English

• Molecular imaging plays an increasingly important role in bio-medical research, enabling a better understanding of fundamental molecular pathways in living organisms. Various different imaging techniques are used in molecular imaging, such as X-ray computer tomography (CT), positron emission tomography (PET), single photon emission tomography (SPECT), magnetic resonance imaging (MRI) and optical imaging (OI). Over the past few decades, the use of MRI has revolutionized diagnostic medicine and soft tissue imaging. In order to improve the specificity of MRI enhancement, contrast agents (CAs) are widely used to assist in the acquisition and interpretation of MRI images. About one-third of the 60 million MRI procedures done annually worldwide use contrast-enhancing agents, mostly containing the element gadolinium.^{[1, 2]}

  The first contrast agent approved for human use was [Gd(DTPA)(H₂O)]^2－ (DTPA: diethylenetriaminepentaacetato) (Chart 1).^{[3, 4]} Up to now, the number of approved CAs has increased to nine, and all of them were gadolinium-based contrast agents (GBCAs) and T1-weighted MRI images. They can be divided into extracellular fluid (ECF) space, organ-specific (or targeted) and blood pool agents.^[5] [Gd(BOPTA)(H₂O)]^2－ and [Gd(EOB-DTPA)(H₂O)]^2－ (Chart 1) are liver-targeted agents with lipophilic side groups. The two targeted agents are all linear based on ligand DTPA.^{[6, 7]} However, it has been proved that linear GBCAs have a risk of nephrogenic systemic fibrosis (NSF) in certain patients with kidney dysfunction. But macrocyclic GBCAs have a lower risk for the development of NSF.^{[2, 8]} Because the macrocyclic agents are more stable than the linear agents. For this reason, a lot of macrocyclic GBCAs have been synthesized and researched.^[9~15]

  Figure Chart 1

  

  Figure Chart 1.  Some clinical contrast agents

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  DOTA (1, 4, 7, 10-tetraazacyclododecan-N, N', N'', N'''- tetraacetic acid) is the most commonly used macrocyclic chelating agent for the functionalization of peptides, proteins and antibodies with contrast agents, forming thermodynamically stable and kinetically inert complexes with most trivalent metal ions.^{[16, 17]} To enable efficient coupling to biomolecules, reactive groups need to be introduced into the DOTA complex. Amine and hydrazide groups have been established as versatile coupling sites in many different applications in the biomedical research. DOTA-hydrazide (DOTAH) is a good chelating agent for Gd³⁺ and has been researched in some literatures.^[18~20] Herein, we reported the synthesis and characterization of a series of Gd-based DOTA-hydrazide derived contrast agents for MRI. For these agents, we introduced a lipophilic group with benzene ring and hope that it can bind to liver. Then we can get a liver targeted agent based on macrocyclic ligand, which can overcome the drawback of NSF resulting from linear agents.

  2.   Results and discussion

  2.1   Design and synthesis

  In this study, DOTAH was chosen as the chelator for Gd³⁺ to build novel GBCAs. As a macrocyclic ligand, DOTAH has higher thermodynamic stability and slower dissociation kinetics to avoid transmetallation in vivo compared to the linear ligand like DTPA. At the same time, the Gd³⁺ binding neutralized the charges of the three acetic groups of DOTAH, leading to nonionic CAs, which is beneficial to low osmotic pressure. Then, kinds of lipophilic groups with benzene ring were introduced, which may make these new MRI contrast agents stay a longer time in liver so that they cloud get liver-target property.

  The desired complexes 5a~5l and their precursors were prepared following the procedures in Scheme 1. Raw material of 1, 4, 7-tris(tert-butoxycarbonylmethyl)-10-hydra- zinylcarbonylmethyl-1, 4, 7, 10-tetraazacyclododecane (t-Bu-DOTAH, 1) was synthesized from cyclen according to the literature we have published before.^[20] The condensation reaction of the first step was complished using t-Bu-DOTAH and different benzaldehyde (2) in absolute ethanol refluxing for about 4 h. Deprotection of compounds 3a~3l was achieved within 24 h by reacting with trifluoroacetic acid (TFA). Complexes 5a~5l were obtained by combining 4a~4l and gadolinium chloride in an aqueous solution at 50 ℃, and the purification of 5a~5l was accomplished by C-18 SPE cartridge.

  Scheme 1

  

  Scheme 1.  Synthesis of 5a~5l

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  2.2   In Vitro MRI

  To evaluate the contrast enhancement of the new agents, longitudinal relaxivity r₁ and transverse relaxivity r₂ of them were calculated from the linear fitting of 1/T₁ and 1/T₂ plot versus metal concentrations. Commercially available Gd-DOTA was used as control. The values of r₁ and r₂ are estimated to be 2.94 to 5.33 and 3.63 to 6.32 L·mmol^－1·s^－1, respectively (Table 1). Among them, The relaxivities r₁ of 5d, 5h and 5l are 4.67, 4.85 and 5.33 L·mmol^－1·s^－1 respectively, which are a little higher than that of the currently used clinical contrast agent Gd-DOTA with relaxivity r₁ of 4.41 L·mmol^－1·s^－1. It means that these three contrast agents may be used at lower dosage of agents and thus reducing the clinical toxicity. At the same time, the ratios of r₂/r₁ are calculated to be 1.14 to 1.28, the low ratios of r₂/r₁ indicate that all the new agents can be used as T₁-weighted MRI CAs. As shown in Figure 1, all new agents present excellent positive T₁ contrast enhancement, and the brightness of MR images enhance with the increasing concentrations of solutions, showing a clear dose-dependent color change, due to the relaxation of water proton increase with the increase of concentration dose.

