Synthesis and study on magnetic resonance imaging performance of Gd(III)-DTPA-bisbenzothiazol hydrazide

Article abstract of DOI:10.4067/S0717-97072016000200003

In this paper, 2-hydrazino-6-methoxy-1,3-benzothiazole was introduced into diethylenetriamine pentaacetic ( DTPA) by acylation reaction. The corresponding non-ion Gd(III) complex holding promise of a single molecule magnetic resonance imaging (MRI) contrast agent was obtained by treating this ligand with GdCl₃·6H₂ O. The efficacy of the contrast agent was assessed by measuring the longitudinal relaxivity (r₁), the r₁ of Gd(III)-DTPA-bisbenzothiazole hydrazide was up to 6.44 mM^-1·s^-1, which was 1.8 times higher than that of the analogous MRI contrast agent Gd(III)-DTPA(r₁ = 3.64 mM^-1·s^-1 ) in commercial use. In addition, in vitro MR images on a 0.5 T magnetic field exhibited a remarkable enhancement of signal contrast for Gd(III)-DTPA-bisbenzothiazole hydrazide than Gd(III)-DTPA. These results demonstrate that this non-ion Gd(III) complex acts as a potentially MRI contrast agent.

Full text of DOI:10.4067/S0717-97072016000200003

J. Chil. Chem. Soc., 61, Nº 2 (2016)
SYNTHESIS AND STUDY ON MAGNETIC RESONANCE IMAGING PERFORMANCE OF GD(III)-DTPA-
BISBENZOTHIAZOL HYDRAZIDE
*
*
FU-XIAN WAN , TIAN-KAI ZHANG, YING LI, CHANG-CHENG LI, LIN JIANG
College of Chemistry and Material Science, Shan Dong Agricultural University, Taian 271018, People’s Republic of China
ABSTRACT
In this paper, 2-hydrazino-6-methoxy-1,3-benzothiazole was introduced into diethylenetriamine pentaacetic ( DTPA) by acylation reaction. The correspond-
ing non-ion Gd(III) complex holding promise of a single molecule magnetic resonance imaging (MRI) contrast agent was obtained by treating this ligand with
GdCl ·6H O. The efficacy of the contrast agent was assessed by measuring the longitudinal relaxivity (r ), the r of Gd(III)-DTPA-bisbenzothiazole hydrazide
3
2
1
1
−
1
−1
−1 −1
was up to 6.44 mM ·s , which was 1.8 times higher than that of the analogous MRI contrast agent Gd(III)-DTPA(r = 3.64 mM ·s ) in commercial use. In
1
addition, in vitro MR images on a 0.5 T magnetic field exhibited a remarkable enhancement of signal contrast for Gd(III)-DTPA-bisbenzothiazole hydrazide than
Gd(III)-DTPA. These results demonstrate that this non-ion Gd(III) complex acts as a potentially MRI contrast agent.
Keywords: MRI contrast agent; benzothiazole; DTPA derivative; gadolinium complex; relaxivity
INTRODUCTION
1). The longitudinal relaxation time (T ) was measured, and spin–lattice
1
relaxivity values (r ) of this Gd(III) complex was higher than that of Magnivest
1
Over forty years later, magnetic resonance imaging (MRI) has become one
of the most powerful tool in preclinical research and in the clinical diagnosis
of various diseases around the word, due to its especially advantageous: high
spatial (< 0.1 mm) and temporal resolutions, 3-dimensional anatomical images,
non-invasive (lack of ionizing radiation), deep tissue penetration and excellent
(Gd(III)-DTPA). In vitro MR images on a 0.5 T magnetic field exhibited that
proton signal intensity increased with Gd(III) complex concentration and a
remarkable enhancement of signal contrast for Gd(III)-DTPA-bisbenzothiazole
hydrazide than Gd(III)-DTPA. Water-solubility tests showed that the solubility
of the Gd(III)-L complex in water is largely and up to 0.8 g mL at room
temperature. These results showed that the complex is a prospective magnetic
resonance imaging agent.
