Picolinic acid based acyclic bifunctional chelating agent and its methionine conjugate as potential SPECT imaging agents: syntheses and preclinical evaluation

Abstract

Bifunctional chelate, 6,6′-(2-aminoethylazanediyl)bis(methylene)dipicolinic acid (H₂pentapa-en-NH₂), has been synthesized and labeled with ^99mTc with a specific activity of 135–140 MBq μmol⁻¹ in >95% yield. The in vitro stability of the labeled chelate in both PBS and human serum shows only <5% dissociation at 24 h. The in vivo distribution pattern of the labeled chelator in normal as well as EAT tumor bearing BALB/c mice suggested renal as the major route of excretion with <2% ID g⁻¹ uptake in other organs. The target specificity of the chelate towards tumor was introduced by conjugating two molecules of methionine. The conjugated probe H₂pentapa-en-met₂ was synthesized in >85% yield and labeled with ^99mTc in 96.2% radiochemical yield with a specific activity of 110–125 MBq μmol⁻¹. The conjugate probe exhibited high serum stability (>94% at 24 h). The in vivo blood kinetic studies of radiocomplexes of H₂pentapa-en-NH₂ and its methionine conjugated derivative exhibited fast clearance with t_1/2(F) = 32 ± 0.14 min, t_1/2(S) = 4 h 20 min ± 0.21 min and t_1/2(F) = 27 ± 0.3 min, t_1/2(S) = 4 h 01 min ± 0.11 min, respectively. In vivo scintigraphy and ex vivo biodistribution studies in EAT tumor bearing mice demonstrated a high retention of H₂pentapa-en-met₂ at the site of the tumor with tumor to muscle ratio of 6.52 at 1 h, indicating the high specificity of ^99mTc-pentapa-en-met₂ toward tumors.

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Introduction

The central role of SPECT imaging in a non-invasive assessment of organ functions and pathology has led to an upsurge in the design and development of molecular probes to visualize the cellular processes in normal and diseased states.¹ There is a concerted effort for new and efficient chelators that form stable complexes with a variety of radiometal isotopes significant for diagnostic imaging.

Classical research in this area is focused almost exclusively on bifunctional chelators (BFC) based on tetraazamacrocyclicpolycarboxylates (DOnA, n = 1, 2, etc.) and penta(carboxymethyl)diethylenetriamine (DTPA) derivatives. Though the stability of the DOTA derivatives is encouragingly good, elevated temperature requirement for radiolabeling limits their use with biovectors that are sensitive towards high temperatures. The DTPA derivatives form complexes with different radioactive elements at ambient temperature, but the in vivo stability of the complexes is generally less when compared to DOTA derivatives.² Therefore, there is an inherent need of developing new acyclic chelators, which can provide in vivo stability similar to DOTA derivatives and fast complexation at ambient temperature as DTPA derivatives with radiometals applied in theranosis. Towards the same direction, currently, chelators belonging to the “pa” family are being explored due to their desirable properties, which include mild labeling conditions and stable complexation with radiometals. The “pa” family includes well-known bifunctional chelators, viz. H₄octapa, H₂dedpa, H₅decapa, H₂azapa complexed with almost all the current medicinally relevant radiometals such as In(III), Yb(III), Lu(III), Re, Cu(II) and Ga(III).³ However, these chelators have not been utilized with ^99mTc, a radiometal, which has always remained the mainstay of diagnostic nuclear medicine with more than 85% utilization in the diagnostic scans performed clinically. The preferential use of ^99mTc-radiopharmaceuticals is justified by its convenient availability in the form of commercial generators and ideal nuclear properties for imaging applications due to favourable half-life (6 h), gamma ray energy (E_γ = 140 keV; 89% abundance) and also low dose burden to patients.

Based on the “pa” scaffold, a chelate, 6,6′-(2-aminoethylazanediyl)bis(methylene)dipicolinic acid, H₂pentapa-en-NH₂ has been synthesized, which contains two pyridine moieties derivatized with carboxylate groups. For the synthesis of the chelator, pyridine-2,6-dicarbonyl chloride was used as a precursor and conjugated to the amine group of ethylene diamine, thus providing the basic skeleton of the chelate. Finally, in the last step, deprotection in 2 M NaOH gave H₂pentapa-en-NH₂ for further exploration. The developed agent was complexed with ^99mTc in high radiochemical purity and yield after optimising the various parameters of radiolabeling. The stability of the labeled complex in PBS and serum was high under physiological conditions. The uptake of ^99mTc-pentapa-en-NH₂ in various organs was checked in mice.

The bifunctional chelating agent H₂pentapa-en-NH₂ was further explored for targeted imaging after conjugation with the amino acid, L-methionine. The amino acids are interesting targets for metabolic tumor imaging due to the increased protein metabolism in cancer cells.⁴ Various transport mechanisms are known to be involved in providing the tumor cells with an adequate amount of amino acids. The increased amino acid transport in malignant transformation occurs through carrier mediated processes due to an overexpression of amino acid transporter genes. Among the different membrane transporter proteins, the most important and ubiquitously found is L-type amino acid transporter 1 and 2 (LAT1 and LAT2), which have been identified for the transport of large neutral amino acids from the extracellular fluids into the cells.⁵ Apart from an increased L-methionine requirement due to elevated protein synthesis in the cancerous cell, it also falls in the category of essential amino acid and is involved in biological activities such as the initiation of translation in cells as a methyl group donor in many biosynthetic pathways.⁶ The oxidation and reduction of methionine also regulates the various functions of cells, thereby making it a desirable candidate for imaging studies.^{7 11}C-Methionine is one of the most useful biomarkers for the PET imaging of various cancers; however, due to the limitation of a short half-life of 20.3 min and the requirement of an onsite cyclotron to produce ¹¹C, a range of methionine derivatives have been developed for tumor imaging with the radioactive element having a longer half-life.⁸ Thus, it was thought worthwhile to assess the application of a developed agent as BFC in targeted imaging by conjugating it with L-methionine. The methionine conjugate of the chelate was radiolabeled with ^99mTc to investigate its tumor targeting efficacy. The in vitro stability, pharmacokinetics behaviour, scintigraphic study and biodistribution profile on Ehrlich ascites tumor (EAT) bearing BALB/c mice were also studied in detail.

Results and discussion

There is a concerted effort to develop a new class of bifunctional chelating agents, and the interest is towards acyclic chelators based on a pyridine carboxylate scaffold as a potential alternative to classical chelators with significance in nuclear medicine.

Here, the focus has been directed towards the development of a “pa” chelate using a simple and versatile synthetic approach and utilized to prepare LAT1 specific molecules. In this work the applicability of ^99mTc with “pa” family chelators for tumor targeting and imaging has been reported as an original finding. The suitability of the developed chelate in targeted imaging was evaluated after linking it with a tumor targeting biomolecule, L-methionine.

