition to localize in the tissue of a mammal.

A sixth aspect of the present invention provides for a method of making a compound of Formula I, F, II or 77'.

A seventh aspect of the present invention provides for a method of radiolabeling a compound of Formula I, F, II or IF with a radionuclide such as Tc-99m, Re-186 or Re-188.

A eighth aspect of the present invention provides for kits for forming Tc- 99m, Re-186 or Re-188 labeled ligands, comprising a compound of Formula I,

F, II or IF and an effective amount of reducing agent. Brief Description of the Drawings

FIG. IA depicts the HPLC profile of Tc-99m trans-P-BAT, 4, using a reverse phase column. The profile shows one peak with a retention time of 4.8 min. (Reverse phase PRP-1 column; eluent: CH₃CN/DMGA = 8.2 flowrate 1 mL/min.)

FIG. IB depicts the HPLC profile of 4 using an AD chiral column (eluent: hexane/EtOH = 3:1, flowrate 1 mL/min). The profile shows one peak.

FIG. 2 depicts separation of diastereomers of [^99mTc] cis-P-BAT using Chiracel-AD column. The HPLC profile (AD-column, hexane/EtOH 3:1, 1 mL/min.) showed a ratio of "A" to "B" = 1:1.

FIG. 3 depicts the synthesis scheme (Scheme 10) followed in Example 4.

FIG. 4 depicts the synthesis scheme (Scheme 11) followed in Example 5.

FIG. 5 depicts the synthesis scheme (Scheme 12) followed in Example 6.

FIG 6 depicts the synthesis scheme (Scheme 13) followed in Example 7.

Detailed Description of the Preferred Embodiments

The present invention is directed to novel compounds of Formula I and II:

where

X is N or CH;

R¹ and R² are selected from the group consisting of hydrogen, alkyl, and aralkyl, provided that at least one of R¹ and R² is hydrogen, where said alkyl and aralkyl may be optionally substituted;

R³ and R⁴ are hydrogen, or are taken together to form a keto group (=O);

R⁵ and R⁶ are hydrogen, or taken together to form a keto group (=O); m and n are independently 1 or 2;

R is hydrogen, alkyl, cycloalkyl, alkenyl, alkynyl, aryl, or aralkyl, any of which is optionally substituted; and

P^a is a sulfur protecting group or hydrogen.

The groups P^a are both, hydrogen or can be any of the variety of protecting groups available for sulfur, including methoxymethyl, methoxy ethoxymethyl, - methoxybenzyl or benzyl. Sulfur protecting groups are described in detail in Greene, T.W. and Wuts, P.G.M., Protective Groups in Organic Synthesis, 2nd Edition, John Wiley and Sons, Inc., New York (1991).

Preferred compounds are those of Formula 7 and 77 where

X is N;

R¹ and R² are selected from the group consisting of hydrogen, C,_₆ alkyl, and C₆.₁₀ar(C,_.4)alkyl, provided that at least one of R¹ and R² is hydrogen;

R³ and R" are both hydrogen;

R⁵ and R⁶ are both hydrogen; m and n are both either 1 or 2;

R is hydrogen, C,.₆ alkyl, C₃.₇ cycloalkyl, C^,,, aryl or C₆.₁₀ ar(C )alkyl, any of which is optionally substituted by an amino, aminoalkyl, guanidinoalkyl, nitro, cyano, carboxy, halo, haloalkyl, hydroxy or hydroxyalkyl group; and P\ in each instance, is hydrogen, methoxymethyl, methoxyethoxymethyl, -methoxy benzyl or benzyl.

Useful values of R¹ and R² include hydrogen, methyl, ethyl, cyclopropyl, isopropyl, benzyl and phenethyl.

Useful values of R include hydrogen, methyl, ethyl, aminoethyl, aminopropyl, aminobutyl, aminocyclohexyl, chloromethyl, bromomethyl, chloroethyl, bromoethyl, chloropropyl, bromopropyl, hydroxyethyl, hydroxypropyl, phenyl, aminophenyl, aminomethylphenyl, halophenyl, benzyl, aminobenzyl, halobenzyl, aminomethylbenzyl, and guanidinomethylbenzyl.

The present invention is also directed to novel compounds of Formula F and 77' :

where

X, R'-R⁶, m, n and Pa are defined as above for Formula 7 and 77; L is a linking group; and B is a targeting group.

As used herein, the term "linking group" refers to a group which is capable of forming a covalent bond with both the metal-complexing portion of the molecule and the targeting group. The linking group can be chosen to provide specific attachment to the targeting group, i.e. bonding to a predictable site on the targeting group or to provide non-specific attachment to the targeting group, i.e., bonding to one or more sites on a targeting group, which site or sites cannot be predicted. Preferred linking groups include straight or branched alkyl, cycloalkyl, aminoalkyl, aminoaryl, carboxyalkyl, thioalkyl, amino, amido, carboxy, -O-,

-S-, dithio, hydrazino or a direct covalent bond.

As used herein, the term "targeting group" refers to a chemical moiety that specifically interacts with a biological target. Examples of targeting groups (and the target) are monoclonal antibody or fragments thereof (targeted to an antigen), protein (targeted to a receptor as substrate, or a regulatory site, e.g., on a DNA

(gene) or RNA sequence), peptide (targeted usually to a receptor), nucleic acid (targeted to a complimentary nucleic acid, e.g., RNA or DNA), steroid (targeted to a steroid receptor), and the like. The invention is not limited by the choice of biological target. Preferred targeting groups include amino acids, amino acid side chains, peptides, proteins, antibodies, nucleic acids, steroids, lipids, saccharides or cell membrane ligands.

A preferred targeting group is guanidinomethylbenzyl linked via a direct covalent bond to the chelating portion of the compounds. meta- Iodobenzylguanidine (MIBG) has been used as an imaging agent for studying norepinephrine neuronal function. As an analog of norepinephrine, radio labeled

[¹³¹I] MIBG localizes in the tumors and provides a useful diagonistic tool of neuronal storage of norepinephrine (Wieland, D.M.,etα/.,J. Med. Chem. 27:149- 155 (1984); Wieland, D.M., J. Nucl. Med. 27:349-353 (1980)). One other important observation of MIBG was that it also localized in myocardium (Wieland, D.M., et al, J. Nucl. Med. 22:22-31 (1981)). It was suggested that assessment of sympathetic nervous system by MIBG imaging maybe important for patients with ventricular tachycardia, cardiomyopathy and arrhythmia (Dae, M.W., J. Nucl. Cardiol 2:151-154 (1995)). The MIBG scan may have predictable value for patients at risk of sudden death (Id.; Fagret, D., et al, Eur. J. Nucl. Med., 75:624-628 (1989); Fagret, D., et al, J. Nucl. Med., 34:57-60

(1993); Merlet, P., et al, J. Nucl. Med, 55:471-477 (1992)) and patients with diabetes (Schnell, O. et al, Diabetic Medicine, 14:57-62 (1997); Schnell, O. et al, Diabetes 45:801-805 (1996)).

A compound of the present invention for which R is guanidinomethylbenzyl will provide a Tc-99m labeled agent that localizes in the myocardium via a norepinephrine transporter-mediated process. Such an agent will provide a much wider clinical acceptance than the presently employed agents .

The uncomplexed compounds according to the invention are useful as carriers for radioactive metals. They can be firmly coordinated with a radioactive metal to form chelate compounds, which are extremely stable in vitro and in vivo and can be used as a radioactive diagnostic agents for imaging various tissues in vivo. In addition, many of the compounds within the scope of the present invention can be employed to label a receptor-specific organic compound.

The present invention is also directed to complexes of a compound of Formula I, II, I' and 77' with a radioactive metal. Useful radioactive metals include radioactive isotopes of Tc, Ga, Th, In, Zn, Ru, Cu, Co, Pt, Fe, Re, Cr, Mo, ,

Mn, Ni, Rh, Pd, Nb or Ta. Preferred radionuclides include technetium-99m, rhenium-186 and rhenium-188.

A preferred aspect of the invention is directed to stereospecific [Tc^vO]⁺³N₂S₂ complexes of Formula 777, IV, IIP and IV:

where X, R, R³-R⁶, n and m are defined as above for Formula 7 and 77 and L and B are defined above as for Formula P and 77'. With respect to R¹ and R² in the radionuclide complexes, one of R¹ and R² is not present, and the other of R¹ and R² is hydrogen, alkyl, and aralkyl, where said alkyl and aralkyl may be optionally substituted. Preferred values of R, R'-R⁶, n and m are as described above for Formula 7, 77, F, and 77'. The present invention avoids the formation of diastereomers based on incorporating [TcO]⁺³N₂S₂ as a chelating moiety. The [TcO]⁺³N₂S₂ complexes of the present invention form only one isomer. Thus, the complication of forming syn- and anti- isomers can be avoided by use of the ligands of the present invention. The [^99mTc] [TcO]⁺³N₂S₂ complexes of the present invention allow for the design of site-specific Tc-99m labeled compounds, where selective binding is often sensitive to specific stereoconformation. By eliminating the potential interference of introducing other isomers, when the [Tc^vO]⁺³N₂S₂ core is attached there is a greater likelihood that the specific binding of the Tc-99m derivatives will be maintained.

A group of preferred ligands of the present invention are referred to as trans-P-B AT, and have the Formula and VI. The corresponding cis-isomer cis- P-BAT is represented by Formula VII.

(VII) When trans-ligands of the present invention are labeled with Tc-99m only one isomer is produced. The chelating ligand forms a neutral and lipophilic [TcO]³⁺N₂S₂ center core. This is illustrated below with reference to the different isomers of P-BAT in Schemes 2-4.

Scheme 2

3R,4R-isomer

Forming only One isomer

Scheme 3

Scheme 4

meso-isomer

meso-isomer meso-isomer Anti- Syn-

Additional preferred trans-[Tc^vO]⁺³N₂S₂ complexes within the scope of the present invention are represented by the following Formulae VIII-XIX:

A77 XIII

where X and R are as defined above for Formulae 7-7K, and R' is hydrogen, alkyl, and aralkyl, where said alkyl and aralkyl may be optionally substituted.

