Synthesis, Characterization and Biodistribution of 99mTc Labeled Salicyldimethyl Propylenediamine Complex as a Lung Scintigraphic Agent.
Summary: 99mTc-labeled salicyldimethyl propylenediamine ligand might be a potential candidate for lung imaging. The purpose of this study was to investigate the uptake of radiolabeled drug in lungs. In this study, salicyldimethyl propylenediamine ligand was synthesized by the reaction of 2- hydroxybenzaldehyde with 2, 2-dimethylpropane-1, 3- diamine and reduced by using NaBH4. The ligand system was characterized by HPLC, FTIR and 1HNMR. Radiolabeling of the complex with 99mTc was performed by stannous reduction method. The radiochemical purity of the radiolabeled complex was determined by Paper Chromatography (PC) and Instant Thin Layer Chromatography (ITLC). Bioevaluation of the 99mTc complex was studied in rabbits. A significant uptake was observed in lungs which was retained significantly up to 4h. The data indicate that the proposed system may be suitable for lungs imaging in future clinical applications.
Key words: Schiff base, Radiolabeling, Biodistribution, Lungs, Imaging.
The carbon-nitrogen double bond chemistry plays an important role in the progress of chemistry . Compounds with the structure -C=NH are called Schiff bases, which are usually synthesized from the condensation of active carbonyl groups and primary amines. Schiff bases are important compounds having wide range of biological activities and industrial applications. They possess the pharmaco- logical properties such as anticancer, antimalarial, antibacterial, antifungal, antitubercular, anti-infla- mmatory, antimicrobial and antiviral, etc. [2-8]. The conjugation of Schiff base ligands with transition metals has been extensively studied because of their conformational variability, preparative diversity and wide use, as an oxidative catalysis .
Technetium-99m (99mTc) is a metastable isomer of technetium-99. It does not change into another element on transmutation. Molybdenum-99/ Technetium-99m generator is used to extract the metastable isotope of technetium from decaying source i.e., molybdenum-99. 80% of the radiopharm- aceuticals used for diagnosis are tagged with technetium-99m because of its highly favorable radiation and physical characteristics. Six hours half- life and small amount of electronic emission allows the administration of 99mTc radioactivity in millicurie amounts, without significant radiation dose to the patient. The technetium-99m is used only for the diagnostic nuclear medicine imaging procedures; it is not involved in any therapeutic procedure. The conjugation of 99mTc with polydentate ligands having donor atoms such as nitrogen, sulfur or oxygen has been extensively investigated [10, 11].
The tetradentate 99mTc-complexes with desirable radio-chemical and suitable imaging qualities were used in clinical applications in diagnostic nuclear medicine.
The aim of the present study is to synthesize novel salicyldimethyl propylenediamine and form radiocomplex with Tc. Chemical properties of the newly synthesized compound, radiochemical characteristics of Tc labeled complex and gamma imaging study on rabbits were studied to evaluate the diagnostic potential of this radiolabeled complex and its application in diagnostic nuclear medicine.
Results and Discussion
Synthesis and Characterization
It is a three step reaction, which involves reaction of 2-hydroxybenzaldehyde with 2, 2- dimethylpropane-1, 3- diamine, reduction of azomethane (C=N) bond and at last condensation of the reduced azomethane with dione to obtain the desired product. The product was characterized by using HPLC, FTIR and 1H-NMR. HPLC was performed by using an isocratic system. Mobile phase consists of acetonitrile/ water in (1: 1) ratio. Reverse phase C-18 (Carbon 18) column was used at a flow rate of 0.8 ml/min. UV/VIS detector was employed to obtain the chromatogram. The retention time of the salicylaldehyde propylene diamine-N- dione Schiff base was at 12.17 min. HPLC chromatogram of Schiff base is shown in Fig. 1.
