FLUORESCENCE-MAGNETIC RESONANCE DUAL-MODALITY CONTRAST AGENT, PREPARATION METHOD THEREFOR AND USE THEREOF

Abstract

A fluorescence-magnetic resonance dual-modality contrast agent, a preparation method thereof and the use thereof. The contrast agent has the following structure: X-L-Y, wherein: formula (I); R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.9, R.sub.10, R.sub.11, and R.sub.12 are each independently selected from H, halogen, OH, NH.sub.2, COOH, CONH.sub.2, NO.sub.2, CN, and low alkyl groups, the low alkyl groups can be substituted with halogen, OH, NH.sub.2, COOH, CONH.sub.2, SO.sub.3H, NO.sub.2, and CN, wherein R.sub.3 and R.sub.4, taken together with the carbon atoms to which they are attached, may form a phenyl group or a heterocyclic group; R.sub.7 and R.sub.8, taken together with the carbon atoms to which they are attached, may form a phenyl group or heterocyclic group; L is a linking group; and Y is a metal chelate.

##STR00001##

Claims

1. A fluorescence-magnetic resonance dual-modality contrast agent, wherein the contrast agent has the following structure:
X-L-Y, wherein: X has the following structure: ##STR00046## R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.9, R.sub.10, R.sub.11, and R.sub.12 are each independently selected from H, halogen, OH, NH.sub.2, COOH, CONH.sub.2, NO.sub.2, CN, and low alkyl groups, wherein the low alkyl groups can be substituted with halogen, OH, NH.sub.2, COOH, CONH.sub.2, SO.sub.3H, NO.sub.2, or CN, wherein: R.sub.3 and R.sub.4, taken together with the carbon atoms to which they are attached, may form a phenyl group or a heterocyclic group; and R.sub.7 and R.sub.8, taken together with the carbon atoms to which they are attached, may form a phenyl group or a heterocyclic group; L is a linking group, having a structure according to the following formula: ##STR00047## A is selected from the group consisting of S, N, and O; L.sub.1 is a carbon chain containing 8 to 20 carbon atoms, wherein the carbon atom(s) in the carbon chain may be replaced with oxygen atom(s) or nitrogen atom(s); the carbon chain can contain double bond(s); the hydrogen atom(s) in the carbon chain can be substituted with 1-5 R.sub.13, wherein each R.sub.13 is independently selected from benzyl, carboxyl, and C.sub.1-3 alkyl, or two adjacent R.sub.13 can form a ring, taken together with the atoms to which they are attached, and the carbon(s) in the carbon chain can be further replaced with carbonyl group(s); and Y is a metal chelate.

2. The dual-modality contrast agent according to claim 1, wherein the X has the following structure: ##STR00048## wherein R.sub.9, R.sub.10, R.sub.11 and R.sub.12 are defined as in claim 1.

3. The dual-modality contrast agent according to claim 1, wherein the X has the following structure: ##STR00049##

4. The dual-modality contrast agent according to claim 1, wherein the L has the following structure: ##STR00050## wherein the definition of A is the same as claim 1; and L.sub.2 has the same definition as L.sub.1.

5. The dual-modality contrast agent according to claim 4, wherein the L.sub.2 has the following formula: ##STR00051## wherein n is 8-20.

6. The dual-modality contrast agent according to claim 1, wherein the metal chelate is complexed with Gd to form a Gd complex.

7. The dual-modality contrast agent according to claim 6, wherein the Gd complex has the following structures: ##STR00052## ##STR00053## ##STR00054##

8. The dual-modality contrast agent according to claim 1, wherein the dual-modality contrast agent is selected from the following structures: ##STR00055## ##STR00056## ##STR00057## ##STR00058##

9. A method of preparing a fluorescence-magnetic resonance dual-modality contrast agent comprising the step of condensing a compound of formula I with a compound of formula II to obtain a fluorescence-magnetic resonance dual-modality contrast agent of formula III, ##STR00059## wherein the compound of formula II is obtained by complexing a compound of formula ##STR00060## with gadolinium chloride hydrate, wherein the definitions of R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.9, R.sub.10, R.sub.11, R.sub.12, Y, A, L, and Z are the same as defined in claim 1; alternatively, a compound of formula III-1 is complexed with gadolinium chloride hydrate to obtain a compound of formula III-2, ##STR00061## wherein a compound of formula I-2 is condensed with a compound of formula II-2 to obtain the compound of formula III-1, ##STR00062## wherein R.sub.1, R.sub.2, R.sub.3, R.sub.4, R.sub.5, R.sub.6, R.sub.7, R.sub.8, R.sub.9, R.sub.10, R.sub.11, R.sub.12, A, L.sub.2, and Y are the same as defined in claim 1.

