Steps toward high specific activity labeling of biomolecules for therapeutic application: preparation of precursor [(188)Re(H(2)O)(3)(CO)(3)](+) and synthesis of tailor-made bifunctional ligand systems

Two kit preparations of the organometallic precursor [(188)Re(H(2)O)(3)(CO)(3)](+) in aqueous media are presented. Method A uses gaseous carbon monoxide and amine borane (BH(3).NH(3)) as the reducing agent. In method B CO(g) is replaced by K(2)[H(3)BCO(2)] that releases carbon monoxide during hydrolysis. Both procedures afford the desired precursor in yields >85% after 10 min at 60 degrees C. HPLC and TLC analyses revealed 7 +/- 3% of unreacted (188)ReO(4)(-) and <5% of colloidal (188)ReO(2). Solutions of up to 14 GBq/mL Re-188 have been successfully carbonylated with these two methods. The syntheses of two tailor-made bifunctional ligand systems for the precursor [(188)Re(H(2)O)(3)(CO)(3)](+) are presented. The tridentate chelates consist of a bis[imidazol-2-yl]methylamine or an iminodiacetic acid moiety, respectively. Both types of ligand systems have been prepared with alkyl spacers of different length and a pendent primary amino or carboxylic acid functionality, enabling the amidic linkage to biomolecules. The tridentate coordination of the ligands to the rhenium-tricarbonyl core could be elucidated on the macroscopic level by X-ray structure analyses and 1D and 2D NMR experiments of two representative model complexes. On the nca level, the ligands allow labeling yields >95% with [(188)Re(H(2)O)(3)(CO)(3)](+) under mild reaction conditions (PBS buffer, 60 degrees C, 60 min) at ligand concentrations between 5 x 10(-4) M and 5 x 10(-5) M. Thus, specific activities of 22-220 GBq pe micromol of ligand could be achieved. Incubation of the corresponding Re-188 complexes in human serum at 37 degrees C revealed stabilities between 80 +/- 4% and 45 +/- 10% at 24 h, respectively, and 63 +/- 3% and 34 +/- 3% at 48 h postincubation in human serum depending on the chelating system. Decomposition product was mainly (188)ReO(4)(-). The routine kit-preparation of the precursor [(188)Re(H(2)O)(3)(CO)(3)](+) in combination with tailor-made ligand systems enables the organometallic labeling of biomolecules with unprecedented high specific activities.     

Mixed-ligand rhenium-188 complexes with tetradentate/monodentate NS3/P ('4 + 1') coordination: relation of structure with antioxidation stability

Development of new radiopharmaceuticals based on rhenium-188 depends on finding appropriate ligands able to give complexes with high in vivo stability. Rhenium(III) mixed-ligand complexes with tetradentate/monodentate ('4 + 1') coordination of the general formula [Re(NS(3))(PRR'R' ')] (NS(3) = tris(2-mercaptoethyl)amine and derivatives thereof, PRR'R' ' = phosphorus(III) ligands) appear to be among the promising tools to achieve this goal. According to this approach, we synthesized and characterized a series of rhenium model complexes. In vitro stabilities of the corresponding rhenium-188 complexes were determined by incubating 2-3 MBq or alternatively 37 MBq of the complexes in phosphate buffer, human plasma, and rat plasma, respectively, at 22 degrees C or 37 degrees C, followed by checking the amount of (188)ReO(4)(-) formed after 1 h, 24, and 48 h by thin-layer chromatography. The rate of perrhenate formation varied over a wide range, depending primarily on the nature of the phosphorus(III) ligand. Physicochemical parameters of the corresponding nonradioactive rhenium complexes were analyzed in detail to find out the factors influencing their different stability and furthermore to design new substitution-inert '4 + 1' complexes. Tolman's cone angle of phosphorus(III) ligands and the lipophilic character of the inner coordination sphere were found to be crucial factors to build up stable rhenium '4 + 1' complexes. Additional information useful to describe electronic and steric properties of these compounds were selected from electronic spectra (wavelength of the Re-->S charge-transfer band), cyclovoltammetric measurements (E degrees of the Re(III)/Re(IV) couple), and NMR investigations ((31)P chemical shift of coordinated P(III) ligands).     

