From Symmetrical to Asymmetrical Nitrido Phosphino-thiol Complexes

Feb 21, 2006 - ICIS - CNR, Corso Stati Uniti, 4, 35127 Padova, Italy, Laboratory of Nuclear Medicine, Department of Clinical &. Experimental Medicine ...
0 downloads 0 Views 237KB Size
Download PDF
Bioconjugate Chem. 2006, 17, 419−428

419

From Symmetrical to Asymmetrical Nitrido Phosphino-thiol Complexes: A New Class of Neutral Mixed-Ligand 99mTc Compounds as Potential Brain Imaging Agents Cristina Bolzati,*,‡ Elisa Benini,† Mario Cavazza-Ceccato,§ Emiliano Cazzola,† Erica Malago`,‡ Stefania Agostini,§ Francesco Tisato,‡ Fiorenzo Refosco,‡ and Giuliano Bandoli§ ICIS - CNR, Corso Stati Uniti, 4, 35127 Padova, Italy, Laboratory of Nuclear Medicine, Department of Clinical & Experimental Medicine, University of Ferrara, Via Borsari, 46, 44100 Ferrara, Italy, and Department of Pharmaceutical Sciences, University of Padova, Via Marzolo, 5, 35131 Padova, Italy. Received December 20, 2005

A general procedure is presented for the preparation of a new class of nitrido asymmetrical Tc-99m complexes containing two different bidentate ligands bound to the same [Tc(N)]2+ core that could be used to design either essential or target specific imaging agents. This procedure is based on the chemical properties of a new monosubstituted [Tc(N)(R2PS)Cl(PPh3)] species composed of a TctN multiple bond and an ancillary phosphine thiol ligand (R2PSH). This intermediate readily reacts with bidentate mononegative ligands (S∧Y) containing soft π-donor coordinating atoms to give neutral pentacoordinate asymmetrical complexes of the type [Tc(N)(R2PS)(S∧Y)]. The ability of several bidentate ligands containing different combination of heteroatoms (S, N, O) to form complexes with the [Tc(N)(R2PS)]+ building block was investigated. It was found that mononegative dithiocarbamate (DTC) or cysteine carboxyl derivate ligands promptly react with the monosubstituted species to form the final mixed compound in high yield. Preliminary biodistribution data in rats of some representative [Tc(N)(R2PS)(DTC)] compounds revealed an interesting initial brain uptake (in the range 0.20 ( 0.01% ID/g and 0.91 ( 0.06% ID/g), indicating their ability to cross in and out of the intact BBB. In these complexes the dithiocarbamate, or more generally the bidentate ligand (S∧Y), can be designed to carry a functional group or a bioactive molecule, which could be involved in a trapping mechanism to increase brain retention for longer time intervals. These results could be conveniently utilized to devise a new procedure for the production of a novel class of brain perfusion and/or brain receptor imaging agents.

INTRODUCTION 99mTc-based

Over the past few years, several radiopharmaceuticals have been approved by the FDA for determining organ functions or assessing disease status by imaging methods. Despite this, the development of new 99mTc-based brain perfusion or brain receptor imaging agents, which can play an important role in the diagnosis of various cerebrovascular, neurological, and neuropsychiatric disorders, remains a subject of great interest in the radiopharmaceutical field. At present, Tc-99m-based radiopharmaceuticals used in the clinic for the assessment of brain perfusion are [99mTc]-HMPAO and [99mTc]-ECD, whereas [99mTc]-TRODAT-1 represents the first successful reported example of a Tc-based tracer for brain receptor imaging. Since these radiopharmaceuticals are far from ideal, the efforts to find more efficient Tc-99m brain perfusion and new CNS receptor imaging agents continue. Generally, these agents must possess certain basic characteristics in order to cross the intact blood-brain barrier (BBB). They must be stable in vitro and in vivo, neutral in charge, lipophilic in character (Log P octanol/water in the range 1.02.5), and a molecular weight lower than 600 Da. Moreover, after crossing the BBB, a potential Tc-99m perfusion imaging agent must be retained in the tissue for a sufficient amount of time to allow SPECT imaging, via a trapping mechanism which * Author for correspondence. Phone +39 049 8275352. Fax +39 049 8275366. E-mail: [email protected]. ‡ ICIS - CNR. † University of Ferrara. § Department of Pharmaceutical Sciences.

typically involves enzymatic or chemical conversion into a more hydrophilic compound that prevents the agent from recrossing the BBB in the opposite direction. In addition, a potential Tc99m receptor imaging agent must have the appropriate molecular shape to fit correctly with the target receptor site (IC50 value 90%), through a two-step procedure (methods 1, 2). The first step involved the preliminary conversion of the starting [99mTcO4]- into a mixture of prereduced species containing the [Tc(N)]2+ multiple bound. The second step, which took place in basic media, required the addition of 0.25-1 mg of the bidentate phosphino-thiol ligand (R2PSH).