Chemistry

The bifunctional chelating system was synthesized according to Scheme 1. A bivalent approach was employed to synthesize H₂pentapa-en-met₂, giving a homodimeric bivalent ligand with a thermodynamically more favourable binding interaction than a monovalent binding of two molecules on independent recognition sites. The strategy used for the synthesis is facile, versatile and high yielding with easy purification steps. Pyridine-2,6-dicarbonyl dichloride (1) was taken as the precursor and was converted to an ester derivative, diethyl pyridine-2,6-di carboxylate (2), for further reactions. One of the ester groups was selectively reduced to alcohol (3) and modified to a chloro group (4) using thionyl chloride. The chloro derivative of pyridine carboxylate was conjugated to the derivative of ethylene diamine wherein one amino functionality was selectively protected by a Boc group. The di-pyridyl carboxylate derivative (7) was obtained and the cleavage of the Boc and ester groups with trifluoroacetic acid and 2 M NaOH, respectively, gave the final bifunctional chelate, 6,6′-(2-aminoethylazanediyl)bis(methylene)dipicolinic acid (H₂pentapa-en-NH₂), in >95% yield from 8 with >84% overall yield. To employ the developed chelate for targeted imaging, the probe was conjugated to the tumor targeting biomolecule, L-methionine. For this purpose, deprotected derivative (8) was linked to methyl 2-amino-4-(methylthio)butanoate (10) via a linker to give 6-(9-carboxy-5-(1-carboxy-3-(methylthio)propylamino)-2-oxoethyl)-2-((6-carboxypyridin-2-yl)methyl)-7-oxo-12thia-2,5,8-triazatridecyl-picolonic acid (H₂pentapa-en-met₂) in 89.1% yield (Scheme 1). The final molecules were purified using preparatory HPLC and purity was found to be >98% (Fig. S30 and S31, ESI† data). In the ¹H NMR spectrum, the peaks in the region of 7.57–7.88 ppm were due to aromatic protons of the chelating agent, whereas the peak at 2.11 ppm confirmed the presence of methionine in the final compound. The molecular ion peak at 709.8 in the mass spectrum further confirmed the formation of the H₂pentapa-en-met₂ (Fig. S1–S27, ESI† data).

Scheme 1 Synthesis of 6-(9-carboxy-5-(1-carboxy-3-(methylthio)propylamino)-2-oxoethyl)-2-((6-carboxypyridin-2-yl)methyl)-7-oxo-12thia-2,5,8 triazatridecyl-picolonic acid (H₂pentapa-en-met₂). Reagents and conditions: a = ethanol, reflux, b = sodium-borohydride, ethanol, reflux, inert atmosphere, c = thionyl chloride, 0 °C, d = di-tert-butyl dicarbonate, NEt₃, ethanol, 0 °C, e = anhyd ACN, K₂CO₃, reflux, f = trifluoroacetic acid, 0 °C, g = 2 M NaOH, methanol, R.T., h = 2-chloroacetyl chloride, K₂CO₃, DCM/H₂O, 0 °C, i = (1) anhyd ACN, K₂CO₃, reflux and (2) 2 M NaOH, methanol, R.T.

DFT studies

Density functional theory (DFT) studies were performed for H₂pentapa-en-NH₂ and H₂pentapa-en-met₂ to predict their stability after complexation with respective metal and also to calculate the electrostatic potential surfaces. During DFT analysis, rhenium was used in place of technetium. There has been a significant interest in using Re as the cold congener of ^99mTc, because Re is the third row group VII transition-metal analogue of Tc. Both Re and Tc exhibit similar chemical properties, size and coordination chemistry.⁹ Hence, it is a standard practice to use Re for studying the size/charge of the compound.¹⁰ Here, DFT analysis was performed on the complexes H₂pentapa-en-NH₂ and H₂pentapa-en-met₂ with Re because computationally DFT optimization of Tc is not tractable, whereas the Re optimization leads to convergence with the fine ground state without being computationally expensive. DFT studies were also performed for the copper complexes of H₂pentapa-en-NH₂ and H₂pentapa-en-met₂ to explore their potential in PET imaging with ⁶⁴Cu.

The final ligand has been optimized under B3LYP hybrid functional of DFT and thus found to be optimized with highly accurate calculations with increased grid density. The coordination geometry and mapping of electrostatic surfaces of 4- and 6-coordinated Cu(II) and Re(V) complexes that were estimated through DFT in silico calculations seems to be not much affected with the incorporation of methionine. The bond lengths, bond angles and torsional angles were found to be quite similar in both the cases of the studied complexes after DFT optimization. To further confirm the suitability of structure selected for metal complex of H₂pentapa-en-met₂ for performing DFT studies, two more plausible structures of the complex (ReO complexed with methionine residues of the pentapa-en-met₂) were studied. After fitting the Re oxocore with methionine instead of chelate, the DFT analyses revealed relatively longer bond lengths between Re–S and Re–O–CO thereby decreasing the possibility of Re complexation with methionine residues (Table S1 and Fig. S33 in ESI† data).

The Re complex was in plane in comparison to the Cu complex where the backbone of methionine is out of the plane as it was in optimized ligand. The overall impression from the bond lengths and bond angles shows that the metal cavity of Re and Cu complexes is quite large to accommodate the metals approximately at an angle of 110°, whereas the methionine ends of the ligand are pushed outwards. The insight into the MO isosurfaces of Cu and Re displays an electron density and electrostatic potential distributed over the complexes with maximum contribution of moderate charge density over the complexes (Fig. 1 and S32, ESI† data). Thus, DFT calculations predict that Re and Cu complexes of the ligand are stable in solution phase in terms of their geometry and charge distribution, which leads to their important imaging applications.

Fig. 1 In silico DFT predicted electrostatic potential map of [ReO(pentapa-en-met₂)]⁺ and Cu-pentapa-en-met₂ varying from −0.07 a.u. to +0.06 a.u. and −0.1189 a.u. to +0.0980 a.u., respectively.

Radiolabeling with ^99mTc

The developed chelate and its methionine conjugate were radiolabeled with ^99mTc to assess their potential in SPECT imaging. The “pa” based ligand has already been explored with ¹¹¹In;³ however, we directed our work towards ^99mTc due to its easy and cost effective production from ⁹⁹Mo/^99mTc generator. Moreover the half-life of ¹¹¹In is too high (t_1/2 = 2.8 days) compared to that of ^99mTc (t_1/2 = 6.1 h); therefore, the usage of ^99mTc can reduce the radiation burden on the subject during biological studies over ¹¹¹In.

The various parameters of radiolabeling were optimized, and the stability of the formed radiocomplexes was checked. The pH plays an important role in the radiolabeling, and the percentage of labeling varied with pH. The optimum pH range was 6.5–7.5 for ^99mTc-pentapa-en-NH₂ and ^99mTc-H₂pentapa-en-met₂. At higher pH, a gradual decrease in labeling yield and increase in the percentage of colloid formation was observed (Fig. S34 in ESI† data). Similarly, the amount of stannous chloride used also had an important role on the labeling yield. The effect of the concentration of stannous chloride on the labeling of H₂pentapa-en-NH₂ and H₂pentapa-en-met₂ was checked using various concentrations of stannous chloride from 50 to 250 μg, keeping other parameters constant. The labeling was observed at its maximum at 150–175 μg (Fig. S35 in ESI† data). The percentage of free and labeled ligand was determined using ITLC-SG strips. On ITLC-SG, labeled complexes (^99mTc-pentapa-en-NH₂, ^99mTc-pentapa-en-met₂) remained at the point of application, whereas free ^99mTc moves towards the solvent front in 100% acetone as a mobile phase. The radiochemical yield of H₂pentapa-en-NH₂ with ^99mTc was found to be 97.2% by ITLC-SG (Fig. 2), and the specific activity was 135–140 MBq μmol⁻¹. A decrease of 7% after 24 h indicates its high stability.