Definitions:

The term "alkyl" as employed herein includes both straight and branched chain radicals of up to 12 carbons, preferably 1-8 carbons, such as methyl, ethyl, propyl, isopropyl, butyl, t-butyl, isobutyl, pentyl, hexyl, isohexyl, 1-ethylpropyl, heptyl, 4,4-dimethylpentyl, octyl, 2,2,4-trimethylpentyl, nonyl, decyl, undecyl and dodecyl.

Useful alkenyl groups are C₂.₆ alkenyl groups, preferably C₂.₄ alkenyl. Typical C₂_₄ alkenyl groups include ethenyl, propenyl, isopropenyl, butenyl, and sec.-butenyl.

Useful alkynyl groups are C₂.₆ alkynyl groups, preferably C₂.₄ alkynyl. Typical C2.4 alkynyl groups include ethynyl, propynyl, butynyl, and 2-butynyl groups.

The term "cycloalkyl" as employed herein includes saturated cyclic hydrocarbon groups containing 3 to 12 carbons, preferably 3 to 8 carbons, which include cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, cyclodecyl and cyclododecyl, any of which groups may be substimted with substituents such as halogen, C,.₆ alkyl, C,^ alkoxy and/or hydroxy group.

The term "aryl" as employed herein by itself or as part of another group refers to monocyclic or bicyclic aromatic groups containing from 6 to 12 carbons in the ring portion, preferably 6-10 carbons in the ring portion, such as phenyl, naphthyl or tetrahydronaphthyl.

The term "aralkyl" or "arylalkyl" as employed herein by itself or as part of another group refers to C,.₆ alkyl groups having an aryl substituent, such as benzyl, phenylethyl or 2-naphthylmethyl. The term "halogen" or "halo" as employed herein by itself or as part of another group refers to chlorine, bromine, fluorine or iodine with chlorine being preferred.

The term "optionally substituted" as employed herein, unless otherwise specified, includes groups as defined above that have one, two or three halo, hydroxy, amino, nitro, cyano, trifluoromethyl, halogen, C,.₆ alkyl, C₆.₁₀ aryl, C_w alkoxy, C,_₆ aminoalkyl, C,_₆ aminoalkoxy, C_2.6 alkoxycarbonyl, carboxy, C,.₆ hydroxyalkyl, C₂.₆ hydroxyalkoxy, C_6.I0 aryl(C,.₆)alkyl, C,_₆ alkylcarbonyl, C₂.₆ carboxyalkyl, C,.₆ guanidinoalkyl, trifluoromethoxy and/or carboxy substituents, provided that said substituents result in a stable molecule. The present invention is also directed to a method for preparing compounds of Formula 7 and 77.

Compounds of Formula 7 and 77 can be prepared according to methods known to those skilled in the are, including the multi-step syntheses illustrated in

Schemes 5 through 13. Choice of the appropriate tartaric acid starting material dictates the stereochemistry of the final product in Schemes 5 and 6. Scheme 5

S-.

DCC, HOBT or Oxalyl chloride

There are three possible isomers of ligand 2 of the present invention: two trans-isomers and one cis-isomer. Using the commercially available starting material, the 2R, 2R- (L-), 2S, 2S- (D-) and the meso- tartaric acids all three of the possible isomers of P-BAT can be prepared. In synthesis of the model system, trans-P-BAT, the 3R, 4R-(trans-) isomer was selected first, because the L-tartaric acid is the naturally occuring isomer and is cheaper and readily available (Scheme 5). Starting with 2R, 2R- (L-) tartaric acid, the key starting material 1 was prepared according to a reported procedure. Subsequent acylation produced 2 in 87% yield. The amide bonds were reduced by borane reduction which gave compound 3 in good to excellent yield (73%). The protecting groups for thiols were removed by mercuric acetate and the free thiols were regenerated by treatment of hydrogen sulfide gas to give the desired compound 4.

It is possible to prepare comparable compounds, such as 3S, 4S-trans-P-BAT series, using more expensive (because L-tartaric acid is the naturally occurring compound) 3S, 4S-D-tartaric acid as the starting material.

Scheme 5a

D-Tartatic acid, (2S.

Compounds for which R is benzyl substituted with a C,.₆ guanidinoalkyl group are prepared by reaction of the corresponding C,_₆ aminoalkyl group with 1 ,3-bis(tert-butoxycarbonyl)-2-methyl-2-thiopseudourea in THF in the presence of a small amount of water (Delle Monache, G., et al, J. Med. Chem. 36:2956- 2963).

Additional compounds of Formula 7 or 77 can be synthesized by the routes illustrated in Schemes 7, 8 and 9.

We have also prepared the meso derivative (or cis-isomer) using a similar scheme (Scheme 6). Compound 6 was prepared from compound 5 with 64% yield. The reduction reaction produced the desired meso (cis-) diamine 7 in 56% yield. Deprotection of the para-methoxybenzyl groups yielded the free thiol, 8. Scheme 6

Scheme 7

Key Intermediate *^■»

Scheme 8

Alternatively, the Sharpless asymmetric synthesis (Li, G., et al, Chem. Int. Ed. Engl. 55:451-453 (1996)) can be used to prepare the asymmetric diamine intermediate:

Scheme 9

Bifunctional conjugates of Formula F and 77' can be prepared by methods known to those skilled in the art. Reaction of 2R,2R-(L-) tartaric acid with a primary amine having a group which can later serve as a linking group to a targeting molecule will provide a starting material analogous to starting material 1 in Scheme 5. The metal chelating N₂S₂ core is then constructed according to known methods, including the methods outlined in Schemes 5-7. Protecting groups may be necessary depending on the choice of linking group. For example, an aminoalkyl or aminoaryl linking group will require protection of the amine during the construction of the metal chelating N₂S₂ core. Alternatively, one can begin with amine intermediate 14 shown in

Scheme 7 and construct the linking group by appropriate modification of the amine.

The functionality of the linking group will dictate how the targeting group will be appended thereto. For example, linking groups having carboxy functionality can be reacted with saccharides to append the targeting group via an ester bond. Likewise, amino functionality present in a linking group provides a means of attachment to the carboxy groups in an amino acid, peptide or protein.

An alternate strategy for constructing bifunctional conjugates of the present invention is to begin with appropriately functionalized targeting groups and metal chelating cores and simultaneously attach the linking group to both.

For example, a carboxy substituted targeting group and a carboxy substituted metal chelating core can be simultaneously reacted with a diamine, such as ethylene diamine, to afford a conjugate of Formula F or 77' in which the linking group L is defined by the ethylene diamine moiety.

Protecting group P^a can be removed by appropriate methods well known in the art of organic synthesis, such as trifiuoroacetic acid, mercuric chloride or sodium in liquid ammonia. In the case of Lewis acid labile groups, including acetamidomethyl and benzamidomethyl, P^a can be left intact. Labeling of the ligand with technetium in this case will cleave the protecting group, rendering the protected diaminedithiol equivalent to the unprotected form. The corresponding Re-complex can be similarly prepared.

Depending upon the chemical properties of the radioactive metal, there may be adapted two different labeling manners. When the radioactive metal is in an oxidation state which is not required to be reduced or oxidized for formation of a stable chelate compound, a compound of Formula 7 or Formula 77 is reacted with the radioactive metal in an aqueous medium. This labeling manner may be applied to gallium-67, indium-I l l, etc. When the radioactive metal is in an oxidation state which is required to be reduced or oxidized for formation of a stable chelate compound, the compound of Formula 7 or Formula 77 is reacted with the radioactive metal in an aqueous medium containing a reducing agent or an oxidizing agent. This labeling manner may be applied to technetium-99m, rhenium-186 and rhenium-188. As a reducing agent, there may be usually employed a stannous salt, i.e., a salt of divalent tin ion (Sn ⁺⁺). Specific examples are stannous halides (e.g., stannous chloride), stannous sulfate, stannous nitrate, stannous acetate, stannous citrate, etc. Examples of the oxidizing agent are hydrogen peroxide, etc.

When, for instance, the radioactive metal is technetium-99m, compound of Formula 7 or 77 may be treated with technetium-99m in the form of pertechnetate in an aqueous medium containing a reducing agent, such as a stannous salt. As to the order of the addition of the above reagents into the reaction mixture, any particular limitation does not exist. Usually, however, the mixing of the stannous salt with the pertechnetate in an aqueous medium in the first place should be avoided. The stannous salt may be used in such an amount as can reduce sufficiently the pertechnetate.

Tc-99m complexes are prepared as follows. A small amount of N₂S₂ ligand of Formula 7, F, II or 77' (1-2 mg) is dissolved in 100 μL EtOH is mixed with 200 μL HCl (1 N) and 1 mL Sn-glucoheptanate solution (containing 8-32 μg

SnCl₂ and 80-320 μg Na-glucoheptanate, pH 6.67) and 50 μL EDTA solution (0.1 N). [^99mTc]Pertechnetate (100-200 μL; ranging from 2-20 mCi) saline solution is then added. The reaction is heated for 30 min at 100° C, then cooled to room temperature. The reaction mixture is analyzed on TLC (EtOHxonc. NH₃9:1) for product formation and purity check. The mixture can be neutralized with phosphate buffer to pH 5.0.

A compound of Formula 7, F, II or 77' when employed as a carrier for radioactive metal may be in the form of a solution. Usually, it is converted into a powder form by lyophilization or distillation at low temperature under reduced pressure and stored in such powder form. When it is time to use the compound, the powder is dissolved in sterilized water, physiological saline solution, buffer, etc. The compound of Formula I, F, II or 77' in a solution or powder form can be incorporated with pharmaceutically acceptable solubilizing agents (e.g., organic solvents), pH regulating agents (e.g., acids, bases, buffers), stabilizers (e.g., ascorbic acid), preservatives (e.g. sodium benzoate), isotonizing agents (e.g., sodium chloride), etc., as well as reducing or oxidizing agents for adjustment of the atomic oxidation state of the radioactive metal.