−
1
1
-3
soft tissue contrast and multiple contrast mechanisms . In MRI, image contrast
can be generated by differences in tissue water content, water relaxation times,
4
flow, or diffusion . Unfortunately the image contrast is often insufficient for
diagnostic purposes. Contrast can be enhanced through the addition of an
exogenous MRI contrast agents(CAs) to increase the relaxation rates of water
protons in tissue in which the agent accumulates. Most commonly, this is a
paramagnetic chelate that shortens the relaxation times of water molecules it
5-6
encounters . At present, the clinical utility of MRI icontrast agents is well
established. Annually, at our medical institutions more than 50% of MRI
7
scans were aided by contrast agents to diagnose disease . Therefore, many
research groups around the world have been studying MRI contrast agents.
To date, more than nine types of the ligand of paramagnetic metal ion-
chelate complexes have been approved by Food and Drug Administration
(FDA) for clinical application. A vast body of literature exists describing
ligands for Gd(III), and the majority are polyaminopolycarboxylate ligands,
like Gd-DTPA (DTPA=diethylenetriamine pentaacetic acid), Gd-DOTA
(DOTA=1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid), Gd(HP-
DO3A) (HP-DO3A=10-(2-hydroxypropyl)-1,4,7,10-tetra-azacyclododecane-
8
-10
1
,4,7-triacetic acid), etc
.
However, most contrast agents have non-specific extracellular distribution
and the disadvantages of low relaxivity, low tissue specificity, and rapid
clearance. Moreover, most of the MRI contrast agents suffer low T relaxivity,
1
which is still far from expectation for highly sensitive MRI. These drawbacks
1
1
have hindered the further development of MRI as a diagnostic tool . Therefore,
an ideal MRI contrast agent should be designed as the tissue or organ-targeting
materials with high relaxivity, low toxicity and side effects, suitable long
intravascular duration and excretion time, and high contrast enhancement
Scheme 1. Synthesis of ligand(H L) and complex(Gd(III)-L)
3
EXPERIMENTAL
1
2
with low dose, in vivo, all coupled to low overall cost . More recently, by
GENERAL
conjugation with a targeting moiety, low molecular weight gadolinium chelates
Melting points were determined on a digital apparatus and are uncorrected.
TLC analysis was performed on glass sheets coated with Merck silica gel 60
1
3-14
provide strategy for new generation of tumor targeting contrast agent
.
1
Benzothiazole and its derivatives have been extensively investigated due
to the high biological activity such as antimicrobial, anti-inflammatory and
F254. Compounds were visualized by I steam or Uv-vis. H-NMR spectra were
2
recorded on Bruker AV-400 spectrometers respectively. Chemical shifts(δ)
were reported in parts per million from internal standard tetramethylsilane
(TMS). Coupling constants (J) were measured in Hertz. Multiplicity was
reported as follows: s(singlet), d(doublet), t(triplet), m (multiplet) and
combination of these signals. Column chromatographies were performed on
silica gel 60 (200-300 mesh). IR spectrometry recorded with a Nicolet 380
FT-IR. Elemental analyses were determined on a Vario EL III(Elementar),
Mass spectra were obtained on a Angilent6110 (Angilent Technologies, USA).
15-17
anticancer activity
. Based on the previous work, a great deal of research
groupsdevotedtodevelopvariousbenzothiazolederivativeswithhighantitumor
activity in the past two decades. Gratifying results have been achieved in this
area, conjugates of benzothiazoles with 1,4,7,10-tetraazacyclododecane-1,4,7-
tristert-butyl acetate (DO3A) have been employed as multimodal imaging
probes such as magnetic resonance imaging (MRI)/optical and MRI/SPECT
18-19
.
The above observations prompted us to design and synthesize a new
The solvent longitudinal relaxation time (T ) for gadolinium complexes in
1
gadoliniumcomplex incorporatingbenzothiazoleunitbythestraightforwardand
efficient reaction of the bicyclic anhydride of diethylenetraiaminepentaacetic
acid (DTPAA) with 2-hydrazino-6-methoxy-1,3-benzothiazole (3) (Scheme
distilled water was determined by a standard inversion-recovery sequence
on the MicroMR imaging & analyzing system at 32 °C and 0.5 T ( Niumag
Technology Co., Ltd., Suzhou, China ). MARS5 microwave digestion system.
e-mail: wfx@sdau.edu.cn
2861

J. Chil. Chem. Soc., 61, Nº 2 (2016)
RT1001-RT1002 ultarsonic cleaning machine.