The FT-IR of the spectrum of the proposed tetradentate N2O2 ligand (Fig. 2) differs from the starting Schiff base notably in the absence of the absorption band at 1634 cm-1, assignable to the - C=N bond which was formed in the first step of synthesis. In the second step, reduction with NaBH4 converted -C=N bond into -C-NH bond, which resulted in new FTIR absorption at 3262.85 cm-1 for -N-H bond. In the third step, condensation of the crude product with pentane dione was performed and a new absorption at 1603.30 cm-1 for the conjugation (C=C-C=O) bond was observed instead of absorption of -C=O bond at 1700 cm-1. Conjugation of -C=O bond lowered the absorption frequency as shown by the absorption of C=C-C=O bond at 1603.30 cm-1.
Tetradentate N2O2 ligand (Fig. 2) was characterized by 300 MHz 1HNMR. The spectra consisted of three distinct regions: multiplet, singlet and broad peak. Methyl proton resonance appeared as singlet in the region at 2.50 and 2.65 ppm, benzyl proton resonance appeared at 4.50 ppm as singlet and aromatic proton resonance appeared at 6.85 and 7.15 ppm as multiplet. Due to slow exchange of the secondary N-H bond, the amino proton was partly decoupled and a broad spectrum resulted at 4.55 ppm. The results of 1HNMR (300 MHz, CD3OH) can be expressed as: d = 6.85, 7.15 (m, 4H, aromatic), 4.5 (s, 2H, -CH2), 4.55, 4.56 [b, 2H, 2(-NH)], 2.10, 3.09 [s, 4H (-N-CH2-C-)2], 0.9, 1.04 [s, 6H, -C(CH3)2], 4.95 (s, 1H, N-C=CH-), 1.9 [s, 3H, -N-C(CH3)=], 2.1 [s, 3H, -C(CH3)=O].
Three species were formed during labeling of the proposed diamine-dione ligand with 99mTc, the bound 99mTc complex, 99mTc-pertechnetate (99mTcO4-) and reduced hydrolyzed 99mTcO2, which were determined by ITLC, and paper chromatography. For paper chromatography, Whatmann No. 3 mm paper was used as a solid support and methyl ethyl ketone (MEK) as a mobile phase. In this system, 99mTcO4- and 99mTc-complex have an Rf value of 0.8 - 0.9, while the reduced or hydrolyzed 99mTcO2 (colloid) appeared at Rf = 0.00 - 0.01. Free 99mTcO4- was separated by ITLC by using saline as a mobile phase and silica gel as a solid support. In this system, the 99mTc-complex and 99mTcO2 remained at the origin, while 99mTcO4-appeared at Rf = 0.9 - 1.0. Labeling yield of 99mTc-complex, determined by using saline as a solvent was greater than 95% while in case of MEK as solvent, it was greater than 99.7%.
In vitro Stability
99mTc complex was formed as a result of binding of Na99mTcO4 with ligand and showed high stability at room temperature. Complex was reasonably stable and no significant change was observed in the %age binding of the complex till 4h. No complex degradation was observed as indicated by the plotting of %age binding against time. Results of %age binding of the 99mTc-complex in saline and methyl ethyl ketone at various time intervals are given in Fig. 3.
Before studying the uptake of the drug in an organ, binding of the labeled complex with plasma proteins was evaluated by developing an in vitro experimental model with human blood plasma from a volunteer donor. Protein binding of 99mTc labeled complex in human blood plasma was 43.4 +- 1.2%. Binding with plasma proteins suggested the rapid clearance of radiopharmaceutical from the blood, and the high accumulation in the organ. As the protein binding of the complex was reasonable so it was retained in the lungs even after 24h.
To evaluate the efficacy of proposed radiolabeled complex as a feasible lung imaging agent, 3 mCi of the 99mTc complex was injected into left ear vein of the rabbit and dynamic study was performed for 15 min (Fig. 4). The most probable route of excretion as observed for this complex was through kidney and bladder. Delayed static images of lungs showed high tracer uptake (Fig. 5). The tracer is not excreted through the hepatobiliary route. Both kidney were visualized and activity was collected in the urinary bladder. This confirms that complex is excreted through the urinary system. In the lungs, retention of radiopharmaceutical was observed even after 24h (Fig. 6). Results indicated that this compound got rapidly extracted and retained by the lungs and it may be used as a potential lung imaging agent in future.