As a radioactive metal, there may be used any metallic element having radioactivity, which has physical and chemical characteristics suitable for nuclear medical diagnosis and can be coordinated easily with the compound of Formula

I, F, II or 77'. Specific examples of the radioactive metallic element are gallium- 67, gallium-68, thallium-201, indium-I l l, technetium-99m, rhenium-186, rhenium- 188, zinc-62, copper-62, etc. They are normally employed in their salt forms, particularly their water-soluble salt forms. Certain metals, including technetium, are capable of forming complexes with ligands at more than one oxidation state. Any such oxidation state is contemplated in the present invention. The technetium-99m is preferably in the form of the Tc'O when complexed with the ligand, although a TcN core may also be employed.

The radioactive diagnostic agent should have sufficient radioactivity and radioactivity concentration which can assure reliable diagnosis. For instance, in case of the radioactive metal being technetium-99m, it may be included usually in an amount of 0.1 to 50 mCi in about 0.5 to 5.0 ml at the time of administration. The amount of a compound of Formula I, F, II or 77' may be such as sufficient to form a stable chelate compound with the radioactive metal.

The thus formed chelate compound as a radioactive diagnostic agent is sufficiently stable, and therefore it may be immediately administered as such or stored until its use. When desired, the radioactive diagnostic agent may contain any additive such as pH controlling agents (e.g., acids, bases, buffers), stabilizers

(e.g., ascorbic acid) or isotonizing agents (e.g., sodium chloride).

A technetium-99m complex according to the present invention is generally used in the form of a composition which is suitable for examining the function of a particular organ. In addition to the radioactive complex, such a radiopharmaceutical composition will usually comprise a liquid, pharmaceutically acceptable carrier material, preferably a physiological saline solution.

A radiodiagnostic examination can be performed with such a composition by administering the composition to a warmblooded living being, in particular a primate, in a quantity of 0.1 to 30 mCi, preferably of 0.5 to 10 mCi, per 70 kg of body weight, and by then recording the radioactive radiation emitted by the living being by a radioactivity recording means, for example, a gamma camera.

The present invention further relates to a method of preparing a technetium-99m complex according to the present invention by reacting technetium-99m in the form of a pertechnetate in the presence of a reducing agent and optionally a suitable chelator with an appropriate compound.

The reducing agent serves to reduce the Tc-99m pertechnetate which is eluted from a molybdenum-technetium generator in a physiological saline solution. Suitable reducing agents are, for example, dithionite, formamidine sulphinic acid, diaminoethane disulphinate or suitable metallic reducing agents such as Sn(II), Fe(II), Cu(I), Ti(III) or Sb(III). Sn(II) has proven to be particularly suitable.

For the above-mentioned complex-forming reaction, technetium-99m is reacted with the above-mentioned compounds according to Formula 7, F, II or 77' as a salt or in the form of technetium bound to comparatively weak chelators.

In the latter case the desired technetium-99m complex is formed by ligand exchange. Examples of suitable chelators for the radionuclide are dicarboxylic acids, such as oxalic acid, malonic acid, succinic acid, maleic acid, orthophtalic acid, malic acid, lactic acid, tartaric acid, citric acid, ascorbic acid, salicylic acid or derivatives of these acids; phosphorus compounds such as pyrophosphates; or enolates. Citric acid, tartaric acid, ascorbic acid, glucoheptonic acid or a derivative thereof are particularly suitable chelators for this purpose, because a chelate of technetium-99m with one of these chelators undergoes the desired ligand exchange particularly easily. The most commonly used procedure for preparing [TcO]⁺³N₂S₂ complexes is based on stannous (II) chloride reduction of [^99mTc]pertechnetate, the common starting material. The labeling procedure normally relies on a Tc-99m ligand exchange reaction between Tc-99m (Sn)-glucoheptonate and the N₂S₂ ligand. Preparation of stannous (II) chloride and preserving it in a consistent stannous (II) form is critically important for the success of the labeling reaction.

To stabilize the air-sensitive stannous ion it is a common practice in nuclear medicine to use a lyophilized kit, in which the stannous ion is in a lyophilized powder form mixed with an excess amount of giucoheptonate under an inert gas like nitrogen or argon. The preparation of the lyophilized stannous chloride/sodium giucoheptonate kits ensures that the labeling reaction is reproducible and predictable. The N₂S₂ ligands are usually air-sensitive (thiols are easily oxidized by air) and there are subsequent reactions which lead to decomposition of the ligands. The most convenient and predictable method to preserve the ligands is to produce lyophilized kits containing 100-500 μg of the ligands under argon or nitrogen. Since the radiopharmaceutical composition according to the present invention can be prepared easily and simply, the preparation can be carried out readily by the user. Therefore, the present invention also relates to a kit, comprising: ( 1 ) A compound of the general Formula 7, F, II or 77' the compound optionally being in a dry condition; and also optionally having an inert, pharmaceutically acceptable carrier and/or auxiliary substances added thereto; and

(2) a reducing agent and optionally a chelator; wherein ingredients (1) and (2) may optionally be combined; and further wherein instructions for use with a prescription for carrying out the above- described method by reacting ingredients (1) and (2) with technetium-99m in the form of a pertechnetate solution may be optionally included.

Examples of suitable reducing agents and chelators for the above kit have been listed above. The pertechnetate solution can be obtained by the user from a molybdenum-technetium generator. Such generators are available in a number of institutions that perform radiodiagnostic procedures. As noted above the ingredients (1) and (2) may be combined, provided they are compatible. Such a monocomponent kit, in which the combined ingredients are preferably lyophilized, is excellently suitable to be reacted by the user with the pertechnetate solution in a simple manner.

The ingredient (1) of the above kits may be delivered as a solution, for example, in the form of a physiological saline solution, or in some buffer solution, but is preferably present in a dry condition, for example, in a lyophilized condition. When used as a component for an injection liquid, it should be sterile, and, if the ingredient ( 1 ) is present in a dry condition, the user should use a sterile physiological saline solution as a solvent. If desired, ingredient (1) may be stabilized in a usual manner with suitable stabilizers such as ascorbic acid, gentisic acid or salts of these acids, or it may be provided with other auxiliary means such as fillers, e.g., glucose, lactose, mannitol, inositol, and the like. It is preferred that these compositions be parenterally administered, most preferably by intravenous bolus injection. Selective preparation of stereoisomers of [TcO]⁺³N₂S₂ compounds is an important requirement for developing receptor or site-specific imaging agents. As most of the biological binding sites are derived from three-dimensional protein structures, they are inherently stereoselective sites. In a previous study of diastereomers of [^99mTc]TRODAT-l, it appeared that different diastereomers displayed distinctively different biological properties, different biodistribution and unique in vitro binding affinity. It is likely that a more selective Tc-99m complex system with high propensity for forming one single isomer will be needed to design site-specific imaging agents. The present invention thus solves a problem in the art by enabling the production of only one Tc-99m compound using the [TcO]⁺³N₂S₂ core.

The following examples are illustrative, but not limiting, of the method and compositions of the present invention. Other suitable modifications and adaptations of the variety of conditions and parameters normally encountered and obvious to those skilled in the art are within the spirit and scope of the invention.

Examples

General

Reagents used in the syntheses were purchased from Aldrich (Milwaukee, WI) or Fluka (Ronkonkoma, NY), and were used without further purification unless otherwise indicated. Anhydrous Na₂SO₄ was used as a drying agent.

Reaction yields are reported without attempts at optimization. Thin layer chromatography was performed on EM Science (Gibbstown, NJ) precoated (0.2 mm) silica gel 60 plates, and the spots were detected with iodine vapor and/or UV light. Silica gel 60 (70-230 mesh), obtained from EM Science (Gibbstown, NJ), was used for column chromatography. 'H NMR spectra were obtained on a

Bruker spectrometer (Bruker AC 300). All samples prepared for NMR analysis were dissolved in CDC1₃, purchased from Aldrich. Chemical shifts are reported as δ values with chloroform or TMS as the internal reference. Coupling constants are reported in Hz. The multiplicity is defined by s (singlet), d (doublet), t (triplet), brs (broad signal), dt (doublet of triplet) and m (multiplet). IR spectra were recorded with a Mattson Polaris FT-IR spectrometer and are reported in cm^" ' . Melting points were determined on a Meltemp apparatus (Cambridge, MA), and are uncorrected. Elemental analyses were performed by Atlantic Microlabs

(Norcross, GA). High resolution mass spectrometry was performed by the Nebraska Center for Mass Spectroscopy, University of Nebraska (Lincoln, NE).

Example I N-benzyl, 3(R),4(R)-di-[N-2-(4-methoxybenzylthio)- methylcarbonyl] amino pyrrolidine (2)

To a solution of N-benzyl, 3(R),4(R)-diamino pyrrolidine (900 mg, 4.7 mmol) and 2-(4-methoxybenzylthio)acetic acid (3 g, 1.5 eq) in a mixed solvent (40 mL, CH₂C1₂ : DMF = 1 : 1) was added dicyclohexylcarbodiimide (DCC) (2.9 g, 1.5 eq) followed by HOBT (1.9 g, 1.5 eq) in solid form. The mixture was stirred at RT overnight. Water was added and the mixture was extracted with mixed solvent (CH₂C1₂ : MeOH = 9:1). The combined organic layers were purified by MPLC (EtOAC:Hex = 3:2 as eluent) to give 2.37 g of product (87%). [α] = -11.6° (c, 2.11, CHC1₃). 'HNMR (300 MHz, δ, CDC1₃): 2.33 (2H, dd, J = 5.7, 9.6 Hz, CH₂-pyrrolidine ring), 2.98 (2H, dd, J = 6.9, 9.6 Hz, C/7₂-pyrrolidine ring), 3.07 (2H, s, COC//₂S), 3.08 (2H, s, COCH₂S), 3.58 (1H, s, benzyl), 3.61

(1H, s, benzyl), 3.66 (4H, s, SCH₂Ar), 3.77 (6H, s, OC7Q, 4.06 (2H, m, CONHCH-pyrrolidine ring), 6.81 (4H, d, t, J = 2.1 , 8.7 Hz, SCH₂ArH), 7.06 (2H, d, J = 6.5 Hz, CONH), 7.17 (4H, d, t, , J = 2.1, 8.7 Hz, SCH₂ArH), 7.25-7.33 (5H, m, NCH₂ArH). HRMS: Calcd for C₃₁H₃₇N₃O₄S₂: 580.2304 (M⁺+H); Found: 580.2300 (M⁺+H).