All reagents and solvents were analytical grade from commercial suppliers
and used without further treatments unless otherwise stated. Reactions were
RESULTS AND DISCUSSION
As mentioned previously, because of the favorable pharmacological
properties of benzothiazole compounds, we have produced the corresponding
DTPA analogues containing benzothiazole. The synthetic scheme of ligand
carried out under N atmosphere, unless otherwise noted.
2
SYNTHESES
Synthesis of 2-hydrazino-6-methoxy-1,3-benzothiazole (2)
Concentrated hydrochloric acid (8.6 mL, 36%, 0.1 mol) was added drop
wise with stirring to hydrazine hydrate (11.5 mL, 85%, 0.2 M) at 0 ºC followed
by ethylene glycol (30 mL), there after 2-amino-6-methoxy-1,3-benzothiazole
(DTPA-bisbenzothiazole hydrazide, H L) is shown in Scheme 1.
3
1
The structure of ligand was characterized by FT-IR, ES-API-MS, H NMR
1
and elemental analysis. H NMR, FT-IR were conformed with the structure
of
diethylenetriamine-N,N”-bis(acetyl-6-methoxy-1,
3-benzothiazole-2-
(
1)( 9 g, 0.05 mol) was added in portions and the resultant mixture was refluxed
hydrazino) - N,N′,N”-triacetic acid (DTPA-bisbenzothiazole hydrazide, H L).
3
1
1
for 12 h and cooled at room temperature. The reaction progress was monitored
by TLC using toluene:ethylacetate ( v:v = 75:25 ) as mobile phase. The reaction
mixture was filtered and resulting precipitate was washed with distilled water,
then recrystallized from ethanol to give 2 as a reddish brown solid. Yield: 6.73g
The H NMR spectrum of the ligand (H L) was shown in Fig. S2. The H
3
NMR spectra showed the characteristic peaks of NCH COOH groups of
2
DTPA structure and benzyl groups of benzothiazole structure, indicating that
DTPA was covalently bound to benzothiazole. The ES-API-MS spectra(Fig.
S3) revealed that the molecular ion peaks were in accordance with the given
(
69 %); mp 176-177.5 ºC. FT-IR (KBr pellet): 3200-3450 (-NHNH ), 1628
2
-1 1
+
(C=N), 1448 (thiazole) cm ; H NMR (400 MHz, CDCl , δ/ppm): 1.24 (brs,
structure of it. ES-API-MS(positive mode): m/z [M+H] Calcd. 748.21 Da;
3
1
H, NH, disappeared on D O exchange), 3.83 (s, 3 H, -OCH ), 4.22 (brs, 2
Obsd. 748.3 Da.
2
3
3
4
H, NH , disappeared on D O exchange), 6.92 (dd, J =8.8 Hz, J =2.4 Hz, 1 H),
7
The Gd(III)-L complex was prepared by reacting the ligand with
stoichiometric amounts of the GdCl ·6H O in high yield. Water-
2
2
4
3
.19 (d, J =2.4 Hz, 1 H), 7.44 (d, J =8.8 Hz, 1 H). Anal. Calcd. for C H N OS
8 9 3
3
2
(
w /%): C, 49.21; H, 4.65; N, 21.52; Found: C, 49.25; H, 4.62; N, 21.48.
solubility tests showed that the solubility of the Gd(III)-L complex in water is
B
−1
Synthesis of
largely and up to 0.8 g mL at room temperature. According to reported method
20
diethylenetriamine-N,N”-bi(acetyl-6-methoxy-1,3-benzothiazole-2-
hydrazino) -N,N′,N”-Triacetic acid (DTPA-bisbenzothiazole Hydrazide)
H L)
: Gd(III)-DTPA-bisbenzothiazole hydrazide was dissolved in distilled water
at concentration of 10.0 mM, then the solution of Gd(III) complex was added
2
−
(
into oxalic ion (C O ) and the molar amount 10 times than that of Gd(III)
3
2 4
DTPA dianhydride (1.428 g, 4.0 mmol) and 2-hydrazino-6-methoxy-1,3-
complex, the mixed solution was stand for 30 days under room temperature.