Fig. 2 ITLC profile of ^99mTc complexes of H₂pentapa-en-NH₂ and H₂pentapa-en-met₂.

Fig. 3 In vitro serum stability profile of ^99mTc-pentapa-en-NH₂ and ^99mTc-pentapa-en-met₂.

The radiolabeling yield of H₂pentapa-en-met₂ with ^99mTc was 96.2% (Fig. 2) at 30 min, and after 24 h, a decrease of 10% was observed. The specific activity of ^99mTc-pentapa-en-met₂ was found to be 110–125 MBq μmol⁻¹.

The results show high radiochemical yield and stability of the radiocomplexes, which are the prerequisites for clinical studies. It demonstrates the good potential of the developed probes in SPECT imaging.

To further confirm the structure of ^99mTc-pentapa-en-NH₂ complex, a cold rhenium complex was prepared and their HPLC profiles were compared. The similar retention time (^99mTc-complex: 7.13 min; ¹⁸⁵Re-complex: 7.06 min) under identical HPLC conditions indicates the formation of the same species¹¹ (Fig. S38 and S39 in ESI† data).

Thermodynamic studies

To explore the utility of developed systems with radiometal ⁶⁴Cu, the stability of the probes with copper was evaluated potentiometrically. The high stability of the metal chelates is imperative for in vivo applications. To determine the stability of the developed ligands with Cu²⁺, thermodynamic studies were performed by potentiometric titrations. The protonation constants of H₂pentapa-en-NH₂ and H₂pentapa-en-met₂ were determined in the pH range of 2–12. Two protonation constants for the ligand H₂pentapa-en-NH₂ (log K^H₁ = 10.95 ± 0.01, log K^H₂ = 7.72 ± 0.04) and three protonation constants for H₂pentapa-en-met₂ (log K^H₁ = 10.80 ± 0.05, log K^H₂ = 8.43 ± 0.04, log K^H₃ = 4.46 ± 0.02) associated to carboxylic acid protons were obtained (Table 1).

Table 1 Protonation constants (log K ± S.D, n = 3) of H₂pentapa-en-NH₂ and H₂pentapa-en-met₂ (298 K, μ = 0.1 M NMe₄Cl)

	H2pentapa-en-NH2	H2pentapa-en-met2
log K^H1	10.95	10.80
log K^H2	7.72	8.43
log K^H3	—	4.46

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The results from DFT and thermodynamics studies encouraged us to complex H₂pentapa-en-NH₂ and H₂pentapa-en-met₂ with copper. H₂pentapa-en-NH₂ and H₂pentapa-en-met₂ were metalated with CuCl₂ to form corresponding cold complexes. The characterization of complexes, Cu(pentapa-en-NH₂) and Cu-(pentapa-en-met₂), using NMR spectroscopy was not feasible because of the paramagnetic nature of copper, and hence the products were characterized through mass spectrometry. In the mass spectrum, the peaks at 392.7 and 772.0 confirmed the formation of corresponding cold complexes of copper, Cu(pentapa-en-NH₂) and Cu(pentapa-en-met₂) with the characteristic isotopic pattern of ⁶³Cu/⁶⁵Cu (Fig. S28 and S29, ESI† data). The complex formation equilibria of H₂pentapa-en-NH₂ and H₂pentapa-en-met₂ with Cu²⁺ was also studied and the stability constant (K_ML) was calculated. The stability constant of H₂pentapa-en-NH₂ and H₂pentapa-en-met₂ with Cu²⁺ was found to be 20.8 and 19.9, respectively. The data was similar to the stability constant of the reported copper complex of H₂dedpa (19.2) (Table 2). However, these encouraging preliminary studies with copper justify its further analysis for validating the suitability of the Cu-64 complex in vitro and in vivo.

Table 2 Stability constants (log β ± S.D, n = 3) of different “pa family” chelators with metal ions

	H2dedpa^a	H4octapa^a	H2decapa^a	H2pentapa-en-NH2	H2pentapa-en-met2^b
a Ref. 14.b 298 K, μ = 0.1 M NMe4Cl.
Cu^2+	19.2	—	—	20.8	19.9
Ga^3+	28.1	—	—	—	—
In^3+	—	26.8	27.5	—	—
Lu^3+	—	20.1	—	—	—

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In vitro studies

Stability studies in PBS buffer. The in vitro stability of ^99mTc-pentapa-en-NH₂ in PBS buffer was assessed. 96.4% and 93.9% of intact tracer was observed on ITLC after 30 min and 24 h of incubation at pH 7.2, respectively. The stability of ^99mTc-pentapa-en-met₂ in PBS was also almost constant over the entire observation period of 24 h and was in the range of 97.2–93.2% (Fig. S36 in ESI† data). This suggested no significant detachment from the radiolabeled complex even after 24 h of incubation at physiological pH.

Stability studies in human serum. The strong binding to serum protein such as albumin often delays blood clearance and lowers the target to background ratio; in addition, human plasma enzymes also tend to decompose the exogenous compounds. Consequently, a binding study with human serum protein is a must for correct prediction of the biological importance of the probe. The serum stability studies were performed to also assess the trans-complexation of chelate (^99mTc-pentapa-en-NH₂) and conjugate (^99mTc-pentapa-en-met₂) with biological metal ions. Under physiological temperature, the stability of ^99mTc-pentapa-en-NH₂ and ^99mTc-pentapa-en-met₂ was evaluated in human serum, and the stability pattern was followed for 24 h (Fig. 3). The stability pattern exhibited >98% stability at 2 h for both complexes, whereas after 24 h, it was 96.1% for ^99mTc-pentapa-en-NH₂ and 94.5% for ^99mTc-pentapa-en-met₂ (Fig. S37 in ESI† data). The high stability percentage of complexes in serum clearly predicts negligible binding with protein and trans-complexation with other biological metal ions present in serum and highlights their suitability for in vivo studies.

In vivo studies

Blood kinetics. Blood kinetic studies were carried out to observe the binding affinity of both the chelate (^99mTc-pentapa-en-NH₂) and conjugate (^99mTc-pentapa-en-met₂) towards serum proteins to further understand its in vivo suitability. In vivo, the blood kinetics study exhibited rapid blood clearance, as observed in the blood kinetics data of radiolabeled drugs at various time intervals, which could be corroborated to the hydrophilic character of the agents (Fig. 4). Approximately, 80% and 92% of drug clearance was observed in 5 and 30 min, respectively. After 4 h, negligible activity was left in the blood. The biological half-lives of ^99mTc-pentapa-en-NH₂ and ^99mTc-pentapa-en-met₂ were found to be t_1/2(F) = 32 ± 0.14 min, t_1/2(S) = 4 h 20 ± 0.21 min and t_1/2(F) = 27 ± 0.3 min, t_1/2(S) = 4 h 01 ± 0.11 min, respectively. The fast clearance of labeled complexes from the blood pool not only results in the minimization of background activity but also validates the low binding affinity of both complexes towards blood serum proteins.