  Table 1

  

  Table 1.  r₁ and r₂ values of the synthesized compounds at 0.5 T, Gd-DOTA is as control

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  Entry	Compd.	r2/ (L·mmol－1·s－1)	r1/ (L·mmol－1·s－1)	r2/r1
  1	Gd-DOTA	5.1289	4.4123	1.1624
  2	5a	3.9125	3.2437	1.2062
  3	5b	5.0427	4.1717	1.2088
  4	5c	3.6309	2.9353	1.2370
  5	5d	5.7423	4.6725	1.2290
  6	5e	4.4016	3.7504	1.1736
  7	5f	4.0837	3.5676	1.1447
  8	5g	4.8747	3.9786	1.2252
  9	5h	6.2079	4.8494	1.2801
  10	5i	4.9583	3.9940	1.2414
  11	5j	5.1293	4.2989	1.1932
  12	5k	4.7857	3.9714	1.2050
  13	5l	6.3216	5.3330	1.1854

  Figure 1

  

  Figure 1.  T₁-weighted images of 5a~5l and Gd-DOTA at 0.5 T

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  2.3   In vivo liver-target MRI

  For exploring the liver-target imaging property of these new synthesized contrast agents, we chose four of them with high relaxivity to do in vivo liver-target MRI test. Five adult male Spraque-Dawley (SD) rats weighing 280~300 g were randomly divided into five groups. All rats were scanned for livers with T₁ weighted imaging (T1WI). Then they were intravenously injected with 5d, 5h, 5j, 5l and Gd-DOTA as control at a dose of 0.1 mmol/kg in the tail vein, respectively. After administration, T1WI of liver was performed at 0 h and 6 h. As shown in Figure 2, immediately after administration of Gd-DOTA and four new contrast agents, the signal intensities (SIs) of normal livers were all strongly enhanced. But after 6 h, only the SI administrated by 5d was still enhanced. The contrast ratios of liver vs normal muscle at pre- and post-injection shown in Figure 3 also demonstrated that only the rat administrated by 5d could get the continuous liver enhancement signal. All the results show the potential of compound 5d used as a new liver-target contrast agent for MRI.

  Figure 2

  

  Figure 2.  MR images of liver at pre- and post-injection

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  Figure 3

  

  Figure 3.  Contrast ratio of liver vs normal muscle at pre- and post-injection

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  3.   Conclusions

  In summary, a series of novel Gd-based DOTA-hydra- zide derived contrast agents have been designed and synthesized. And we explored their feasibilities as T₁ MRI CAs. The results showed that the thus-prepared agents have good positive imaging effect and low r₂/r₁ ratios suitable as T₁-weighted MRI CAs. Moreover, three new agents exhibited a little higher r₁ relaxivities and the ideal contrast enhancements in T₁-weighted MR images, which were comparable to the clinical contrast agent Gd-DOTA. It means that they may be used at lower dosage of agents and thus reducing the clinical toxicity. In vivo liver-target MRI shows that complex 5d may be used as a novel liver-target contrast agent for MRI.

  4.   Experimental section

  4.1   General procedures

  All chemicals and reagents were used as received without further purification. Glassware was dried in an oven at 130 ℃ and purged with a dry atmosphere prior to use. Unless otherwise mentioned, reactions were performed open to air. Reactions were monitored by thin-layer chromatography (TLC) and visualized by a dual short/long wavelength UV lamp. Flash column chromatography was performed using 200~400 mesh silica gel (Fisher). NMR spectra were recorded on a Bruker AVANCE AV-300 or a Bruker AVANCE AV-500 NMR spectrometer at room temperature. Chemical shifts for ¹H NMR and ¹³C NMR were reported as δ. Spectra were referenced tointernal standards (TMS in CDCl₃ and TMSP in D₂O). Multiplicity and coupling constants (J) were calculated automatically on MestReNova 10.0, a NMR processing software from Mestrelab Research. MS (ESI) was determined by using a microTOF-Q II HRMS/MS instrument (Bruker). Element analysis was determined by using a Perkin-Elmer 240c elemental analysis instrument. In vitro MRI testing and T₁ relaxation time measurements were tested at a 0.5 T NMR120-Analyst NMR Analyzing and Imaging system (Niuman Corporation, Shanghai, China). In vivo MRI was performed on a clinical 1.5 T MR magnet (Echo speed; GE Co., NY).

  4.2   General method for the synthesis of 3a~3l

  To a 100 mL round-bottom flask fitted with a magnetic stir bar were added compound 1 (1.17 g, 2 mmol), different benzaldehyde (2 mmol) and 20 mL of absolute ethanol. The mixture was stirred at 75 ℃ for 4 h. After completion of the reaction, ethanol was removed in vacuo, and the resulting residue was recrystallized with ethyl acetate. Filtered, and washed with cold ethyl acetate, the resulting solid was used without further purification.

  Tri-tert-butyl 2, 2', 2''-(10-(2-(2-(benzylidene)hydrazineyl)-2-oxoethyl)-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7-triyl)triacetate (3a): White solid, yield 88%. m.p. 204.5~205.8 ℃; ¹H NMR (300 MHz, CDCl₃) δ: 12.99 (s, 1H), 8.76 (s, 1H), 7.83~7.70 (m, 2H), 7.39~7.25 (m, 3H), 3.62 (s, 2H), 3.29~2.81 (br, 6H), 2.74~1.97 (br, 16H), 1.49 (s, 15H), 1.46 (s, 12H); ¹³C NMR (75 MHz, CDCl₃) δ: 171.9, 171.6, 168.3, 148.1, 134.6, 129.1, 128.2, 127.8, 127.3, 126.8, 81.7, 81.6, 55.6, 55.4, 55.2, 50.0, 27.7, 27.6; ESI-MS m/z: 675.5 [M+H]⁺, 697.4 [M+Na]⁺. Anal. calcd for C₃₅H₅₈N₆O₇: C 62.29, H 8.66, N 12.45; found C 62.23, H 8.68, N 12.47.

