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.     

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.     

Radiosynthesis and evaluation of novel [99mTc(I)]+ and [99mTc(I)(CO)3]+ complexes with a 4-nitroimidazole isocyanide for imaging tumor hypoxia

[^99mTc(I)]⁺ and [^99mTc(I)(CO)₃]⁺ complexes with isocyanide exhibit high stability, which makes them suitable platforms to develop novel ^99mTc radiopharmaceuticals. To develop novel ^99mTc radiotracers for imaging hypoxia, in this study, a novel L ligand (4-nitroimidazole isocyanide derivative) was synthesized and labelled using [^99mTc(I)]⁺ core and [^99mTc(I)(CO)₃]⁺ core to produce [^99mTc(L)₆]⁺ and [^99mTc(CO)₃(L)₃]⁺ with high yields. To verify the structure of the ^99mTc complexes, corresponding rhenium analogues were synthesized and characterized. Both of the ^99mTc complexes were stable and hydrophilic. in vitro cellular uptake results showed they could exhibit good hypoxic selectivity. The evaluation of biodistribution in mice bearing S180 tumors indicated both of them could accumulate in tumor. Between them, [^99mTc(L)₆]⁺ exhibited higher tumor uptake and tumor/non-target ratio than [^99mTc(CO)₃(L)₃]⁺. Further, single photon emission computed tomography (SPECT) imaging studies of [^99mTc(L)₆]⁺ indicated an obvious accumulation in tumor and the value of the region-of-interest (ROI) ratio of the uptake for the tumor site to the corresponding non-tumor region was 5.64 ± 0.52. The above results suggested [^99mTc(L)₆]⁺ would be a potential tracer for imaging tumor hypoxia.     

Two novel procedures of preparation for [Tc(CO)<Subscript>2</Subscript>(NO)]<Superscript>2+</Superscript>labeled by EHIDA and its biodistribution

To develop potential new Tc radiopharmaceuticals, a novel compound [^99mTc(CO)₂(NO)(EHIDA)]⁰ (EHIDA: 2,6-diethylphenylcarbamoylmethyliminodiacetic acid) has been prepared by reacting [^99mTc(CO)₃)(EHIDA)]⁻ with NOBF₄ both in water and acetonitrile. The conversion of [^99mTc(CO)₃)(EHIDA)]⁻ to [^99mTc(CO)₂(NO)(EHIDA)]⁰ was supported by TLC, HPLC and eletrophoresis. The radiochemical purity (more than 99%) was proved by TLC and HPLC. The biodistribution in mice demonstrated that [Tc(CO)₂(NO)(EHIDA)]⁰ showed higher uptake in blood, kidney and lung (15 min, blood: 19.24±2.95; kidney: 13.61±3.49; lung: 10.81±1.09.) but a lower uptake in liver (15 min, 5.73±0.74). The slower clearances (120 min, blood: 12.75±1.34; kidney: 13.61±3.49) from blood and kidney were also found. This research describes two methods for the conversion of [^99mTc(CO)₃]⁺ into [^99mTc(CO)₂)(NO)]²⁺ by using NOBF₄ as the source of NO⁺ both in organic solvent and water. The latter method offers the possibility to introduce the NO-group in high yield in water.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

[M(CO)2(NO)], a new core in bioorganometallic chemistry: model complexes of [Re(CO)2(NO)] and [Tc(CO)2(NO)]

In this study selected bidentate (L²) and tridentate (L³) ligands were coordinated to the Re(I) or Tc(I) core [M(CO)₂(NO)]²⁺ resulting in complexes of the general formula fac-[MX(L²)(CO)₂(NO)] and fac-[M(L³)(CO)₂(NO)] (M = Re or Tc; X = Br or Cl). The complexes were obtained directly from the reaction of [M(CO)₂(NO)]²⁺ with the ligand or indirectly by first reacting the ligand with [M(CO)₃]⁺ and subsequent nitrosylation with [NO][BF₄] or [NO][HSO₄]. Most of the reactions were performed with cold rhenium on a macroscopic level before the conditions were adapted to the n.c.a. level with technetium (^99mTc). Chloride, bromide and nitrate were used as monodentate ligands, picolinic acid (PIC) as a bidentate ligand and histidine (HIS), iminodiacetic acid (IDA) and nitrilotriacetic acid (NTA) as tridentate ligands. We synthesised and describe the dinuclear complex [ReCl(μ-Cl)(CO)₂(NO)]₂ and the mononuclear complexes [NEt₄][ReCl₃(CO)₂(NO)], [NEt₄][ReBr₃(CO)₂(NO)], [ReBr(PIC)(CO)₂(NO)], [NMe₄][Re(NO₃)₃(CO)₂(NO)], [Re(HIS)(CO)₂(NO)][BF₄], [⁹⁹Tc(HIS)(CO)₂(NO)][BF₄], [^99mTc(IDA)(CO)₂ (NO)] and [^99mTc(NTA)(CO)₂(NO)]. The chemical and physical characteristics of the Re and Tc-dicarbonyl-nitrosyl complexes differ significantly from those of the corresponding tricarbonyl compounds.     

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.     

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.     

Cyclopentadienyl tricarbonyl complexes of Tc for the in vivo imaging of the serotonin 5-HT1A receptor in the brain

Technetium and rhenium tricarbonyl complexes with derivatized cyclopentadienyl ligands were prepared starting from pertechnetate and an appropriate ferrocene ligand. Furthermore, the complexes (M(CO)₃cp-COOC₅H₉N-R, M = Tc, Re; R = Me, isopropyl) could be obtained starting from the precursor complexes [^99mTc(CO)₃(H₂O)₃]⁺ and [Re(CO)₃Br₃]²⁻. Their chemical identity was confirmed by chromatographic methods and electron spray mass spectrometry. The biodistribution of the ^99mTc complexes (cytectrene I and cytectrene II) in Wistar rats was studied. Both compounds show high uptake in the brain and fast blood clearance. The pattern of regional distribution in the brain demonstrated in autoradiographic studies indicates binding to the 5-HT_1A and α₁ adrenergic receptors.     

Using Tc-Re chemical analogy to synthesize and identify new99mTc complexes

The strong chemical resemblance between Tc and Re is applied to design and evaluate experiments with^99mTc complexes. A combination of spectrophotometric and electrophoretic techniques allows to propose the formula [TcO₂/amine/₂]⁺ for compounds prepared by reduction of^99mTcO ₄ ^– with Zn /solid phase/ in presence of several /bidentate/ amines.     

Radiosynthesis and in vitro evaluation of 99mTc(CO)3-labeled folic acid derivative

The over-expression of folate receptors in variety of neoplastic tissues makes radiolabeled folate conjugates potential agents for imaging and therapy of such cancers. With the aim of preparing an imaging agent for targeting folate receptors, folic acid has been conjugated with homocysteine for complexation with [^99mTc(CO)₃(H₂O)₃]⁺ core. The radiolabeled complex of the homocysteine-folate could be obtained in >95% radiochemical yield as observed by HPLC. Stability of complex in saline was studied and challenge studies with histidine and cysteine revealed kinetic stability of the complex. Lipophilicity of the radiolabeled complex (log P) was found to be 0.45. In vitro uptake of ^99mTc(CO)₃-labeled folic acid derivative was studied in KB cells and inhibition studies were carried out using ³H-folic acid and cold homocysteine–folate conjugate. The in vitro studies indicated loss of binding affinity of the derivative towards folate receptors.