Tctagged compounds

Because it is difficult to rationally design Tc-essential radiopharmaceuticals that can target specific receptors, there has been a shift toward using biomol-ecules as a means of directing the biodistribution of Tc. This approach is non-trivial because Tc is a transition metal which complicates the labeling chemistry. In order to tag a biomolecule with Tc, the parent agent must have the appropriate arrangement of donor atoms which can form a stable metal complex or the targeting agent must be derivatized with a ligand that is capable of coordinating the radiometal.

Preparing Tc-tagged compounds requires that labeling not have a detrimental effect on the affinity of the parent targeting agent for the target receptor and that it not significantly alter the pharmacokinetics of the parent in an unfavorable manner. Furthermore, it is essential that the Tc complex remain intact in vivo so that the resulting images represent the distribution of the Tc biocon-jugate and not a fragment of the parent radiopharmaceutical.

Unlike 11C and 18F, which are commonly used positron-emitting radionu-clides, or 123I which (like 99mTc) is used for single photon emission computed tomography (SPECT), Tc cannot be directly bound to the carbon backbone of a targeting agent by replacing a hydrogen atom. For compounds possessing the appropriate arrangement of good donor atoms such as thiol, amino or amido groups, it is possible to perform a direct labeling with technetium. This approach has been used to tag proteins and monoclonal antibodies; however, the regioselectivity of labeling is typically low, resulting in the formation of multiple species. Direct labeling can also disrupt the targeting ability of the parent compound and is therefore not widely employed.

A more effective approach to tagging a targeting agent with Tc is to use a bifunctional chelate (BFC). A BFC is a ligand that has the appropriate combination of donor atoms and structure to form a well-defined and stable/inert metal (Tc) complex while also possessing an additional functional group for bioconjugation. Labeling can be performed by first forming the chelate-Tc complex and then undertaking a bioconjugation reaction (known as the pre-labeling approach) or by appending the free ligand to the molecule and then labeling with Tc (post-labeling). Post-labeling is the more attractive of the two methods for clinical applications because it reduces the number of steps associated with handling radioactive materials and it is more amenable to the development of instant kits.

There is a virtual cornucopia of BFCs for Tc that have been developed.27 30 Tc has eight accessible oxidation states which affords the opportunity to design structurally diverse ligand systems. The two main oxidation states that are most widely utilized to produce BFC complexes for nuclear medicine applications are Tc(V) and more recently Tc(I).

Bifunctional ligands of Tc(V)31 involve, predominately, coordination complexes of the {TcO}3+ core. This particular core is stabilized by a wide range of donors but has a preference for thiolate, amido and ^-donating alkoxide ligands, which help satisfy the high formal charge at the metal center. Chelates are typically tetradentate and form complexes having square pyramidal geometries. These include ligands that contain all-nitrogen donors, like the N4 propylene diamine dioxime (PnAO) type ligands and the oft-employed mixed nitrogen-sulfur ligands. Examples of the latter are triamidomonothiol (N3S), monoamine monoamide (N2S2) (Figure 18.3a),32,33 diamidodithiols (Figure 18.3b)34 and diaminodithiol ligands (Figure 18.3c).35 Amino acid analogues

Figure 18.3 Tc-N2S2 Chelate complexes

of these compounds have also been prepared; however, once a stereogenic center is introduced into the backbone of the ligand, coordination to Tc results in the formation of a mixture of isomers.36 These diastereomers, in which substituents about the stereogenic center are located syn or anti to the Tc-oxo bond, are often difficult to separate even at the macroscopic scale. For substituted PnAO37 and certain amino acid-based monoamide monamino Tc(V) complexes,38 even when the isomers are separated they have been shown to interconvert in solution.

A large number of different small-molecule-Tc(V)-chelate complexes have been prepared as novel radiotracers.39 These include derivatives of steroids, which are designed to target estrogen receptors expressed on breast cancers, and biological dyes, which are designed to target amyloid plaques associated with Alzheimer's disease. 99mTc TRODAT-1 (Figure 18.4),40 which is a tropane analogue containing a diaminodithiol ligand, deserves special notation as it is the first Tc complex to target specific receptors in the human brain. 99mTc TRODAT-1 has been used for imaging the central nervous system's (CNS) dopamine transporters (DAT) which is particularly useful for studying patients with Parkinson's disease.41

A limitation of tetradentate Tc(V) ligands arises when trying to label proteins and peptides, which are attractive targeting agents for delivering Tc to specific receptor systems (discussed later). Peptides can contain sequences of amino acids which are ideally set up to bind Tc(V) in a manner analogous to the ligands described above. This creates the same set of problems encountered with direct labeling in that the regioselectivity of labeling can be low thereby resulting in the formation of complex mixtures of products.