Fig. 4 Blood kinetics graph of ^99mTc-pentapa-en-NH₂ and ^99mTc-pentapa-en-met₂ in rabbit.

Scintigraphy and biodistribution studies. The scintigraphy studies were carried out on a normal New Zealand rabbit by imaging the animal at different time intervals. It aided in visualizing the distribution pattern of ^99mTc labeled complexes in various organs. Both ^99mTc-pentapa-en-NH₂ and ^99mTc-pentapa-en-met₂ showed fast and appreciable accumulation in the kidneys suggesting a renal route of excretion (Fig. 5). Biodistribution studies are helpful for the screening of the biological profile and to anticipate the potential of the developed probe at the target site. Biological distribution was studied in mice and percent injected activity (%IA) associated with each organ was calculated based on the activity measured per gram of organ or tissue.

Fig. 5 γ-Scintigraphic images of (a) ^99mTc-pentapa-en-NH₂ and (b) ^99mTc-pentapa-en-met₂ in normal rabbit at 1 h post injection.

Biodistribution studies were first performed with ^99mTc-pentapa-en-NH₂ on normal BALB/c mice to authenticate the distribution profile obtained through scintigraphy. The accumulation in kidney at 1 h and 2 h post injection was 20.03 ± 0.94 and 8.47 ± 0.42 ID g⁻¹, respectively (Fig. 6a). Early kidney activity predominantly results from the rapid urinary excretion of ^99mTc-pentapa-en-NH₂ and indicates a renal route of excretion. It will lead to low absorbed doses of radiation to the body. The accumulation of the radioactivity in the liver, stomach and small intestine was <2% ID g⁻¹; thus, the complex remained essentially inert with fast blood clearance and no retention in the organs. To better understand the target specific behaviour of radiolabeled H₂pentapa-en-met₂, the distribution pattern of the radiolabeled H₂pentapa-en-NH₂ was also understood on tumor bearing mice and both patterns were compared. The pattern of distribution of the labeled chelate in the tumor bearing mice in different organs was similar to that of normal mice and exhibits high uptake in the kidney (18.98% ± 1.28% ID g⁻¹ at 1 h; 7.98% ± 0.58% ID g⁻¹ at 2 h) and <2% ID g⁻¹ uptake in other organs (Fig. 6b). In tumors, the accumulation of the complex was like other organs with fast clearance rate. It suggested that the chelate has low specificity towards tumors. However, the distribution of the methionine conjugate, ^99mTc-pentapa-en-met₂, in tumor bearing mice was different from the parent chelate. The uptake in the kidney was 5.6% ± 0.87% ID g⁻¹ and 3.06% ± 0.58% ID g⁻¹ at 1 h and 2 h post injection, respectively. The biodistribution pattern of “pa” family known chelates H₄octapa, H₂dedpa, H₅decapa and H₂azapa complexed with radioisotopes, such as ⁶⁴Cu, ¹¹¹In, ¹⁷⁷Lu, and ⁶⁸Ga, have demonstrated a renal route of excretion in mice with low uptake in other organs.³ The same trend was followed by the synthesized probes, H₂pentapa-en-NH₂ and H₂pentapa-en-met₂, after labeling with ^99mTc.

Fig. 6 Biodistribution profile of ^99mTc-pentapa-en-NH₂ in (a) normal (b) EAT BALB/c mice at 0.5, 1 and 2 h after intravenous injection through tail vein.

The retention of ^99mTc-pentapa-en-met₂ in liver was for a longer period possibly because it is the inherent nature of amino acids based derivative to metabolise in the liver.⁸ The uptake of ^99mTc-pentapa-en-met₂ conjugate in the other non-target organs got significantly lower than the chelate as conjugation with methionine changed its final chemical properties which corroborated to the decreased accumulation in non-target organs along with a fast clearance rate from the organs. The accumulation in the target organ tumor was there for a longer time period.

At the site of the tumor, the accumulation and residence time of ^99mTc-H₂pentapa-en-met₂ increased with time, leading to the retention of the radio complex till 2 h. Such behaviour increased its tumor to muscle ratio (T/M) 6.52% ± 0.24% ID g⁻¹ at 1 h and 6.13% ± 0.47% ID g⁻¹ at 2 h (Fig. 7) in comparison to 1.60% ± 0.25% ID g⁻¹ and 1.39% ± 0.28% ID g⁻¹ for the chelate. Furthermore, biodistribution analysis was supported by scintigraphic studies carried out in EAT tumor bearing BALB/c mice (Fig. 8a and b).

Fig. 7 Biodistribution profile of ^99mTc-pentapa-en-met₂ in EAT tumor bearing BALB/c mice at 0.5, 1, 2, 3 and 4 h after intravenous injection through tail vein.

Fig. 8 γ-Scintigraphic image of (a) ^99mTc-pentapa-en-NH₂ and (b) ^99mTc-pentapa-en-met₂in EAT tumor bearing BALB/c mice at 1 h post injection.

Experimental section

Materials and methods

Chemicals. Pyridine-2,6-dicarbonyl dichloride, ethanol, sodium-borohydride, thionyl chloride, potassium carbonate, sodium sulphate, sodium bicarbonate, di-tert-butyl dicarbonate, 2-chloroacetyl chloride, trifluoroacetic acid, acetonitrile, chloroform, dichloromethane, water, stannous chloride, methanol, sodium hydroxide, hydrochloric acid, and cupric chloride were purchased from Sigma-Aldrich Co., USA. Column chromatography was carried out using silica MN60 (60–120 μm), TLC on aluminium plates coated with silica gel 1160, and F₂₅₄ (Merck, Germany). ^99mTc was collected from Regional Centre for Radiopharmaceuticals (Northern Region), Board of Radiation and Isotope Technology (BRIT), Department of Atomic Energy, India.

Instrumentation. Spectroscopic studies were carried out using the instruments Bruker Avance II 400 MHz system (Switzerland) and Agilent 6310 ion trap (USA, mass accuracy ± 0.2 u). Elemental analysis was performed using an elemental analyser system GmbH Vario EL-III instrument. Radiolabeling and biodistribution studies were performed using γ-scintillation counter (GRS230, ECIL, India). Potentiometric measurements were carried out with an automatic titration system consisting of Metrohm 713 pH meter equipped with a Metrohm A.60262.100 electrode and 800 Dosino autoburet. SPECT studies were performed using γ-camera HAWKEYE (USA, Siemens). HPLC analysis was performed using an Agilent 1200 LC coupled to a UV detector (λ = 254 nm). Flow rate for analytical HPLC was 0.4 mL min⁻¹, whereas for preparative HPLC, it was 10 mL min⁻¹. HPLC analysis of compounds was undertaken using C-18 RP columns (5 μm, 4.6 mm × 250 mm and 5 μm, 9.4 mm × 250 mm) and mobile phase (System A: 20% water and 80% acetonitrile; System B: 30% water and 70% acetonitrile) were used.

