Synthesis and investigation on water soluble mono(aquated) and bis(aquated) Gd(III) and Mn(II) complexes as potential MRI contrast agents

I would like to thank all the people who contributed in one way or another to the work described in this thesis. First of all, I would like to express my sincere thanks and appreciation to my supervisor Dr. I would like to acknowledge SAIF (IIT Bombay), CIF (IIT Guwahati), and Department of Chemistry (IIT Guwahati) for the instrument facilities.

Bedika Phukan

Thesis Title

Date of Submission of Thesis

List of Publications

List of Conferences/Symposiums

Doctoral Committee

Synthesis of complex 2A

These anions can form ternary adducts by replacing the inner-sphere water molecules of the complex, leading to a sharp decrease in relaxation value. T1-weighted phantom MR images of complex 2A at four different concentrations of the complex at 1.5 T are shown in Figure 1 . water molecules (q) was found to be 2.22±0.1.

Synthesis of complex 3A

A New Water-coordinated seven-coordinated Mn(II) complex as a dual-mode T1 and T2 MRI contrast agent. The discovery of the disease Nephrogenic Systemic Fibrosis (NSF) has led to new research into the development of Mn(II)-based high-spin MRI contrast agents as an alternative to classical Gd(III)-based MRI contrast agents. Phantom MR images of complex 4A measured at 1.5 T and 14.1 T consolidated its potential use as a dual-mode MRI contrast agent.

A) Synthesis of complex 5B, (B) Optimised structure of the complex with two inner sphere water molecules

Magnetic Resonance Imaging (MRI)
MRI Contrast Agents

Theoretical calculations showed a significant increase in the structural stability of the complex with two water molecules. However, the difference in number is very small and depends on the strength of the applied magnetic field. Transverse relaxation is also called spin-spin relaxation, which is the time required to dissipate 69% of the magnetization along its transverse plane.

Commercially available Gd(III)‒based MRI CAs. The number of coordinated water molecule in all cases is one

Relaxivity of Metal Complexes
High Relaxivity at High Field Strength
Introduction

The development of a safe alternative to Gd(III)-based MRI CA for patients with renal impairment is one of the great challenges in the field of MRI CA. However, current design of contrast agents focuses primarily on achieving a higher inner-sphere longitudinal relaxivity, r1p, from the protons of the water molecules in the first coordination sphere of the paramagnetic metal. The electronic relaxation time of the metal center also plays an important role in the design of the chelate.

Ligands containing picolinate group(s)

Synthesis and Characterisation of Ligand H 4 peada, C 15 H 19 N 3 O 8
Synthesis and Characterisation of Gd(III) Complex of Ligand H 4 peada, 2A
Xylenol Orange Test for Determination of Free Gd(III) in Complex 2A
Determination of Number of Coordinated Water Molecules (q)
Stability of Complex 2A
Longitudinal Relaxivity Measurement of Complex 2A at 1.41 T
Affinity for Physiological Anions
Affinity for Physiological Cations
Phantom MR Images of Complex 2A at 1.5 T
Conclusion
Introduction

Electrospray ionization mass spectrum measurement of the ligand in positive mode in HPLC-grade MeOH provided a 100% molecular ion peak at m/z amu. The color of an aqueous solution of xylenol orange changes as the pH of the solution changes. Using commercially available DTPA as a competitive ligand, following the same protocol used for the determination of the pGd value of the H4peada ligand, the pZn and pCu values of the H4peada ligand were determined at pH = 7.4 and 25 ºC.

Examples of Gd(III) complexes having two inner sphere water molecules

Synthesis and Characterisation of Ligand H 4 bedik, C 15 H 18 N 2 O 8
Synthesis and Characterisation of Bis(aquated) Gd(III) Complex of Ligand H 4 bedik, 3A
Xylenol Orange Test for Determination of Free Gd(III) in Complex 3A
Determination of Number of Coordinated Water Molecules (q)
Stability of complex 3A
Relaxivity Measurements of Complex 3A at 1.41 T and 14.1 T
Affinity for Physiological Anions
Affinity for Physiological Cations
Conclusion
Introduction

ESI measurement of the ligand mass spectrum in positive mode in HPLC grade MeOH provided a 100% molecular ion peak at m/z amu. The signals in the range 7.57–7.22 ppm were due to the four aromatic protons of the benzene ring in the ligand backbone. The resulting white solid was washed thoroughly with MeOH and the formation of the complex was confirmed using mass spectrometry and FTIR spectroscopy.