  Tri-tert-butyl 2, 2', 2''-(10-(2-(2-(2-nitrobenzylidene)hy-drazinyl)-2-oxoethyl)-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7-triyl)triacetate (3b): White solid, yield 90%. m.p. 194.7~195.2 ℃; ¹H NMR (300 MHz, CDCl₃) δ: 13.31 (s, 1H), 8.90 (s, 1H), 8.73 (s, 1H), 8.14 (d, J＝7.8 Hz, 1H), 7.95 (d, J＝7.5 Hz, 1H), 7.50 (t, J＝7.8 Hz, 1H), 3.62 (s, 2H), 3.44~2.92 (br, 6H), 2.86~1.99 (br, 16H), 1.51 (s, 15H), 1.47 (s, 12H); ¹³C NMR (75 MHz, CDCl₃) δ: 172.2, 168.9, 148.4, 145.3, 137.0, 133.9, 129.1, 123.5, 121.1, 82.2, 82.0, 55.8, 55.7, 55.4, 50.3, 28.0, 27.9; ESI-MS m/z: 720.6 [M+H]⁺. Anal. calcd for C₃₅H₅₇N₇O₉: C 58.40, H 7.98, N 13.62; found C 58.45, H 7.96, N 13.60.

  Tri-tert-butyl 2, 2', 2''-(10-(2-(2-(4-methylbenzylidene)-hydrazinyl)-2-oxoethyl)-1, 4, 7, 10-tetraaza-cyclododecane-1, 4, 7-triyl)triacetate (3c): White solid, yield 87%. m.p. 188.3~189.3 ℃; ¹H NMR (300 MHz, CDCl₃) δ: 12.88 (s, 1H), 8.71 (s, 1H), 7.66 (d, J＝7.8 Hz, 2H), 7.12 (d, J＝7.8 Hz, 2H), 3.61 (s, 2H), 3.47 (s, 2H), 3.30~2.87 (br, 5H), 2.74~1.99 (br, 18H), 1.48 (s, 14H), 1.46 (s, 13H); ¹³C NMR (75 MHz, CDCl₃) δ: 172.1, 168.4, 148.7, 139.4, 132.1, 128.8, 127.6, 82.1, 82.0, 56.0, 55.7, 55.5, 50.3, 28.0, 27.9, 21.3; ESI-MS m/z: 689.5 [M+H]⁺. Anal. calcd for C₃₆H₆₀N₆O₇: C 62.77, H 8.78, N 12.20; found C 62.71, H 8.80, N 12.22.

  Tri-tert-butyl 2, 2', 2''-(10-(2-(2-(4-nitrobenzylidene)-hydrazinyl)-2-oxoethyl)-1, 4, 7, 10-tetraaza-cyclododecane-1, 4, 7-triyl)triacetate (3d): Yellow solid, yield 87%. m.p. 138.9~140.5 ℃; ¹H NMR (300 MHz, CDCl₃) δ: 13.26 (s, 1H), 8.87 (s, 1H), 8.18 (d, J＝8.6 Hz, 2H), 7.93 (d, J＝8.7 Hz, 2H), 3.61 (s, 2H), 2.39~2.87 (br, 6H), 2.81~2.15 (br, 16H), 1.49 (s, 14H), 1.47 (s, 13H); ¹³C NMR (75 MHz, CDCl₃) δ: 172.17, 169.2, 148.0, 145.5, 141.3, 128.0, 123.5, 82.2, 82.1, 55.9, 55.7, 55.5, 50.5, 28.0, 27.9; ESI-MS m/z: 720.5 [M+H]⁺, 742.5 [M+Na]⁺. Anal. calcd for C₃₅H₅₇N₇O₉: C 58.40, H 7.98, N 13.62; found C 58.44, H 7.97, N 13.60.

  Tri-tert-butyl 2, 2', 2''-(10-(2-(2-(4-chlorobenzylidene)-hydrazinyl)-2-oxoethyl)-1, 4, 7, 10-tetraaza-cyclododecane-1, 4, 7-triyl)triacetate (3e): White solid, yield 95%. m.p. 182.1~183.9 ℃; ¹H NMR (300 MHz, CDCl₃) δ: 13.08 (s, 1H), 8.75 (s, 1H), 7.70 (d, J＝8.3 Hz, 2H), 7.29 (d, J＝8.0 Hz, 2H), 3.61 (s, 2H), 3.38~2.87 (br, 16H), 2.83~1.95 (br, 16H), 1.48 (s, 15H), 1.46 (s, 12H); ¹³C NMR (75 MHz, CDCl₃) δ: 172.1, 168.6, 147.1, 135.1, 133.5, 128.7, 128.3, 114.5, 82.1, 82.0, 55.9, 55.7, 55.5, 50.6, 28.6, 27.9; ESI-MS m/z: 709.5 [M+H]⁺, 731.4 [M+Na]⁺. Anal. calcd for C₃₅H₅₇ClN₆O₇: C 59.27, H 8.10, N 11.85; found C 59.21, H 8.11, N 11.87.

  Tri-tert-butyl 2, 2', 2''-(10-(2-(2-(2, 4-dichlorobenzyli-dene)hydrazinyl)-2-oxoethyl)-1, 4, 7, 10-tetraaza-cyclodo-decane-1, 4, 7-triyl)triacetate (3f): White solid, yield 90%. m.p. 164.6~166.1 ℃; ¹H NMR (300 MHz, CDCl₃) δ: 13.26 (s, 1H), 9.08 (s, 1H), 8.02 (d, J＝8.5 Hz, 1H), 7.33 (s, 1H), 7.17 (d, J＝8.4 Hz, 1H), 3.62 (s, 2H), 3.40~2.88 (s, 6H), 2.84~2.15 (br, 16H), 1.48 (s, 15H), 1.46 (s, 12H). ¹³C NMR (126 MHz, CDCl₃) δ: 172.2, 169.1, 144.1, 135.4, 135.2, 131.5, 129.4, 128.4, 126.7, 82.1, 82.0, 56.0, 55.8, 55.6, 28.11, 28.0; ESI-MS m/z: 765.4 [M+Na]⁺. Anal. calcd for C₃₅H₅₆Cl₂N₆O₇: C 56.52, H 7.59, N 11.30; found C 56.57, H 7.58, N 11.28.