Ethics statement. The human serum study was approved by the institutional ethical committee. Blood samples were collected from volunteers who were informed that their role was completely voluntary. From all participants, written consent was obtained, and participants were above 18 years of age.

Animal model. Animal protocols have been approved by the institute's animal ethics committee (Reg. no. 8/GO/a/99/CPCSEA). New Zealand rabbits (2–3 kg) were used for the blood clearance study and BALB/c mice (22–28 g) for scintigraphy and biodistribution studies. Rabbits and mice were housed under the conditions of controlled temperature of 22 ± 2 °C and normal diet. BALB/c mice were inoculated subcutaneously with 0.1 mL of cell suspension of EAT (1.5 × 10⁷ cells) in the fore/hind limb under sterile conditions. Mice were used when the tumor volume reached 200–400 mm³ for biodistribution and scintigraphy studies.

Synthesis and characterisation

Diethyl pyridine-2,6-di carboxylate (2). Pyridine-2,6-dicarbonyl dichloride (1) (5.0 g, 24.50 mmol) was dissolved in ethanol (20 mL) in a round bottom flask and the reaction mixture was refluxed. After 16 h, the solvent of the reaction mixture was evaporated to dryness under reduced pressure. The residue was dissolved in dichloromethane, and the organic layer was washed with water (2 × 50 mL). The organic layer was dried over sodium sulphate, filtered and evaporated under reduced pressure to give a solid residue 2 as a pure product. Yield: 91.2%.

¹H NMR (400 MHz, CDCl₃) δ (ppm): 1.44 (t, 6H, J = 7.2, 2 × –CH₃), 4.47 (q, 4H, J = 6.8, 2 × –O–CH₂–), 7.99 (t, 1H, J = 7.6, –CH–) and 8.27 (d, 2H, J = 7.6, 2 × –CH–); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 14.20 (–CH₃), 62.38 (–CH₂–O–CO–), 127.82 (–CH–), 139.50 (–CH–CH–CH–), 148.58 (–C–) and 164.61 (–CO–); ESI-MS (m/z): C₁₁H₁₃NO₄Na found: 246.1 (M + Na)⁺, calculated: 246.0 (M + Na)⁺.

Ethyl 6-(hydroxylmethyl)picolinate (3). NaBH₄ (0.15 g, 4.03 mmol) was added to a solution of 2 (1.50 g, 6.72 mmol) in ethanol (20 mL). The reaction mixture was heated at 80 °C for 16 h. After completion of the reaction, solvent was evaporated under reduced pressure. The residue was dissolved in dichloromethane and washed with water (2 × 50 mL) to remove excess NaBH₄. The organic layer was dried over sodium sulphate, filtered and evaporated under reduced pressure to give a pure product of 3. Yield: 61.3%.

¹H NMR (400 MHz, CDCl₃) δ (ppm): 1.46 (t, 3H, J = 7.2, –CH₃), 3.52 (s, 1H, –OH), 4.50 (q, 2H, J = 6.8, –O–CH₂–), 4.87 (s, 2H, –CH₂OH), 7.51 (d, 1H, J = 8.0, –CH–), 7.86 (t, 1H, J = 8.0, –CH–) and 8.05 (d, 1H, J = 7.6, –CH–); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 14.26 (–CH₃), 61.92 (–CH₂–CH₃), 64.54 (–CH₂OH), 123.66 (–C–CH–CH–), 124.05 (–CH–CH–C–), 137.41 (–CH–CH–CH–), 147.23 (–CH–C–CO–), 160.30 (–C–CH₂OH) and 165.05 (–CO–); ESI-MS (m/z): C₉H₁₂NO₃ found: 182.1 (M + H)⁺, calculated: 182.0 (M + H)⁺.

Ethyl 6-(chloromethyl)picolinate (4). Compound 3 (1.80 g, 9.94 mmol) was dissolved in an excess of thionyl chloride (6.0 mL) at 0 °C under gentle stirring, and the reaction was left to stir at room temperature for another 18 h. After completion of the reaction, an excess of thionyl chloride was removed by distillation. The residue was dissolved in dichloromethane and washed with 20% sodium bicarbonate solution (2 × 10 mL). The organic layer was dried over sodium sulphate and concentrated under reduced pressure to get pure compound 4. Yield: 85.6%.

¹H NMR (400 MHz, CDCl₃) δ (ppm): 1.43 (t, 3H, J = 7.2, –CH₃), 4.48 (q, 2H, J = 6.8, –O–CH₂–), 4.78 (s, 2H, –CH₂–Cl), 7.73 (d, 1H, J = 7.6, –CH–), 7.89 (dd, 1H, J = 8.0, –CH–) and 8.06 (d, 1H, J = 7.6, –CH–); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 14.30 (–CH₃), 46.31 (–CH₂–Cl), 62.12 (CH₃–CH₂–O–), 124.37 (–CH–CH–C–), 126.06 (–C–CH–CH–), 138.12 (–CH–CH–CH–), 147.73 (–CH–C–CO–), 157.25 (–C–CH₂OH) and 164.83 (–CO–); ESI-MS (m/z): C₉H₁₁ClNO₂ found: 200.1 (M + H)⁺, calculated: 200.0 (M + H)⁺.

tert-Butyl 2-aminoethylcarbamate (6). A solution of di-tert-butyl dicarbonate (1 g, 4.58 mmol) in ethanol was added dropwise to a stirring solution of ethylene diamine (4.59 mL, 68.8 mmol) and triethyl amine (0.76 mL, 5.5 mmol) in ethanol (30 mL) at 0 °C. The reaction mixture was stirred for 18 h. The solvent was evaporated under reduced pressure and residue obtained was dissolved in dichloromethane. The organic layer was washed with water (3 × 20 mL), dried over sodium sulphate and was evaporated under reduced pressure to get crude oil 6. The compound was purified using column chromatography (silica gel, 2% methanol in dichloromethane). Yield: 87.5%.

¹H NMR (400 MHz, CDCl₃) δ (ppm): 1.44 (s, 9H, –C(CH₃)₃), 2.78 (t, 2H, J = 6.0, –CH₂–), 3.16 (q, 2H, J = 5.6, –CH₂–) and 4.95 (s, 1H, –NH–CO–); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 28.75 (–C(CH₃)₃), 52.18 (–O–C(CH₃)₃), 61.60 (NH₂–CH₂–), 79.97 (–CH₂–CO–) and 156.25 (–CO–); ESI-MS (m/z): C₇H₁₇N₂O₂ found: 161.1 (M + H)⁺, calculated: 161.1 (M + H)⁺.