Scheme 1

Table 2. Variation of % RCY in Formation of [99mTc(N)(R2PS)2] Complexes, at Different Times, as Function of pH, Temperature, and Amount of R2PSH Ligand [Tc(N)(PScy)2] (% RCY) PScyH (mg)

pH

temp (°C)

15 min

30 min

60 min

0.050 0.050 0.050 0.050 0.050 1

9 7 5 2 2 2

RT RT RT RT 100 RT

73.0 60.4 57.3 40.5 85.0

78.0 78.5 69.0 57.9 65.1

91.0 87.2 85.8 85.0 94.9

[Tc(N)(PSiso)2] (% RCY) PSisoH (mg)

pH

temp (°C)

15 min

30 min

60 min

0.025 0.025 0.025 0.025 0.025 1 1

9 7 5 2 2 9 2

RT RT RT RT 100 RT RT

73.0 86.4 84.2 40.5 78.1 95.0

75.0 88.5 87.2 57.9 80.1 95.1

71.0 87.2 87.4 85.0 78.9 94.9

The reaction was carried out using different procedures depending on the reducing agents, the source of nitride (N3-), and the pH employed (Scheme 1). Coordination of R2PSH was achieved at basic pH after 15 min at 100 °C. Lower temperatures (50 °C) required prolonged reaction times. Acidic media caused the reaction to be incomplete; in particular when SDH and PPh3 were used to produce the intermediate nitrido compounds, different species were obtained (vide infra). Following this latter procedure, the influence of pH, temperature, and concentration of R2PSH on the RCY formation of [99mTc(N)(R2PS)2] (R ) C6H11 and CH(CH3)2) was monitored at different times. The results are illustrated in Table 2. Alternatively, due to the intrinsic reducing power of the phosphino-thiol ligands, disubstituted symmetrical compounds may be obtained directly with a one-step procedure (method 3) using these ligands as reducing and coordinating agents. In this case an increased amount of R2PSH (ca. 1-2 mg) was required to guarantee a radiochemical yield >85%. However, this reduction-substitution procedure gave low yield of formation (90% purity of the injected radiolabeled compounds. Tissue distribution data of neutral disubstituted symmetrical compounds [99mTc(N)(R2PS)2] are summarized in Table 3. Our results show that the substituents at the phosphorus atom of the R2PSH ligand influence the biodistribution pattern. Notable differences were observed in blood, liver, and kidney accumulations and especially in the brain uptake. For all these compounds a rapid blood clearance is generally observed. The high liver uptake suggests that the hepatobiliary system is the major route of excretion of the administered agents. In detail, the [99mTc(N)(PSiso)2] complex revealed the highest brain uptake (1.28 ( 0.02% ID/g) at 2 min postinjection (p.i.), with a significant brain-to-blood ratio (B/bl ) 4), though, the initial high brain uptake was followed by a rapid wash-out and at 20 min p.i. the brain activity dropped to 0.20 ( 0.02% ID/g. The more lipophilic [99mTc(N)(PStbu)2] complex showed initial brain uptake (0.71 ( 0.02% ID/g; 2 min p.i.), but a slower brain elimination (0.23 ( 0.06% ID/g; 60 min p.i.), indicating that by increasing the lipophilicity of the complex it is possible to reduce the wash-out from the brain tissue. Biodistribution of some representative neutral [99mTc(N)(R2PS)(S∧S)] mixed compounds are summarized as % of dose/g in Table 4. The data show that, at 2 min p.i., all the investigated compounds were distributed throughout the body with a similar pattern, independently from the nature of the two bidentate ligands. The complexes exhibited a rapid blood clearance and, despite their high lipophilicity, the radioactivity was eliminated through the liver and intestines as well as the kidneys and the urine. A significant initial high extraction by the myocardial tissue was observedfor all [99mTc(N)(R2PS)(S∧S)] compounds (in the range 5.48 ( 0.09%ID/g; 2.48 ( 0.35% ID/g) followed by a rapid wash-out (0.55 ( 0.09% ID/g; 0.29 ( 0.01% ID/g). All investigated complexes show a transient but significant initial brain uptake at 2 min p.i. with the activity in the range 0.20 ( 0.01% ID/g and 0.91 ( 0.06% ID/g. A brain-to-blood ratio > 1 was observed at 2 and 10 min p.i., demonstrating that these compounds quickly diffuse across the BBB, probably through passive diffusion due to their lipophilicity.