10. Use of a fluorescence-magnetic resonance dual-modality contrast agent according to claim 1 for medical magnetic resonance-enhanced imaging, determination of liver function, assisting in preoperative planning, intraoperative fluorescence navigation, prediction of fluorescence distribution of viscera by magnetic resonance images, determination of liver function, determination of renal function, monitoring of the vivo circulation as well as labeling of cells, and markers for in vitro cellular analysis.

11. The dual-modality contrast agent according to claim 1, wherein A is N or O.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0040] FIG. 1(A) is the compound PL-001 in mice magnetic resonance T1 enhancement imaging;

[0041] FIG. 1(B) is the bar chart of the mean signal values of T1-enhanced liver and kidney magnetic resonance images;

[0042] FIG. 1(C) is the liver and kidney fluorescence imaging images after 6 h of PL-001 administration;

[0043] FIG. 1(D) is the liver and kidney fluorescence efficiency values bar chart.

[0044] FIG. 2 is the results of flow cytometry to measure the rate of contrast agent entry in H22 cells.

[0045] FIG. 3 is cross-sectional images of magnetic resonance T1-enhanced imaging of mice liver in situ tumors before and after contrast agent injection, with tumor tissue in the dashed line.

[0046] FIG. 4 is the results of fluorescence imaging of major tissues in mice with liver in situ tumors.

[0047] FIG. 5 is the PL-003 HPLC purity chromatogram.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0048] In order to better understand the technical solution of the present invention, the present invention is further illustrated by following embodiments. The embodiments are intended to facilitate understanding of the present invention and should not be deemed as a limitation to the present invention.

Embodiment 1: Preparation of Compound PL-001

[0049] ##STR00022##

[0050] The synthesis scheme is as follows:

##STR00023## ##STR00024## ##STR00025##

Reaction Steps:

[0051] 1. IR-820 (new indocyanine green) and 3,6,9-trioxaundecamethylenediamine (1:1-1:5 eq.) were added to the DMSO solution. And then the triethylamine as the deacid reagent was added to the mixture. The mixture reacted at 20-80? C. for 2 h. The desired product B01 was obtained after precipitation. [0052] 2. B01 and DOTA-NHS (NBS is N-hydroxysuccinimide) (1:1-1:3 eq.) were dissolved in DMSO solution, and 1-20 equivalents of triethylamine was added to the mixture. The mixture reacted at 10-40? C. for 24 h. The desired product B02 was obtained after precipitation. [0053] 3. B02 was reacted with 0.5-2 equivalents of gadolinium chloride hydrate in DMSO at 10-50? C. for 10 h-50 h, and the desired product PL-001 was obtained after precipitation, yield of 83%, purity of 91%. [0054] 4. The final product was dissolved in pure water to create a solution. The product was administered to ICR mice by tail intravenous injection at doses of 5 mg/kg and 30 mg/kg, respectively. Magnetic resonance T1-enhanced imaging (SE sequence) was performed on the liver and kidney of the mice before and after the administration of the product. After the magnetic resonance imaging was completed, the livers and kidneys were removed for fluorescence imaging. The obtained images were analyzed by ImageJ software, and the results are shown in FIG. 1: A is the compound PL-001 in mice magnetic resonance T1-enhanced imaging, with significant enhancement of liver and kidney; Figure B is the bar chart of the mean signal value of the T1-enhanced liver and kidney magnetic resonance image; Figure C is the fluorescence imaging image of the liver and kidney 6 hours after administration of PL-001, with significant fluorescence of the liver and kidney; and Figure D is the value of liver and kidney fluorescence efficiency bar chart. From the figure, it can be seen that the magnetic resonance enhancement and fluorescence imaging results of the liver and kidney were basically consistent.

Embodiment 2: Preparation of Compound PL-002

[0055] ##STR00026##

[0056] The synthesis scheme is as follows:

##STR00027## ##STR00028##

Reaction Steps:

[0057] 1. IR-820 and sodium p-oxide phenylpropionate (1:1-1:5 eq.) were dissolved in DMSO and reacted at 20-80? C. for 1-8 h. The desired intermediate C01 was obtained after precipitation. [0058] 2. C01 and dipyrrolidino(N-succinimidyloxy)carbenium hexafluorophosphate (1:05-1:3 eq.) were dissolved in DMSO, 1 equivalent of N, N-diisopropylethylamine was added, and the mixture reacted at 10-50? C. for 10-30 h. The desired intermediate C02 was obtained after precipitation. [0059] 3. 5,8,11-Trioxa-2-azatridecanoic,13-amino,1,1-dimethylethyl ester and DOTA-NHS (1:1-1:3 eq.) were dissolved in DMSO, 1-3 equivalents of triethylamine was added to the mixture. The reaction reacted at room temperature for 24 h. The desired intermediate D01 was obtained after precipitation. [0060] 4. D01 was added to a mixed trifluoroacetic acid and dichloromethane solution and reacted at 0-40? C. for 24 h. The desired intermediate D02 was obtained after ether precipitation. [0061] 5. D02 and gadolinium chloride hydrate (1:0.5-1:3 eq.) were dissolved in water and reacted at room temperature for 1-5 days, and the intermediate D03 was obtained after purification by column chromatography. [0062] 6. D03 and C02 (1:0.5-1:3 eq.) were dissolved in DMSO, 1-5 equivalents of N, N-diisopropylethylamine was added to the mixture. The reaction reacted at 0-50? C. for 8 h and the desired product PL-002 was obtained after purification by column chromatography, purity of 91.2%, yield of 55%.