  Tri-tert-butyl 2, 2', 2''-(10-(2-(2-(4-fluorobenzylidene)-hydrazinyl)-2-oxoethyl)-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7-triyl)triacetate (3g): Light yellow solid, yield 93%. m.p. 184.6~186.0 ℃; ¹H NMR (300 MHz, CDCl₃) δ: 12.92 (s, 1H), 8.73 (s, 1H), 7.75 (s, 2H), 7.00 (t, J＝8.2 Hz, 2H), 3.59 (s, 2H), 3.39~2.89 (br, 6H), 2.78~2.00 (br, 16H), 1.48 (s, 14H), 1.46 (s, 13H); ¹³C NMR (75 MHz, Chloroform-d) δ: 172.1, 168.6, 165.2, 161.8, 147.3, 131.1 (d), 129.4, 129.3, 115.3, 115.0, 82.1, 82.0, 55.9, 55.7, 55.4, 50.3, 28.0, 27.9; ESI-MS m/z: 693.5 [M+H]⁺, 715.5 [M+Na]⁺. Anal. calcd for C₃₅H₅₇FN₆O₇: C 60.67, H 8.29, N 12.13; found C 60.61, H 8.30, N 12.15.

  Tri-tert-butyl 2, 2', 2''-(10-(2-(2-(3-fluorobenzylidene)-hydrazinyl)-2-oxoethyl)-1, 4, 7, 10-tetraaza-cyclododecane-1, 4, 7-triyl)triacetate (3h): Light yellow solid, yield 91%. m.p. 185.3~186.7 ℃; ¹H NMR (300 MHz, CDCl₃) δ: 12.97 (s, 1H), 8.56 (d, J＝9.8 Hz, 1H), 7.45 (d, J＝7.7 Hz, 1H), 7.28 (d, J＝6.4 Hz, 1H), 7.00 (t, J＝7.3 Hz, 1H), 3.59 (s, 2H), 3.36~2.83 (br, 6H), 2.77~1.96 (br, 16H), 1.49 (s, 14H), 1.46 (s, 13H); ¹³C NMR (75 MHz, CDCl₃) δ: 172.17, 169.2, 148.0, 145.5, 141.3, 128.0, 123.5, 82.2, 82.1, 55.9, 55.7, 55.5, 50.5, 28.0, 27.9; ESI-MS m/z: 720.5 [M+H]⁺, 742.5 [M+Na]⁺. Anal. calcd for C₃₅H₅₇FN₆O₇: C 60.67, H 8.29, N 12.13; found C 60.70, H 8.28, N 12.11.

  Tri-tert-butyl 2, 2', 2''-(10-(2-(2-(2-fluorobenzylidene)-hydrazinyl)-2-oxoethyl)-1, 4, 7, 10-tetraaza-cyclododecane-1, 4, 7-triyl)triacetate (3i): White solid, yield 89%. m.p. 193.4~195.0 ℃; ¹H NMR (300 MHz, CDCl₃) δ: 13.10 (s, 1H), 8.93 (s, 1H), 8.00 (t, J＝7.2 Hz, 1H), 7.28 (d, J＝6.5 Hz, 1H), 7.13~6.91 (m, 2H), 3.63 (s, 2H), 3.41~2.93 (br, 6H), 2.83~1.95 (br, 16H), 1.48 (s, 14H), 1.46 (s, 13H); ¹³C NMR (75 MHz, CDCl₃) δ: 172.1, 168.9, 163.1, 159.7, 141.9, 141.9, 130.8, 127.4, 123.6, 123.5, 115.7, 115.4, 82.0, 81.9, 55.9, 55.7, 55.5, 50.2, 28.0, 27.9; ESI-MS m/z: 693.5 [M+H]⁺, 715.5 [M+Na]⁺. Anal. calcd for C₃₅H₅₇FN₆O₇: C 60.67, H 8.29, N 12.13; found C 60.62, H 8.30, N 12.14.

  Tri-tert-butyl 2, 2', 2''-(10-(2-(2-(3, 4-difluorobenzylide-ne)hydrazinyl)-2-oxoethyl)-1, 4, 7, 10-tetraazacyclodode-cane-1, 4, 7-triyl)triacetate (3j): White solid, yield 92%. m.p. 191.2~193.1 ℃; ¹H NMR (300 MHz, CDCl₃) δ: 13.10 (s, 1H), 8.73 (s, 1H), 7.69 (t, J＝9.0, 1H), 7.39 (s, 1H), 7.10 (dd, J＝17.8, 8.5 Hz, 1H), 3.59 (s, 2H), 3.42~2.88 (br, 6H), 2.86~1.93 (br, 16H), 1.49 (s, 15H), 1.46 (s, 12H); ¹³C NMR (75 MHz, CDCl₃) δ: 172.1, 168.7, 145.9, 124.6, 117.0, 116.8, 115.2, 114.9, 82.1, 82.0, 55.94, 55.76, 55.49, 50.19, 28.04, 27.92; ESI-MS m/z: 733.5 [M+Na]⁺. Anal. calcd for C₃₅H₅₆F₂N₆O₇: C 59.14, H 7.94, N 11.82; found C 59.10, H 7.95, N 11.84.

  Tri-tert-butyl 2, 2', 2''-(10-(2-(2-(4-methoxybenzylidene)-hydrazinyl)-2-oxoethyl)-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7-triyl)triacetate (3k): White solid, yield 85%. m.p. 177.5~178.9 ℃; ¹H NMR (300 MHz, CDCl₃) δ: 12.80 (s, 1H), 8.70 (s, 1H), 7.71 (d, J＝8.3 Hz, 2H), 6.84 (d, J＝8.3 Hz, 2H), 3.81 (s, 3H), 3.59 (s, 2H), 3.36~2.87 (br, 6H), 2.81~1.91 (br, 16H), 1.49 (s, 14H), 1.46 (s, 13H); ¹³C NMR (75 MHz, CDCl₃) δ: 172.1, 168.2, 160.7, 148.3, 129.1, 127.6, 114.5, 113.6, 82.0, 81.9, 56.0, 55.7, 55.5, 55.2, 50.3, 28.0, 27.9; ESI-MS m/z: 727.5 [M+Na]⁺. Anal. calcd for C₃₆H₆₀N₆O₈: C 61.34, H 8.58, N 11.92; found C 61.39, H 8.57, N 11.90.