Development of a rhenium-186-labeled MAG3-conjugated bisphosphonate for the palliation of metastatic bone pain based on the concept of bifunctional radiopharmaceuticals

Rhenium-186-1-hydroxyethylidene-1,1-diphosphonate (186Re-HEDP) has been used for the palliation of metastatic bone pain. Delayed blood clearance and high gastric uptake of radioactivity have been observed upon injection, due to the instability of (186)Re-HEDP in vivo. In this study, on the basis of the concept of bifunctional radiopharmaceuticals, we designed a stable 186Re-mercaptoacetylglycylglycylglycine (MAG3) complex-conjugated bisphosphonate, [[[[(4-hydroxy-4,4-diphosphonobutyl)carbamoylmethyl]carbamoylmethyl]carbamoylmethyl]carbamoylmethanethiolate]oxorhenium(V) (186Re-MAG3-HBP). As a precursor, [1-hydroxy-1-phosphono-4-[2-[2-[2-(2-tritylmercaptoacetylamino)acetylamino]acetylamino]acetylamino]butyl]phosphonic acid (Tr-MAG3-HBP) was synthesized by the conjugation of N-[(tritylmercapto)acetyl]glycylglycylglycine (Tr-MAG3) with the bisphosphonate analogue. After deprotection of the trityl group of Tr-MAG3-HBP, 186Re-labeling was performed by reacting 186ReO4- with SnCl2 in citrate buffer. After purification by HPLC, 186Re-MAG3-HBP showed a radiochemical purity of over 95%. To compare the stability of 186Re-MAG3-HBP and 186Re-HEDP, these (186)Re complexes were incubated in phosphate buffer. No measurable decomposition of 186Re-MAG3-HBP occurred over a 24-h period, while only approximately 30% of 186Re-HEDP remained intact 24 h postincubation. In biodistribution experiments, the radioactivity level of 186Re-MAG3-HBP in bone was significantly higher than that of (186)Re-HEDP. Blood clearance of 186Re-MAG3-HBP was faster than that of 186Re-HEDP. In addition, the gastric accumulation of 186Re-MAG3-HBP radioactivity was lower than that of 186Re-HEDP. In conclusion, 186Re-MAG3-HBP is expected to be a useful radiopharmaceutical for the palliation of metastatic bone pain.     

Synthesis, characterization, and labeling with 99mTc/188Re of peptide conjugates containing a dithia-bisphosphine chelating agent

Radiolabeling of small receptor-avid peptides at specific predetermined chelation sites with radioactive metals has been an effective approach for production of target-specific radiopharmaceuticals for diagnosis and therapy of diseases. Among various electron-donating groups found on chelator frameworks, phosphines are unique because they display versatile coordination chemistry with a wide range of transition metals. We have recently reported the utility of a dithia-bis(hydroxymethyl)phosphine-based (P2S2) bifunctional chelating agent (BFCA) containing air-stable primary phosphine groups to form 99mTc-labeled receptor-avid peptides by the preconjugation approach. Here we report a novel strategy for labeling small peptides with both 99mTc and 188Re using the P2S2-COOH (6,8-bis[3-(bis(hydroxymethyl)phosphanyl)propylsulfanyl]octanoic acid) BFCA by a postconjugation radiolabeling approach. The first step in this approach involves the coupling of the corresponding (PH2)2S2-COOH intermediate to the N-terminus of the peptide(s). Formylation of P-H bonds with aqueous formaldehyde in the presence of HCl in ethanol affords the corresponding (hydroxymethyl)phosphine-P2S2-peptide conjugates in the form of an oxidatively stable phosphonium salt. The P2S2-peptide conjugates are generated (where the PH2 groups are converted to P(CH2OH)2 groups) by treatment of the P2S2-peptide phosphonium salt(s) with 1 M sodium bicarbonate solution at pH 8.5. Complexation of BFCA conjugates with 99mTc is achieved by direct reduction with Sn(II) tartarate to yield the 99mTc-P2S2-peptide conjugate in near quantitative yields. Complexation of the BFCA conjugates with 188Re is achieved by transchelation with 188Re citrate in yields of >/=90%. In this study, (PH2)2S2-COOH BFCA was conjugated to model peptides. The glycineglycine ethyl ester (GlyGlyOEt)-(PH2)2S2-COOH BFCA conjugate was converted to the hydroxymethylene phosphine form and complexed with 99mTc to produce the 99mTcO2-P2S2-GlyGlyOEt conjugate 8 in RCPs of >/=95%. This singular 99mTc product is stable over 24 h in aqueous solution as confirmed by HPLC. Identical retention times of the 99mTcO2-P2S2-GlyGlyOEt complex and its cold rhenium analogue (ReO2-P2S2-GlyGlyOEt) on HPLC indicates similarity in structures at the macroscopic and the tracer levels. The utility of this postconjugation strategy was further demonstrated by synthesizing a P2S2-D-Lys6-LHRH conjugate and producing its corresponding 99mTc complex in RCPs of >/=88%. Finally, the P2S2-5-Ava-BBN[7-14]NH2 bombesin (BBN) analogue was synthesized, the PH2 groups converted to P(CH2OH)2 groups and subsequently labeled with 188Re to yield a 188Re-labeled bombesin analogue with a RCP of >/=90%. The biological integrity of this conjugate was demonstrated in both in vitro and in vivo. The results of this investigation demonstrate that the (PH2)2S2-COOH BFCA can be conveniently used as a precursor for labeling small receptor-avid peptides with diagnostic (99mTc) and therapeutic (188Re) radionuclides via the postconjugation approach in high yields.     