DISCUSSION A systematic investigation on the reactivity of bidentate phosphino-thiol ligands (R2PSH where R ) alkyl groups) toward

426 Bioconjugate Chem., Vol. 17, No. 2, 2006

Bolzati et al.

Table 3. Biodistribution of Some Representative Disubstituted [Tc(N)(R2PS)2] Compounds, as % Dose/g in Rats (n ) 3) at Different Times [99mTc(N)(PSme)2]

[99mTc(N)(PSiso)2]

organ

0 min

2 min

20 min

0 min

2 min

20 min

blood brain heart lungs liver kidneys intestine muscle brain/blood

1.94( 0.82 0.64( 0.02 2.96 ( 0.19 1.92 ( 0.04 0.86( 0.03 4.05 ( 0.96 1.35 ( 0.43 0.13( 0.04 0.33

0.90 ( 0.21 0.65 ( 0.02 0.98 ( 0.12 0.90 ( 0.09 1.62 ( 0.20 1.32 ( 0.22 1.14 ( 0.22 0.34 ( 0.07 0.72

0.44 ( 0.03 0.19 ( 0.03 0.56( 0.01 0.59 ( 0.02 1.19 ( 0.08 0.87 ( 0.03 2.20( 0.26 0.45 ( 0.02 0.43

0.59( 0.14 1.18 ( 0.17 2.44 ( 1.03 1.46 ( 0.24 1.73 ( 0.30 3.43 ( 0.91 1.47 ( 0.43 0.15 ( 0.03 2.00

0.32 ( 0.04 1.28 ( 0.09 1.36 ( 0.39 0.90 ( 0.13 2.95 ( 0.40 1.56 ( 0.42 2.48 ( 0.50 0.17 ( 0.05 4.00

0.23 ( 0.01 0.08 ( 0.01 0.28 ( 0.04 0.35 ( 0.06 1.81 ( 0.30 0.56 ( 0.13 13.69 ( 6.10 0.25 ( 0.05 0.35

[99mTc(N)(PSibu)2]

[99mTc(N)(PStbu)2]

organ

0 min

2 min

20 min

0 min

2 min

20 min

blood brain heart lungs liver kidneys intestine muscle brain/blood

1.38 ( 0.23 0.20 ( 0.04 2.90 ( 0.08 1.94 ( 0.23 2.68 ( 0.41 2.24 ( 0.01 0.58 ( 0.04 0.28 ( 0.06 0.14

0.32 ( 0.04 0.15 ( 0.01 1.97 ( 0.08 1.14 ( 0.15 4.43 ( 0.26 1.94 ( 0.11 1.74 ( 0.06 0.37 ( 0.18 0.47

0.20 ( 0.06 0.09 ( 0.01 0.62 ( 0.03 0.45 ( 0.04 2.75 ( 0.09 0.47 ( 0.01 10.91 ( 3.63 0.41 ( 0.06 0.45

2.80 ( 0.77 0.83 ( 0.01 3.60 ( 0.75 3.27 ( 0.89 4.75 ( 0.78 4.25 ( 0.53 1.68 ( 0.17 0.48 ( 0.12 0.29

1.06 ( 0.13 0.71 ( 0.02 1.93 ( 0.03 1.62 ( 0.02 7.02 ( 0.62 3.03 ( 0.37 4.30 ( 0.18 0.65 ( 0.06 0.66

0.43 ( 0.01 0.36 ( 0.08 0.97 ( 0.23 0.83 ( 0.16 5.23 ( 0.95 1.16 ( 0.22 13.9 ( 3.03 0.50 ( 0.04 0.83

Table 4. Biodistribution of Some Representative Asymmetrical [Tc(N)(S∧Y)(R2PS)] Compounds, as %Dose/g, in Rats (n ) 3) at Different Times [99mTc(N) (PScy) (DTC1)]