  Tri-tert-butyl 2, 2', 2''-(10-(2-(2-(4-phenylbenzylidene)-hydrazinyl)-2-oxoethyl)-1, 4, 7, 10-tetraazacyclodeane-1, 4, 7-triyl)triacetate (3l): White solid, yield 88%. m.p. 148.3~150.2 ℃; ¹H NMR (300 MHz, CDCl₃) δ: 12.85 (s, 1H), 8.77 (s, 1H), 7.84 (d, J＝7.9 Hz, 2H), 7.58 (t, J＝8.6 Hz, 4H), 7.42 (t, J＝7.0 Hz, 2H), 7.34 (d, J＝7.1 Hz, 1H), 3.60 (s, 2H), 3.39~2.86 (br, 6H), 2.80~1.96 (br, 16H), 1.49 (s, 16H), 1.46 (s, 11H); ¹³C NMR (75 MHz, CDCl₃) δ: 172.1, 168.6, 148.2, 142.0, 133.8, 128.6, 128.1, 127.6, 127.3, 127.0, 126.8, 82.1, 82.0, 56.0, 55.7, 55.5, 50.3, 28.0, 27.9. HRMS (ESI⁺) calcd for C₄₁H₆₂N₆O₇ [M+H]⁺ 751.4753, found 751.4752.

  4.3   General method for the synthesis of 4a~4l

  A solution of 3 (1.5 mmol) in trifluoroacetic acid (TFA) (10 mL) was stirred at room temperature for 24 h. TFA was removed under reduced pressure and the residue was dissolved in ethyl acetate (5 mL). After freezing for 12 h, the mixture was filtered to obtain solid compounds quantitatively.

  2, 2', 2''-(10-(2-(2-(Benzylidene)hydrazinyl)-2-oxoethyl)-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7-triyl)triacetic acid (4a): White solid, yield 100%. m.p. 180.4~182.3 ℃; ¹H NMR (300 MHz, D₂O) δ: 8.18 (s, 1H), 7.77 (s, 2H), 7.52 (s, 3H), 3.96~2.92 (m, 24H); ¹³C NMR (75 MHz, D₂O) δ: 173.1, 172.6, 154.0, 135.7, 133.9, 133.6, 131.7, 130.5, 130.0, 59.1, 58.2, 56.6, 54.2, 53.5, 53.1, 51.2; ESI-MS m/z: 543.2 [M+K－2H]^－. Anal. calcd for C₂₃H₃₄N₆O₇: C 54.54, H 6.77, N 16.59; found C 54.50, H 6.78, N 16.62.

  2, 2', 2''-(10-(2-(2-(2-Nitrobenzylidene)hydrazinyl)-2-oxoethyl)-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7-triyl)tri-acetic acid (4b): White solid, yield 100%. m.p. 169.9~172.0 ℃; ¹H NMR (300 MHz, D₂O) δ: 8.47 (s, 1H), 8.25 (d, J＝9.0 Hz, 1H), 8.14 (s, 1H), 8.03 (d, J＝8.5 Hz, 1H), 4.06~2.87 (m, 24H); ¹³C NMR (126 MHz, D₂O) δ: 148.5, 148.1, 148.0, 134.8, 133.8, 130.1, 125.2, 122.3, 117.6, 55.4, 54.1, 50.9, 48.6; ESI-MS m/z: 552.2 [M+H]⁺, 550.0 [M－H]^－. Anal. calcd for C₂₃H₃₃N₇O₉: C 50.09, H 6.03, N 17.78; found C 50.14, H 6.02, N 17.76.

  2, 2', 2''-(10-(2-(2-(4-Methylbenzylidene)hydrazinyl)-2-oxoethyl)-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7-triyl)-triacetic acid (4c): White solid, yield 100%. m.p. 185.7~187.3 ℃; ¹H NMR (500 MHz, D₂O) δ: 8.14 (s, 1H), 7.66 (d, J＝7.0 Hz, 2H), 7.33 (d, J＝6.4 Hz, 2H), 3.96~2.96 (m, 24H), 2.38 (s, 3H); ¹³C NMR (126 MHz, D₂O) δ: 170.4, 163.1, 162.8, 151.6, 142.3, 130.3, 129.7, 127.9, 127.4, 117.6, 115.3, 56.5, 54.1, 51.6, 50.9, 48.6, 48.0, 20.7; ESI-MS m/z: 521.2 [M+H]⁺, 519.1 [M－H]^－. Anal. calcd for C₂₄H₃₆N₆O₇: C 55.37, H 6.97, N 16.14; found C 55.41, H 6.96, N 16.12.

  2, 2', 2''-(10-(2-(2-(4-Nitrobenzylidene)hydrazinyl)-2-oxoethyl)-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7-triyl)-triacetic acid (4d): Yellow solid, yield 100%. m.p. 183.8~185.6 ℃. ¹H NMR (300 MHz, D₂O) δ: 8.23 (t, J＝8.0 Hz, 2H), 8.14 (s, 1H), 7.87 (d, J＝8.2 Hz, 1H), 7.81 (d, J＝8.2 Hz, 1H), 4.04~2.97 (m, 24H); ¹³C NMR (126 MHz, D₂O) δ: 163.0, 162.7, 148.3, 139.5, 128.5, 128.0, 124.0, 117.6, 115.2, 55.3, 53.8, 50.4, 48.6; ESI-MS m/z: 588.2 [M+K－2H]^－. Anal. calcd for C₂₃H₃₃N₇O₉: C 50.09, H 6.03, N 17.78; found C 50.05, H 6.04, N 17.80.