Carbohydrate conjugates for molecular imaging and radiotherapy: 99mTc(I) and 186Re(I) tricarbonyl complexes of N-(2'-Hydroxybenzyl)-2-amino-2-deoxy-D-glucose

An approach to a new class of potential radiopharmaceuticals is demonstrated by the labeling of a glucosamine derivative with the tricarbonyls of 99mTc and 186Re. The proligand HL2 (N-(2'-hydroxybenzyl)-2-amino-2-deoxy-D-glucose) was produced by hydrogenation of the corresponding Schiff base and reacted with [NEt4]2[Re(CO)3Br3] to form the neutral complex [(L2)Re(CO)3] in 40% yield. 1H and 13C NMR spectra indicate that the [Re(CO)3] core is bound in a tridentate fashion via the amino N, phenolato O, and C-3 hydroxyl O atoms of the ligand. At the tracer-level, labeling of HL2 with [99mTc(CO)3(H2O)3]+ and [186Re(CO)3(H2O)3]+ was achieved in aqueous conditions in 95 +/- 2% and 94 +/- 3% average radiochemical yields, respectively.     

Expression Profile of Vascular Cell Adhesion Molecule-1 (CD106) in Inflammatory Foci using Rhenium-188 Labelled Monoclonal Antibody in Mice

Rhenium (Re)-188 is a generator (W-188/Re-188) produced high energy β-emitter suitable for radionuclide therapy (T_1/2 is 16.9 hrs and E_max 2.1 MeV (range 11 mm)). We have labelled monoclonal antibody (MAb) raised against vascular cell adhesion molecule-1 (VCAM-1) with Re-188 using glucoheptonate chelation technique and SnCl₂ as reducing agent. The labelling efficiency, free perrhenate and reduced Re were controlled with thin layer chromatography and the purification of Re-188-MoAbs was performed using gel filtration. Our results indicate that Re-188-labelled antibodies remain in vitro stable and the labelling purity is >90%. We also have applied these Re-188-MoAbs for detection of inflammatory disease in a mouse. The effective half-lives of organs of interest after an injection of Re-188-anti-VCAM1 were as follows: blood 5.2 hr, kidney 4.7 hr, and liver 9.6 hr. Re-188-anti-VCAM-1 was found to accumulate mainly in kidney and liver. One hour after the injection, the kidney contained in average as high as 12.5% and the liver 2.8 ID/g tissue. After 6 hr, the kidney contained 5.5% ID/g and the liver 2.6% ID/g. At 24 hr, the kidney uptake was 0.5% ID/g and the liver uptake 0.8 % ID/g, respectively. The inflamed foci, subcutaneous lesions in the footpad skin, were visualized using gamma camera. From the distribution data the upakes in the inflamed foci as follows: at 1 hr 2.18 (inflammation) and 1.72% ID/g (control), at 6 hr 1.42 (inflammation) and 0.85% ID/g (control), and at 24 hr 0.17 (inflammation) and 0.084% ID/g (control), respectively. Anti-VCAM-1 MAb showed better targeting as compared to control MoAbs in inflammation (caused by E. coli lipoplysaccaride). In conclusion, Re-188 is suitable for MAb labelling, and MAb against VCAM-1 may be used for detection of local inflammatory disease.     

Chemical and biological characterization of technetium(I) and Rhenium(I) tricarbonyl complexes with dithioether ligands serving as linkers for coupling the Tc(CO)(3) and Re(CO)(3) moieties to biologically active molecules