After careful observation, the mixed solution was keep clear and transparent. It
illustrated that this Gd(III) complex is preliminarily stable in vitro.
benzothiazole (2) (1.56 g, 8.0 mmol) were dissolved in 20 mL dry pyridine
and stirred at 60 ºC for 4 h under N2 atmosphere. After removal of the
solvent, the solid residue was washed with iced water and dried in vacuo, then
In the FT-IR spectra of the ligand(Fig. 1a), strong and broad absorption
-
1
-1
recrystallized from methanol:water( v:v = 3:1 ) to give H L as a yellowish
peaks at 3446 cm were attributed to vOH and vNH . The peaks at 1725 cm (A)
3
-1
solid. Yield: 2.4g (80 %); mp 138-140 ºC. FT-IR (KBr pellet): 3446, 3045,
and 1660 cm (B) , were attributed to vCOOH and vCONH- , respectively. For
Gd(III)-L complex(Fig. 1b), the band (A) disappeared in the complex, showing
that the carboxyl proton dissociates and the oxygen atom was coordinated to
metal. The band (B) was shifted to 1610 cm , suggesting that the oxygen atom
of the amide is coordinated to metal . The ligand provided three nitrogen
-1 1
2
964, 2883, 1729, 1650, 1473, 1385, 1230, 1133, 619, 544 cm ; H NMR (400
MHz, DMSO-d , δ/ppm): 2.73 (t, 4 H, 2×N-CH ), 2.89 (t, 4 H, 2×N-CH ),
6
2
2
-1
2
.95 (s, 4 H, 2×N-CH -CO-NH ) 3.27 (s, 4 H, 2×N-CH -COOH ), 3.42 (s, 2 H,
2
2
21
N-CH -COOH ), 3.74 (s, 6 H, 2×O-CH ), 4.12 (brs, 2 H, NH disappeared on
2
3
D O exchange), 6.87-6.84 (dd, 2 H, Ar-H), 7.34 (m, 2 H, Ar-H), 7.36 (m, 2 H,
atoms, five carboxyl oxygen atoms bonding to metal.
2
Ar-H), 7.95 (s, 2 H, -CONH-).
Anal. Calcd. for C H N O S ·CH CH OH·H O(w /%): C, 47.34; H,
30
37
9
10
2
3
2
2
B
5
.59; N, 15.53. found C, 47.43; H, 5.47; N, 15.37. ES-API-MS(positive mode):
+ 2+
m/z [M+H] Calcd. 748.21 Da; Obsd. 748.3 Da and [M+2H] Calcd. 374.6 Da;
Obsd. 374.7 Da.
Synthesis of Gd(III)- DTPA-bisbenzothiazole Hydrazide(Gd(III)-L)
Diethylenetriamine-N,N”-bi(acetyl-6-methoxy-1,
hydrazino) - N,N′,N”-Triacetic acid (DTPA-bisbenzothiazole Hydrazide)
H L) (0.748 g, 1.0 mmol) was dissolved in water (15 mL) to which was added
3-benzothiazole-2-
(
3
gadolinium(III) chloride hexahydrate (0.37 g, 1.0 mmol). The mixture was
stirred at room temperature for 24 h, during which time pH of the solution was
periodically adjusted to 7.0-7.5 with NaOH (1.0 M). Until no free gadolinium
ions was detected by the xylenol orange test, water was removed by evaporation,
and the remaining oily product was taken up in a minimum amount of water (∼5
mL) to be added dropwise to acetone for precipitation. The white precipitate
thus formed was removed by filtration, washed with acetone, and dried under
vacuum to yield an off-white solid. Yield: 0.874g (95%); mp > 250 ºC. FT-IR
(
5
KBr pellet): 3434, 2975, 2883, 1610, 1464, 1406, 1386, 1232, 1125, 1092,
-1
45, 519 cm ; Anal. calcd for C H GdN NaO S (w /%): C, 36.14; H, 4.25;
30 42 9 14 2 B
N, 12.64. found C, 36.43; H, 4.17; N, 12.57.