  2, 2', 2''-(10-(2-(2-(4-Chlorobenzylidene)hydrazinyl)-2-oxoethyl)-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7-triyl)tra-cetic acid (4e): Light yellow solid, yield 100%. m.p. 186.4~188.6 ℃; ¹H NMR (500 MHz, D₂O) δ: 8.03 (s, 1H), 7.55 (d, J＝45.1 Hz, 2H), 7.32 (d, J＝34.5 Hz, 2H), 3.98~2.96 (m, 24H); ¹³C NMR (126 MHz, D₂O) δ: 162.9, 162.6, 149.7, 136.2, 131.9, 129.2, 129.0, 117.6, 115.3, 55.3, 53.9, 50.9, 48.6, 48.1; ESI-MS m/z: 541.2 [M+H]⁺, 539.0 [M－H]^－. Anal. calcd for C₂₃H₃₃ClN₆O₇: C 51.06, H 6.15, N 15.53; found C 51.10, H 6.14, N 15.50.

  2, 2', 2''-(10-(2-(2-(2, 4-Dichlorobenzylidene)hydrazinyl)-2-oxoethyl)-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7-triyl)tri-acetic acid (4f): Light yellow solid, yield 100%. m.p. 184.5~186.4 ℃; ¹H NMR (500 MHz, D₂O) δ: 8.34 (s, 1H), 8.11 (s, 1H), 7.16 (d, J＝24.4 Hz, 1H), 7.02 (s, 1H), 3.97~2.85 (m, 24H); ¹³C NMR (126 MHz, D₂O) δ: 162.9, 162.6, 136.4, 135.8, 134.8, 129.6, 127.3, 117.6, 115.3, 55.7, 50.9, 48.6, 48.1; ESI-MS m/z: 575.1 [M+H]⁺, 573.0 [M－H]^－. Anal. calcd for C₂₃H₃₂Cl₂N₆O₇: C 48.01, H 5.61, N 14.60; found C 48.05, H 5.60, N 14.58.

  2, 2', 2''-(10-(2-(2-(4-Fluorobenzylidene)hydrazinyl)-2-oxoethyl)-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7-triyl)tria-cetic acid (4g): White solid, yield 100%. m.p. 163.6~166.5 ℃; ¹H NMR (300 MHz, D₂O) δ: 8.13 (s, 1H), 8.00 (s, 1H), 7.78 (s, 2H), 7.22 (s, 1H), 4.00~2.95 (m, 24H); ¹³C NMR (75 MHz, D₂O) δ: 168.4, 165.1, 152.9, 132.7, 132.6, 132.2, 132.0, 118.8, 118.5, 58.8, 57.7, 56.6, 53.43, 51.9, 50.7; ESI-MS m/z: 561.2 [M+K－2H]^－. Anal. calcd for C₂₃H₃₃FN₆O₇: C 52.66, H 6.34, N 16.02; found C 52.70, H 6.33, N 16.01.

  2, 2', 2''-(10-(2-(2-(3-Fluorobenzylidene)hydrazinyl)-2-oxoethyl)-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7-triyl)tri-acetic acid (4h): White solid, yield 100%. m.p. 170.0~171.5 ℃; ¹H NMR (300 MHz, D₂O) δ: 8.12 (s, 1H), 7.96 (s, 1H), 7.49 (d, J＝14.8 Hz, 3H), 7.23 (d, J＝7.5 Hz, 1H), 3.99~3.04 (m, 24H); ¹³C NMR (75 MHz, D₂O) δ: 173.0, 166.9, 163.7, 152.5, 138.0, 133.4, 126.9, 120.6, 116.1, 115.6, 58.9, 57.9, 56.6, 53.5, 51.2; ESI-MS m/z: 525.2 [M+H]⁺, 523.1 [M－H]^－. Anal. calcd for C₂₃H₃₃FN₆O₇: C 52.66, H 6.34, N 16.02; found C 52.62, H 6.35, N 16.04.

  2, 2', 2''-(10-(2-(2-(2-Fluorobenzylidene)hydrazinyl)-2-oxoethyl)-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7-triyl)tria-cetic acid (4i): White solid, yield 100%. m.p. 170.1~172.5 ℃; ¹H NMR (500 MHz, D₂O) δ: 8.34 (s, 1H), 7.90 (t, J＝7.1 Hz, 1H), 7.56~7.46 (m, 1H), 7.32~7.17 (m, 2H), 3.95~3.09 (m, 24H); ¹³C NMR (126 MHz, D₂O) δ: 170.5, 162.4, 160.4, 144.7, 133.1, 127.4, 124.8, 120.7, 117.6, 116.2, 55.5, 53.8, 50.9, 48.6; ESI-MS m/z: 525.2 [M+H]⁺, 523.1 [M－H]^－. Anal. calcd for C₂₃H₃₃FN₆O₇: C 52.66, H 6.34, N 16.02; found C 52.69, H 6.33, N 16.00.

  2, 2', 2''-(10-(2-(2-(3, 4-Difluorobenzylidene)hydrazinyl)-2-oxoethyl)-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7-triyl)tri- acetic acid (4j): White solid, yield 100%. m.p. 182.8~184.6 ℃; ¹H NMR (300 MHz, D₂O) δ: 8.04 (s, 1H), 7.72~7.22 (m, 3H), 4.01~3.06 (m, 24H); ¹³C NMR (126 MHz, D₂O) δ: 173.3, 155.8, 154.4, 151.6, 148.1, 133.1, 127.9, 120.6, 118.3, 1177, 58.8, 57.8, 56.5, 53.0, 51.5; ESI-MS m/z: 543.2 [M+H]⁺, 541.1 [M－H]^－. Anal. calcd for C₂₃H₃₂F₂N₆O₇: C 50.92, H 5.95, N 15.49; found C 50.95, H 5.94, N 15.47.

