Evaluation and comparison of 99mTc-labeled d-glucosamine derivatives with different 99mTc cores as potential tumor imaging agents

Isocyanide is a strong coordination ligand that can coordinate with [^99mTc(I)(CO)₃]⁺ core and [^99mTc(I)]⁺ core to produce stable ^99mTc complexes, therefore developing a ^99mTc-labeled isocyanide complex for single-photon emission computed tomography (SPECT) imaging is considered to be of great interest. In order to develop potential tumor imaging agents with satisfied tumor uptake and suitable pharmacokinetic properties in vivo, a novel d -glucosamine isocyanide derivative, 4-isocyano-N-(2,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-3-yl)butanamide (CN3DG), was synthesized and radiolabeled with [^99mTc(I)]⁺ and [^99mTc(CO)₃]⁺ cores to obtain [^99mTc(CN3DG)₆]⁺ and [^99mTc(CO)₃(CN3DG)₃]⁺ in high radiolabeling yields (>95%). Both of the complexes showed good hydrophilicity and great stability in vitro. Cell uptake studies performed in S180 cells demonstrated they were transported into cells by glucose transporters. Biodistribution studies of the two complexes in mice bearing S180 tumor showed they had high tumor uptakes and rapid clearance from muscle and blood so that the tumor/blood and tumor/muscle ratios were high. By comparison, [^99mTc(CN3DG)₆]⁺ was superior to [^99mTc(CO)₃(CN3DG)₃]⁺ in regard to tumor uptake, tumor/blood and tumor/liver ratios. S180 tumors could be seen clearly from the SPECT/CT images with [^99mTc(CN3DG)₆]⁺. Considering its favorable properties, [^99mTc(CN3DG)₆]⁺ would be a promising tumor imaging agent and needs to be further studied.     

Synthesis of 99mTc(CO)3-NOET via [99mTc(OH2)3(CO)3]+ precursor and comparative biological studies with 99mTcN-noet

Summary The organometallic precursor fac-[^99mTc(CO)₃(H₂O)₃]⁺ was reacted with N-ethoxy, N-ethyl dithiocarbamate (NOET) in phosphate buffered saline (pH 7.4) at room temperature for 30 minutes to produce the ^99mTc(CO)₃-NOET complex. The radiochemical purity (RCP) of the product was over 90% as measured by thin layer chromatography (TLC). No decomposition of the complex at room temperature (RT) was observed over a period of 6 hours. Its partition coefficient indicated that it was a lipophilic complex. The biodistribution comparison in mice of the ^99mTc(CO)₃-NOET complex and the ^99mTcN-NOET complex showed that the former had a lower heart and brain uptake as compared to that of the latter, suggesting the incorporation of the [^99mTc(CO)₃]⁺ core into the NOET ligand does not improve the biological features as a myocardial imaging agent.     

Synthesis, radiolabeling and biodistribution studies of [99mTc(CO)3(MN-TZ-BPA)]+ in tumor-bearing mice

This study reports the synthesis, radiolabeling and preliminary biodistribution results of [^99mTc(CO)₃(MN-TZ-BPA)]⁺ in tumor-bearing mice. The novel nitroimidazole derivative was successfully synthesized by conjugation of bis(pyridin-2-ylmethyl)amine (BPA) to 2-methyl-5-niroimidazole via “click” reaction. The ligand could be labeled by [^99mTc(CO)₃]⁺ core in high yield to get [^99mTc(CO)₃(MN-TZ-BPA)]⁺, which was very hydrophilic and was stable at room temperature. Biodistribution studies in tumor-bearing mice showed that [^99mTc(CO)₃(MN-TZ-BPA)]⁺ accumulated in the tumor with certain initial uptake while poor retention. The rapid clearance from normal organs with favorable tumor/muscle ratios warrants further research to improve tumor targeting efficacy and pharmacokinetic profile of radiolabeled nitroimidazoles by structural modification.     