The organometallic precursor (NEt(4))(2)[ReBr(3)(CO)(3)] was reacted with bidendate dithioethers (L) of the general formula H(3)C-S-CH(2)CH(2)-S-R (R = -CH(2)CH(2)COOH, CH(2)-C&tbd1;CH) and R'-S-CH(2)CH(2)-S-R' (R' = CH(3)CH(2)-, CH(3)CH(2)-OH, and CH(2)COOH) in methanol to form stable rhenium(I) tricarbonyl complexes of the general composition [ReBr(CO)(3)L]. Under these conditions, the functional groups do not participate in the coordination. As a prototypic representative of this type of Re compounds, the propargylic group bearing complex [ReBr(CO(3))(H(3)C-S-CH(2)CH(2)-S-CH(2)C&tbd1;CH)] Re2 was studied by X-ray diffraction analysis. Its molecular structure exhibits a slightly distorted octahedron with facial coordination of the carbonyl ligands. The potentially tetradentate ligand HO-CH(2)CH(2)-S-CH(2)CH(2)-S-CH(2)CH(2)-OH was reacted with the trinitrato precursor [Re(NO(3))(3)(CO)(3)](2-) to yield a cationic complex [Re(CO)(3)(HO-CH(2)CH(2)-S-CH(2)CH(2)-S-CH(2)CH(2)-OH)]NO(3) Re8 which shows the coordination of one hydroxy group. Re8 has been characterized by correct elemental analysis, infrared spectroscopy, capillary electrophoresis, and X-ray diffraction analysis. Ligand exchange reaction of the carboxylic group bearing ligands H(3)C-S-CH(2)CH(2)-S-CH(2)CH(2)-COOH and HOOC-CH(2)-S-CH(2)CH(2)-S-CH(2)-COOH with (NEt(4))(2)[ReBr(3)(CO)(3)] in water and with equimolar amounts of NaOH led to complexes in which the bromide is replaced by the carboxylic group. The X-ray structure analysis of the complex [Re(CO)(3)(OOC-CH(2)-S-CH(2)CH(2)-S-CH(2)-COOH)] Re6 shows the second carboxylic group noncoordinated offering an ideal site for functionalization or coupling a biomolecule. The no-carrier-added preparation of the analogous (99m)Tc(I) carbonyl thioether complexes could be performed using the precursor fac-[(99m)Tc(H(2)O)(3)(CO)(3)](+), with yields up to 90%. The behavior of the chlorine containing (99m)Tc complex [(99m)TcCl(CO)(3)(CH(3)CH(2)-S-CH(2)CH(2)-S-CH(2)CH(3))] Tc1 in aqueous solution at physiological pH value was investigated. In saline, the chromatographically separated compound was stable for at least 120 min. However, in chloride-free aqueous solution, a water-coordinated cationic species Tc1a of the proposed composition [(99m)Tc(H(2)O)(CO)(3)(CH(3)CH(2)-S-CH(2)CH(2)-S-CH(2)CH(3))](+) occurred. The cationic charge of the conversion product was confirmed by capillary electrophoresis. By the introduction of a carboxylic group into the thioether ligand as a third donor group, the conversion could be suppressed and thus the neutrality of the complex preserved. Biodistribution studies in the rat demonstrated for the neutral complexes [(99m)TcCl(CO)(3)(CH(3)CH(2)-S-CH(2)CH(2)-S-CH(2)CH(3))] Tc1 and [(99m)TcCl(CO)(3)(CH(2)-S-CH(2)CH(2)-S-CH(2)-C&tbd1;CH)] Tc2 a significant initial brain uptake (1.03 +/- 0.25% and 0.78 +/- 0.08% ID/organ at 5 min. p.i.). Challenge experiments with glutathione clearly indicated that no transchelation reaction occurs in vivo.     

Synthesis and structural studies of mixed-ligand rhenium(V) complexes anchored by tridentate pyrazole-based ligands

The novel oxorhenium dichlorides mer-[ReO(L1)Cl₂] (1) and fac-[ReO(L2)Cl₂] (2) (L1 = 2-[2-(pyrazol-1-yl)ethyliminomethyl]phenolate; L2 = 2-[2-(pyrazol-1-yl)ethylaminomethyl]phenolate) were synthesized by reacting [NBu₄][ReOCl₄] with L1H and L2H, respectively. X-ray structural analysis of 1 and 2 has shown that L1 and L2 act as (N,N,O)-tridentate chelators coordinating to the Re(V) centre in a meridional and in a facial fashion, respectively. The reactivity of 2 towards potential bidentate/dianionic substrates is strongly dependent on the donor atom set, being observed that the presence of sulphur favours the displacement of the ancillary ligand (L2). By contrast, complex 2 reacted with (O,O)-bidentate substrates (1,2-ethanediol and oxalic acid) providing the mixed-ligand complexes fac-[ReO(L2)(OCH₂CH₂O)] (3) and fac-[ReO(L2)(C₂O₄)] (4). Complexes 3 and 4 are air and water-stable and have been characterized by the common spectroscopic techniques (IR, ¹H and ¹³C NMR) and by X-ray diffraction analysis.     

Design, synthesis, and evaluation of [188Re]organorhenium-labeled antibody fragments with renal enzyme-cleavable linkage for low renal radioactivity levels