Materials and Methods
Salicylaldehyde and molecular sieves 40A were purchased from Fluka, USA. 2, 2- Dimethylpropane-1, 3-diamine and SnCl2.2H2O were purchased from Aldrich, USA. Pentane Dione was taken from Acros Organics, USA. Ethyl acetate was purchased from Panreac, France. Methanol was purchased from Merck, Germany. Methyl ethyl ketone was purchased from Fisher Scientific, UK. Acetonitrile was purchased from J. T. Beakers, Holland. Trichloroacetic acid (TCA) was purchased from BDH laboratory reagents, England. 99mTc generator was taken from PINSTECH, Islamabad.
Whole process of synthesis of Schiff base was carried out in three steps:
i) Reaction of 2-hydroxybenzaldehyde with 2, 2- dimethylpropane-1, 3- diamine
0.1 mole (10.7 ml) of 2-hydroxy- benzaldehyde (commonly known as salicylaldehyde) was dissolved in 80 ml of methanol and added slowly during one hour to 0.6 mole (72.2 ml) of 2, 2- dimethylpropane-1, 3-diamine. The resultant mixture was then stirred with a magnetic bar for 30 min. Afterwards, 7g molecular sieve (4 A) was added and stirring was continued overnight at room temperature.
The solution was filtered and used for the next step without purification.
ii) Reduction of azomethane (C=N) bond
0.2 moles (7.56g) of NaBH4 was slowly added to the above solution in small portions within 30 min at constant stirring in the ice bath. After completing addition of the reducing agent (NaBH4), the reaction mixture was stirred for 2h at room temperature. The solvent was evaporated by rotary evaporator and 100 ml of distilled water was added into reaction mixture. The aqueous phase was extracted with CH2Cl2 (3 x 50ml).
iii) Condensation with dione
61.4 mmole (13.3g) of crude product of the previous reaction was dissolved in 61.4 ml of acetonitrile, mixed slowly with 128 mmole (13.2 ml) of pentane 2, 4-dione in 61.4 ml acetonitrile and stirred with a magnetic bar for 3h at room temperature. The solvent was evaporated and a reddish brown product was obtained. The reaction scheme is shown in Fig. 7.
Characterization of Schiff base
High Performance Liquid Chromatography (HPLC), Nuclear Magnetic Resonance Spectroscopy (1HNMR), and Fourier Transform Infrared Spectroscopy (FTIR), were used for the characterization of the product.
Kit Formulation and Radiolabeling
As an experimental procedure, 10 mg ligand (Schiff base) was dissolved in 10 ml of ethanol with continuous stirring (A). Afterwards, 50 mg of SnCl2.2H2O was dissolved in 0.3 ml conc. HCl (on heating) and diluted to 10 ml by distilled water (B).
100 ul out of solution (B) was added in solution (A) and pH was adjusted at 8.5 by using 1N NaOH, 0.1N NaOH, 1N HCl and 0.1N HCl. The resultant solution was dispensed in 1 ml volume into 10 ml serum vials. For radiolabeling, 20 mCi Na99mTcO4 (0.4 ml) was added in a vial and incubated at room temperature for 15 min. Radiochemical purity of the complex (amount of radioisotope bound to ligand) was determined by chromatographic techniques.
Stability of radiolabeled complex at room temperature
Stability of the radiolabeled complex was studied at different time intervals of 0, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, and 4h at room temperature. Change in the stability of radiolabeled complex was monitored with the passage of time. In case of significant dissociation of the ligand-radiometal complex, the ligand cannot be suggested for use as a radiopharmaceutical.