Synthesis and preliminary biological evaluation of 99mTc(CO)3-labeled pegylated 2-nitroimidazoles

This work reports the synthesis, radiolabeling and preliminary biodistribution results in tumor-bearing mice of ^99mTc(CO)₃-labeled pegylated (PEG) 2-nitroimidazoles for tumor hypoxia imaging. The novel 2-nitroimidazole derivatives were successfully synthesized by conjugation of tridendate chelators to 2-nitroimidazole via PEG₃ linker. Radiolabeling was performed in high yield with [^99mTc(CO)₃]⁺ core to get cationic [^99mTc(CO)₃(BPA-PEG₃-NIM)]⁺, neutral [^99mTc(CO)₃(AOPA-PEG₃-NIM)] and anionic [^99mTc(CO)₃(IDA-PEG₃-NIM)]^? respectively, all of which were hydrophilic and stable at room temperature. Biodistribution studies in tumor-bearing mice showed that ^99mTc(CO)₃-labeled pegylated 2-nitroimidazoles accumulated in the tumor with low uptake. ^99mTc-chelate and charge had significant impact on partition coefficient, radiotracer tumor uptake and pharmacokinetic properties. The results indicate the need for synthetic modification of the parent 2-nitroimidazole derivatives and the ^99mTc-chelate with a view to improve the tumor targeting efficacy and in vivo kinetic profiles.     

Chemical and biological evaluation of 99mTc (CO)3 and 99mTc complexes of some IDA derivatives

The confirmation that N-substituted imidodiacetic acids, as small and simple ligand systems containing amines and carboxylic acids, could be coordinated to the tricarbonyl core and form inert complexes with [^99mTc (CO)₃(H₂O)₃]⁺, is demonstrated. The HPLC quality control results of ^99mTc-carbonyl tagged IDA molecules, performed by gradient HPLC, have shown that HIDA, EHIDA and p-butyl-IDA form complexes with [^99mTc(CO)₃(H₂O)₃]⁺, with a labeling yield of ~90% for each of ^99mTc(CO)₃ IDA derivatives. However, the changes in the structure of labeled compounds, e.g., EHIDA, influence the changes in the biological behavior. In comparison with ^99mTc-EHIDA, the biliary excretion of ^99mTc(CO)₃ EHIDA was lower, but the urinary excretion higher. This revised version was published online in July 2006 with corrections to the Cover Date.     

Characterization of 99mTc(CO)3-iminodiacetic acid (IDA) and its comparison with 99mTc-(IDA)2 using compounds for hepatobiliary scintigraphy

The organometallic precursor of fac-[^99mTc(CO)₃(H₂O)₃]⁺ has attracted much attention because of the robustness and small size of Tc(I)-tricarbonyl complexes compared to Tc(V) complexes and the good labeling affinity with a variety of donor atoms. Among various ligand systems, an iminodiacetic acid (IDA) was proven as a good chelating group to form a Tc(III)-compelx as well as has been shown its potential as a chelating system for fac-[^99mTc(CO)₃] precursor. In an attempt to confirm the similarity and the difference between ^99mTc(CO)₃-IDA and ^99mTc-(IDA)₂-complex, M(CO)₃-IDA (M = ^99mTc, Re) complexes of disofenin, mebrofenin and N-(3-iodo-2,4,6-trimethyl phenylcarbamoylmethyl) iminodiacetic acid were prepared, and the biological evaluation of ^99mTc(CO)₃-disofenin was performed. The ^99mTc(CO)₃-IDA complexes were prepared with a high radiolabeling yield (>98%) in a quantitative manner and showed a negative charge. The in vivo pharmacokinetic behavior of ^99mTc(CO)₃-disofenin showed a similar biological activity to ^99mTc-(disofenin)₂ in that those complexes were quickly cleared from the blood by the hepatocytes and excreted into the gallbladder and intestine. Accordingly, the ^99mTc(CO)₃-IDA derivatives of disofenin and mebrofenin might be used as hepatobiliary imaging agents. Since an IDA is a promising chelator for ^99mTc-based radiopharmaceutical and the biological properties of ^99mTc(CO)₃-IDA derivative shows similar to that of ^99mTc-complex, a biomolecule containing IDA can be freely radiolabeled with fac-[^99mTc(CO)₃]-precursor or ^99mTc. However, the radiolabeling efficiency and the biological behavior demonstrates the favorable properties of ^99mTc(CO)₃-IDA compound for the development of a new imaging agent.     