Since the thermodynamic stability of the Gd(III) xylenol orange complex (log K = 5.8)11 is lower than that of commercially available MRI CAs based on Gd(III) and their derivatives, xylenol orange is expected to only chelate with free ions of Gd(III) present in the complex soln. The value indicated that most of the complex (80%) present in the solution was in the bis(aqueous) form. Similar to the longitudinal relaxivity, the transverse relaxation times were measured at four different complex concentrations and from the slope of the transverse relaxation rate (R2 = 1/T2) vs.

Comparison of image intensities using ImageJ Software in the same area of images also proved the complex as a better contrast agent even at very low concentration (Figure 3.23). Comparison of image intensity using ImageJ software in the same area of images is as shown in Figure 3.25. MR phantom images of complex 3A at both 1.5 T (under a clinical MRI scanner) and 14.1 T (under a BRUKER NMR microimager) imply the utility of the complex as a high-field MRI CA.

The discovery of the disease nephrogenic systemic fibrosis (NSF) has prompted new research to develop safer MRI contrast agents (CAs).

Ligands studied for synthesising mono(aquated) seven–coordinate Mn(II) complex

Synthesis and Characterisation of Ligand H 4 bedik, C 15 H 18 N 2 O 8
Synthesis and Characterisation of Water Coordinated Mn(II) Complex of Ligand H 4 bedik, 4A
Stability of Complex 4A
Relaxivity Measurements of Complex 4A at 1.41 and 14.1 T
Conclusion
Introduction

A competitive batch titration with EDTA (ethylenediaminetetraacetic acid) as a competitive ligand was performed to determine the pMn value of the H4bedik ligand at 25 C. Using the inversion recovery method, the longitudinal relaxation times T1 were measured at four different complex concentrations and the slope of the longitudinal relaxation rate curve (R1 = 1/T1) compared to a similar 1.41 T, the longitudinal relaxation time, T1 was measured at four different concentrations of complex 4A, at 25 C and pH ~ 7.4, and the slope of the longitudinal relaxation rate curve (R1 = 1/T1) vs.

Here, a comparative study of MR image intensities was also performed with a commercially available Gd(III)-based CA MRI, MultiHance, and when image intensities were compared using ImageJ software while maintaining constant area under imaging , it was found that the image intensity with 0.5 mM concentration of complex 4A was similar to that of. It was observed that in the case of T1-weighted images (Figure 4.13), as the complex concentration increases, the intensity of the MR image of the corresponding image increases. In the case of T2-weighted images, it was observed that as the concentration of the complex increased from 0.25 mM to 1.00 mM, the signal intensity of T2-weighted MR images gradually decreased and became almost zero at concentration 1, 00 mM.

The comparison of the image intensities was done using ImageJ software and is as shown in Figure 4.19. The phantom MR images of 1.00 mM concentration of the complex at selected echo times are also depicted in Figure 4.20. Mn(II) ion with its five unpaired electrons, slow electronic relaxation rate and fast water exchange rate is the best candidate in place of Gd(III) based MRI-CAs.5.

This complex is the absence of the inner-sphere water molecule, and the enhanced contrast is obtained by the in vivo release of the free Mn(II) ion from the chelate.6 Recently, another Mn(II)-based oral contrast agent CMC‒ 001 has been developed which is a mixture of MnCl2, alanine and vitamin D3.7 The relaxation in this case is due to the free Mn(II) ion.

Ligands coordinating with Mn(II), and have two inner sphere water molecules

Synthesis and Characterisation of Ligand H 2 pmpa, C 13 H 17 N 3 O 4
Synthesis and Characterisation of Bis(aquated) Mn(II) Complex of Ligand H 2 pmpa, 5B
Stability of Complex 5B
Longitudinal Relaxivity Measurement of Complex 5B at 1.41 T
Longitudinal Relaxivity of Complex 5B at 1.41 T in the Presence of Physiological Anions
Phantom MR Images of Complex 5B at 1.5 T
Conclusion
Methods and Equipments Chemicals and Solvents
Experimental Section

Synthesis of Ligand H 4 peada

1H NMR spectrum (Figure 5.3) of the ligand showed signals in the region due to three aromatic protons. Mn-N bonding (as shown in Figure 5.7) was the key factor for the overall structural stability of the complexes. The theoretical calculations showed a significant improvement in the structural stability of the complex with two water molecules in the inner sphere.