[99mTc(N) (PSiso)(DTC1)]

organ

0 min

2 min

10 min

20 min

0 min

2 min

20 min

blood brain heart liver kidneys intestine brain/blood

1.52( 1.13 0.26( 0.03 5.48 ( 0.09 3.89( 0.22 4.42 ( 0.47 1.45 ( 0.01 0.166

0.23 ( 0.05 0.20 ( 0.01 3.79 ( 0.52 5.53 ( 1.0.35 4.29 ( 0.55 1.50 ( 0.30 0.87

0.16 ( 0.03 0.18 ( 0.03 1.88 ( 0.06 5.43 ( 0.90 2.58 ( 0.05 0.29(0.25 1.13

0.15 ( 0.03 0.10 ( 0.01 0.55( 0.07 4.52 ( 1.06 1.25 ( 0.26 5.87(0.43 0.66

4.24 ( 1.90 0.31( 0.04 2.48 ( 0.35 0.18 ( 0.04 1.26 ( 0.21 0.33 ( 0.06 0.07

0.33 ( 0.02 0.56 ( 0.03 1.24 ( 0.07 3.81 ( 0.09 1.87 ( 0.03 1.57 ( 0.07 1.69

0.17 ( 0.02 0.08 ( 0.01 0.29 ( 0.01 2.98 ( 0.09 0.56 ( 0.02 4.38 ( 0.91 0.47

[99mTc(N) (PSiso)(DTC4)]

[99mTc(N) (PSiso)(PSS2)]

organ

0 min

2 min

20 min

0 min

2 min

20 min

blood brain heart liver kidneys intestine brain/blood

1.14 ( 0.59 0.61 ( 0.28 4.81 ( 0.73 0.42 ( 0.20 2.22 ( 1.29 1.40 ( 1.08 0.54

0.27 ( 0.07 0.96 ( 0.14 1.92 ( 0.47 3.94 ( 1.85 2.37 ( 0.57 3.90 ( 2.59 3.56

0.17 ( 0.03 0.13 ( 0.02 0.36 ( 0.03 3.30 ( 0.33 0.68 ( 0.03 7.61 ( 2.22 0.76

0.99( 0.35 0.83 ( 0.09 3.70 ( 0.66 1.84 ( 0.69 4.73 ( 0.96 1.77 ( 0.16 0.84

0.53 ( 0.02 0.70 ( 0.04 2.05 ( 0.36 3.89 ( 0.52 2.39 ( 0.40 2.29 ( 0.35 1.32

0.36 ( 0.05 0.26 ( 0.01 0.44 ( 0.01 4.90 ( 0.25 1.00 ( 0.10 5.60 ( 0.59 0.72

[99mTc(N) (PSiso)(DTC3)]

[99mTc(N) (PSibu)(DTC3)]

organ

0 min

2 min

20 min

0 min

2 min

20 min

blood brain heart liver kidneys intestine brain/blood

0.58 ( 0.09 0.86 ( 0.06 4.78 ( 0.76 1.63 ( 0.74 4.04 ( 0.40 1.60 ( 0.13 1.48

0.25 ( 0.06 0.91 ( 0.06 1.25 ( 0.43 3.20 ( 0.39 1.61 ( 0.14 2.18 ( 0.21 3.64

0.20 ( 0.05 0.11 ( 0.03 0.39 ( 0.08 3.34 ( 0.20 0.65 ( 0.10 5.53 ( 0.14 0.55

1.42 ( 0.40 1.11 ( 0.14 4.02 ( 1.37 1.99 ( 0.66 5.54 ( 1.21 1.35 ( 0.38 0.78

0.30 ( 0.02 0.76 ( 0.11 2.17 ( 0.61 4.19 ( 0.72 2.93 ( 0.31 1.78 ( 0.15 2.53

0.26 ( 0.01 0.23 ( 0.01 0.80 ( 0.01 3.71 ( 0.24 1.05 ( 0.08 4.59 ( 3.63 0.88

nitrido-99mTc precursors led to the preparation of pentacoordinate symmetrical [99mTc(N)(R2PS)2] and asymmetrical [99mTc(N)(R2PS)(S∧Y)] complexes. [99mTc(N)(R2PS)2] complexes were prepared following the methods reported in Scheme 1. The best labeling procedure involved ligand-exchange reactions performed in basic media (methods 1, 2) using a very low amount of phosphino-thiol (∼10-4 mmol). The basic pH was essential in guaranteeing the quantitative formation of the disubstituted products, indicating that the coordination of the R2PSH ligand probably occurred through the initial nucleophilic attachment of the deprotonated thiol sulfur to the metal center. Lower radiochemical yields were obtained using the R2PSH ligand as both a reducing and coordinating agent (one-step procedure, method 3), because of the concomitant formation of the corresponding Tc(III) compounds [99mTc(R2PS)3] (20).