Diethyl 6,6′-2(tert-butoxycarbonylamino)(ethylazanediyl)bis(methylene)dipicolinate (7). tert-Butyl 2-aminoethylcarbamate (0.79 g, 4.90 mmol) and potassium carbonate (2.73 g, 19.81 mmol) were added to a solution of 4 (1.97 g, 9.90 mmol) in anhydrous acetonitrile (25.0 mL). The reaction mixture was stirred and refluxed at 80 °C for 16 h. After completion of the reaction, solvent was evaporated under reduced pressure. The crude compound was dissolved in dichloromethane and washed with water (2 × 30 mL). The organic layer was dried over sodium sulphate and evaporated under reduced pressure to get yellow coloured oil. Column chromatography (silica gel, 3% methanol in dichloromethane) afforded the desired product 7 as brown coloured oil. Yield: 84.5%.

¹H NMR (400 MHz, CDCl₃) δ (ppm): 1.30 (s, 9H, –C(CH₃)₃), 1.44 (t, 6H, J = 7.2, 2 × –CH₃), 2.73 (t, 2H, J = 5.6, –CH₂–), 3.27 (t, 2H, J = 5.2, –CH₂–), 3.95 (s, 4H, 2 × –CH₂–), 4.45 (q, 4H, J = 7.2, 2 × –O–CH₂–), 7.70 (d, 2H, J = 7.6, 2 × –CH–), 7.77 (t, 2H, J = 8.0, 2 × –CH–) and 7.97 (d, 2H, J = 7.6, 2 × –CH–); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 14.31 (–CH₃), 28.35 (–C(CH₃)₃), 38.55 (–CH₂–), 54.34 (–CH₂–), 60.10 (–CH₂–C–), 61.87 (–CH₂–CH₃), 78.90 (–C(CH₃)₃, 123.53 (–C–CH–CH–), 126.18 (–CH–CH–C–), 137.70 (–CH–CH–CH–), 147.69 (–CH–CH–CH–), 156.09 (–CO–C–CH–), 160.06 (–NH–CO–O–) and 165.26 (–O–CO–CH–); ESI-MS (m/z): C₂₅H₃₅N₄O₆ found: 487.3 (M + H)⁺, calculated: 487.2 (M + H)⁺.

Diethyl 6,6′-(2-aminoethylazanediyl)bis(methylene)dipicolinate (8). Compound 7 (2.0 g, 4.11 mmol) was dissolved in trifluoroacetic acid (2.0 mL) at 0 °C with gentle stirring. It was allowed to stir at 0 °C for another 4 h and then left at room temperature. After 18 h, the solvent was evaporated under vacuum. The residue was dissolved in methanol (3.0 mL) and precipitated using diethyl ether at 0 °C. It was allowed to stir for 1 h. After 1 h it was dried under reduced pressure and purified by HPLC (System B, λ = 254 nm). The compound was obtained as a brown coloured solid 8. Yield: 88.7%.

¹H NMR (400 MHz, CDCl₃) δ (ppm): 1.39 (t, 6H, J = 7.2, 2 × –CH₃), 2.49 (s, 2H, –NH₂), 3.63 (t, 2H, J = 5.6, –CH₂–), 3.81 (t, 2H, J = 5.2, –CH₂–), 4.42 (q, 4H, J = 7.2, 2 × –O–CH₂–), 4.67 (s, 4H, 2 × –CH₂–), 7.94 (dd, 2H, J = 7.6, 2 × –CH–), 7.59 (d, 2H, J = 8.0, 2 × –CH–) and 8.08 (d, 2H, J = 7.2, 2 × –CH–); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 13.83 (–CH₃), 36.31 (–CH₂–), 53.17 (–CH₂–), 58.17 (–CH₂–), 62.81 (–O–CH₂–), 125.19 (–C–CH–CH–), 127.33 (–CH–CH–C–), 139.81 (–CH–CH–CH–), 146.75 (–CO–C–CH–), 152.21 (–CH–C–CH₂–) and 164.21 (–CO–); ESI-MS (m/z): C₂₀H₂₇N₄O₄ found: 387.8 (M + H)⁺, calculated: 387.2 (M + H)⁺.

6,6′-(2-Aminoethylazanediyl)bis(methylene)dipicolinic acid, (H₂pentapa-en-NH₂). NaOH solution (2 M) was added slowly to a stirred solution of 8 (0.5 g, 1.29 mmol) in methanol (5.0 mL) till the pH of the reaction mixture reached 12. The reaction was stirred at room temperature. After 12 h, the reaction mixture was neutralized to pH 7 by the dropwise addition of 2 M HCl solution and evaporated under reduced pressure. The residue obtained was dissolved in chloroform and layer was extracted with water. The organic fractions were collected, dried over sodium sulphate and filtered. The filtrate obtained was concentrated under reduced pressure to give the desired compound H₂pentapa-en-NH₂. The compound was purified by preparative HPLC (System A, λ = 254 nm; t_R = 5.33 min). Yield: 95.1%.

¹H NMR (400 MHz, D₂O) δ (ppm): 2.99 (t, 2H, J = 5.4, –CH₂–), 3.15 (t, 2H, J = 5.2, –CH₂–), 3.88 (s, 4H, 2 × –CH₂–), 7.21 (d, 2H, J = 5.6, 2 × –CH–) and 7.66 (m, 4H, 4 × –CH–); ¹³C NMR (100 MHz, D₂O) δ (ppm): 37.19 (–CH₂–NH₂), 52.05 (–N–CH₂–), 59.64 (–C–CH₂–N–), 122.21, 125.45, 138.09, 152.68, 158.04 (–CH–) and 173.05 (–COOH); ESI-MS (m/z): C₁₆H₁₉N₄O₄ found: 331.0 (M + H)⁺, calculated: 331.1 (M + H)⁺; elemental analysis: calcd for C₁₆H₁₈N₄O₄: C, 58.17; H, 5.49; N, 16.96%; found: C, 58.25; H, 5.39; N, 16.89%.

Methyl 2-(2-chloroacetamido-4(methylthio)butanoate) (10). 2-Chloroacetyl chloride (0.39 g, 5.0 mmol) in dichloromethane (10.0 mL) and potassium carbonate (0.76 g, 5.50 mmol in 10 mL of water) were added simultaneously dropwise through dropping funnels to the round bottom flask containing a solution of 9 (0.50 g, 2.50 mmol) in dichloromethane (35.0 mL) maintained at 0 °C. The reaction was stirred for 2 h. The reaction was further left to stir at room temperature for an additional 16 h. After completion of the reaction, the organic layer was separated and dried over sodium sulphate. It was filtered and evaporated under reduced pressure to give the crude product, which was purified by column chromatography (silica gel, 1% methanol in dichloromethane) to give 10. Yield: 92.0%.

¹H NMR (400 MHz, CDCl₃) δ (ppm): 2.06 (m, 1H, –CH–CH₂–CH₂–), 2.11 (s, 3H, –S–CH₃), 2.20 (m, 1H, –CH–CH₂–CH₂–), 2.55 (t, 2H, J = 7.2, –CH₂–CH₂–S–), 3.79 (s, 3H, –O–CH₃), 4.09 (s, 2H, Cl–CH₂–CO–) and 4.75 (m, 1H, –CH–); ¹³C NMR (100 MHz, CDCl₃) δ (ppm): 15.47 (–S–CH₃), 29.85 (–CH₂–S–CH₃), 31.32 (–CH–CH₂–CH₂–), 42.41 (–CH–), 51.83 (–OCH₃), 52.71 (Cl–CH₂–CO–), 165.90 (–CO–O–) and 171.69 (–CO–); ESI-MS (m/z): C₈H₁₄ClNO₃SNa found: 262.0 (M + Na)⁺, calculated: 262.0 (M + Na)⁺.