Renal localization of radiolabeled antibody fragments constitutes a problem in targeted imaging and radiotherapy. We have reported that Fab fragments labeled with 3'-[131I]iodohippuryl Nepsilon-maleoyl-lysine (HML) showed markedly low renal radioactivity levels even shortly after injection, due to a rapid and selective release of m-[131I]iodohippuric acid by the action of brush border enzymes. To estimate the applicability of the molecular design to metallic radionuclides, [188Re]tricarbonyl(cyclopentadienylcarbonate)rhenium ([188Re]CpTR-COOH) was conjugated with Nepsilon-tert-butoxycarbonyl-glycyl-lysine or Nepsilon-maleoyl-glycyl-lysine to prepare [188Re]CpTR-GK-Boc or [188Re]CpTR-GK. The cleavage of the glycyl-lysine linkage of the two compounds generates a glycine conjugate of [188Re]CpTR-COOH ([188Re]CpTR-Gly), which possesses in vivo behaviors similar to those of m-iodohippuric acid. The hydrolysis rate of the peptide bond in [188Re]CpTR-GK-Boc was compared with that in 3'-[125I]iodohippuryl Nepsilon-Boc-lysine ([125I]HL-Boc) using brush border membrane vesicles (BBMVs) prepared from rat kidneys. [188Re]CpTR-GK was conjugated to thiolated Fab fragments to prepare [188Re]CpTR-GK-Fab. The biodistribution of radioactivity after injection of [188Re]CpTR-GK-Fab was compared with that of [125I]HML-Fab and [188Re]CpTR-Fab prepared by conjugating N-hydroxysuccinimidyl ester of [188Re]CpTR-COOH with antibody fragments. While [188Re]CpTR-GK-Boc liberated [188Re]CpTR-Gly in BBMVs, [125I]HL-Boc liberated m-[125I]iodohippuric acid at a much faster rate. In addition, although [125I]HL-Boc was hydrolyzed by both metalloenzymes and nonmetalloenzymes, metalloenzymes were responsible for the cleavage of the peptide linkage in [188Re]CpTR-GK-Boc. In biodistribution studies, [188Re]CpTR-GK-Fab exhibited significantly lower renal radioactivity levels than did [188Re]CpTR-Fab. However, the renal radioactivity levels of [188Re]CpTR-GK-Fab were slightly higher than those of [125I]HML-Fab. The analysis of urine samples collected for 6 h postinjection of [188Re]CpTR-GK-Fab showed that [188Re]CpTR-Gly was the major radiometabolite. In tumor-bearing mice, [188Re]CpTR-GK-Fab significantly reduced renal radioactivity levels without impairing the radioactivity levels in tumor. These findings indicate that the molecular design of HML can be applied to metallic radionuclides by using a radiometal chelate of high inertness and by designing a radiometabolite of high urinary excretion when released from antibody fragments following cleavage of a glycyl-lysine linkage. This study also indicates that a change in chemical structure of a radiolabel attached to a glycyl-lysine linkage significantly affected enzymes involved in the hydrolysis reaction. Since there are many kinds of enzymes that cleave a variety of peptide linkages on the renal brush border membrane, selection of a peptide linkage optimal to a radiometal chelate of interest may provide radiolabeled antibody fragments that exhibit renal radioactivity levels similar to those of [131I]HML-labeled ones. The in vitro system using BBMVs might be useful for selecting an appropriate peptide linkage.     

Molecular structures and redox properties of oxorhenium(V) ‘3+1’ mixed ligand complexes with heterocyclic thiolates containing nitrogen

Reactions of chloro(3-thiapentane-1,5-dithiolato)oxorhenium(V) [ReO(SSS)Cl] with N-methyl-1H-imidazole-2-thiol (HL¹) and 2-pyrimidinethiol (HL²) have been studied to form ‘3+1’ oxorhenium(V) complexes. In the absence of triethylamine, [Re(SSS)(HL¹)]Cl (1a) was formed, while in the presence of triethylamine [Re(SSS)L¹] (1b) and [Re(SSS)L²] (2) were produced. Molecular structures of complexes 1a and 2 were determined to be distorted square pyramidal by single crystal X-ray analytical method. From cyclic voltammetric studies, furthermore, it was proposed that complexes 1b and 2 are irreversibly oxidized to Re(VI) at around 0.84 and 1.01 V versus Ag/AgNO₃, respectively, and are reduced to Re(IV) at −1.55 and −1.51 V with the dissociation of L¹ or L², followed by the quasi-reversible reductions to Re(III) at around −1.69 V, respectively.     

Mixed-ligand technetium(III) complexes with tetradendate/monodendate NS(3)/isocyanide coordination: a new nonpolar technetium chelate system for the design of neutral and lipophilic complexes stable in vivo.

Starting from the tripodal ligand 2,2',2' '-nitrilotris(ethanethiol) (NS(3)) and isocyanides (CNR) as co-ligands, neutral mixed-ligand technetium(III) complexes of the general formulation [Tc(NS(3))(CNR)] have been synthesized and characterized. The (99)Tc complexes can be( )()obtained by a two-step reduction/substitution procedure starting from [TcO(4)](-) via the phosphine-containing precursor complex [Tc(NS(3))(PMe(2)Ph)]. As shown by X-ray structural analyses, the complexes adopt a nearly ideal trigonal-bipyramidal geometry with the trigonal plane formed by the three thiolate sulfurs of the tripodal ligand. The central nitrogen atom of the chelate ligand and the monodendate isocyanides occupy the apical positions. The no-carrier-added preparation of the corresponding (99m)Tc complexes was performed by a one-step procedure starting from (99m)[TcO(4)](-) with stannous chloride as reducing agent. Biodistribution studies in the rat demonstrated for the nonpolar, lipophilic compounds a significant initial brain uptake. In vitro challenge experiments with glutathione clearly indicated that no transchelation reaction occurs. Furthermore, there were no indications for reoxidation of Tc(III) to Tc(V) species or pertechnetate. We propose this type of complexes as a useful tool in the design of lipophilic (99m)Tc or (186)Re/(188)Re radiopharmaceuticals.     