The chemical identity of [99mTc(N)(R2PS)2] complexes was established by HPLC comparison with the corresponding compounds prepared at macroscopic level with the long-lived isotope Tc-99g (Figure 3a). The remarkable in vitro stability of the disubstituted compounds is determined by the combination of two π-acceptor (P) and two π-donor (S-) atoms around the [Tc(N)]2+ core which confer high thermodynamic stability and kinetic inertness. Biological evaluation of these agents showed encouraging results. The [99mTc(N)(PSiso)2] compound showed the best brain uptake of the series with a significant brain-to-blood ratio (B/ bl ) 4) at 2 min p.i. (Table 3). However, the initial high brain uptake was followed by a rapid wash-out, and at 20 min p.i. brain activity dropped to 1/6 of its initial value. Data collected for the more lipophilic [99mTc(N)(PStbu)2] species revealed that

Bioconjugate Chem., Vol. 17, No. 2, 2006 427

Nitrido Phosphino-thiol Complexes

it was possible to modulate the kinetic of the wash-out, increasing the permanence of the complex in the tissue. These results suggested that the diffusion across the BBB was a reversible process for this class of complexes, and that these compounds moved freely in and out of the brain. This behavior could be attributed to the absence of an efficient trapping mechanism that prevents the complex from crossing the BBB in the opposite direction. In connection, while the lipophilicity of [Tc(N)(R2PS)2] symmetrical compounds can be modified by varying the substituents on the phosphorus donor or on the R2PSH backbone, the introduction of pendant groups that could be involved in trapping mechanisms required instead quite complicated and difficult synthesis. Considering the recent demonstration that other disubstituted complexes of the type [99mTc(N)(S∧Y)2], where S∧Y is a dithiocarbamate (DTC) or a dithiophosphinate (PSS) ligand, showed an appreciable accumulation in the cerebral tissue (25, 27, 28), we decided to design a new class of CNS agents which combined the biological properties of [99mTc(N)(S∧Y)2] and [99mTc(N)(R2PS)2] compounds through the preparation of mixed pentacoordinate species of the type [99mTc(N)(R2PS)(S∧Y)]. In these complexes the co-ligand S∧Y offered the possibility of being modified with an appropriate functional group designed for trapping mechanism in order to increase the brain retention for longer time intervals. The possibility of introducing only one R2PSH ligand onto a [Tc(N)]2+ group was accomplished with bulky aliphatic substituents to the phosphine phosphorus under particular reaction conditions. In fact, the existence of a monosubstituted phosphino thiolato complex [99gTc(N)Cl(R2PS)(PPh3)] has already been demonstrated at macroscopic level using the encumbering 2-(dicyclohexylphosphino)ethanethiol (PScyH) ligand (21). The subsequent replacement of the monodentate chloride and PPh3 ligands with a mononegative chelate (S∧Y) such as dithiocarbamate (DTC) permitted the preparation of neutral disubstituted asymmetrical complexes of the type [99gTc(N)(R2PS)(DTC)]. They represent prototypes of a new class of complexes characterized by the presence of the [Tc(N)(R2PS)]+ building block (22). Identical asymmetrical monosubstituted [99mTc(N)Cl(R2PS)(PPh3)] and disubstituted [99mTc(N)(R2PS)(DTC)] species were efficiently prepared at tracer level, following the procedures indicated in Scheme 2. The acidic medium was essential in obtaining the monosubstituted compounds. In fact, high proton concentration prevented the thiol group of the R2PSH ligand from being deprotonated, thus reducing its affinity toward the metal center. On the contrary, as described above, basic conditions favored the substitution of chlorine and PPh3 ligands giving the substitution inert [99mTc(N)(R2PS)2] symmetrical complexes. In addition, the use of PPh3 was also found to be a key factor in the redox synthesis and in the stabilization of the monosubstituted compounds. In particular in the one step procedure (method b), the complex was obtained though a redox-exchange reaction, by adding pertechnetate to a vial containing SDH, PPh3 and the R2PSH, in the presence of H+. Despite the recognized ability of the phosphino thiol ligands to work both as reducing and coordinating agents, the small amount (∼10-4 mmol) of R2PSH used in this preparation was not enough to guarantee reduction of the pertechnetate. Thus, reduction was performed by PPh3 that simultaneously acts with R2PSH as coordinating agent to form [99mTc(N)Cl(R2PS)(PPh3)] compounds. Addition of the appropriate bidentate mononegative ligand (S∧Y) to the reaction vial containing the monosubstituted species (15 min, at room temperature) gave the final neutral [99mTc(N)(R2PS)(S∧Y)] complexes in high yield. These results suggest that the monosubstituted [Tc(N)Cl(R2PS)(PPh3)] complex rep-

resents the key intermediate for the synthesis of this new class of mixed technetium species. In general, the intermediate complex prefers to react with S∧Y ligands having ‘soft’ σ-,π-donor combinations such as [S-, S] or [S-, NH2] pairs, whereas ligands containing at least one ‘hard’ σ-donor atom, such as [O-, S] or [S-, O] combinations, do not react at all. The ability to form the [99mTc(N)(R2PS)(S∧Y)] species (R ) CH(CH3)2), expressed as % of RCY, is as follows:

DTC[94%] > PSS[90%] > DTCOX[88%] > SN[85%] g SN-Cys[83%] In vitro stability studies demonstrated that these mixed agents were stable for hours in aqueous solution in the presence of β-HPC and inert toward tranchelation in the presence of GSH (10 mM) or cysteine (1 mM). This indicates that the combination of π-acceptor and π-donor coordinating atoms is responsible for the stability and kinetic inertness of the resulting mixed complexes in this case as well. No significant interaction with the serum protein was observed. In vivo biodistribution data of some representative [99mTc(N)(R2PS)(S∧Y)] compounds displayed a similar pattern, comparable to those exhibited by the symmetrical [99mTc(N)(PSiso)2] complex. All investigated compounds showed a transient brain uptake and encouraging B/bl ratio >1, indicating that this class of mixed complexes retains the ability to freely cross the intact BBB. The initial brain uptake of these tracers was apparently driven by their lipophilic character as we have already demonstrated for the disubstituted compounds. Data collected for the complexes having a Log k0′ value around 4.4 ([99mTc(N)(PSibu)(DTC3)] and [99mTc(N)(PSiso)(PSS2)]) revealed that the kinetic of the wash-out from the brain tissue was decreased. Prolonged brain retention must still be achieved, but the possibility of introducing specific groups onto the S∧Y ligand in mixed complexes allows for the designing of updated agents which may be subject to the trapping mechanism inside the cerebral tissue. Studies aimed toward this goal are currently in progress (29).

CONCLUSIONS This study devises a general procedure for the preparation of a new class of nitrido asymmetrical Tc-99m complexes containing two different bidentate ligands bound to the same [Tc(N)]2+ core, which could be used to design either essential or target specific imaging agents. This procedure is based on the chemical property of a new monosubstituted [Tc(N)Cl(R2PS)(PPh3)] species which represents the key intermediate for the development of a new class of neutral pentacoordinate asymmetrical nitrido compounds of the type [Tc(N)(R2PS)(S∧Y)], characterized by the presence of a TctN terminal group, an ancillary P∧S ligand and a bidentate mononegative co-ligand (S∧Y), such as a dithiocarbamate. In these complexes the phosphino-thiol ligand stabilizes the metal oxidation state, whereas the S∧Y chelate, designed to carry a functional group or a bioactive molecule, completes the metal coordination sphere. The molecular weight of the [Tc(N)(R2PS)]+ building block (about 300 Da) allows the molecular weight of the final compound to be maintained within the limit required to cross the BBB. Moreover, the bidentate ligand S∧Y may be used to carry additional functional groups which are useful for the trapping mechanism such as ‘pH shift′, enzymatic degradation mechanism, and receptor specific interactions, to increase brain retention for longer time intervals. The flexibility of this system due to the possibility of changing the substituents at the P atoms and/or the bidentate ligand may

428 Bioconjugate Chem., Vol. 17, No. 2, 2006

be utilized to design a wide range of TctN mixed compounds and to modulate their biological properties. The discovery that the amino acid cysteine exhibited encouraging coordinating properties toward the [Tc(N)(R2PS)]+ intermediate suggests the use of this chelating system for incorporating a short peptide in a Tc-99m asymmetrical complex.

ACKNOWLEDGMENT The authors are grateful to Mr. Mariano Schiavon for his assistance in the animal studies.