By using same procedure compound 6 was made in 64% yield (222 mg) starting from 5 (1 14 mg, 0.6 mmol) which was prepared from racemic tartaric acid in five steps. 'HNMR (200 MHz, δ, CDC1₃): 2.34 (2H, dd, J = 5.5, 9.8 Hz, CH₂-pyrrolidine ring), 2.99 (2H, dd, J = 6.7, 9.8 Hz, CH₂-pyrrolidine ring), 3.07 (2H, s, COCH ), 3.08 (2H, s, COCH₂S), 3.60 (IH, s, benzyl), 3.62 (IH, s, benzyl), 3.67 (4H, s, SCH₂Ar), 3.77 (6H, s, OCH₃), 4.07 (2H, m, CONHCH-pyrrolidine ring), 6.82 (4H, d, L J = 2.4, 8.7 Hz, SCH₂ArH), 7.04 (2H, δ, J = 6.2 Hz, CON/fj, 7.18 (4H, d, t, J = 2.4, 8.7 Hz, SCH₂Ar£Q, 7.25-7.33 (5H, m, NCH₂ArH).

Example 2 N-benzyl, 3(R),4(R)-di-[N-2-(4-methoxybenzylthio)ethyl]amino pyrrolidine (3)

To a solution of diamide 2 (366 mg, 0.63 mmol) in THF (10 mL) was added dropwise a solution of BH₃- THF (10 mL, 1M in THF) at RT. The mixture was stirred at reflux overnight. Water was added with caution after the reaction mixture was cooled. THF was removed and 10% of HCl solution (6 mL) was added and the mixture was stirred at reflux for 1 h. The reaction mixture was adjusted to basic with NaOH (1 M) and extracted with mixed solvent (CH₂C1₂ : MeOH = 9:1). Usual work up gave crude product which was purified by PTLC

( CH₂C1₂ : MeOH = 9: 1 as developing solvent) to give 255 mg (73%) of product, [α] = -12.4° (c, 1.05, MeOH). 'HNMR (200 MHz, δ, CDC1₃): 2.30 (2H, dd, J = 2.9, 6.3 Hz, CH₂-pyrrolidine ring), 2.55 (4H, t, J = 4.0 Hz, NHCH₂CH₂S), 2.68 (4H, t, J = 4.0 Hz, NHCH₂CH₂S), 2.78 (2H, dd, J = 4.1, 6.3 Hz, C^-pyrrolidine ring), 2.88 (2H, m, NHCH-pyrrolidine ring), 3.55 (IH, s, benzyl), 3.58 (IH, s, benzyl), 3.64 (4H, s, SCH₂Ar), 3.77 (6H, s, OC/Q, 6.82 (4H, δ,t, J = 1.4, 5.8 Hz, SCH₂ArH), 7.20 (4H, δ,t, , J = 1.4, 5.8 Hz, SCH₂ArH), 7.24-7.31 (5H, m, NCH₂Ar/ ). HRMS: Calcd for C₃₁H₄₁N₃O₂S₂: 552.2718 (M⁺+H); Found: 552.2713 (M⁺+H). By using same procedure compound 7 was made in 56% yield (80 mg) starting from 6 (150 mg, 0.26 mmol). 'HNMR (200 MHz, δ, CDC1₃): 2.32 (2H, dd, J - 3.9, 9.0 Hz, CHrpyrrolidine ring), 2.56 (4H, t, J = 5.7 Hz, NHCH₂CH₂S), 2.69 (4H, t, J = 5.7 Hz, NHCT^CH^), 2.80 (2H, dd, J = 6.7, 9.0 Hz, CHrpyrrolidine ring), 2.88 (2H, m, NHC/f-pyrrolidine ring), 3.57 (1 H, s, benzyl), 3.60 (IH, s, benzyl), 3.64 (4H, s, SCN₂Ar), 3.78 (6H, s, OC ₃), 6.82 (4H, δ,t, J = 1.4, 5.8 Hz, SCH₂Ar7Q, 7.21 (4H, δ,t, , J = 1.9, 6.7 Hz, SCH₂ArH), 7.24-7.31 (5H, m, NCH₂ArH).

Example 3 N-benzyl, 3(R),4(R)-di-(N-2-mercaptoethyl)amino pyrrolidine (4)

To a solution of diamine 3 (130 mg, 0.24 mmol) and anisole (2 drops) in TFA (5 mL) was added Hg(OAc)₂ (180 mg, 1.2 eq) in solid form at 0 °C in an ice water bath. The mixture was stirred at 0 °C for 30 min. TFA was removed and ether was added. The resulting solid was collected by suction filtration and washed with ether to which a mixed solvent (20 mL, EtOH:EtOAc = 1 :1) was added. H₂S gas was bubbled through for 5 min. The black mixture was filtered through CELITE (diatomaceous earth) and washed with MeOH. The filtrate was concentrated to give 124 mg of oil. 'HNMR (200 MHz, δ, MeOD): 2.74-2.88 (4H, m, HSCH₂), 3.04-3.18 (4H, m, NHC ₂), 3.67-3.77 (2H, m, CH₂NCH_ϊH_b),

3.85-3.95 (2H, m, CH₂NCH_aH_b), 4.29 (IH, δ, j = 12.8 Hz, ArCH-H_b), 4.38 (IH, δ, j = 12.8 Hz, ArCHJi_t,), 4.54 (2HY, br, NHCH), 7.46 (5H, m, ArH). HRMS: Calcd for C₁₅H₂₅N₃S₂: 312.1568 (M⁺+H); Found: 312.1573 (M⁺+H).

By using same procedure 60 mg of compound 8 was obtained starting from 7 (75 mg, 0.14 mmol). 'HNMR (200 MHz, δ, MeOD): 2.82-2.89 (4H, m,

HSCH₂), 3.02-3.25 (4H, m, NHCH₂), 3.72-3.78 (2H,m, CH₂NCH_aH_b), 3.86-3.98 (2H, m, CH₂NCH.H_b), 4.28 (IH, δ, j = 12.8 Hz, ArCHT^)^', 4.36 (IH, δ, j = 12.8 Hz, ArCH/i), 4.69 (2HY, br, NHCH), 7.47 (5H, m, ArH). HRMS: Calcd for C₁₅H₂₅N₃S₂: 312.1568 (M⁺+H); Found: 312.1563 (M⁺+H). Example 4 3(R),4(R)-di-[N-2-(4-methoxybenzylthio)methylcarbonyl]amino pyrrolidine (14)

N-benzyl, 3(R),4(R)-di-(N-t-butoxylcarbonyl)amino pyrrolidine (9): To a solution of diamine 1 (100 mg, 0.52 mmol) and Et₃N (0.6 mL, 4 eq) in methylene chloride (10 mL) was added a solution of (Boc)₂O (274 mg, 1.2 eq) in methylene chloride (10 mL) dropwise at 0 °C in an ice bath. The mixture was stirred at 0 °C for 1 h. then at RT for another 2 h. Water was added and the resulting mixture was extracted with CH₂C1₂. The organic phase was dried, concentrated and purified by PTLC (Hex:EtOAc = 1:1 as developing solvent) to give 177 mg

(87%) of product. 'HNMR (200 MHz, δ, CDC1₃): 1.43 (18H, s, tert-CC//₃), 2.39 (2H, δ,δ, J = 5.2, 9.1 Hz, NC ₂-pyrrolidine ring), 2.99 (2H, δ,δ, J = 5.2, 9.5 Hz, NCH₂-pyrrolidine ring), 3.58 (2H, s, benzyl), 3.83 (2H, m, NHCH-pyrrolidine ring), 4.85 (2H, br, NH), 7.28 (5H, m, ArH).

3(R),4(R)-di-(N-t-butoxylcarbonyl)aminopyrrolidine (10): The mixture of protected diamine 9 ( 540 mg, 1.39 mmol) and Pd/C (250 mg, 10%) in a mixed solvent (EtOAc:EtOH = 5:1, 60 mL) was hydrogenated in a Parr appartus at 45 psi overnight. The mixture was filtered and washed with MeOH. The filtrate was concentrated to give 420 mg of product which was pure enough to run the next reaction without further purification. 'HNMR (200 MHz, δ, MeOD): 1.44 (18H, s, tert-CCH₃), 2.67 (2H, δ,δ,δ, J = 2.0, 5.8, 11.8 Hz, NCH₂-pyrrolidine ring), 3.17 (2H, δ,δ,δ, J = 2.2, 6.6, 11.8 Hz, NCH₂-pyrrolidine ring), 3.82 (2H, m, NHC/7-pyrrolidine ring).

N-Fmoc, 3(R),4(R)-di-(N-t-butoxylcarbonyl)amino pyrrolidine (11): To a suspension of amine 10 (100 mg) in a mixed solvent (6 mL, dioxane: 10% Na₂CO₃ saturated solution = 1 :2) was added a solution of FmocCl (103 mg) in dioxane (4 mL) dropwise at 0 °C in an ice bath. The mixture was stirred at RT for 2 h. Water was added and the mixture was extracted with CH₂C1₂. The organic layer was dried, concentrated and purified by PTLC (Hex:EtOAc = 4: 1 as developing solvent) to give 163 mg (94%, 2 steps) of product. 'HNMR (200 MHz, δ, CDC1₃): 1.46 (18H, s, tert-CCTQ, 3.13 (2H, m, NCH₂-pyrrolidine ring), 3.86 (2H, m, NCN₂-pyrrolidine ring), 3.99 (2H, m, NHCH-pyrrolidine ring), 4.22 (IH, t, J = 6.6 Hz, COCH₂CH), 4.40 (2H, δ, J = 6.3 Hz, COC ₂CH), 4.83 (IH, br, NH), 4.96 (IH, br, NH), 7.31 (2H, δ,t, J = 1.4, 7.4 Hz, ArH), 7.40 (2H, δ,t, J = 1.0, 7.1

Hz, ArH), 7.58 (2H, δ,δ, J = 0.7, 7.2 Hz, AτH , 7.76 (2H, δ, J = 7.0 Hz, ArH).