Experimental pretargeting studies of cancer with a humanized anti-CEA x murine anti-[In-DTPA] bispecific antibody construct and a (99m)Tc-/(188)Re-labeled peptide

The aim of this study was to localize (99m)Tc and (188)Re radionuclides to tumors, using a bispecific antibody (bsMAb) in a two-step approach where the radionuclides are attached to novel peptides incorporating moieties recognized by one arm of the bsMAb. A chemically cross-linked human/murine bsMAb, hMN-14 x 734 (Fab' x Fab'), anti-carcinoembryonic antigen [CEA] x anti-indium-DTPA was prepared as a prelude to constructing a fully humanized bsMAb for future clinical application. N,N'-o-Phenylenedimaleimide was used to cross-link the Fab' fragments of the two antibodies at their hinge regions. This construct was shown to be >92% pure and fully reactive with CEA and a divalent (indium)DTPA-peptide. For pretargeting purposes, a peptide, IMP-192 [Ac-Lys(In-DTPA)-Tyr-Lys(In-DTPA)-Lys(TscG-Cys-)-NH(2) ?TscG = 3-thiosemicarbazonylglyoxyl?], with two indium-DTPAs and a chelate for selectively binding (99m)Tc or (188)Re, was synthesized. IMP-192 was formulated in a "single dose" kit and later radiolabeled with (99m)Tc (94-99%) at up to 1836 Ci/mmol and with (188)Re (97%) at 459-945 Ci/mmol of peptide. [(99m)Tc]IMP-192 was shown to be stable by extensive in vitro and in vivo testing and had no specific uptake in the tumor with minimal renal uptake. The biodistribution of the hMN-14 x murine 734 bsMAb was compared alone and in a pretargeting setting to a fully murine anti-CEA (F6) x 734 bsMAb that was reported previously [Gautherot, E., Bouhou, J., LeDoussal, J.-M., Manetti, C., Martin, M., Rouvier, E., and Barbet, J. (1997) Therapy for colon carcinoma xenografts with bispecific antibody-targeted, iodine-131-labeled bivalent hapten. Cancer 80 (Suppl.), 2618-2623]. Both bsMAbs maintained their integrity and dual binding specificity in vivo, but the hMN-14 x m734 was cleared more rapidly from the blood. This coincided with an increased uptake of the hMN-14 x m734 bsMAb in the liver and spleen, suggesting an active reticuloendothelial cell recognition mechanism of this mixed species construct in naive mice. Animals bearing GW-39 human colonic cancer xenografts were injected with bsMAb (15 microg) and after allowing 24 or 72 h for the bsMAb constructs to clear from the blood (hMN-14 and murine F6 x 734, respectively), [(188)Re]IMP-192 (7 microCi) or [(99m)Tc]IMP-192 (10 microCi) was injected at a bsMAb:peptide ratio of 10:1. Tumor uptake of [(99m)Tc] or [(188)Re]IMP-192 was 12.6 +/- 5.2 and 16.9 +/- 5.5% ID/g at 3 h postinjection, respectively. Tumor/nontumor ratios were between 5.6 and 23 to 1 for every major organ, indicating that early imaging with (99m)Tc will be possible. Radiation absorbed doses showed a 4.8-, 7.2-, and a 12.6 to 1.0 tumor to blood, kidney, and liver ratios when (188)Re was used. Although this new bsMAb pretargeting approach requires further optimization, it already shows very promising targeting results for both radioimmunodetection and radioimmunotherapy of colorectal cancer.     

Investigations of the {ReO} core: A '2+2' complex from bidentate and potentially trident ligands: [ReO(η-HOC(6)H(4)-2-CH(2)NC(6)H(4)S)(η-SC(5)H(4)N)(PPh(3))

The reaction of [ReOCl(3)(PPh(3))(2)] with N-(2-hydroxybenzyl)-2-mercaptoaniline (H(3)hbma) (2) and 2-mercaptopyridine in hot CHCl yields [ReO(η(2)-HOC(6)H(4)-2-CH(2)NC(6)H(4)S)(η(2)-SC(5)H(4)N)(PPh(3))] (3). The structure of 3 consists of distorted octahedral Re(V) monomers. The coordination geometry at the rhenium is defined by a terminal oxo-group, the nitrogen and sulfur donors of the chelating mercaptopyridine, the nitrogen and sulfur donors of a bidentate (Hhbma)(2-) ligand, and the phosphorus of the PPh(3) group. The -C(6)H(4)OH arm of (Hhbma)(2-) is pendant, and the coordinated nitrogen of this ligand is present as a deprotonated amido nitrogen.     