LITERATURE CITED (1) Kung, H. F., Kung, M. P., and Choi, S. R. (2003) Radiopharmaceuticals for Single-Photon Emission Computed Tomography Brain Imaging. Sem. Nucl. Med. 33, 2-13. (2) Kung, H. F., Yu, C. C., Billings, J., Molnar, M., and Blau, M. (1985) Synthesis of New Bis(Aminoethanethiol) (BAT) Derivatives: Possible Ligands for 99mTc Brain Imaging Agents. J. Med. Chem. 28, 1280-1284. (3) Hom, R. K., and Katzenellenbogen, J. A. (1997) Technetium-99mTcLabeled Receptor-Specific Small-Molecule Radiopharmaceuticals: Recent Developments and Encouraging Results. Nucl. Med. Biol. 24, 485-498. (4) Boschi, A., Uccelli, L., Duatti, A., Bolzati, C., Refosco, F., Tisato, F., Romagnoli, R., Baraldi, P. G., Varani, K., and Borea, P. A. (2003) Asymmetrical Nitrido Tc-99m Heterocomplexes as Potential Imaging Agents for Benzodiazepine Receptors. Bioconjugate Chem. 14, 1279-1288. (5) Alberto, R., Schibli, R., Waibel, R., Abram, U., and Schubiger, A. P. (1999) Basic aqueous chemistry of [M(OH2)3(CO)3]+ (M ) Re, Tc) directed towards radiopharmaceutical application. Coord. Chem. ReV. 190-192, 901-919. (6) Egli, A., Alberto, A., Tannahill, L., Schibli, R., Abram, U., Schaffland, A., Waibel, R., Tourwe’, D., Jeannin, L., Iterbeke, K., and Schubiger, P. A. (1999) Organometallic 99mTc-aquaion labels peptide to an unprecedent high specific activity. J. Nucl. Med. 40, 1913-1917. (7) Spies H., Fietz Th., Glaser M., Pietzsch H. J., and Johannsen, B. (1995) The ‘n + 1’ concept in the synthesis strategy of novel technetium and rhenium tracer. In Technetium and Rhenium in Chemistry and Nuclear Medicine 4 (Nicolini, M., Bandoli, G., and Mazzi, U., Eds.) pp 243-246, SGEditoriali, Padova, Italy. (8) Johannsen, B., Berger, R., Brust, P., Pietzsch, H. J., Scheunemann, M., Seifert, S., Spies, H., and Syher, R. (1997) Structural modification of receptor-binding technetium-99m complexes in order to improve brain uptake. Eur. J. Nucl. Med. 24, 316-319. (9) Bolzati, C., Boschi, A., Duatti, A., Prakash, S., Uccelli, L., Refosco, F., Tisato, F., and Bandoli, G. (2000) Geometrically Controlled Selective Formation of Nitrido Technetium(V) Asymmetrical Heterocomplexes with Bidentate Ligands. J. Am. Chem. Soc. 122, 45104511. (10) Bolzati, C., Boschi, A., Uccelli, L., Tisato, F., Refosco, F., Cagnolini, A., Duatti, A., Prakash, S., Bandoli, G., and Vittadini, A. (2002) Chemistry of the strong electrophilic metal fragment [99Tc(N)(PXP)]2+ (PXP ) Diphosphine ligand). A novel tool for the selective labeling of small molecules. J. Am. Chem. Soc. 124, 11468-11479. (11) Boschi, A., Bolzati, C., Benini, E., Malago`, E., Uccelli, L., Duatti, A., Piffanelli, A., Refosco, F., and Tisato, F. (2001) A novel approach to the high-specificic-activity labeling of small peptides with the technetium-99m fragment [99mTc(N)(PXP)]2+ (PXP ) Diphosphine ligand). Bioconjugate Chem. 12, 1035-1042. (12) Boschi, A., Bolzati, C., Uccelli, L., Duatti, A., Benini, E., Refosco, F., Tisato, F., and Piffanelli, A. (2002) A class of asymmetrical nitrido 99mTc heterocomplexes as heart imaging agents with improved biological properties. Nucl. Med. Commun. 23, 689-693. (13) Boschi, A., Uccelli, L., Bolzati, C., Duatti, A., Sabba, N., Moretti, E., Di Domenico, G., Zavattini, G., Refosco, F., and Giganti, M. (2003) Biological Evaluation of monocationic Asymmetrical Nitride Tc-99m Heterocomplexes Showing high Heart Uptake and Improved Imaging Properties. J. Nucl. Med. 44, 806-814.