Relaxation Measurements
In this experiment, the concentration of gadolinium ion [Gd ] was
Fig. 1 FT-IR spectrum of H L(a) and Gd(III)-L complex (b)
3
+
3
measured by an ICP Atomscan-2000 spectrometer. The solvent longitudinal
The effects of paramagnetic ions on the T relaxation nuclear spins were
1
relaxation time (T ) for gadolinium complex was carried out on a solution of
1
first formulated by Bloembergen and Solomon and subsequently extended by
gadolinium complex in distilled water at concentrations ranging from 0.0 to 2.0
mM (0.4, 0.8, 1.2, 1.6 and 2.0 mM) were measured by a standard inversion-
recovery sequence on the MicroMR imaging & analyzing system at 32 °C and
22-23
several authors
. On the basis of this theory, the longitudinal relaxation time
T1 in the presence of Gd complex is given by Eq. (1):
0
.5 T (Niumag Technology Co., Ltd., Suzhou, China). Thus the relaxivity r for
1
1/T1,obsd = 1/T1,d + r [M] (1)
1
gadolinium complex in distilled water can be calculated. This Gd(III)-complex
was compared with Magnevist (Gd(III)-DTPA) at the same condition.
where (1/T1)obsd is the observed solvent relaxation rate in the presence of a
paramagnetic species, (1/T ) is the solvent relaxation rate in the absence of a
T -Weight imaging in vitro
1
1
d
In vitro imaging effect of Gd(III)-DTPA-bisbenzothiazole hydrazide
is visualized by FLASH images in phantoms. Multislice spin echo (MSE)
sequence(TR=100 ms, TE=12.5 ms, NS=32, Slice thickness=3.0 mm) on
the MR23-060H-I imaging & analyzing system(0.5 T) was employed for the
acquisition of the in vitro imaging.
paramagnetic species, [M] denotes the concentration of gadolinium ion in the
-1 -1
measured solution, and r is the relaxivity of the agent in a unit of mM s ,
1
which is the most important parameter in evaluating a contrast agent and was
calculated from the slope of the plots of (1/T1)obsd versus the Gd concentrations.
Fig. 2 illustrates that the relaxivity of gadolinium complex produced in our
−
1
−1
present work was 6.44 mM ·s , which was 1.8 times higher than that of the
2
862

J. Chil. Chem. Soc., 61, Nº 2 (2016)
−
1
−1
analogous MRI contrast agent Gd(III)-DTPA(r = 3.64 mM ·s ) in commercial
REFERENCES
1
use at the same condition. Possible explanations for increased relaxivity,
relative to clinical contrast agent Gd(III)-DTPA, include: (a) increasing the
molecule size by introducing an aromatic group causes a decrease in rotational
correlation rate(relatively long rotational correlation time); (b) an increase in
the number of outer sphere coordinated water molecules via hydrogen bonds to
the nitrogen atoms of the thiarizonaole groups; (c) hydrophilic hydrazide group
may be able to transmit water molecule from outer-sphere to the inner-sphere
easily.
1. P.C. Lauterbur, Nat. 242, 190, (1973).
2. J. Lee, J. E. Burdette, K.W. MacRenaris, D. Mustafi, T.K. Woodruff, T.
Meade, J. Chem. Biol. 14, 824, (2007).
3. L. Frullano, P. Caravan, Curr. Org. Synth. 8, 535, (2011).
4. K.M. Payne, M. Wood, Bioconjugate Chem. 26, 338, (2015).
5. P. Caravan, J.J. Ellison, T.J. McMurry, R.B. Lauffer, Chem. Rev. 99, 2293,
(1999).
6
.