微生物转化法生产度洛西汀中间体手性醇菌株的鉴定

【目的】获得以DKTP为底物合成度洛西汀关键中间体手性醇(S)-DHTP的菌株。【方法】采用常规及改进的微生物转化法从土壤中进行筛选。【结果】筛选获得一株菌株,能够将底物DKTP对映选择性地还原为(S)-DHTP,且具有较高的转化率(>90%)和几乎绝对的对映体过量值(e.e.>99%),改进的筛选方法更为简便高效。形态学特征和26S rDNA序列分析综合判断,该菌株属于红酵母属,命名为红酵母Rhodotorula sp.507。【结论】供试菌株能够高效地、不对称地生物还原DKTP成度洛西汀前体物(S)-DHTP,使大量获得度洛西汀前体原料变得经济可行。     

Synthesis and in vivo evaluation of [18F]-4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide as a PET imaging probe for COX-2 expression

Synthesis of [18F]4-[5-(4-methylphenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzenesulfonamide ([18F]celecoxib), a selective COX-2 inhibitor, is achieved via a bromide to [18F]F- exchange reaction. Synthesis of the precursor for radiolabeling was achieved from 4'-methylacetophenone in four steps with 22% overall yield. Under non-radioactive conditions, fluorination was achieved using TBAF in DMSO at 135 degrees C in 80% yield. Synthesis of [18F]celecoxib was achieved using [18F]TBAF in DMSO at 135 degrees C in 10+/-2% yield (EOS) with >99% chemical and radiochemical purities. The specific activity was 120+/-40 mCi/micromol (EOB). [18F]celecoxib was found to be stable in ethanol, however, de[18F]fluorination (6.5%) was observed after 4 h in 10% ethanol-saline solution. Rodent PET studies show bone labeling indicating in vivo de[18F]fluorination of [18F]celecoxib. PET studies in baboon indicated a lower rate of de[18F]fluorination than rat and retention of radioactivity in brain regions consistent with the known distribution of COX-2. A radiolabeling method that can generate consistent high specific activity is needed for routine human use.     

Reductive thiocyanolysis of tetraoxorhenate (VII): Synthesis, crystal structure, catalytic oxidation and kinetic studies of (PPh4)2[Re(NCS)6] and (PPh4)2[ReO(NCS)5]

Thiocyanate ions reduce perrhenate in aqueous acidic solution, and on addition of a suitable countercation (PPh₄Cl) afford (PPh₄)₂[Re(NCS)₆] (1) and (PPh₄)₂[ReO(NCS)₅] (2), which have been confirmed by X-ray crystallography. The kinetics of the above reaction has been studied. Both the complexes exhibit efficient and highly selective catalytic epoxidation ability in the presence of NaHCO₃ as a co-catalyst and competent catalytic properties in the selective oxidation of alcohols to the corresponding aldehydes or ketones in the presence of pyrazole as an additive and sulfides to sulfoxides and sulfones. H₂O₂ was used as the terminal oxidant in all the above-mentioned oxidation reactions.     

The reaction of the cis-dioxorhenium(V) core with N,O-donor ligands: mono and bidentate coordination of 3-methyl-2-aminophenol

The reaction of cis-[ReO₂I(PPh₃)₂] with 3-methyl-2-aminophenol (H₂map) in ethanol led to the formation of trans-[Re(map)(Hmap)I(PPh₃)₂]I. The X-ray crystal structure shows that the ligand map is coordinated monodentately through the doubly deprotonated amino nitrogen and is therefore present as an imide. The chelate Hmap is coordinated via the neutral amino nitrogen and the singly deprotonated phenolate oxygen, which is coordinated trans to the imido nitrogen. In benzene equimolar quantities of cis-[ReO₂I(PPh₃)₂] and 4-methyl-2-aminophenol (H₂amp) produced the five-coordinate cis-[ReO₂(Hamp)(PPh₃)]. A twofold molar excess of 2-aminophenol (H₂ap) yielded the six-coordinate [ReO(Hap)(ap)(PPh₃)]. The chelate ap is coordinated through the deprotonated phenolate oxygen and the singly deprotonated amino nitrogen (amide).     