N-Fmoc, 3(R),4(R)-diaminopyrrolidine (12): The mixture of starting material 11 (5.6 g, 10.7 mmol) in HCl-EtOAc solution (3 M 60 mL) was stirred at RT for 30 min. The solvent was removed to give 4.4 g of white solid which was pure enough to use in the next reaction without further purification. 'HNMR

(200 MHz, δ, MeOD): 3.62 (2H, m, NCH₂-pyrrolidine ring), 3.99 (2H, δ,δ,J = 6.5, 12.4 Hz, NCH₂-pyrrolidine ring), 4.13 (2H, m, NHCH-pyrrolidine ring), 4.24 (IH, t, J = 6.2 Hz, COCH₂CH), 4.46 (2H, δ, J = 6.3 Hz, COCH,CH), 7.31 (2H, δ,t, J = 1.2, 7.4 Hz, ArH), 7.38 (2H, δ,t, J = 0.9, 7.3 Hz, ArH), 7.63 (2H, δ, J = 7.1 Hz, ArH), 7.76 (2H, δ,δ, J = 1.1, 7.6 Hz, ArH).

N-Fmoc, 3(R),4(R)-di-[N-2-(4-methoxybenzylthio)methylcarbonyl]amino pyrrolidine (13): To a solution of diamine 12 (4.2 g) and 2-(4-methoxybenzyl)thioacetic acid (6.75 g, 1.5 eq) in a mixed solvent (100 mL, CH₂C1₂ : DMF = 2:3) was added DCC (6.9 g, 1.5 eq) followed by HOBT (4.6 g, 1.5 eq) in solid form. The mixture was stirred at RT overnight. Water was added and the mixture was extracted with mixed solvent (CH₂C1₂ : MeOH = 9:1). The combined organic layers were dried over Na₂SO₄ and concentrated, purified by MPLC (EtOAC:Hex = 3:1 as eluent) to give 5.94 g of product (81%, 2 steps). 'HNMR (200 MHz, δ, CDC1₃): 3.05 (2H, m, NCH₂-pyrrolidine ring), 3.12 (4H, s, COCH₂S), 3.67 (4H, s, SCH₂Ar), 3.75 (3H, s, OCH₃), 3.78 (3H. s, OCH₃), 3.86

(2H, m, NCH₂-pyrrolidine ring), 4.06-4.27 (3H, m, NHCH-pyrrolidine ring, COCΗ₂CH), 4.41 (2H, δ, J = 6.0 Hz, COCH₂CH), 6.84 (4H, δ, J = 8.5 Hz, CH₂ArH), 7.19 (4H, δ, J = 8.5 Hz, CH₃OArH), 7.31 (2H, δ,t, J = 0.9, 7.3 Hz, ArH), 7.40 (2H, δ,t, J = 1.0, 7.1 Hz, ArH), 7.58 (2Η, δ,δ, J = 0.4, 7.6 Hz, ArH), 7.76 (2Η, δ,δ, J = 0.9, 6.8 Hz, ArH).

3(R),4(R)-di-[N-2-(4-methoxybenzylthio)methylcarbonyl]amino pyrrolidine (14):

The mixture of starting material 13 (1.5 g, 2.1 mmol) in piperidine (25 mL) was stirred at RT for 1 h. Solvent was removed and the residue was purified by flash chromatography (CΗ₂Cl₂:MeOΗ = 9:1 as eluent) to give 530 mg (51%) of product. 'HNMR (200 MHz, δ, CDC1₃): 2.70 (2H, δ,δ, J = 6.0, 10.3 Hz,

NCH₂-pyrrolidine ring), 3.05 (4Η, s, COCH₂S), 3.30 (2Η, δ,δ, J = 6.3, 11.2 Hz,

NCH₂-pyrrolidine ring), 3.65 (4Η, s, SCH₂Ar), 3.73 (6Η, s, OCH₃), 4.03 (2Η, hex, J = 5.3 Hz, NHCH-pyrrolidine ring,) 6.78 (4H, δ, J = 8.5 Hz, CH₂ArH), 7.15 (4H, δ, J - 8.5 Hz, CH₃OArH), 7.36 (2Η, δ, J = 5.6 Hz, CONH).

Example 5

N-2-[4-(2-meth oxyph enyl)piperazinyl] ethyl, 3 (R),4(R) -di-(N-2- mercapto ethyl) amino pyrrolidine (18)

N-2-[4-(2-methoxyphenyl)piperazinyl]ethyl,3(R),4(R)-di-[N-2-(4- methoxybenzylthio) methylcar bony I] amino pyrrolidine (16):

4-(2-Methoxyphenyl)piperazine was reacted with 2-bromoethanol then converted to 2-[4-(2-methoxyphenyl)piperazinyl]ethyl bromide 15 by CBr₄ and triphenylphosphine in CC1₄. The mixture of 15 (164 mg, 0.55 mmol), pyrrolidine 14 (267 mg, 0.55 mmol) and K₂CO₃ (302 mg, 4 eq) in DMF (10 mL) was stirred at RT overnight, water was added and the mixture was extracted with CΗ₂C1₂. The organic layer was dried, filtered, concentrated and purified by flash chromatography (CH₂Cl₂:MeOH=95:5 as elunet) to give 302 mg (70%) of white solid. ⁽¹HNMR (200 MHz, δ, CDC1₃): 2.41 (2H, d,d, J=5.5, 9.6 Hz, NCH,-pyrrolidine ring), 2.35-2.73 (8Η, m, NCH₂), 3.03 (2H, d,d, J=6.8, 9.6 Hz, NCH₂-pyrrolidine ring), 3.10 (4Η, s, COCH₂S), 3.69 (4Η, s, SCH₂Ar), 3.78 (6Η, s, OCH₃), 3.86 (3Η, s, OCH_j), 4.08 (2Η, hex, J=5.6 Hz, NHCH-pyrrolidine ring), 6.83 (4Η, d,t, J=2.7, 8.6 Hz, CH₂ArH), 6.90-7.13 (4Η, 4H, m, ArH), 7.20 (4Η, d,t, J=2.7, 8.5 Hz, CH₃OArH).)

N-2-[4-(2-methoxyphenyl)piperazinyl]ethyl, 3(R),4(R)-di-[N-2-(4- methoxybenzylthio)ethyl]-amino pyrrolidine (17): To a solution of diamide 16 (290 mg, 0.41 mmol) in TΗF (10 mL) was added dropwise a solution of BΗ₃ THF

(6 mL, 1 M_in THF) at RT. The mixture was stirred at reflux overnight. Water was added with caution after the reaction mixture was cooled down. THF was removed and 10% HCl solution (10 mL) was added and the mixture stirred at reflux for 1 h. The reaction mixture was adjusted to basic with NaOH (50% solution) and extracted with mixed solvent ( CH₂C1₂ : MeOH = 9:1). Usual work up gave crude product which was purified by PTLC ( CH₂C1₂ : MeOH = 9:1 as developing solvent) to give 150 mg (54%) of product. ('HNMR (200 MHz, δ, CDC1₃): 2.27-2.38 (4H, m, CH₂), 2.57 (8Η, m, NCH₂), 2.68 (8H, m, CH₂), 2.85 (2Η, m, CH₂), 3.08 (4Η, m, CH₂), 3.64 (4Η, s, SCH₂Ar), 3.76 (6Η, s, OCH₃), 3.83 (3Η, s, OCH₃), 6.81 (4Η, d, J=8.1 Hz, CH₂ArH), 6.90 (4Η, 4H, m, ArH),

7.20 (4Η, d, J=8.1 Hz, CH₃OArH).)

N-2-[4-(2-methoxyphenyl)piperazinyl]ethyl, 3(R),4(R)-di-(N-2- mercaptoethyl) amino pyrrolidine (18): To a solution of diamine 17 (100 mg, 0.15 mmol) and anisole (2 drops) in TFA (4 mL) was added Ηg(OAc)₂ (113 mg, 1.2 eq) in solid form at 0 °C in an ice water bath. The mixture was stirred at

0 °C for 30 min. TFA was removed and ether was added. The resulting solid was collected by suction filtration and washed with ether. To the solid was added a mixed solvent (12 mL, EtOH:EtOAc=l : 1). H₂S gas was bubbled through for 5 min. The black mixture was filtered through Celite and washed with MeOH. The filtrate was concentrated to give 120 mg of oil. ('HNMR (200 MHz, δ, CDC1₃):

2.85-3.76 (24H, m, CH₂), 3.83 (3Η, s, OCH₃), 4.08 (2Η, m, NCH), 6.77-7.11 (4Η, m, ArH),) Example 6

N^!-3-[3(R),4(R)-di-(N-2- mercaptoethyl)aminopyrrolidinyl]methylbenzyl guanidine (24)

N- 3 -p h th a lim ido m e thylb e n zy l, 3 (R) , 4 (R) - di-[N- 2 - (4- methoxybenzylthio)methylcarbonyl] amino pyrrolidine (20): A mixture of pyrrolidine 14 (270 mg, 0.55 mmol), 3-phthalimidomethylbenzyl chloride 19 (190 mg, 1.2 eq) (which was made from monoalkylation of m-α,cc'-dichloroxylene and phthalimide potassium salt) and K₂CO₃ (302 mg, 4 eq) in DMF (4 mL) was stirred at RT overnight. Water was added and the mixture was extracted with CH₂C1₂. The organic layer was dried, filtered, concentrated and purified by PTLC

(Hex:EtOAc=l :2 as developing solvent) to give 177 mg (43%) of product. ⁽'HNMR (200 MHz, δ, CDC1₃): 2.95 (2H, d,d, J=5.5, 9.6 Hz, NCH₂-pyrrolidine ring), 2.98 (2Η, d,d, J=6.5, 9.6 Hz, NCH₂-pyrrolidine ring), 3.09 (4Η, s, COCH₂S), 3.52 (lΗ, d, J=13.1 Ηz,NCH_abAr), 3.64 (lΗ, d, J=13.1 Hz, NCH^Ar), 3.68 (4Η, SCH₂Ar), 3.76 (6Η, s, OCH₃), 4.07 (2Η, m, CONHCH-pyrrolidine ring), 4.83 (2Η, br, CONCH₂Ar). 6.80 (4Η, d,t, J=2.4, 8.7 Hz, SCH₂ArH), 7.18 (4Η, d,t, J=2.2, 8.7 Hz, CH₂OArH), 7.22-7.40 (4Η, m, ArH), 7.68 (2Η, m, ArH), 7.83 (2Η, m, ArH).)