To Sandwich Technetium: Highly Functionalized Bis-Arene Complexes [99mTc(η6-arene)2]+ Directly from Water and [99mTcO4]−

The labeling of (bio)molecules with metallic radionuclides such as ^99mTc demands conjugated, multidentate chelators. However, this is not always necessary since phenyl rings can directly serve as integrated, organometallic ligands. Bis-arene sandwich complexes are generally prepared by the Fischer–Hafner reaction. In extension of this, we show that [^99mTc(η⁶-C₆R₆)₂]⁺-type complexes are directly accessible from water and [^99mTcO₄]⁻, even using arenes incompatible with Fischer–Hafner conditions. To unambiguously confirm the nature of these unprecedented ^99mTc complexes, their rhenium homologous have been prepared by substituting naphthalene ligands in [Re(η⁶-C₁₀H₈)₂]⁺ with the corresponding phenyl groups. The ease with which highly stable [^99mTc(η⁶-C₆R₆)₂]⁺ complexes are formed under standard labeling conditions enables a multitude of new potential imaging agents based on commercial pharmaceuticals or lead structures.     

To Sandwich Technetium: Highly Functionalized Bis‐Arene Complexes [99mTc(η6‐arene)2]+ Directly from Water and [99mTcO4]−

The labeling of (bio)molecules with metallic radionuclides such as ^99mTc demands conjugated, multidentate chelators. However, this is not always necessary since phenyl rings can directly serve as integrated, organometallic ligands. Bis‐arene sandwich complexes are generally prepared by the Fischer–Hafner reaction. In extension of this, we show that [^99mTc(η⁶‐C₆R₆)₂]⁺‐type complexes are directly accessible from water and [^99mTcO₄]^?, even using arenes incompatible with Fischer–Hafner conditions. To unambiguously confirm the nature of these unprecedented ^99mTc complexes, their rhenium homologous have been prepared by substituting naphthalene ligands in [Re(η⁶‐C₁₀H₈)₂]⁺ with the corresponding phenyl groups. The ease with which highly stable [^99mTc(η⁶‐C₆R₆)₂]⁺ complexes are formed under standard labeling conditions enables a multitude of new potential imaging agents based on commercial pharmaceuticals or lead structures.     

Preparation and preliminary evaluation of a tris-metronidazole-<Superscript>99m</Superscript>Tc(CO)<Subscript>3</Subscript> complex for targeting tumor hypoxia

Conventional ^99mTc-radiopharmaceuticals for the detection of tumor hypoxia generally possess a single nitroimidazole moiety. Herein, we report the synthesis and evaluation of a ^99mTc-complex with three-nitroimidazole moieties in an attempt to improve hypoxic cell detection. Isocyanide derivative of metronidazole (MetroNC) was synthesized and subsequently radiolabeled with [^99mTc(CO)₃(H₂O)₃]⁺ precursor complex, wherein the three labile water molecules were replaced with MetroNC ligand to form a pseudo-octahedral [^99mTc(CO)₃(MetroNC)₃]⁺ complex. Analysis of corresponding Re(CO)₃-analog prepared in macroscopic scale confirmed the formation of expected complex. Cyclic voltammetric studies of [Re(CO)₃(MetroNC)₃]⁺ complex showed no significant change in single-electron reduction potential (SERP) of MetroNC ligand (??0.96 V) upon forming the [Re(CO)₃(MetroNC)₃]⁺ complex (??0.90 V). In vitro studies in Chinese hamster ovary (CHO) cells showed three-fold preferential accumulation of [^99mTc(CO)₃(MetroNC)₃]⁺ complex in hypoxic cells over normoxic cells. Biodistribution studies of [^99mTc(CO)₃(MetroNC)₃]⁺ complex in Swiss mice bearing fibrosarcoma tumor showed tumor uptake and steady retention till 60 min post injection. Present study constitutes a novel design approach towards development of a ^99mTc-radiopharmaceutical for hypoxia imaging application, which could be extended to other potential nitroimidazole ligands.     

Preparation and biological evaluation of Tc-CO-MIBI as myocardial perfusion imaging agent

^99mTc-Sestamibi has been playing an important role in the cardiac imaging for the last decades. Previously, we reported that [^99mTc(CO)₃(MIBI)₃]⁺ demonstrated a significant location in myocardium with a lower liver uptake as compared with ^99mTc-Sestamibi. In this work, we found that new [^99mTc(CO)₂(MIBI)₄]⁺ could be prepared with high radiochemical purity. The inter-transformations between [^99mTc(CO)₃(H₂O)(MIBI)₂]⁺, [^99mTc(CO)₃(MIBI)₃]⁺, and [^99mTc(CO)₂(MIBI)₄]⁺ were investigated and biodistribution was performed to evaluate the [^99mTc(CO)₂(MIBI)₄]⁺ as a myocardial perfusion imaging agent. The results showed that one more CO was replaced by MIBI slowing down the pharmacokinetics. The structure characterization was performed on their corresponding rhenium complexes, and the results indicated that there were differences between ^99mTc-CO-MIBI and Re-CO-MIBI in preparation and hydrophobic characteristics.     

