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Synthesis, Characterization and Study of Water Soluble Gd(III) and Mn(II) Complexes as MRI Contrast Agents ” is an authentic record of the results obtained from the research work done by Miss Mahmuda Khannam (Roll No. under my supervision in the Department of Chemistry, Indian Institute of Technology Guwahati, India I will always be grateful to the Indian Institute of Technology, Guwahati for giving me the opportunity to pursue my research work and encouraging me with PhD scholarship.

Mahmuda Khannam Thesis Title

List of Publications

List of Conferences/Symposiums

Doctoral Committee

Synthesis and Characterization of Ligand Li 3 cbda 30

Synthesis and Characterization of Ligand Li 2 hbda 34

Synthesis and Characterization of Ligand H 3 cpmda 38

Syntheses and Characterizations of Mn(II) Complexes of Ligand H 3 cpmda 80

Synthesis and Characterization of Mn(II) Complex of Ligand Li 3 cbda 108

Synthesis and Characterization of Mn(II) Complex of Lligand Li 2 hbda 126

Magnetic Resonance Imaging (MRI)

In the case of T2-weighted imaging, tissues with a short T2 appear dark due to loss of full signal. Therefore, cerebrospinal fluid with a long T2 appears brighter on T2-weighted images compared to fat with a short T2 [Figure 1.4(B)].

Figure 1.3.  Variation of magnetization in returning back to equilibrium position, (A) on z-axis  in longitudinal relaxation, and (B) on xy-plane in transverse relaxation
Figure 1.3. Variation of magnetization in returning back to equilibrium position, (A) on z-axis in longitudinal relaxation, and (B) on xy-plane in transverse relaxation

MRI Contrast Agents

Whereas, tissues with long T1 appear dark as they are unable to regain much of their longitudinal magnetization, thus producing weak signal. They are classified again according to their sizes, 13. a) Super paramagnetic iron oxides (SPIO), average diameter  50 nm and.

Figure 1.5. Effect of contrast agent on the images detecting blood-brain barrier after stroke
Figure 1.5. Effect of contrast agent on the images detecting blood-brain barrier after stroke

Relaxivity and Solomon-Bloembergen Equations

Here PM refers to the mole fraction of the paramagnetic metal ion, q is the number of coordinated water molecules per metal ion, T1M and τM represent the relaxation time and residence time of the bound water molecule in the metal ion, respectively. In equations (5)(7), I is the gyromagnetic ratio of the proton, g is the electronic g-factor, S is the total electron spin of the metal ion,  is the Bohr magneton, r is the distance from proton of the metal ion are S and I electronic and Larmor precession frequencies respectively, A/p is the electron-nuclear hyperfine coupling constant, T1e is the longitudinal electron spin relaxation time, τM is the residence time of the water molecule, and τR is the rotation time of the entire molecule.

Figure 1.6.  Schematic representation of various parameters affecting relaxivity.
Figure 1.6. Schematic representation of various parameters affecting relaxivity.

Contrast Agents with Gd(III) and Mn(II) ions

Further evaluation shows that NSF affects patients with severe kidney problems and after administration of Gd(III)-based contrast agents.21 Risk of NSF introduces new research to develop safer MRI contrast agents. Consequently, the design of suitable ligand frameworks that can form thermodynamically stable and kinetically inert Mn(II) complexes as potential MRI contrast agents must be carefully evaluated.27.

Introduction

Examples of ligands containing picolinate moieties in their structures, along with the longitudinal relaxivity value and thermodynamic stability constant of their corresponding Gd(III) complexes are given in Table 2.1. Recent report on a series of chiral derivatives of H4dota (Scheme 2.4)11(c) reveals that their corresponding Gd(III) complexes show impressive thermodynamic stability.