N-3-aminomethylbenzyl, 3(R),4(R)-di-[N-2-(4-methoxybenzylthio)- methylcarbonyl] amino pyrrolidine (21): The mixture of compound 20 (160 mg, 0.22 mmol) and hydrazine (0.2 mL) in MeOΗ (10 mL) was stirred at RT overnight. The solid was removed by filtration and the filtrate was concentrated and purified by PTLC (MeOΗ:CΗ₂Cl₂=l :9 as developing solvent) to give 100 mg (76% yield) of white solid. ⁽¹HNMR (200 MHz, δ, CDC1₃): 2.34 (2H, d,d, J=5.5, 9.7 Hz, NCH.-pyrrolidine ring), 2.86 (2Η, m, NCH₂-pyrrolidine ring), 3.05 (4Η, s, COCH₂S), 3.56 (1Η, s, NCH,_bAr), 3.57 (1Η, s, NCH,_bAr), 3.66 (4Η, SCH₂Ar), 3.75 (6Η, s, OCH₃), 3.86 (2Η, br, H₂NCH₂Ar). 4.10 (2Η, m, CONHCH-pyrrolidine ring), 6.79 (4Η, d,t, J=2.0, 8.7 Hz, SCH₂ArH), 7.17 (4Η, d,t, J=2.0, 8.7 Hz, CH₂OArH), 7.23-7.28 (4Η, m, ArH).) N-3-aminomethylbenzyl, 3(R),4(R)-di-[N-2-(4-methoxybenzylthio)ethyl]amino pyrrolidine (22): To a solution of starting material 21 (100 mg, 0.16 mmol) in THF (3 mL) was added a solution of BH₃ (3 mL, 1M in THF) dropwise at RT. The mixture was stirred at reflux overnight. Water was added with caution after cooling and the THF was removed in vacuo. HCl solution (5 mL, 10%) was added and the resulting mixture was stirred at reflux for 1 h. The solution was made basic with 50% KOH solution after cooling and extracted with mixed solvent (CH₂Cl₂:MeOH=9:l). The combined solvent was dried, concentrated to give 78 mg (82% yield) of product which was pure enough to use in the next reaction without purification. ('HNMR (200 MHz, δ, CDC1₃): 2.27-3.09 (14H, m, CH₂), 3.53 (1Η, s.NCH^Ar), 3.56 (IH, s,NC^_bAr), 3.63 (4H, SCH₂Ar), 3.76 (6Η, s, OCH₃), 3.83 (2Η, br, H₂NCH₂Ar). 6.80 (4Η, d, J=8.3 Hz, SCH₂ArH), 7.18 (4Η, d, J=8.3 Hz, CH₂OArH), 7.23 (4Η, m, ArH).)

N¹-3-{3 (R),4(R)-di-[N-2-(4-methoxybenzylth io) ethyl]amino- pyrrolidinyl methylbenzyl, N², N³-di-tert-butoxycarbonylguanidine (23): The mixture of 22 (78 mg, 0.13 mmol) and l,3-bis(tert-butoxycarbonyl)-2-methyl-2- thiopseudourea (46 mg, 1.2 eq) in TΗF (3 mL) containing 4 drops of water was stirred at 65 °C for 3 h. The solvent was removed and the residue was purified by PTLC (CΗ₂Cl₂:MeOΗ=93:7 as developing solvent) to give 42 mg (38% yield) of product. ('HNMR (200 MHz, δ, CDC1₃): 1.47 (9H, s, tert-CCH₃), 1.51 (9H, s, tert-CCH₃), 2.28-2.89 (14H, m, CH₂), 3.57 (2Η, s, NCH^Ar), 3.64 (4Η, SCH₂Ar), 3.77 (6Η, s, OCH₃), 4.61 (2Η, d, J=4.9 Hz, HNCH₂Ar). 6.81 (4Η, d,d, J=1.9, 8.6 Hz, SCH₂ArH), 7.20 (4Η, d,d, J=1.9, 8.6 Hz, CH₂OArH), 7.29 (4Η, m, ArH), 8.55 (1Η, br, NHC(=NBoc)NΗBoc), 11.54 (IH, s, NHBoc).)

N'-3-[3(R),4(R)-di-(N-2-mercaptoethyl)aminopyrrolidinyl]methylbenzyl guanidine (24): To starting material 23 (42 mg, 0.05 mmol) was added

TFA (3 mL) and the mixture was stirred at RT for lh. Anisole (2 drops) was added followed by Hg(OAc)₂ (49 mg, 1.2 eq) in solid form after the mixture was cooled down at 0 °C in an ice water bath. The mixture was stirred at 0 °C for 1 h. TFA was removed and ether was added. The resulting solid was collected by suction filtration and washed with ether. To the solid was added a mixed solvent (12 mL, EtOH:EtOAc=l :1). H₂S gas was bubbled through for 5 min. The black mixture was filtered through Celite and washed with MeOH. The filtrate was concentrated and washed with chloroform to give 26 mg of oil. ('HNMR (200

MHz, δ, CDC1₃): 2.78-2.89 (4H, m, CH_), 3.22-3.34 (8Η, m, CH₂), 3.87 (2Η, NCH₂Ar), 4.04 (2Η, br, NHCH-pyrrolidine-ring), 4.40 (2Η, s, HNCH₂Ar). 7.35 (4Η,ArH).)

Example 7 N -2-[3(R), 4 (R)-di-(2-mercapto ethyl) amino pyrrolidinyl] ethyl,

2,3-dimethoxy-5-iodo-benzamide (32)

N-Fmoc, 3(R),4(R)-di-[N-2-(4-methoxvbenzvlthio)ethvl]amino pyrrolidine 25:

To a solution of starting material 13 (600 mg, 0.84 mmol) in TΗF (10 mL) was added a solution of BΗ₃ (18 mL, 1M in THF) dropwise at RT. The mixture was stirred at reflux overnight. Water was added with caution after cooling and the THF was removed in vacuo. HCl solution (15 mL, 10%) was added and the resulting mixture was stirred at reflux for 1 h. The solution was made basic with 50% KOH solution after cooling and extracted with mixed solvent (CH₂Cl₂:MeOH=9:l). The combined solvent was dried, concentrated and purified by PTLC (CH₂Cl₂:MeOH=9: 1 as the developing solvent) to give 253 mg

(44% yield) of product. ('HNMR (200 MHz, δ, CDC1₃): 2.58 (4H, t, J=5.9 Hz, CH₂), 2.74 (4Η, t, J=6.2 Hz, CH₂), 2.94 (2Η, m, CH_), 3.13 (2H, m, CH₂), 3.67 (2Η, s, SCH₂Ar), 3.68 (2Η, SCH₂Ar), 3.69 (2Η, m, NCH), 3.78 (6H, s, OCH₃), 4.24 (1Η, t, J=6.7 Ηz, COCΗ₂CH), 4.39 (2Η, d,d, J=1.5, 7.6 Hz, COCH_jCH), 6.84 (4H, d, J=8.6 Hz, SCH₂ArH), 7.22 (4Η, d, J=8.6 Hz, CH₂OArH), 7.30 (2Η, d,t, J=1.2, 7.5 Hz, ArH), 7.39 (2Η, d,t, J=1.2, 7.5 Hz, ArH), 7.60 (2Η, d, J=7.2 Hz, ArH), 7.76 (2Η, d, J=7.2 Hz, ArH). )

N-Fmoc, 3(R),4(R)-di-[N-2-(4-methoxybenzylthio)ethyl, N-tert- butoxycarbonyljamino pyrrolidine 26: To a solution of starting material 25 (383 mg, 0.56 mmol) and Et₃N (0.8 mL) in CH₂C1₂ (20 mL) was added a solution of (Boc)₂O (2.46 g, 10 eq) in CH₂C1₂ (5 mL) dropwise at RT. The resulting mixture was stirred at reflux for 2 h. Water was added and usual work up and purification by PTLC (EtOAc:Hex=2:l as developing solvent) to give 420 mg (85% yield) of product. ('HNMR (200 MHz, δ, CDC1₃): 1.46 (18H, s, tert-

CCH₃), 2.52 (4H, m, CH₂), 3.18 (4Η, m, CH₂), 3.58 (2Η, m, CH₂), 3.71 (2Η, s, SCH₂Ar), 3.74 (3Η, s, CH₃OAr), 3.76 (3Η, s, OCH₃), 4.25 (1Η, t, J=6.7 Ηz, COCΗ₂CH), 4.40 (2Η, br, COCH₂CH), 4.50 (2H, br, BocNHCH), 6.82 (4Η, d,d, J=3.8, 8.2 Hz, SCH₂ArH), 7.26 (4Η, d, J=8.6 Hz, CH₂OArH), 7.31 (2Η, t, J=7.2 Hz, ArH), 7.41 (2Η, t. J=7.2 Hz, ArH), 7.61 (2Η, d, J=7.2 Hz, ArH), 7.77

(2Η, d, J=7.2 Hz, ArH). )