S‐Functionalized Cysteine: Powerful Ligands for the Labelling of Bioactive Molecules with Triaquatricarbonyltechnetium‐99m(1+) ([99mTc(OH2)3(CO)3]+)

S‐Alkylated cysteines are used as efficient tridentate N,O,S‐donor‐atom ligands for the fac‐[M(CO)₃]⁺ moiety (M=^99mTc or Re). Reaction of (Et₄N)₂[ReBr₃(CO)₃] ( 3 ) with the model S‐benzyl‐L ‐cysteine ( 2 ) leads to the formation of [Re( 2′ )(CO)₃] ( 4 ) as the exclusive product ( 2′ =C‐terminal anion of 2 ). The tridentate nature of the alkylated cysteine in 4 was established by X‐ray crystallography. Compound 2 reacts with [^99mTc(OH₂)₃(CO)₃]⁺ under mild conditions (10⁻⁴ M , 50°, 30 min) to afford [^99mTc( 2′ )(CO)₃] ( 5 ) and represents, therefore, an efficient chelator for the labelling of biomolecules. L ‐Cysteine, S‐alkylated with a 3‐aminopropyl group (→ 7 ), was conjugated via a peptide coupling sequence with Coα‐[α‐(5,6‐dimethyl‐1H‐benzimidazolyl)]‐Coβ‐cyanocobamic b‐acid ( 6 ), the b‐acid of cyanocob(III)alamin (vitamin B₁₂) (Scheme 3). More convenient was a one‐pot procedure with a derivative of vitamin B₁₂ comprising a free amine group at the b‐position. This amine 15 was treated with NHS (N‐hydroxysuccinimide)‐activated 1‐iodoacetic acid 14 to introduce an I‐substituent in vitamin B₁₂. Subsequent addition of unprotected L ‐cysteine resulted in nucleophilic displacement of the I‐atom by the S‐substituent, affording the vitamin B₁₂ alkylated cysteine fragment 17 (Scheme 4). The procedure was quantitative and did not require purification of intermediates. Both cobalamin–cysteine conjugates could be efficiently labelled with [^99mTc(OH₂)₃(CO)₃]⁺ ( 1 ) under conditions identical to those of the model complex 5 . Biodistribution studies of the cobalamin conjugates in mice bearing B10‐F16 melanoma tumors showed a tumor uptake of 8.1±0.6% and 4.4±0.5% injected dose per gram of tumor tissue after 4 h and 24 h, respectively (Table 1).     

Synthesis and biological evaluation of novel 99mTc‐oxo and 99mTc‐tricarbonyl complexes with C3′‐functionalized thymidine dithiocarbamate for tumor imaging

A novel C3′‐functionalized thymidine dithiocarbamate derivative (3’DTC‐TdR) was successfully synthesized and labelled using [^99mTcO]³⁺ core and [^99mTc(CO)₃(H₂O)₃]⁺ core with high yields. The structures of the ^99mTc complexes were verified by preparation and characterization of the corresponding stable rhenium complexes. Both of the complexes were lipophilic and stable in vitro. Cell internalization experiments indicated that the uptakes of ^99mTcO‐3’DTC‐TdR were related to nucleoside transporters. Biodistribution of these complexes in mice bearing tumor showed that they had high tumor uptakes, good tumor/muscle ratios and tumor/blood ratios. Especially for ^99mTcO‐3’DTC‐TdR, it exhibited the highest tumor/muscle ratio and tumor/blood ratio at 4 h post‐injection. SPECT/CT imaging studies indicated clear accumulation in tumor, suggesting ^99mTcO‐3’DTC‐TdR would be a promising candidate for tumor imaging.     