Table  2.1.  Picolinate-based  ligands  forming  Gd(III)  complexes,  number  of  inner-sphere  water  molecules (q), longuitudinal relaxivity value and stability constant
Table 2.1. Picolinate-based ligands forming Gd(III) complexes, number of inner-sphere water molecules (q), longuitudinal relaxivity value and stability constant

Synthesis and Characterization of Ligand Li 3 cbda

The 1H-NMR spectrum of the Li3cbda ligand in CD3OD solvent is shown in Figure 2.5. The spectrum showed 10 peaks characteristic for 10 different types of carbon atoms present in the ligand.

Figure 2.3. FTIR spectrum of ligand Li 3 cbda.
Figure 2.3. FTIR spectrum of ligand Li 3 cbda.

Synthesis and Characterization of Ligand H 2 hbda

Electron spray ionization mass spectrum of the H2hbda ligand in positive mode in Mili Q water provided the 100% molecular ion peak at m/z values ​​of 332.1245. In the spectrum, 9-characteristic peaks appeared for 9-different types of C atoms present in the H2hbda ligand.

Figure 2.7. FTIR spectrum of ligand H 2 hbda.
Figure 2.7. FTIR spectrum of ligand H 2 hbda.

Synthesis and Characterization of Ligand H 3 cpmda

The electrospray ionization mass spectrum of ligand H3cpmda in positive mode in HPLC grade MeOH provided a 100% molecular ion peak at m/z value 289.1395 (Figure 2.12). The multiplet that appeared in the region of ppm was due to hydrogen atom present in the ring attached with the methylamine arm.

Figure 2.11. FTIR spectrum of ligand H 3 cpmda.
Figure 2.11. FTIR spectrum of ligand H 3 cpmda.

Introduction

Syntheses and Characterizations of Gd(III) Complexes of Hexadentate Picolinate-Based Ligands

-MS (+ve) mass spectrum of aqueous solution of complex 3B. Simulated spectra have been indicated as inset. The FTIR spectrum of complex 3B is shown in Figure 3.3. OH) stretching of coordinated water molecules was observed at 3431 cm-1.4 The band for.

Figure 3.1. FTIR spectrum of complex 3A.
Figure 3.1. FTIR spectrum of complex 3A.

Xylenol Orange Test to Investigate the Presence of Free Gd(III) ion

However, with increasing concentration of Gd(III) ion, the absorbance intensity of these two bands changes. Calibration curve obtained from the absorbance ratio of known concentrations of Gd(III) ion in xylenol orange.

Figure 3.5.  UV-Vis spectral change during addition of various concentrations of Gd(III) ion to  xylenol orange in acetate buffer at pH  5.8
Figure 3.5. UV-Vis spectral change during addition of various concentrations of Gd(III) ion to xylenol orange in acetate buffer at pH  5.8

Determination of Number of Coordinated Water Molecules (q)

The electrospray ionization mass spectrum of an aqueous solution of complex 3C yielded a 100% molecular peak at an m/z value of 516.0248 in positive mode. The electrospray ionization mass spectrum of an aqueous solution of complex 3D in positive mode yielded a 100% molecular ion peak at an m/z value of 488.0313.

Figure 3.8. ESI-MS (+ve) mass spectrum of aqueous solution of complex 3C. Simulated spectra  has been given as inset
Figure 3.8. ESI-MS (+ve) mass spectrum of aqueous solution of complex 3C. Simulated spectra has been given as inset

Thermodynamic Stability

According to previously reported analogous systems, the protonation constant at log K1H = 7.68 and log K2H = 4.63 was assigned to the protonation of the amine nitrogen atom and the pyridine nitrogen atom, respectively. Here, complex 3A was stable due to the presence of a chiral methyl group in the backbone of the ligand, which provided a certain degree of rigidity to the complex.

Table 3.2. Protonation constants, stability constants, and pGd value of its analogues
Table 3.2. Protonation constants, stability constants, and pGd value of its analogues

Longitudinal Relaxivity

The overall tumbling rate may vary with the pH of the medium.18 A higher relaxivity value of complex 3B at pH  4 could be due to an increase in rotational correlation time. The relaxation value of complex 3B was found to be lower in the basic pH range of 8–10, which may be due to the formation of hydroxyl species.