Synthesis and reactions of [M(CO)4(Ph2PSiMe3)X] complexes (M = Mn,Re; X = Halogen)

The silylphosphine ligand Ph₂PSiMe₃ reacts readily with a slurry of [Re(CO)₅X] (X  Cl, Br) in polar and in non-polar solvents to yield soluble cis-[Re(CO)₄- (Ph₂PSiMe₃)X] (Ia, X  Cl;Ib, X  Br) via CO substitution. Compound I is readily hydrolyzed by water or silica gel to cis-[Re(CO)₄(Ph₂PH)X]. Compound Ib reacts with [Re(CO)₅Br] to yield [Re₂(CO)₈(μ-PPh₂)- (μ-Br)] (II), and with [Mn(CO)₅Br] to yield [MnRe- (CO)₈(μ-PPh₂)(μ-Br)] (III).The reaction of Ph₂PSiMe₃ with [Mn(CO)₅X] (X=Cl,Br,I) is highly dependent upon reaction conditions.In polar and in non-polar solvents, an excess of ligand gives mainly cis-[Mn(CO)₄(Ph₂PSiMe₃)X] (IVa, X  Cl;IVb, X  Br;IVc, X I). With ligand: [Mn(CO)₅X] reacting ratios in the range 0.5–1.0:1, the products from the three respective halomanganese complexes in THF were: (a) mainly [Mn₂(CO)₈(μ- PPh₂)(μ-Cl) (Va); (b) both [Mn(CO)₄(Ph₂PSiMe₃)Br] and [Mn₂(CO)₈(μ-PPh₂)(μ-Br)] (Vb); and (c) exclusively [Mn(CO)₄(Ph₂PSiMe₃)I]. The compounds IVa-c are stable in solution at ambient temperatures and are readily hydrolyzed by water or methanol to [Mn(CO)₄(Ph₂PH)X]. Compound IVb reacts at room temperature with [Mn(CO)₅Cl] to yield only [Mn₂- (CO)₈(μ-PPh₂)(μ-Br)] (Vb); compound IVc reacts in hot toluene with [Mn(CO)₅Cl] to yield mainly [Mn₂(CO)₈(μ-PPh₂)(μ-I)] (Vc), together with a small amount of the chloro-bridged analog.The dinuclear species II, III and Va-c appear to be formed mainly via an intermolecular elimination of Me₃SiX from the appropriate [M(CO)₄(Ph₂PSiMe₃)X] and metalpentacarbonylhalide (chloride or bromide) complexes.     

Syntheses and properties of organocyanamide, cyanoguanidine and dinitrogen complexes of rhenium. Crystal structure of mer-[ReCl2(NCNEt2) (PMePh2)3]

Treatment of a THF solution of trans-[ReCl(N₂)L₄] (L = PMePh₂) with a cyanamide, NCNR₂ (R = Me, Et or H) or with cyanoguanidine, NCNC(NH₂)₂ , yields mer-[ReCl(N₂)(NCNR₂)L₃] (1) or mer-[Re(N₂)[Re(N₂){NCNC(NH₂)₂}₂L₃]Cl (2), respectively, which, to our knowledge, are the first mixed dinitrogen-cyanamide-type complexes to be reported. The former products (1, R=Me or Et) can also be obtained from reaction of the benzoyldiazenido complex [ReCl₂(NNCOPh)L₃] with NCNR₂ in refluxing methanol; the Re(II) complex mer-[ReCl₂(NCNEt₂)L₃] (3) is also formed (conceivably via an unusual homolysis of the C---N bond of the benzoyldiazenido ligand) and its crystal structure is reported. It shows an unusual pyramidal conformation at the amine N atom of the diethylcyanamide ligand which also exhibits a significant structural trans influence on the phosphine, behaving as a stronger net electron donor than the latter ligand.     

Functionalization of glucose at position C-3 for transition metal coordination: organo-rhenium complexes with carbohydrate skeletons

Novel 3-O-[1,2;5,6-di-O-isopropylidene-alpha-D-glucofuranose] and 3-O-[D-glucose] derivatives with an iminodiacetate (N,O,O), a histidinate, and an N-(acetetyl)picolylamine (N,N,O) chelating system for tridentate coordination of the organometallic M(CO)(3)-fragment (M = Tc, Re) have been prepared. The chelates were introduced and assembled through reductive amination starting from 3-O-[1,2;5,6-di-O-isopropylidene-alpha-D-glucofuranose]-acetaldehyde. After deprotection, the pyranose derivatives were reacted with the precursor [NEt(4)](2)[ReBr(3)(CO)(3)] to afford the corresponding organometallic complexes in yields between 54% and 94%. The NMR, MS, and IR analyses corroborated the tridentate coordination of the organometallic metal center exclusively via the synthetic chelates. In the case of the N-(acetyl)picolylamine derivative, the coordinative properties were further confirmed by X-ray structure analysis of the first Re(CO)(3)-D-glucofuranose complex. All glucose complexes unveiled good stability and solubility in organic and aqueous media.