Preparation and Evaluation of Novel Folate Isonitrile 99mTc Complexes as Potential Tumor Imaging Agents to Target Folate Receptors

In order to seek novel technetium-99m folate receptor-targeting agents, two folate derivatives (CN5FA and CNPFA) were synthesized and radiolabeled to obtain [^99mTc]Tc-CN5FA and [^99mTc]Tc-CNPFA complexes, which exhibited high radiochemical purity (>95%) without purification, hydrophilicity, and good stability in vitro. The KB cell competitive binding experiments indicated that [^99mTc]Tc-CN5FA and [^99mTc]Tc-CNPFA had specificity to folate receptor. Biodistribution studies in KB tumor-bearing mice illustrated that [^99mTc]Tc-CN5FA and [^99mTc]Tc-CNPFA had specific tumor uptake. Compared with [^99mTc]Tc-CN5FA, the tumor/muscle ratios of [^99mTc]Tc-CNPFA were higher, resulting in a better SPECT/CT imaging background. According to the results, the two ^99mTc complexes have potential as tumor imaging agents to target folate receptors.     

Chemistry at High Dilution: Dinuclear 99 mTc Complexes

The formation of di- or polynuclear complexes at nanomolar concentrations is generally too slow to be observed with ^99mTc. It is reported in this communication that an appropriate choice of potentially bridging ligands, herein thiols HS-R, accelerates the dimerization reaction to an extent that dinuclear complexes are formed at very high dilution. The dinuclear nature of [^99mTc₂(μ₂-SR)₃(CO)₆]⁻ is shown by chromatographic comparison, not only with its rhenium homologue as commonly done, but also with the true ⁹⁹Tc analogue.     

The case for fractional solubility profiles

This work reports the synthesis, radiolabeling and preliminary biodistribution results in tumor-bearing mice of [^99mTc(CO)₃(PA-TZ-CHC)]⁺. The novel colchicine (CHC) ligand was successfully synthesized via “click” reaction. Radiolabeling was performed in high yield with [^99mTc(CO)₃]⁺ core to get [^99mTc(CO)₃(PA-TZ-CHC)]⁺, which was hydrophilic and cationic, and was stable at room temperature. Biodistribution studies in tumor-bearing mice showed that [^99mTc(CO)₃(PA-TZ-CHC)]⁺ accumulated in the tumor with good uptake while comparatively low retention. The clearance of the ^99mTc-complex from normal organs was fast, resulting in increasing tumor/blood and tumor/muscle ratios. The promising results in preliminary biodistribution studies warrant further research to improve tumor targeting efficacy and pharmacokinetic profile of radiolabeled CHC derivative by structural modification.     

From TcVII to TcI; facile syntheses of bis-arene complexes [99(m)Tc(arene)2]+ from pertechnetate

Bis-arene complexes of technetium represent a fundamental class of organometallic compounds. Due to complex synthetic routes, no detailed insights into their properties have been reported so far. Reacting [⁹⁹TcO₄]^– with arenes in the exclusive presence of AlCl₃ gives highly stable [⁹⁹Tc(arene)₂]⁺ in good yields. These complexes have extraordinarily high stabilities, where oxidation is found to occur at potentials higher than +1.3 V and reduction at potentials below –2 V vs. Fc/Fc⁺. The ^99mTc analogues are similarly synthesised by applying a novel ionic liquid extraction pathway. Complexes of ^99mTc with suitably functionalized arenes will represent new building blocks for bioorganometallic pharmaceuticals in molecular imaging.     

Imaging Hydrogen Sulfide in Hypoxic Tissue with [99mTc]Tc-Gluconate

Hydrogen sulfide (H₂S) is the third gasotransmitter and is generated endogenously in hypoxic or inflammatory tissues and various cancers. We have recently demonstrated that endogenous H₂S can be imaged with [^99mTc]Tc-gluconate. In the present study, we detected H₂S generated in hypoxic tissue, both in vitro and in vivo, using [^99mTc]Tc-gluconate. In vitro uptake of [^99mTc]Tc-gluconate was measured under hypoxic and normoxic conditions, using the colon carcinoma cell line CT26, and was higher in hypoxic cells than that in normoxic cells. An acute hindlimb ischemia-reperfusion model was established in BALB/c mice by exposing the animals to 3 h of ischemia and 3 h of reperfusion prior to in vivo imaging. [^99mTc]Tc-gluconate (12.5 MBq) was intravenously injected through the tail vein, and uptake in the lower limb was analyzed by single-photon emission computed tomography/computed tomography (SPECT/CT). SPECT/CT images showed five times higher uptake in the ischemic limb than that in the normal limb. The standard uptake value (SUVmean) of the ischemic limb was 0.39 ± 0.03, while that of the normal limb was 0.07 ± 0.01. [^99mTc]Tc-gluconate is a novel imaging agent that can be used both in vitro and in vivo for the detection of endogenous H₂S generated in hypoxic tissue.     