Figure  3.15. r 1   relaxivity changes  in  the pH range 4-10  for;  (A) complex  3A, and (B) complex  3B; at 1.41 T, and 25 °C
Figure 3.15. r 1 relaxivity changes in the pH range 4-10 for; (A) complex 3A, and (B) complex 3B; at 1.41 T, and 25 °C

Affinity for Physiological Anions

This was supported by the species distribution diagram (Figure 3.13) of complex 3B, and also by luminescence lifetime measurements at pH. Due to overall positive charge of the complex, it was more affected by these physiological anions.14(g).

Figure 3.18. Variation of relaxivity in the presence of 100 equivalents of physiological anions at  1.41 T, 25 °C, pH  7.4; [complex 3A] = 0.5 mM, and [physiological anions] = 50 mM
Figure 3.18. Variation of relaxivity in the presence of 100 equivalents of physiological anions at 1.41 T, 25 °C, pH  7.4; [complex 3A] = 0.5 mM, and [physiological anions] = 50 mM

Phantom MR Imaging

The image intensity plot also revealed an increase in image intensity with increasing concentration of complex 3A. The obtained images showed an increase in the brightness of the images with an increase in the concentration of complex 3B.

Figure  3.20.  T 1 -weighted  phantom  images;  (A)  of  micro-centrifuge  tubes  with  different  concentrations of complex 3A (W = water, A = 0.25 mM, B = 0.50 mM, C = 0.70 mM, and D =  1.00 mM) at 1.5 T, 25 °C, pH  7.4, TR = 468 ms, and TE = 8.2 ms; R
Figure 3.20. T 1 -weighted phantom images; (A) of micro-centrifuge tubes with different concentrations of complex 3A (W = water, A = 0.25 mM, B = 0.50 mM, C = 0.70 mM, and D = 1.00 mM) at 1.5 T, 25 °C, pH  7.4, TR = 468 ms, and TE = 8.2 ms; R

Conclusion

Introduction

Syntheses and Characterizations of Mn(II) Complexes of Ligand H 3 cpmda

The molecular structure of complex 4B is shown in Figure 4.6, and selected bond lengths and bond angles are given in Table 4.3. In the molecular structure of complex 4B, two Li(I) ions were present outside the coordination sphere instead of the Mn(II) ion.

Figure 4.2. ESI-MS (ve) mass spectrum of aqueous solution of complex 4A. Simulated spectrum  has been given as inset
Figure 4.2. ESI-MS (ve) mass spectrum of aqueous solution of complex 4A. Simulated spectrum has been given as inset

Thermodynamic Stability

The protonation constants indicated the stepwise protonation of the amine nitrogen atom, the piperidine ring nitrogen atom, and the carboxylate groups of the ligand. Below pH  5, protonated species were appearing; which was due to the protonation of one of the carboxylate groups present in the ligand framework.

Figure 4.8. Species distribution diagram of H 3 cpmda:Mn(II) (1:1) solution, where [H 3 cpmda] =  [Mn(II)] = 1 mM (L in the figure represents cpmda 3- )
Figure 4.8. Species distribution diagram of H 3 cpmda:Mn(II) (1:1) solution, where [H 3 cpmda] = [Mn(II)] = 1 mM (L in the figure represents cpmda 3- )

Longitudinal Relaxivity

The increase in relaxivity value in the presence of BSA solution for complex 4B was due to macromolecular association. The r1 values ​​remained consistent in the pH range of 5-9; suggesting the presence of complex 4B most abundantly in this pH range.