6-(9-Carboxy-5-(1-carboxy-3-(methylthio)propylamino)-2-oxoethyl)-2-((6-carboxypyridin-2-yl)methyl)-7-oxo-12thia-2,5,8-triazatridecyl-picolonic acid, (H₂pentapa-en-met₂). Potassium carbonate (0.70 g, 5.70 mmol) was added to a solution of 8 (1.0 g, 2.60 mmol) in dry acetonitrile under an inert atmosphere (20 mL) and was stirred. Compound 10 (0.75 g, 3.12 mmol) was dissolved in acetonitrile (10 mL) and added dropwise to the reaction mixture. The reaction mixture was then heated at 80 °C for 18 h. The progress of the reaction was monitored using thin layer chromatography (DCM/MeOH: 9/1). On the completion of the reaction, the reaction mixture was filtered and solvent was evaporated to dryness. The crude product was purified by column chromatography to give the desired compound. The purified compound (1.0 g, 1.26 mmol) was dissolved in methanol and 2 M NaOH solution was added to it dropwise with constant stirring till the pH of the reaction mixture reached 12. The solution was left for overnight stirring at room temperature. The pH of the solution was adjusted to neutral by the dropwise addition of 2 M HCl. The reaction mixture was filtered, the filtrate was evaporated to dryness and the crude product obtained was purified by preparative HPLC (System A, λ = 254 nm; t_R = 5.55 min) to give the desired compound, H₂pentapa-en-met₂. Yield: 89.1%.

¹H NMR (400 MHz, D₂O) δ (ppm): 1.99–2.28 (m, 10H, 2 × –CH₂–, 2 × CH₃–S–), 2.54 (m, 4H, 2 × –CH₂N–), 2.80–2.98 (m, 4H, 2 × –CH₂S–), 3.40 (s, 4H, 2 × –CH₂–), 4.07 (s, 4H, 2 × –CH₂–), 4.35 (t, 2H, 2 × –CH–), 7.57 (dd, 2H, 2 × –CH–), 7.84 (t, 2H, J = 7.2, 2 × –CH–) and 7.88 (dd, 2H, 2 × –CH–); ¹³C NMR (100 MHz, D₂O) δ (ppm): 14.32 (CH₃–S–), 31.12 (–CH₂S–), 51.74, 52.21, 54.22, 57.42, 60.13, 61.21 (–CH₂–), 122.33, 125.77, 137.90 (–CH–), 152.64, 172.69, 178.59 (–CO–); ESI-MS (m/z): C₃₀H₄₁N₆O₁₀S₂ found: 709.8 (M + H)⁺, calculated: 709.2 (M + H)⁺; elemental analysis: calcd for C₃₀H₄₀N₆O₁₀S₂: C, 50.84; H, 5.69; N, 11.86; S, 9.05%; found: C, 50.87; H, 5.71; N, 11.83; S, 9.07%.

Cu-pentapa-en-NH₂. Compound H₂pentapa-en-NH₂ (0.3 g, 0.90 mmol) was dissolved in 1 mL of water. CuCl₂ (0.12 g, 0.90 mmol) was dissolved in 1 : 1 mixture of methanol and water (total volume 1 mL) and added dropwise to the ligand solution. The pH was adjusted to 6–6.5 by the dropwise addition of 0.1 M NaOH. After 1 h, the colour of the solution became greenish blue and solvent was evaporated under reduced pressure. The residue obtained was dissolved in a minimum amount of water and loaded on a C18 Sep-Pak cartridge. The cartridge was flushed with 5–10 mL of water and the complex was eluted from the cartridge with ethanol as a blue color solution. The solvent was removed under reduced pressure to give Cu(pentapa-en-NH₂).

ESI-MS (m/z): C₁₆H₁₇CuN₄O₄ found: 392.7 (M + H)⁺, calculated: 392.0 (M + H)⁺.

Cu-pentapa-en-met₂. The methionine complex was afforded through the same synthetic procedure as Cu(pentapa-en-NH₂) from H₂pentapa-en-met₂ and CuCl₂.

ESI-MS (m/z): C₃₀H₄₁CuN₆O₁₀S₂ found: 772.0 (M + 3H)⁺, calculated: 772.1 (M + 3H)⁺.

Re-pentapa-en-NH₂. The synthesis of Re-complex was performed according to the reported literature.¹² KReO₄ (65.75 mg, 0.22 mmol) was added to a solution of SnCl₂ (71.81 mg, 0.37 mmol) in citric acid (0.5 M, 5 mL) and stirred for 30 min at room temperature followed by the dropwise addition of H₂pentapa-en-NH₂ (50 mg, 0.15 mmol) dissolved in water. The reaction mixture was further allowed to stir at room temperature for 3 h. After completion of the reaction, the pH of the solution was increased to 7 using 1 M NaOAc. The crude complex was purified.

¹H NMR (400 MHz, D₂O) δ (ppm): 2.92 (t, 2H, J = 5.4, –CH₂–), 3.09 (t, 2H, J = 5.2, –CH₂–), 3.77 (s, 4H, 2 × –CH₂–), 7.92 (m, 2H, J = 9.6, 2 × –CH–), 7.99 (d, 1H, J = 8.0, –CH–), 8.38 (m, 2H, J = 8.0, 2 × –CH–) and 8.47 (m, 1H, J = 8.0, –CH–).

IR (cm⁻¹): 3054 (N–H), 2910 (C–H), 1685 (C=O), 926 (m, Re=O).

ESI-MS (m/z): C₁₆H₂₁N₄O₆Re found: 549 (M + H₂O)⁺ (isotopic distribution of the molecular ion peak), calculated: 549 (M + H₂O)⁺.

HPLC: λ = 254 nm; 50% solvent A (H₂O), 50% solvent B (ACN); flow rate: 0.5 mL min⁻¹; t_R = 7.06 min.

DFT studies

All the computational studies were performed using Schrödinger Software, Maestro 9.7 (Schrödinger, LLC, NewYork, NY, 2014).¹³

The ligands and respective copper and rhenium complexes were subjected to stable conformer search by random changes in torsion angle using the Monte Carlo method implemented in Macro model suite in OPLS2005 force field. Lowest energy conformer of ligands and copper and rhenium complex structures were fully optimized in vacuum and Poisson–Boltzmann solvent phase at DFT level using Jaguar 8.3. DFT level of calculations were employed without imposing constraints using B3LYP hybrid functional with basis set psLACV3P** (which is a triple-zeta contraction of the LACVP basis set) 6-31G set developed by Pople and co-workers. Effective core potential used for heavy atoms and non-ECP atoms uses 6-311G basis. The calculated stationary points were characterized by calculating vibrational frequencies with the Hessian obtained during the geometry optimization.