  2, 2', 2''-(10-(2-(2-(4-Methoxybenzylidene)hydrazinyl)-2-oxoethyl)-1, 4, 7, 10-tetraazacyclododeca-ne-1, 4, 7-triyl)tria- cetic acid (4k): Yellow solid, yield 100%. m.p. 152.9~154.1 ℃; ¹H NMR (300 MHz, D₂O) δ: 8.09 (s, 1H), 7.71 (d, J＝7.8 Hz, 1H), 7.65 (d, J＝7.9 Hz, 1H), 7.05 (d, J＝7.2 Hz, 2H), 3.98~2.98 (m, 27H); ¹³C NMR (75 MHz, D₂O) δ: 197.5, 164.0, 163.7, 153.8, 135.4, 132.3, 131. 8, 128.9, 128.6, 117.2, 58.9, 58.2, 56.6, 55.0, 53.5, 51.2; ESI-MS m/z: 537.2 [M+H]⁺, 535.1 [M－H]^－. Anal. calcd for C₂₄H₃₆N₆O₈: C 53.72, H 6.76, N 15.66; found C 53.75, H 6.75, N 15.65.

  2, 2', 2''-(10-(2-(2-(4-Phenylbenzylidene)hydrazinyl)-2-oxoethyl)-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7-triyl)tri-acetic acid (4l): White solid, yield 100%. m.p. 180.5~181.7 ℃; ¹H NMR (300 MHz, D₂O) δ: 8.01 (s, 1H), 7.65~6.57 (m, 9H), 4.07~2.68 (m, 24H); ¹³C NMR (75 MHz, D₂O) δ: 177.4, 173.1, 165.2, 152.1, 148.5, 144.5, 144.1, 142.0, 141.8, 135.2, 131.3, 129.2, 121.0, 117.2, 59.2, 55.7, 54.3, 53.4, 51.2, 50.7. HRMS (ESI⁺) calcd for C₂₉H₃₈N₆O₇ [M+H]⁺ 605.2694, found 605.2701.

  4.4   General method for the synthesis of 5a~5l

  To a solution of 4a~4l (1.5 mmol) in water (20 mL) was added a solution of GdCl₃ in water (0.05 mol/L, 36 mL, 1.8 mmol) while maintaining the pH at 6.5 by addition of 1 mol/L NaOH. The solution was stirred at 50 ℃ for 24 h. The pH was raised to 8.0. The solution was filtered through a 0.2 μm filter, and the pH was lowered to 7.0 by adding 1 mol/L HCl. The solution was loaded on a Hypersep C-18 cartridge, and the stationary phase was washed extensively with water. The product was recovered after gradient elution with MeOH/H₂O from 1:9 to 3:2. The fractions containing the product were evaporated under reduced pressure to give 5a~5l as solid compounds.

  Gadolinium 2, 2', 2''-(10-(2-(2-(benzylidene)hydrazin-yl)-2-oxoethyl)-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7-triyl)-triacetic acetate (5a): White solid, yield 85%; ESI-MS m/z: 684.2 [M+Na]⁺. A correct Gd isotope pattern was observed. Anal. calcd for C₂₃H₃₁GdN₆O₇: C 41.81, H 4.73, N 12.72; found C 41.74, H 4.74, N 13.19.

  Gadolinium 2, 2', 2''-(10-(2-(2-(2-nitrobenzylidene)-hydrazinyl)-2-oxoethyl)-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7-triyl)triacetic acetate (5b): Yellow solid, yield 90%; ESI-MS m/z: 729.1 [M+Na]⁺. A correct Gd isotope pattern was observed. Anal. calcd for C₂₃H₃₀GdN₇O₉: C 39.14, H 4.28, N 13.89; found C 39.19, H 4.27, N 13.87.

  Gadolinium 2, 2', 2''-(10-(2-(2-(4-methylbenzylidene)-hydrazinyl)-2-oxoethyl)-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7-triyl)triacetic acetate (5c): White solid, yield 88%; ESI-MS m/z: 698.2 [M+Na]⁺. A correct Gd isotope pattern was observed. Anal. calcd for C₂₄H₃₃GdN₆O₇: C 42.72, H 4.93, N 12.45; found C 42.66, H 4.94, N 12.47.

  Gadolinium 2, 2', 2''-(10-(2-(2-(4-nitrobenzylidene)hydra-zinyl)-2-oxoethyl)-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7-triyl)triacetic acetate (5d): Light yellow solid, yield 92%; ESI-MS m/z: 729.1 [M+Na]⁺. A correct Gd isotope pattern was observed. Anal. calcd for C₂₃H₃₀GdN₇O₉: C 39.14, H 4.28, N 13.89; found C 39.18, H 4.27, N 13.86.

  Gadolinium 2, 2', 2''-(10-(2-(2-(4-chlorobenzylidene)-hydrazinyl)-2-oxoethyl)-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7-triyl)triacetic acetate (5e): Light yellow solid, yield 95%; ESI-MS m/z: 718.2 [M+Na]⁺. A correct Gd isotope pattern was observed. Anal. calcd for C₂₃H₃₀ClGdN₆O₇: C 39.74, H 4.35, N 12.09; found C 39.69, H 4.36, N 12.12.

  Gadolinium 2, 2', 2''-(10-(2-(2-(2, 4-dichlorobenzylidene)-hydrazinyl)-2-oxoethyl)-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7-triyl)triacetic acetate (5f): Light yellow solid, yield 90%; ESI-MS m/z: 729.2 [M]⁺. A correct Gd isotope pattern was observed. Anal. calcd for C₂₃H₂₉Cl₂GdN₆O₇: C 37.86, H 4.01, N 11.52; found C 39.65, H 4.02, N 11.55.

  Gadolinium 2, 2', 2''-(10-(2-(2-(4-fluorobenzylidene)-hydrazinyl)-2-oxoethyl)-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7-triyl)triacetic acetate (5g): Light yellow solid, yield 93%; ESI-MS m/z: 702.1 [M+Na]⁺. A correct Gd isotope pattern was observed. Anal. calcd for C₂₃H₃₀FGdN₆O₇: C 40.70, H 4.46, N 12.38; found C 40.75, H 4.45, N 12.36.