Figure  4.10. 1/T 1  vs [Mn(II)] plot in the presence of BSA solution at 1.41 T, 37 °C, and pH    7.4
Figure 4.10. 1/T 1 vs [Mn(II)] plot in the presence of BSA solution at 1.41 T, 37 °C, and pH  7.4

Affinity for Physiological Anions

The most obvious reasons could be either release of free Mn(II) ion from the complex or due to formation of aggregation.20 The dissociation of complex 4B could take place during formation of Mn(II) aqua ion due to interaction between ligand and HPO42 anion .21 To investigate this possibility, UV-Vis spectra of complex 4B were recorded in the presence of various equivalents of HPO42 anion. No appreciable changes were noticeable in UV-Vis spectra, even in the presence of 200 equivalents of HPO42 anion (Figure 4.13).

Figure 4.13. Changes in UV-Vis spectra of complex 4B in the presence of various equivalents of  HPO 4 2-  anion at 25 °C and pH  7.4
Figure 4.13. Changes in UV-Vis spectra of complex 4B in the presence of various equivalents of HPO 4 2- anion at 25 °C and pH  7.4

Phantom MR Imaging

The image intensity of the acquired images was further compared using ImageJ software considering the same area for all images. From the image intensity graph, it was confirmed that the brightness of the images was increasing with increasing concentration of complex 4B.

Figure 4.15. Relative image intensity plot using ImageJ Software.
Figure 4.15. Relative image intensity plot using ImageJ Software.

Conclusion

Introduction

Synthesis and Characterization of Mn(II) Complex of Ligand Li 3 cbda

The dianionic unit of complex 5A consisted of two seven-coordinate Mn(II) coordination units, which were connected to the carbonyl-oxygen atom O22. The oxidation state of the central Mn ion was determined by X-band EPR measurement of the aqueous solution of complex 5A at room temperature.

Thermodynamic Stability

By pH potentiometric titration of ligand Li3cbda with Mn(II) ion in 1:1 molar equivalent under the same experimental conditions, a stability constant of 11.90 was obtained for complex 5A. The observed value for complex 5A was comparable to already reported analogous mono(aquated) Mn(II) complexes.

Table  5.3.  Protonation  constants,  stability  constants  and  pMn  value  of  some  six-coordinated  ligands
Table 5.3. Protonation constants, stability constants and pMn value of some six-coordinated ligands

Longitudinal Relaxivity

This was possibly due to the replacement of the coordinated water molecule with a hydroxyl group.

Figure 5.6. 1/T 1  vs [Mn(II)] plot at 1.41 T, 25 °C, and pH  7.4.
Figure 5.6. 1/T 1 vs [Mn(II)] plot at 1.41 T, 25 °C, and pH  7.4.

Affinity for Physiological Anions

This implied that neither the stability of the complex was challenged nor that the coordinated water molecule was replaced by the anions. A low positive charge on Mn ion (+II) and an overall negative charge of the complex possibly promoted a repulsive effect between the complex and the anions.

Phantom MR Imaging

A comparison of image intensities was performed using ImageJ software considering the same image area. The image intensity plot justified the fact that images became brighter with increase in the concentration of complex 5A.

Conclusion

Introduction

Synthesis and Characterization of Mn(II) Complex of Ligand H 2 hbda

Single-crystal X-ray diffraction measurement was performed at 293(2) K to determine the molecular structure of complex 6A. The molecular structure of complex 6A is given in Figure 6.3, and selected bond lengths and bond angles are shown in Table 6.1.

Thermodynamic Stability

The aqueous solution of complex 6A showed a six-line spectrum, which is given in Figure 6.4. For single-core hyperfine coupling with I = 5/2(Mn), the six-line spectrum is usually predicted and displayed as such. . Under physiological conditions, the stability constant of each complex is more accurately described in terms of the pM value; which is given by log[M]free at pH  7.4, and 25 °C, where [M] = [L]total = 10 M.12 The calculated pMn value for the H2hbda ligand was found to be 9.00, which was significantly higher compared to previously reported hexadentate ligands, examples are given in Table 6.3.7(a) The alcoholic group stabilizes the corresponding Ln(III) complexes ( log K = 2.9; where  log K refers to the energy change free upon binding).12 Here it was observed that due to the presence of the alcoholic group, the stability of complex 6A was also found to be increased.