For tagging proteins and peptides with Tc, substituted hydrazine ligands, specifically hydrazinonicotinamides, were developed.42 These ligands form stable organohydrazino complexes with Tc (Figure 18.5a)43 even in the presence of competing thiolate ligands from cysteine-containing amino acids. The remaining coordination sites on Tc are occupied by a co-ligand (L). There are a wide variety of co-ligands that can be employed,44,45 including tricine, mannitol and trisodium triphenylphosphine-3',3",3"'-trisulfonate (TPPTS), which is a convenient means of modifying the hydrophilicity/hydrophobicity of the Tc conjugate.46

Figure 18.4 99mTc TRODAT-1

O MHR

NH N

NH N

NHBoc

Figure 18.5 (a) Hydrazinonicotinamide complex of Tc-containing co-ligands (L, L') and (b) active ester derivative of a protected form of the ligand for tagging amino groups

N-hydroxysuccinimidylhydrazinonicotinamide, which is commonly referred to as HYNIC, is the best-known Tc-hydrazine ligand. The active ester of a Boc-protected form of the ligand (Figure 18.5b) provides a convenient means for tagging amino groups on biomolecules. HYNIC conjugates of a variety of targeting agents including chemotactic peptides,47,48 somatostatin analogues,49 leukotriene B4 (LTB4)50 and vitronectin receptor antagonists,51 and large biomolecules like human IgG52 have been reported. It is therefore somewhat surprising that there are no approved radiopharmaceuticals derived from this particular ligand. One of the drawbacks of HYNIC is that producing macroscopic quantities of reference standards (99Tc or Re complexes) has proven to be difficult, which is a result of the complex coordination chemistry of the ligand.53 Notwithstanding, HYNIC continues to be one of the most widely employed ligands in Tc radiopharmaceutical chemistry.

Because of their large size, traditional Tc-chelate/ligand complexes can influence the 'homing' ability of targeting agents particularly for low molecular weight compounds. Furthermore, conventional Tc ligands can undergo redox and/or transchelation reactions in vivo resulting in premature loss of the radionuclide. These characteristics along with the increasing demand for radiopharmaceuticals that target specific receptor systems has motivated a search for new Tc synthons, which are smaller, more robust and have better-defined structures.

The Tc(I) isonitrile complexes on which Cardiolite™ is based are inert owing in part to their low-spin d6 electronic configuration. Tc(I) complexes would therefore appear to be ideal synthons from which to prepare receptor-targeted radiophar-maceuticals. Unfortunately the poly-substituted isonitrile complexes are difficult to monosubstitute and they are relatively non-reactive making them unsuitable starting materials from which to prepare Tc-tagged radiopharmaceuticals.

Alberto and co-workers reported the synthesis of [Tc(CO)3(OH2)3]+ as a practical and versatile Tc(I) synthon.54 In this complex, the three facially oriented water molecules are sufficiently labile that they can be readily displaced by a variety of mono-, bi- and tridentate ligands (discussed later). The synthesis of the Tc(I) precursor involves reduction of TcO4 with borohydride in the presence of CO (Scheme 18.1). The Tc(CO)^ core can be prepared at the macroscopic scale with 99Tc and at the tracer level using 99mTc. A further innovation was the development of a kit for [99mTc(CO)3(OH2)3]+, which contains potassium boranocarbonate (K2H3BCO2); a compound that acts as both a reducing agent and a source of CO.55

TcO4

NaBH4, CO

Scheme 18.1

The [Tc(CO)3]+ core interacts with a broad range of different ligands including thioureas, isonitriles and phosphines. The resulting complexes are for the most part octahedral, d®-low spin and are therefore typically inert, which is ideal for radiopharmaceutical applications. Aliphatic amines and carboxylates coordinate rapidly with [Tc(CO)3]+; however, the resulting complexes are more reactive than complexes containing soft donors. Thioethers, for example, form inert complexes but do so at a very slow rate. A compromise between the rapid coordination of hard donors and the inertness of complexes containing soft donors can be found for ligands that contain aromatic amines, including substituted pyridines and imidazoles.56

Access to [Tc(CO)3(OH2)3]+ is transforming Tc radiopharmaceutical chemistry because it provides a means to append inert and low molecular weight Tc complexes to targeting vectors.57,58 There are a number of bifunctional Tc(I) ligands that are now available for preparing bioconjugates (Figure 18.6). These include N2O, N3 and NSN type ligands which form neutral or cationic complexes with the [Tc(CO)3]+ core.59,60 Schibli and co-workers investigated the reaction of the [Tc(CO)3]+ core with a variety of bi- and tridentate pyridine- or imidazole-type ligands to gain insight into the optimal chelate type and 'denticity' for radiopharmaceutical development.61 Their study revealed that oc„ i -N

X CO2H 0C...Jc^ C02HocJxnb

Figure 18.6 Examples of tridentate ligand complexes of Tc(I)

both the bidentate and tridentate chelates bound rapidly with the [Tc(CO)3]+core on a macroscopic scale and at the tracer level. The tridentate systems were shown to be superior for developing Tc-tagged compounds because they demonstrated enhanced stability in ligand challenge experiments and the complexes remained intact in vivo. The bidentate analogues, in contrast, decomposed after being incubated with histidine and cysteine for 24 h and they showed significant binding to plasma after one hour of incubation in human blood.

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