  Gadolinium 2, 2', 2''-(10-(2-(2-(3-fluorobenzylidene)-hydrazinyl)-2-oxoethyl)-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7-triyl)triacetic acetate (5h): Light yellow solid, yield 92%; ESI-MS m/z: 702.1 [M+Na]⁺. A correct Gd isotope pattern was observed. Anal. calcd for C₂₃H₃₀FGdN₆O₇: C 40.70, H 4.46, N 12.38; found C 40.66, H 4.47, N 12.40.

  Gadolinium 2, 2', 2''-(10-(2-(2-(2-fluorobenzylidene)-hydrazinyl)-2-oxoethyl)-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7-triyl)triacetic acetate (5i): White solid, yield 96%; ESI-MS m/z: 702.1 [M+Na]⁺. A correct Gd isotope pattern was observed. Anal. calcd for C₂₃H₃₀FGdN₆O₇: C 40.70, H 4.46, N 12.38; found C 40.73, H 4.45, N 12.36.

  Gadolinium 2, 2', 2''-(10-(2-(2-(3, 4-difluorobenzylidene)-hydrazinyl)-2-oxoethyl)-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7-triyl)triacetic acetate (5j): White solid, yield 90%; ESI-MS m/z: 720.1 [M+Na]⁺. A correct Gd isotope pattern was observed. Anal. calcd for C₂₃H₂₉F₂GdN₆O₇: C 39.65, H 4.20, N 12.06; found C 39.70, H 4.19, N 12.03.

  Gadolinium 2, 2', 2''-(10-(2-(2-(4-methoxybenzylidene)-hydrazinyl)-2-oxoethyl)-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7-triyl)triacetic acetate (5k): Light yellow solid, yield 88%; ESI-MS m/z: 714.1 [M+Na]⁺. A correct Gd isotope pattern was observed. Anal. calcd for C₂₄H₃₃GdN₆O₈: C 41.73, H 4.82, N 12.17; found C 41.68, H 4.83, N 12.20.

  Gadolinium 2, 2', 2''-(10-(2-(2-(4-phenylbenzylidene)-hydrazinyl)-2-oxoethyl)-1, 4, 7, 10-tetraazacyclododecane-1, 4, 7-triyl)triacetic acetate (5l): Light yellow solid, yield 85%; ESI-MS m/z: 760.2 [M+Na]⁺. A correct Gd isotope pattern was observed. Anal. calcd for C₂₉H₃₅GdN₆O₇: C 47.27, H 4.79, N 11.41; found C 47.33, H 4.78, N 11.38.

  4.5   In vitro MRI

  To obtain T₁ relaxation times of the aqueous solution of the new agents at different concentrations, following measurement parameters were used: repetition time (TR) is 6000 ms, number of data is 25, and number of averages (NA) is 2. While the T₂ relaxation times were determined with the following parameters: TR is 6000 ms, echo time (TE) is 1 ms, echo count is 6000 and NA is 2. Both r₁ and r₂ relaxivities were calculated by the linear fitting of 1/T₁ or 1/T₂ as a function of metal concentration. For T₁- weighted MR images, the instrument parameters were set as follows: TR＝100 ms, TE＝18.2 ms, imaging matrix＝192×256, slice thickness＝5 mm, field of view (FOW)＝100 mm×100 mm and NA＝2.

  4.6   Animals

  Adult male Sprague-Dawley (SD) rats weighing 280~300 g were provided by the Experimental Animal Center, Jiangsu Province Academy of Traditional Chinese Medicine. All the animal experiments were implemented with the approval the institutional animal care and research advisory.

  4.7   In vivo MRI and imaging analyses

  MRI was performed on a clinical 1.5 T MR magnet (Echo speed; GE Co., NY) by using a rat coil. All animals were anaesthetized with isoflurane in a mixture of 80% room air and 20% oxygen. Spin-echo (SE) T1-weighted imaging (T1WI) was performed with the following parameters: TR＝550 ms, TE＝24 ms, FOV＝100 mm×100 mm, imaging acquisition matrix＝224×192, a total examination time of 185 s. Contract agents of 5d, 5h, 5j, 5l and Gd-DOTA were used at the same dosage of 0.1 mmol/kg. MRI analyses were performed by using the built-in software of the system (GE, AW 4.3, GE Healthcare Bio-Sciences, Pittsburgh, PA). The signal intensities (SIs) of tissue were measured with an operator-defined circular region of interest (ROI) with 3 mm² on T1WI. Contrast ratio was used to express the visual conspicuity of adjacent component on the one image and calculated with the formula: CR＝SI_A/SI_B.

  Supporting Information   ¹H NMR and ¹³C NMR spectra, mass spectra of the synthesized compounds. Longitudinal and transverse relaxivities of Gd-DOTA and 5a~5l. The Supporting Information is available free of charge via the Internet at http://sioc-journal.cn/.

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  Figure Chart 1  Some clinical contrast agents

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  Scheme 1  Synthesis of 5a~5l

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  Figure 1  T₁-weighted images of 5a~5l and Gd-DOTA at 0.5 T

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  Figure 2  MR images of liver at pre- and post-injection

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  Figure 3  Contrast ratio of liver vs normal muscle at pre- and post-injection

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  Table 1.  r₁ and r₂ values of the synthesized compounds at 0.5 T, Gd-DOTA is as control

  Entry	Compd.	r2/ (L·mmol－1·s－1)	r1/ (L·mmol－1·s－1)	r2/r1
  1	Gd-DOTA	5.1289	4.4123	1.1624
  2	5a	3.9125	3.2437	1.2062
  3	5b	5.0427	4.1717	1.2088
  4	5c	3.6309	2.9353	1.2370
  5	5d	5.7423	4.6725	1.2290
  6	5e	4.4016	3.7504	1.1736
  7	5f	4.0837	3.5676	1.1447
  8	5g	4.8747	3.9786	1.2252
  9	5h	6.2079	4.8494	1.2801
  10	5i	4.9583	3.9940	1.2414
  11	5j	5.1293	4.2989	1.1932
  12	5k	4.7857	3.9714	1.2050
  13	5l	6.3216	5.3330	1.1854

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