Table 6.3. Protonation constants, stability constants, and pMn value of its analogues
Table 6.3. Protonation constants, stability constants, and pMn value of its analogues

Longitudinal Relaxivity

To investigate the interaction between complex 6A and plasma protein, the longitudinal relaxation times of complex 6A were measured in the presence of 4.5% BSA ( 0.66 mM, physiological concentration). The increase in relaxivity value of complex 6A in the presence of BSA solution was expected to be due to macromolecular association.

Affinity for Physiological Anions

No change in the relaxivity value in the presence of HCO3 and F anion suggested that the complex was inert towards these physiological anions. To understand the likely reason, the absorption spectra of complex 6A were recorded in the presence of various equivalents of HPO42 anion (Figure 6.10).

Figure  6.9. Variation  of  longitudinal  relaxivity  in  the  presence  of  200  equivalents  of  physiological anions at 1.41 T, 25 °C, pH   7.4; [complex  6A] = 0.5 mM, and [physiological  anions] = 100 mM
Figure 6.9. Variation of longitudinal relaxivity in the presence of 200 equivalents of physiological anions at 1.41 T, 25 °C, pH  7.4; [complex 6A] = 0.5 mM, and [physiological anions] = 100 mM

Phantom MR Imaging

Conclusion

With three coordinated water molecules, both complexes showed sufficient thermodynamic stability and could be used as future T1-weighted contrast agents. In aqueous solution, the neutral complex showed high efficiency with a longitudinal relaxivity value of 3.74 mM-1s-1 at 1.41 T, 25 °C and pH  7.4; which is almost equivalent to clinically used Gd(III) contrast agents.

Methods and Equipments

  • Experimental Section
    • Synthesis of Ligand Li 3 cbda

After a delay time, known as the inversion recovery time (TI), the system is exposed to another 90° pulse. During this delay time (TR), the magnetization begins to return to its original position, which is detected in the xy plane by the application of another 90° pulse.

Figure 7.1. Pulse sequence in inversion recovery method.
Figure 7.1. Pulse sequence in inversion recovery method.

The filtrate was concentrated to dryness to obtain a white solid compound, which was further purified by column chromatography on silica gel using ethyl acetate/hexane (1:1) as the eluent. The filtrate was concentrated to complete dryness to obtain off-white solid compound, which was purified by column chromatography on silica gel with ethyl acetate/hexane (1:3) as the eluent.

Figure 7.4.  1 H-NMR spectrum of [C 9 H 9 NO 4 ], (ii).
Figure 7.4. 1 H-NMR spectrum of [C 9 H 9 NO 4 ], (ii).
  • Synthesis of Ligand H 2 hbda
  • Synthesis of Ligand H 3 cpmda
  • Synthesis of Complex 3A
  • Synthesis of Complex 3B
  • Synthesis of Complex 3C

The organic phase was evaporated to dryness to yield a yellowish liquid, which was purified by column chromatography on silica using a mixture of methanol/ethyl acetate (gradient = 1%) as eluent to give compound E as a white solid. The organic phase was evaporated to dryness to yield an oily residue, which was further purified by column chromatography on silica using a mixture of hexane/ethyl acetate (5:1) as eluent to give the compound as a light brown oil.

Figure 7.9.  1 H-NMR spectrum of [C 18 H 21 N 3 O 5 ], (E).
Figure 7.9. 1 H-NMR spectrum of [C 18 H 21 N 3 O 5 ], (E).

Figure

Figure  2.1.  Ligand  backbones  used  in  commercially  available  Gd(III)-based  MRI  contrast  agents
Figure 2.2. Relative affinity of different donor atoms for Ln(III) ions.
Figure  2.4.  ESI-MS  (+ve)  mass  spectrum  of  aqueous  solution  of  ligand  Li 3 cbda
Figure 2.6.  13 C-NMR spectrum of ligand Li 3 cbda in CD 3 OD solvent.
+7

References

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