Radiolabeling and in vitro stability studies of H₂pentapa-en-NH₂ and H₂pentapa-en-met₂

1.0 mL of distilled water was added to H₂pentapa-en-NH₂ (5.0 μmol) in a shielded vial and shaken gently until a clear solution was obtained. A solution of stannous chloride (4.0 mg mL⁻¹) was prepared by dissolving stannous chloride (2.0 mg) in 10% acetic acid, and an aliquot of stannous chloride solution (10 μL) was added to the vial containing H₂pentapa-en-NH₂ solution (1.0 mL). The pH of the mixture was adjusted to 7–7.5 by adding 0.1 M NaHCO₃ solution dropwise, and the mixture was filtered using a 0.22 μm filter. Finally, the compound was added to the TcO₄⁻ (7.4 MBq) solution, and then samples were kept at room temperature for 10–15 min for the completion of the reaction.

The labeling efficiency and chemical purity of the product were measured by ascending ITLC on SG strip as a stationary phase and 100% acetone was used as the solvent (mobile phase). After developing the ITLC, it was cut into 0.5 cm segments and counts of each segment were taken using well-type γ-counter. Free ^99mTcO₄⁻, reduced or hydrolysed ^99mTc move with the solvent front in acetone while leaving ^99mTc-pentapa-en-NH₂ at the point of application. The labeling efficiency of a compound with radioactive metal was calculated using ITLC. A similar radiolabeling procedure was adopted for H₂pentapa-en-met₂.

Thermodynamic studies

The protonation constants of H₂pentapa-en-NH₂ and H₂pentapa-en-met₂ and stability constants of the corresponding copper complexes were determined by potentiometric titrations. Potentiometric measurements were carried out with an automatic titration system consisting of a Metrohm 713 pH meter equipped with a Metrohm A.60262.100 electrode, 800 Dosino autoburet. The electrode of the pH meter was calibrated using standard buffers. Protonation constants were determined by titrating 1 mM of ligand with 10 mM tetramethylammoniumhydroxide (TMAOH) in the pH range of 2–12. The titrations were performed in tetramethylammoniumchloride (TMACl, 0.1 M) at 25 °C for maintaining the constant ionic strength.

The stability constants of Cu(II) complexes were determined under the conditions similar to the protonation constants (2 mM metal and 2 mM ligand solution), I = 0.1 M using TMACl. The metal solutions were prepared by dissolving their corresponding salts in deionized water. A solution was prepared containing ligand and metal in a 1 : 1 ratio of ligand/metal and complexes were titrated with TMAOH (0.1 M). The experiments were performed in triplicate.

In vitro studies

Stability studies in PBS buffer. The in vitro stability of radiolabeled complexes was calculated by adding 100 μL of labeled compounds to 2–2.5 mL of PBS (pH 7.2). The solution was incubated at room temperature and the change in radiochemical activity was checked using ITLC at different time intervals up to 24 h using pyridine/acetic acid/water (3 : 5 : 1.5) as the mobile phase.

Stability studies in human serum. The in vitro stability study was carried out in human serum. The serum was collected from a fresh blood sample by incubating it for 50 min at 37 °C at 95% air and 5% CO₂ and then centrifuging at 400 g. The supernatant was filtered through a 0.22 μm syringe filter. The in vitro stability of the complexes, ^99mTc-pentapa-en-NH₂ and ^99mTc-pentapa-en-met₂, was estimated in serum by incubating 100 μL of the complex with 900 μL of human serum at 37 °C up to 24 h. Aliquots at different time intervals were applied on ITLC and run in pyridine/acetic acid/water (3 : 5 : 1.5) to check the stability of H₂pentapa-en-NH₂ and H₂pentapa-en-met₂ after labeling with ^99mTc in serum.

In vivo studies

Blood kinetics. The blood clearance of the complex was studied in rabbits after the intravenous administration of 250 μL (10 MBq) of ^99mTc-pentapa-en-NH₂ through the dorsal ear vein. At different time intervals (starting from 15 min to 24 h), blood samples from the vein of the other ear of the animal were withdrawn and collected in heparinized tubes. The samples were weighed and radioactivity was measured using a gamma counter. The data were expressed as the percent administered dose at each time point considering the entire body blood as 7% of the body weight. A similar procedure was adopted to study the blood kinetics of ^99mTc-pentapa-en-met₂ in rabbits.

Scintigraphy studies. The scintigraphic study of ^99mTc-pentapa-en-NH₂ and ^99mTc-pentapa-en-met₂ was performed in normal rabbits weighing 2–2.5 kg at different intervals of time (30 min, 1 and 2 h) to visualize the distribution pattern and study the retention time in the body by the intravenous injection of 10 MBq of radioactivity through the dorsal ear vein of the rabbit.

Biodistribution studies. The biodistribution study of the ^99mTc-pentapa-en-NH₂ was carried out in normal and Ehrlich ascites tumor bearing BALB/c mice. The mice were injected with 4.0 MBq activity through the tail vein, and the mice were sacrificed at different time points (0.5, 1 and 2 h) post injection. Similarly, the biodistribution study of ^99mTc-pentapa-en-met₂ was performed in BALB/c mice xenografted with Ehrlich ascites tumor. The organs of interest were removed, made free from adhering tissues, weighed and activity was counted using a gamma well counter. The uptake of radioactivity in each organ was calculated and expressed as percentage injected dose per gram of the organ.

Conclusions

A suitable acyclic bifunctional chelating agent, H₂pentapa-en-NH₂, based on pyridine carboxylate scaffold was synthesized in >95% yield. The developed chelate was labeled with ^99mTc with a facile radiolabeling procedure for the applications in SPECT imaging. It is an ideal bifunctional chelating agent for further elaboration and application after conjugation with a biomolecule, L-methionine. After conjugation with L-methionine, the compound H₂pentapa-en-met₂ was also studied with ^99mTc. The ^99mTc based radio complexes were prepared in high radiochemical yield with specific activity 110–140 MBq μmol⁻¹. In vitro competition experiments with human serum and PBS buffer show that the ^99mTc remained predominantly chelate bound with only 7–10% transchelated to serum proteins after 24 h. Biodistribution experiments in mice reveal uptake was largely in the kidney and <2% ID g⁻¹ activity was found in other organs. H₂pentapa-en-met₂ was targeted towards the tumor with T/M of 6.52. The work demonstrates the exploration of “pa” chelate with ^99mTc possibly for the first time. With the encouraging DFT and potentiometric stability studies, the advance studies of the copper complex to confirm the versatility of the developed probes and their potential as copper based radiopharmaceuticals will be useful.

Acknowledgements

We are highly thankful to Dr R. P. Tripathi, Director, Institute of Nuclear Medicine and Allied Sciences and Department of Chemistry, University of Delhi, for providing necessary facilities. This project was supported by Council of Scientific and Industrial Research (CSIR) and Defence Research and Development Organization, Ministry of Defence, under R&D project INM-311.3.1.

Notes and references

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ra13690j

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