3(R),4(R)-di-[N-2-(4-metlιoxybenzylthio)ethyl, N-tert-butoxycarbonylJamino pyrrolidine 27: Starting material 26 (302 mg, 0.34 mmol) in piperidine (5 mL) was strred at RT for 1 h. Solvent was removed and the residue was purified by PTLC (CΗ₂Cl₂:MeOΗ=93 :7 as developing solvent) to give 220 mg (97%) of product. ('HNMR (200 MHz, δ, CDC1₃): 1.41 (18H, s, tert-CCH₃), 2.42 (4H, m, CH₂), 2.92 (2Η, m, CH₂), 3.26 (4Η, m, CH₂), 3.67 (4Η, s, SCH₂Ar), 3.78 (6Η, s, CH₃OAr), 4.13 (2Η, br, BocNHCH), 6.83 (4Η, d,d, J=3.8, 8.2 Hz. SCH₂ArH), 7.23 (4Η, d, J=8.6 Hz, CH₂OArH). )

N-cyanomethyl, 3(R),4(R)-di-[N-2-(4-methoxybenzylthio)-etlιyl, N-tert- butoxycarbonyl] amino pyrrolidine 28: The mixture of starting material 27

(150 mg, 0.23 mmol), chloroacetonitrile (35 mg, 2 eq), K₂CO₃ (156 mg, 5 eq) and KI (50 mg) in DMF (3 mL) was stirred at RT overnight. Water was added and the resulting mixture was extracted with CΗ₂C1₂. The combined organic layers were dried, concentrated and purified by PTLC (Hex:EtOAc=2:l as developing solvent) to give 126 mg (80%) of product. ('HNMR (200 MHz, δ, CDC1₃): 1.43 (18H, s, tert-CCH₃), 2.42-2.92 (8H, m, CH₂), 3.53 (2Η, s, NCCH₂). 3.69 (4Η, s, SCH₂Ar), 3.78 (6Η, s, CH₃OAr), 4.40 (2Η, br, BocNHCH), 6.83 (4Η, d,d, J=3.8, 8.3 Hz, SCH₂ArH), 7.24 (4Η, d, J=8.3 Hz, CH₂OArH). ) N-2-aminoethyl, 3(R),4(R)-di-[N-2-(4-methoxybenzylthio)ethyl, N-tert- btoxy car bony I] amino pyrrolidine 29: To a suspension of lithium aluminum hydride (35 mg, 0.9 mmol) in THF (5 mL) was added to a solution of starting material 28 (126 mg, 0.18 mmol) in THF (5 mL) dropwise at 0 °C in an ice bath. The mixture was stirred at RT for 30 min. H₂O (0.1 mL), NaOH (0.1 mL, \M) and H₂O (0.3 mL) were added successively. The resulting mixture was stirred at RT for 10 min, filtered, and washed with mixed solvent (CH₂Cl₂:MeOH=9:l). The filtrate was concentrated and purified by PTLC (CH₂Cl₂:MeOH=9: 1 as developing solvent) to give 55 mg (43% yield) of product. ('HNMR (200 MHz, δ, CDC1₃): 1.42 (18H, s, tert-CCH₃), 2.49-2.84 (12H, m,

CH₂), 3.42 (2Η,m, CH₂), 3.70 (4Η, s, SCH₂Ar), 3.78 (6Η, s, CH₃OAr), 4.40 (2Η, br, BocNHCH), 6.83 (4Η, d, J=8.4 Hz, SCH₂ArH), 7.24 (4Η, d, J=8.4 Hz, CH₂OArH).)

N-2-{3(R),4(R)-di-[N-2-(4-methoxybenzylthio)-ethyl, N-tert- butoxycarbonyl]aminopyrrolidinyl}ethyl, 2,3-dimethoxy-5-iodo-benzamide 31:

The mixture of starting material 29 (55 mg, 0.08 mmol), 2,3-dimethoxy-5- iodobenzoic acid 30 (36 mg, 1.5 eq, S.Chumpradit, et al. J.Med. Chem. 1993, 36, 221), DCC (16 mg, 1 eq) and ΗOBT (10.6mg, 1 eq) in CH₂C1₂ (5 mL) containing 1 drop of DMF was stirred at RT overnight. Water was added and the mixture was extracted with mixed solvent (CH₂Cl₂:MeOH=9: 1 ). The organic layers were dried, concentrated and purified by PTLC (CH₂Cl₂:MeOH=9:l as developing solvent) to give 45 mg (58% yield) of product. ('HNMR (200 MHz, δ, CDC1₃): 1.42 (18H, s, tert-CCH₃), 2.53-2.83 (10H, m, CH₂), 3.29-3.54 (6Η,m, CH₂), 3.64 (4Η, s, SCH₂Ar), 3.78 (6Η, s, CH₃OAr), ), 3.81 (3Η, s, CH₃OAr), ), 3.86 (3Η, s, CH₃OAr), 4.48 (2Η, br, BocNHCH), 6.81 (4Η, d, J=8.4 Hz, SCH₂ArH), 7.20

(4Η, d, J=8.4 Hz, CH₂OArH), 7.23 (1Η, d, J=2.1 Ηz, ArH), 7.98 (1Η, d, J=2.1 Ηz, ArH), 8.18 (1Η, br, CONH). )

N-2-[3(R),4(R)-di-(2-mercaptoethyl)aminopyrrolidinyl]ethyl,2,3-dimethoxy-5- iodo-benzamide 32: To starting material 31 (45 mg, 0.05 mmol) was added TFA (2 mL) and the mixture was stirred at RT for lh. Solvent was removed and another 2 mL of TFA was added. Anisole (2 drops) was added followed by Hg(OAc)₂ (49 mg, 1.2 eq) in solid form after the mixture was cooled to 0°C in an ice/water bath. The mixture was stirred at 0°C for 1 h. TFA was removed and ether was added. The resulting solid was collected by suction filtration and washed with ether. To the solid was added a mixed solvent (4 mL, EtOH:EtOAc=l : 1). H₂S gas was bubbled through for 5 min. The black mixture was filtered through Celite and washed with MeOH. The filtrate was concentrated and washed with chloroform to give 23 mg of product. ('HNMR (200 MHz, δ, CDC1₃): 2.85 (4H, m, CH₂), 3.04 (6Η,m, CH₂), 3.37 (4Η, m. CH₂), 3.79 (2Η, m,

CH₂), 3.88 (6Η, s, CH₃OAr), ), 4.20 (2Η, br, BocNHCH). 7.31 (1Η. br, ArH), 7.90 (1Η, br, ArH), 8.35 (1Η, br, CONH). )

Example 8 Radiolabeling with Tc-99m

The free thiol ligand (0.2-0.4 μmol) of Example 3 was dissolved in 100 μL of EtOΗ and 100 μL of ΗC1 (IN). ΗC1 (500 μL, IN), 1 mL of Sn-glucoheptanate solution (containing 136 μg of SnCl, and 200 μg of Na-glucoheptanate, pΗ 6.67) and 50 μL of EDTA solution (0.1 N) were successively added. [""TcJPertechnetate (100-200 μL; ranging from 1-20 mCi) in saline solution was then added. The reaction was heated for 30 min at 100 °C

(or heated at 121 °C in an autoclave for 30 min), cooled to room temperature and neutralized with a sat. NaΗCO₃ solution. After extracting the complex from the aqueous reaction medium with ethyl acetate (1 x 3 mL, 2 x 1.5 mL) and passing it through a small column of Na₂SO₄, the ethyl acetate extracts were condensed under a flow of N₂. The residue was dissolved in 200 μL of EtOH and purified by

HPLC (PRP-1 column, 250 x 4.1 mm, CH₃CN/3,3-dimethylglutarate buffer, 5 mM, pH 7, volume ratio 8:2, flow rate 1 mL/min; respectively; radiochemical yield 88%, radiochemical purity >97%). The diastereomers were then separated by a Chiralpak AD column eluted with hexane:EtOH in a ratio of 3 : 1 and a flow rate of 1 mL/min. After separation the complexes displayed in vitro stability at 4 and 24 h after preparation. Little change in radiochemical purity was observed. Using Scheme 5, described above, the desired 3R,4R-(trans-)isomer of trans-P-BAT was prepared and ["^mTc](3R,4R)-trans-P-BAT was successfully produced. The corresponding Re complex can be prepared using the same methodology.

Initial preparation of Tc-99m complex using 3R,4R-trans-P-BAT produced ["^mTc]trans-P-BAT, ["^mTc]4, which showed only one peak on HPLC profile using a reverse-phase column: PRP-1 column, CH₃CN/DMGA (pH 7) 80:20, flowrate 1 mL/min; the retention time was 4.8 min (FIG.l). As predicted using a chiral AD HPLC column, again only one isomer for produced [^99mTc]trans-P-BAT, [^99mTc]4, was observed, (retention times = 11.9 min) (FIG. 1).

Initial preparation of [""Tcjcw-P-B AT showed one peak on HPLC profile using the reverse-phase column: PRP-1 column, CH₃CN/DMGA (pH 7) 80:20, flow rate 1 mL/min; retention time was 5.3 min. Using a chiral AD column, the diastereomers were separated (retention times were 13 and 17 min for peak A and peak B, respectively). HPLC profile (AD-column, hexane/EtOH 3:1, 1 mL/min) showed a ratio of "A" to "B" = 1:1. As expected, the HPLC profile (using chiral column for analysis) of the trans-P-BAT displayed only one isomer, while the cis-P-BAT showed two isomers.

Example 9 Partition coefficients

Partition coefficients were measured by mixing each [^99mTc] compound with 3 g of 1 -octanol and 3 g of buffer (pH 7.0 or 7.4, 0.1 M phosphate) in a test tube. The test tube was vortexed for 3 min at room temperature, then centrifuged for 5 min. Two weighed samples (0.5 g each) from the 1 -octanol and buffer layers were counted in a well counter. The partition coefficient was determined by calculating the ratio of cpm/g of octanol to that of buffer. Samples from the octanol layer were re-partitioned until consistent partition coefficient values were obtained. The measurement was repeated three times.