Synthesis and biodistribution of 99mTc(CO)3-DMSA-MIBI in mice

Mixed ligand fac-tricarbonyl complex of [^99mTc(CO)₃-DMSA-MIBI] has been prepared starting from the precursor [^99mTc(OH₂)₃(CO)₃]⁺. The complex can be obtained in good yield and purity in a two-step procedure by first attaching meso-2,3-dimercaptosuccinic acid (DMSA, HOOCCH(SH)CH(SH)COOH) with [^99mTc(OH₂)₃(CO)₃]⁺, followed by addition of MIBI [tetrakis-2-methoxyisobutylisonitrile (CH₃OC(CH₃)₂CH₂-N≡C) copper(I) tetrafluoroborate] solution. The complex was characterized by TLC and HPLC and was studied by means of octanol-water partition coefficient, electrophoresis, stability in vitro, and normal mice experiment. Biodistribution in mice demonstrated that the complex showed higher myocardial uptake after 0.5-hour p.i. The ratios of heart/liver (%ID/g) in the case of ^99mTc(CO)₃-DMSA-MIBI was higher (1.88) than that observed in case of ^99mTc-MIBI¹ (0.93) after 0.5-hour p.i. (P<0.05). Results showed that the complex may be developed to a novel myocardial perfusion-imaging agent.     

Exploring the nitrosyl-approach: “Re(CO)2(NO)”- and “Tc(CO)2(NO)”-complexes provide new pathways for bioorganometallic chemistry

Nitrosylation reactions are rare in the context of low valent Re(I)- and Tc(I)-tricarbonyl complexes so far. We herein describe a method for the conversion of a “M(CO)₃-moiety” (M = Re, Tc) into a dicarbonyl-nitrosyl moiety “M(CO)₂NO”, the synthesis of important precursor complexes and intermediates and possible applications for this new kind of Re- and Tc-chemistry.The behavior of the complex [ReCl₃(CO)₂(NO)]⁻ in water was studied in detail and compared to that of [ReCl₃(CO)₃]²⁻. Contrary to the conversion of [ReCl₃(CO)₃]²⁻ to the mixed aquo-carbonyl complex [Re(OH₂)₃(CO)₃]⁺ in water, one chloride remains initially bound to the metal center in the dicarbonyl-nitrosyl complex, making [ReCl(OH₂)₂(CO)₂(NO)]⁺ the main species for further reactions. In this context, we isolated and characterized the complex [Re(μ₃-O)(CO)₂(NO)]₄. Examples of complexes with different bi- and tridentate ligands based on ReCl₃(CO)₂(NO)]⁻ are discussed.For the development of potential new radiopharmaceuticals we also adapted the nitrosylation technique to the n.c.a. level with ^99mTc. [^99mTc(OH₂)₃(CO)₃]⁺ served as starting material to form a ^99mTc(CO)₂(NO)-core. Labelling reactions with ligands such as iminodiacetic acid (IDA), nitrilotriacetic acid (NTA) and diethylenetriamine pentaacetic acid (DTPA) were performed, resulting in the complexes [^99mTc(IDA)(CO)₂(NO)], [^99mTc(NTA)(CO)₂(NO)] and [^99mTc(DTPA)(CO)₂(NO)]. In this way, the “nitrosyl-approach” adds a new and challenging synthetic tool to the already established organometallic chemistry of Re- and Tc-tricarbonyl complexes.     

Synthesis, Radiolabelling and in vitro Stability Study of 99mTc(CO)+3 Labeled Dendrimer PAMAM-Folic Acid Conjugate

Dendrimer polyamidoamine generation five‐folic acid conjugate was synthesesed and radiolabelled with fac‐[^99mTc(CO)₃(H₂O)₃]⁺, its in vitro stability was evaluated further. Both of the labeling yield and radiochemical purity of the G5‐FA‐DTPA‐^99mTc(CO)₃ conjugate exceeded 95%. More than 95.7% and 93.1% of the conjugate still keeps its original structure in PBS and new‐born calf serum solution respectively.