Bolzati et al. (14) Hatada, K., Riou, L. M., Ruiz, M., Yamamichi, Y., Duatti, A., Lima, R. L., Goode, A. R., Watson, D. D., George A., Beller, G. A., and Glover, D. K. (2004) 99mTc-N-DBODC5, a New Myocardial Perfusion Imaging Agent with Rapid Liver Clearance: Comparison with 99mTc-Sestamibi and 99mTc-Tetrofosmin in Rats. J. Nucl. Med. 45, 2095-2101. (15) Bolzati, C., Benini, E., Cazzola, E., Jung, C. M., Tisato, F., Refosco, F., Pietzsch, H. J., Spies, H., Uccelli, L., and Duatti, A. (2004) Synthesis, Characterization and Biological Evaluation of Neutral Nitrido Technetium(V) Mixed Ligand Complexes Containing Dithiolates and Aminodiphosphines. A Novel System for Linking Technetium to Biomolecules. Bioconjugate Chem. 15, 628-637. (16) Bolzati, C., Refosco, F., Cagnolini, A., Tisato, F., Boschi, A., Duatti, A., Uccelli, L., Dolmella, A., Marotta, E., and Tubaro, M. (2004) Synthesis, Solution- State and Solid-State Structural Characterization on Monocationic Nitrido Heterocomplex [M(N)(DTC)(PNP)]+ (M ) 99Tc, Re; DTC ) Dithiocarbammate; PNP ) Heterodiphosphine) Eur. J. Inorg. Chem. 1902-1913. (17) Tisato, F., Refosco, F., Porchia, M., Bolzati, C., Bandoli, G., Dolmella, A., Duatti, A., Boschi, A., Jung, C. M., Pietzsch, H. J., and Kraus, W. (2004) The Crucial Role of the Diphosphine Heteroatom X in the Stereochemistry and Stabilization of the Substitution-Inert [M(N)(PXP)]2+ Metal Fragments (M ) Tc, Re; PXP ) Diphosphine Ligand) Inorg. Chem. 43, 8617-8625. (18) Bolzati, C., et al. Unpublished data. (19) Bolzati, C., Boschi, A., Uccelli, L., Malago`, E., Bandoli, G., Tisato, F., Refosco, F., Pasqualini, R., and Duatti, A. (1999) Synthesis of a Novel Class of Trigonal Bipyramidal Nitrido Tc(V) Complexes with Phosphino-thiol Ligands. Crystal Structure of [99gTc(N)(L1)2] [L1 ) 2-(Diphenylphosphino)-ethanethiolato] and [99gTc(N)(L5)2] [L5 ) 2-(Diphenylphosphino)-propanethiolato]. Inorg. Chem. 38, 44734479. (20) Bolzati, C., Uccelli, L., Boschi, A., Malago`, E., Duatti, A., Tisato, F., Refosco, F., Pasqualini, R., and Piffanelli, A. (2000) Synthesis of a Novel Class of Nitrido Tc-99m Radiopharmaceutical with Phosphino-Thiol Ligands Showing Transient Heart Uptake. J. Nucl. Med. Biol. 27, 369-374. (21) Bolzati, C., Malago`, E., Boschi, A., Cagnolini, A., Porchia, M., and Bandoli, G. (1999) Symmetric bis-substituted and asymmetric mono-substituted nitridotechnetium complexes with heterofunctionalized phosphino-thiolate ligands. New J. Chem. 23, 807-809. (22) Bolzati, C., et al., manuscript in preparation. (23) Duatti, A., Marchi, A., and Pasqualini, R. (1990) Formation of the TctN multiple bond from ammonium pertechnetate with S-methyl dithiocarbazate and its application to the preparation of technetium-99m radiopharmaceuticals. J. Chem. Soc., Dalton Trans. 3729-3733. (24) Pasqualini, R., Duatti, A., Bellande, E., Comazzi, V., Brucato, V., Hoffschir, D., Fagret, D., and Comet, M. (1994) Bis(dithiocarbamato) nitrido technetium-99m radiopharmaceuticals: a class of neutral myocardial imaging agents. J. Nucl. Med. 35, 334-341. (25) Bellande, E., Comazzi, V., Laine, J., Lecayon, M., Pasqualini, R., Duatti, A. and Hoffschir, D. (1995) Synthesis and biodistribution of nitrido technetium-99m radiopharmaceuticals with dithiophosphinate ligands: a class of brain imaging agents. Nucl. Med. Biol. 22, 315-320. (26) Demaimay, F., Noiret, N., Roucoux, A., Patin, H., Bellande, E., Pasqualini, R., and Moisan, A. (1997)New bis(dithiocarboxylato)nitridotechnetium-99m radiopharmaceuticals for leucocyte labelling: In Vitro and In ViVo studies. Nucl. Med. Biol. 24, 439-445. (27) Pasqualini, R., Bellande, E., Comazzi, V., Laine´, J., Le´cayon, M., Hoffschir, D., and Duatti, A. (1993) Synthesis of a neutral TcNdithiocarbamate complex bearing ester groups: A new potential imaging agent for cerebral perfusion. J. Nucl. Med 34, 18 P. No 63 abstract. (28) Zhang, J., Wang, X., and Li, C. Y., (2002) Synthesis and biodistribution of a new 99mTc nitrido complex for cerebral imaging. J. Nucl. Med. Biol. 29, 665-669. (29) Bolzati, C., et al., manuscript in preparation. BC050358Q