5 ml of fresh blood was taken in the tube. The kit was prepared according to the procedure as already mentioned for radiolabeling. 250 ul (3 mCi) of the radioactive kit was added in the blood and incubated at room temperature for 1h. The reaction was terminated by placing the tube in water bath set at 37oC for 10 min. Contents of the tube were centrifuged for 10 min, serum and blood cells were separated in the form of layers. Equal volume of 10% TCA (trichloro acetic acid) solution was added into separated serum. TCA was used as a precipitating agent for the serum proteins. Tube containing serum and TCA solution was placed on the shaker for 10 min and then centrifuged at 4000 rpm for another 10 min time interval. Residue was separated from the supernatant and both layers were counted for radioactivity.
For the evaluation of the developed radiopharmaceutical as an imaging agent, the biodistribution study was carried out in rabbit. The kit was prepared under sterilized conditions in a laminar flow hood, and 300 ul (3 mCi) of it was injected intravenously to left ear vein of the animal. The rabbit was scanned on dual head SPECT gamma camera to study the uptake of radiopharmaceutical and its route of excretion.
The novel 99mTc labeled complex of salicyldimethyl propylenediamine was synthesized, characterized and biologically evaluated in a rabbit model. The preliminary studies with this ligand encourage further in vivo experiments to study the uptake in lungs and develop it as a potential lung imaging agent.
1. S. Patai, The Chemistry of the carbon-nitrogen double bond, John Wiley and Sons Ltd, London, (1970).
2. Y. Li, Z. S. Yang, H. Zhang, B. J. Cao, F. D. Wang, Y. Zhang, Y. L. Shi, J. D. Yanga and B. A. Wu, Bioorganic and Medicinal Chemistry, 11, 4363 (2003).
3. R. Villar, I. Encio, M. Migliaccio, M. J. Gil and V. Martinez-Merino, Bioorganic and Medicinal Chemistry, 12, 963 (2004).
4. L. Wang, Y. Feng, J. Xue and Y. Li, Journal of Serbian Chemical Society, 73, 1 (2008).
5. K. N. Venugopal and B. S. Jayashree, Indian Journal of Pharmaceutical Science, 70, 88 (2008).
6. M. A. Bhat, M. Imran, S. A. Khan and N. Siddiqui, Indian journal of Pharmaceutical Science, 67, 151 (2005).
7. M. S. Karthikeyan, D. J. Parsad, B. Poojary, K. S. Bhat, B. S. Holla and N. S Kumari, Bioorganic and Medicinal Chemistry, 14, 7482 (2006).
8. S. N. Pandey, V. S. Lakshmi and A. Pandey, Indian Journal of Pharmaceutical Science, 65, 213 (2003).
9. M. Javed, I. U. Khan, M. Naeem, S. Asghar, H. Naseer, M. S. Iqbal and J. Anwar, Journal of Radioanalytical and Nuclear Chemistry, 277, 303 (2008).
10. H. F. Kung, Seminars in Nuclear Medicine. 20, 150 (1990).
11. S. Jurisson, D. Berning, W. Jia and D. Ma, Chemical Review, 93, 1137 (1993).
1Radiopharmacy and PET Radiochemistry Division, Institute of Nuclear Medicine and Oncology (INMOL), New Campus Road, P. O. Box 10068, Lahore- 54600, Pakistan, 2Institute of Chemistry, University of the Punjab, Quaid-e-Azam Campus, Lahore 54600, Pakistan., 3Gujranwala Institute of Nuclear Medicine (GINUM), Gujranwala, Pakistan, 4School of Biological Sciences (SBS), University of the Punjab, Quaid-e-Azam Campus, Lahore 54600, Pakistan, email@example.com
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|Author:||Mobeen, Saima; Khan, Irfan Ullah; Ahmad, Fayyaz; Javed, Muhammad; Ali, Muhammad; Dar, Ume Kalsoom; H|
|Publication:||Journal of the Chemical Society of Pakistan|
|Date:||Oct 31, 2012|
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