The concentration of ligand H2pmpa and metal was kept constant, while the concentration of the competing ligand was increased progressively during the process. After confirming sufficient stability of the complex, the r1 relaxivity of complex 5B was measured at 1.41 T in a BRUKER minispec mq60 NMR analyzer. However, high relaxivity value was observed at lower pH = 4, which was due to the partial dissociation of the complex and the formation of water complex of Mn(II).

The increase in relaxivity due to dissociation of the complex and formation of an aquacomplex of Mn(II) ions in the presence of phosphate anions could be discarded from the UV-visible spectra of the complex (Figure 5.13). Therefore, the increase in relaxivity could be due to aggregation of the complex through its interaction with the phosphate without replacing the water molecules in the inner sphere. DFT-based structural optimization of the complex implied that its pentagonal bipyramidal geometry had two inner-sphere water molecules in the axial positions and the coordinating ligand in the equatorial plane.

First derivative X-band EPR spectrum of the sample solution in water was measured using.

Synthetic route of ligand H 4 peada

Synthesis of Ligand H 4 bedik

The reaction mixture was stirred under reflux conditions for 48 hours, and the insoluble powder was completely dissolved under this condition. The reaction mixture was then cooled to room temperature (25 °C) and kept at room temperature for ∼2 h. The aqueous solution was then extracted with CHCl 3 (3 x 10 mL) and the combined organic layers were dried over anhydrous.

The crude product was purified by column chromatography on silica gel using hexane/ethyl acetate (1:1) as eluent. The reaction mixture was then quenched using aqueous solution of NaHCO 3 (20 mL) and extracted with CHCl 3 (3 × 10 mL). The solvent was removed in vacuo and the residue was partitioned between CH2Cl2 (10 mL) and saturated water.

The aqueous phase was then washed with CH 2 Cl 2 (3 x 10 mL) and the combined organic extracts were dried over anhydrous. The solvents were evaporated in vacuo and the crude compound was purified by column chromatography on silica gel (hexane/ethyl acetate, 1:1) to give the ester. The solvent was then completely evaporated to dryness and the ligand H4peada was obtained as a pale yellow solid by addition of excess diethyl ether.

The ligand was then filtered, washed thoroughly with diethyl ether (3 × 5 mL) and dried under high vacuum.

Synthetic route of ligand H 4 bedik

Synthesis of Ligand H 2 pmpa

The solvent was then evaporated in vacuo and the product was obtained as a white solid by addition of excess diethyl ether. The solid was then filtered, washed with diethyl ether (10 mL) and dried under high vacuum.

Synthetic route of ligand H 2 pmpa

Synthesis of Complex 2A
Synthesis of Complex 2B

The solvent was then evaporated in vacuo and a white solid was obtained by adding excess diethyl ether. The white solid was then filtered, washed with diethyl ether (10 mL) and used in the next reaction without further purification. The solvent was then completely evaporated to dryness and the ligand was obtained as a light brown solid by addition of excess diethyl ether.

The solution was refluxed for 24 h and the resulting clear solution was kept at room temperature (25 °C) for slow evaporation of water. The solution was refluxed for 24 h and the resulting clear solution was kept at room temperature (25 °C) for slow evaporation of water. The resulting clear solution was kept at room temperature for slow evaporation to obtain crystal.

A pale brown solid was obtained after complete evaporation of the water, which was then thoroughly washed with MeOH. Transverse relaxation times (T2) were obtained with the Carr-Purcell-Meiboom-Gill spin-echo technique.2 Technique e. 17O NMR measurements on Gd(III) complexes are described elsewhere.3 Samples were sealed in glass spheres placed in 10 mm NMR tubes to avoid chemical shift sensitivity corrections.4 To improve sensitivity, 17O-enriched water ( 10% H217.O, CortectNet) was added to the solutions to achieve about 1% enrichment.

Least-squares fitting of 1H NMRD data was performed using Visualiseur/Optimiseur running on a MATLAB 8.3.0 (R2014a) platform.5 The variable temperature 17O T2 measurements provide access to the water exchange rate, kex.