Y. Huang, B. Cao, X. Yang, Q. Zhang, X. Han, Z. Guo, Magn.Reson.Imag.
1, 604, (2013).
3
7
8
.
.
E. Boros, E. M. Gale, P. Caravan, Dalton Trans. 44, 4804, (2015).
K. Yu, Y. Hamdan, F. Wan, Y. Li, K. Huang, J. Zhou, Aust. J. Chem. 60,
2
18, (2007).
9
.
C.Q. Tu, E.A. Osborne, A.Y. Louie, Tetrahedron 65, 1241, (2009).
1
0. F. Wan, K.Yu, Chin. J. Org. Chem. 34, 613, (2014).
11. X. Zhou, L. Yang, G. Yan, W. Xu, C. Zhou, Q. Zhang, L. Li, F. Liu, J.
Guo, Q. Zhao, J. Rare. Earth. 30, 884, (2012).
1
1
1
2. D. Zhang, X. Zhang, Z. Lin, H. Zhang, Y. Chen, S. He, Inorg. Chem.
Comm. 40, 66, (2014).
3. C. Salt, A.J. Lennox, M. Takagaki, J.A. Maguire, N.S. Hosmane, Russ.
Chem. Bull. Int. Ed. 53, 1871, (2004).
4. C.A. Mathis, Y. Wang, D.P. Holt, G.F. Huang, M.L. Debnath, W.E.
Klunk, J. Med. Chem. 46, 2740, (2003).
1
1
5. T.D. Bradshaw, A.D. Westwell, Curr. Med. Chem. 11, 1009, (2004)
6. X. Chen, P. Yu, L. Zhang, B. Liu, Bioorg. Med. Chem. Lett. 18, 1442,
(2008).
1
1
7. N. Saini, R. Varshney, A.K. Tiwari, A. Kaul, M. Allard, M.P. Ishar, A.K.
Mishra, Dalton Trans. 42, 4994, (2013).
8. E.L. Crossley, J. B. Aitken, S. Vogt, H.H. Harris, L.M. Rendina, Angew.
Chem. Int. Ed. Engl. 49, 1231, (2010).
Fig. 2 Plots of 1/T vs the Gd concentrations of Gd(III)-L complex as well
as Gd-DTPA in water solution
1
19. T. Yamamoto, K. Nakai, A. Matsumura, Cancer.Lett. 262,143, (2008).
2
0. A.D. Sherry, R.D. Brown, C. F.G.C. Geraldes, S.H. Koenig, K-T. Kuan,
M. Spiller, Inorg. Chem. 28, 620, (1989).
The Fig. 3 showed in vitro MR images prepared by Gd(III)-DTPA(b) and
Gd(III)-L (a) solutions in phantoms. It was seen that proton signal intensity
increased with Gd(III) complex concentration. Besides, the imaging effect of
Gd(III)-L was superior to that of Gd(III)-DTPA in the same condition.
2
2
2
1. H.-A. Tang, Y. Sheng, R.-D. Yang, Inorg. Chem. Comm. 6, 1213, (2003).
2. I. Solomon, Phys. Rev. 99, 559, (1955).
3. N. Bloembergen, E.M. Purcell, R.V. Pound, Phys. Rev. 73, 679, (1948)
Fig. 3 T -weighted phantom MR images for Gd(III)-L complex (a) and
1
Gd-DTPA(b) with a concentration of 0-2.0 mM
CONCLUSIONS
In summary, We have successfully synthesized a new gadolinium
complex(Gd(III)-L complex by a simple and efficient reaction. The longitudinal
relativity (r ) of this Gd(III)-L complex is higher than that of Gd(III)-DTPA,
1
and the in vitro imaging effect of Gd(III)-L was superior to that of Gd(III)-
DTPA in the same condition, which makes it possible to reduce the risk of the
toxicity by lowering the dose of Gd(III) chelates. Thus Gd(III)-L might be
considered as a potential MRI contrast agent.
ACKNOWLEGEMENTS
This project was supported by Shandong Province Science and Technology
Development Plan (No. 2013GZX20109) and Shandong Province Natural
Science Foundation (No. ZR2014BM030).
2
863