Example 10 Biodistribution in rats

Male Sprague-Dawley rats (225-300 g) allowed free access to food and water were used for in vivo biodistribution studies. While under ether anesthesia, 0.2 mL of a saline solution containing Tc-99m labeled agents (5-10 μCi) was injected directly into the femoral vein of rats, and the rats were sacrificed by cardiac excision at various time points post-injection. The organs of interest were removed and weighed, and the radioactivity was counted with an automatic gamma counter (Packard 5000). The percentage dose per organ was calculated by a comparison of the tissue counts to suitably diluted aliquots of the injected material. Total activities of blood and muscle were calculated under the assumption that they were 7% and 40% of the total body weight, respectively. For dual isotope experiment, [^99mTc] compound (5-10 μCi) were co-injected together to the rats and the biodistribution was performed. The percentage dose per organ was calculated by a comparison of the tissue counts to suitably diluted aliquots of the injected material. Total activities of blood and muscle were calculated under the assumption that they were 7% and 40% of the total body weight, respectively.

The "pure" P-BAT isomers were collected (see Example 1) and a biodistribution study was performed in rats. Preliminary biodistribution study of these Tc-99m labeled compounds showed good heart uptake in rats. Initial heart uptake of these compounds appears to be adequate. The [^99mTc]trans-P-BAT complex showed low heart retention (at 2 hr post i.v. injection only 0.06% dose remained in the heart indicating no trapping of this lipophilic compound). Table 1. Biodistribution in rats after i.v. injection of [^99mTc]trans P-BAT and [^99mTc]cis P-BAT, peak A and peak B

(percentage dose/organ, average of 3 rats ± SD)

Heart/blood ratio = percentage dose/gram in heart divided by the same in blood (avg. st. heart 1.0 g; blood 20 g). P.C. Partition coefficient - measured between 1-octanol/pH 7.4 buffer.

Example 11 Radiolabeling Using Stannous(H) Giucoheptonate and Ligand Kits

A stock solution of stannous chloride/sodium glucoheptanate (per 100 mL) is prepared. Initial ingredients consist of 0.8-3.2 mg of stannous chloride anhydrous, 8-32 mg of sodium glucoheptanate, 5 cc of 0.1 M sodium EDTA and

20 cc of 2 N HCl solution. Stannous chloride is dissolved in 1-2 cc of 2N HCl in a 100 cc volumetric flask. To this solution is added a solution (about 1 cc) containing giucoheptonate and EDTA. This mixture is filled to 100 cc with argon-purged water. The final solution is clear and the pH is 6.7. Each cc of kit solution contains:

8-32 μg of Sn (II)Cl₂-anhydrous; 80-320 μg of sodium glucoheptanate; 0.336 mg of sodium EDTA; 0.06 mL Ethanol; and 0.2 mL HCl (2N).

Water is added to bring the total volume to 1 mL.

The solution is dispensed into about 100 each of 3 mL brown vials and dried under vacuum (lyophilizer). To the dry lyophilized vial,

[⁹⁹ Tc]pertechnetate (1-30 mCi) is added and 1-3 mg of the ligand is added and the resulting vial is heated. The product is evaluated by TLC and the radiochemical purity is calculated.

Example 12

Preparation of kits containing stannous chloride/sodium giucoheptonate and ligand in one vial

N₂S₂ ligand solution (a total of 25 cc of solution) is prepared. Each cc of solution contains:

1.0 mg of N₂S₂ ligand (25 mg); 0.03 mg of Sn (II)Cl₂-dihydrate; 0.3 mg of sodium giucoheptonate;

0.2 mg of sodium EDTA;

0.06 mL Ethanol; and

0.1 mL HCl (2N). The solution is dispensed into about 25 each of 3 mL brown vials and dried under vacuum (lyophilizer). To the dry lyophilized vial, [^99mTc]pertechnetate (1-30 mCi) is added and heated as described above. The product is evaluated by TLC and the radiochemical purity is calculated.

Having now fully described this invention, it will be understood to those of ordinary skill in the art that the same can be performed within a wide and equivalent range of conditions, formulations, and other parameters without affecting the scope of the invention or any embodiment thereof. All patents, patent applications, and publications cited herein are fully incorporated by reference herein in their entirety.

Claims

Wh at is Claimed Is:

1. A compound of Formula 7 or Formula 77:

or a salt thereof, wherein

X is N or CH;

R¹ and R² are selected from the group consisting of hydrogen, alkyl, and aralkyl, provided that at least one of R¹ and R² is hydrogen, where said alkyl and aralkyl may be optionally substituted;

R³ and R⁴ are hydrogen, or are taken together to form a keto group;

R⁵ and R⁶ are hydrogen, or taken together to form a keto group; m and n are independently 1 or 2;

R is hydrogen, C,_-6 alkyl, C₃.₇ cycloalkyl, C_6-K) aryl or C_M0 a^ ^alkyl, any of which is optionally substituted with one or two of amino, aminoalkyl, guanidinoalkyl, nitro, cyano, carboxy, halo, haloalkyl, trifluoromethyl, alkyl, alkoxy, hydroxy or hydroxyalkyl group; and

P^a is a sulfur protecting group or hydrogen.

2. A compound of claim 1, wherein R is hydrogen, methyl, ethyl, aminoethyl, aminopropyl, aminobutyl, aminocyclohexyl, chloromethyl, bromomethyl, chloroethyl, bromoethyl, chloropropyl, bromopropyl, hydroxyethyl, hydroxypropyl, phenyl, 4-aminophenyl, 4-amino-3- methylphenyl, 4-amino-3-chlorophenyl, 4-amino-3-bromophenyl, 4-aminomethylphenyl, 4- aminomethyl-3-methylphenyl, 4-aminomethyl-3-chlorophenyl, 4-amino-3-fluoromethylphenyl, halophenyl, benzyl, 4-aminobenzyl, 4-halobenzyl, 4-aminomethylbenzyl, 4-guanidinomethylbenzyl, 4-aminomethyl-3-fluorobenzyl, 4-aminomethyl-3-iodobenzyl, and 4-aminomethyl-3-methylbenzyl.

3. A compound of Formula F or Formula 77':

or a salt thereof, wherein

X is N or CH;

R¹ and R² are selected from the group consisting of hydrogen, alkyl, and aralkyl, provided that at least one of R¹ and R² is hydrogen, where said alkyl and aralkyl may be optionally substituted;

R³ and R⁴ are hydrogen, or are taken together to form a keto group;

R⁵ and R⁶ are hydrogen, or taken together to form a keto group; m and n are independently 1 or 2; P^a is a sulfur protecting group or hydrogen; L is a linking group; and B is a targeting group

4. A compound of claim 3, wherein

L is selected from alkyl, cycloalkyl, aminoalkyl, aminoaryl, carboxyalkyl, thioalkyl, amino, amido, carboxy, -O-, -S-, dithio, hydrazino, or a direct covalent bond; and

B is selected from an amino acid, amino acid side chain, peptide, protein, antibody, nucleic acid, steroid, lipid, saccharide, or cell membrane ligand.

5. A compound of claim 3 , wherein P^a are both hydrogen or both a sulfur protecting group selected from the group consisting of methoxymethyl, methoxyethoxymethyl, p- methoxybenzyl and benzyl.

6. A compound of claim 3, wherein R¹ and R² are selected from the group consisting of hydrogen, C .₆ alkyl, and

provided that at least one of R¹ and R² is hydrogen.

7. A compound of claim 3, wherein R¹ and R² together from a keto group; and R⁵ and R⁶ are both hydrogen.

8. A radiopharmaceutical complex, comprising a compound of claim 1, and a radioactive metal coordinated therewith.

9. A complex of claim 8, wherein the radionuclide is technetium-99m, rhenium- 186, or rhenium- 188.

10. A complex of claim 9 having Formula 777 or Formula IV:

where

X is N or CH; one of R' and R² is not present, and the other of R¹ and R² is hydrogen, alkyl, or aralkyl, where said alkyl and aralkyl may be optionally substituted;

R³ and R⁴ are hydrogen, or are taken together to form a keto group;

R⁵ and R⁶ are hydrogen, or taken together to form a keto group; m and n are independently 1 or 2; and R is hydrogen, or alkyl, cycloalkyl, alkenyl, alkynyl, aryl, or aralkyl, any of which is optionally substituted

11 A radiopharmaceutical complex, comprising a compound of claim 3, and a radioactive metal coordinated therewith

12 A complex of claim 11 , wherein the radionuclide is technetium-99m, rhenium- 186, or rhenium- 188

13 A process of radioimaging, comprising

(i) administering to a mammal an effective amount of a radiopharmaceutical complex of claim 11 in a pharmaceutically acceptable carrier or diluent, and

(ii) radioimaging said mammal after allowing sufficient time for said composition to localize in a tissue of said mammal

14 A kit for forming an injectable radiopharmaceutical composition, comprising (i) a compound of claim 3; and

(ii) a reducing agent and optionally a chelator

15 A process for forming a single stereoisomer of a radioactive metal complex of claim 11, comprising reacting a compound of claim 3 wherein P^a is hydrogen or a Lewis acid labile sulfur protecting group, with a radioactive metal in a oxidation state which is not required to be reduced or oxidized for formation of said complex, in an aqueous medium

16 A process for forming a complex of claim 12, wherein the radionuclide is technetium-99m, comprising

(a) dissolving a compound of claim 3 in a mixture of ethanol and hydrochloric acid containing a reducing agent selected from stannous chloride, stannous sulfate, stannous nitrate, stannous acetate, and stannous citrate,

(b) adding a salt of technetium-99m to said solution, and (c) heating said resulting reaction mixture for a predetermined time at a predetermined temperature, and then allowing the reaction solution to cool to room temperature.

17. A process according to claim 16, wherein said salt of technetium -99m is (⁹⁹ Tc) pertechnetate.

18. A method of performing a radiodiagnostic examination of a warm blooded living being, comprising:

(a) administering a radiopharmaceutical complex of claim 11 in a quantity of 0.1 to 30 mCi to said warm blooded living being and:

(b) recording the radioactive radiation emitted by said warm blooded living being by a radioactivity recording means.

19. A process according to claim 18, wherein said radioactivity recording means is a gamma camera.

20. A process according to claim 19, wherein said radiopharmaceutical complex is administered parenterally.

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