Embodiment 3: Preparation of Compound PL-003

[0063] ##STR00029##

##STR00030## ##STR00031##

Reaction Steps:

[0064] N-Boc-1,10-diaminodecane and DOTA-NHS (1:1-1:3 eq.) were dissolved in DMSO, and 1-3 equivalents of triethylamine was added and reacted at room temperature for 24 h. The desired intermediate E01 was obtained after precipitation. [0065] D01 was added to a mixed solution of trifluoroacetic acid and dichloromethane and reacted at 0-40? C. for 24 h, and the desired intermediate E02 was obtained after ether precipitation. [0066] D02 and gadolinium chloride hydrate (1:0.5-1:3 eq.) were dissolved in water and reacted at room temperature for 1-5 days, and the intermediate E03 was obtained after purification by column chromatography. [0067] D03 and C02 (1:0.5-1:3 eq.) were dissolved in DMSO, and 1-5 equivalents of N, N-diisopropylethylamine was added, and the reaction reacted at 0-50? C. for 8 h. The desired product PL-003 was obtained after purification by column chromatography, purity of 96.3%, yield of 51%, and its HPLC chromatogram is shown in FIG. 5, and its corresponding data are shown in Table 1:

TABLE-US-00001 TABLE 1 number retention time peak area % peak area 1 27.09 8450 0.24 2 27.647 2744 0.08 3 27.698 2510 0.07 4 27.851 8510 0.24 5 28.021 13452 0.38 6 28.224 14853 0.42 7 28.4 20736 0.58 8 28.554 3438604 96.3 9 28.815 32122 0.9 10 29.108 6220 0.17 11 29.166 9161 0.26 12 29.304 3534 0.1 13 29.402 9883 0.28

[0068] The HPLC method was as follows: flow rate of 0.8 mL/min, injection volume of 10 ?L, detection wavelength of 254 nm, a chromatographic column of Xtimate C18 Welch with 250?4.6 mm?3 um, eluent A was a mixed solvent (pH 6.0) of 120 mM acetamide and 5 mM citric acid solution, eluent B was acetonitrile, and the diluent was methanol, the flow phase gradient was as follows:

TABLE-US-00002 times/min A % B % 0 100 0 4 100 0 8 80 20 13 80 20 16 70 30 21 70 30 26 30 70 29 30 70 33 100 0 38 100 0

Embodiment 4: Preparation of Compound PL-004

[0069] ##STR00032##

##STR00033## ##STR00034##

Reaction Steps:

[0070] N-Boc-2,2-(ethylenedioxy)bis(ethylamine) and DOTA-NH (1:1-1:3 eq.) were dissolved in DMSO, 1-3 equivalents of triethylamine was added and reacted at room temperature for 24 h. The desired intermediate F01 was obtained after precipitation; [0071] F01 was added to the mixed solution of trifluoroacetic acid and dichloromethane, reacted at 0-40? C. for 24 h, and the desired intermediate F02 was obtained after ether precipitation; [0072] F02 and gadolinium chloride hydrate (1:0.5-1:3 eq.) were dissolved in water and reacted at room temperature for 1-5 days, and the intermediate F03 was obtained after purification by column chromatography. [0073] F03 and C02 (1:0.5-1:3 eq.) were dissolved in DMSO, 1-5 equivalents of N, N-diisopropylethylamine was added, reacted at 0-50? C. for 8 h, and the desired product PL-003 was obtained, purity of 93.3%, yield of 43%.

Embodiment 5: Preparation of Compound PL-005

[0074] ##STR00035##

[0075] The synthesis scheme is as follows:

##STR00036## ##STR00037##

Reaction Steps:

[0076] N1-Boc-N4-N9-Dimethylspermine and DOTA-NHS (1:1-1:3eq.) were dissolved in DMSO, 1-3 equivalents of triethylamine was added, reacted at room temperature for 24 h, and the desired intermediate J01 was obtained after precipitation; [0077] J01 was added to the mixed solution of trifluoroacetic acid and dichloromethane, reacted at 0-40? C. for 24 h, and the desired intermediate J02 was obtained after ether precipitation. [0078] J02 and gadolinium chloride hydrate (1:0.5-1:3 eq.) were dissolved in water and reacted at room temperature for 1-5 days, and the intermediate J03 was obtained after purification by column chromatography; [0079] J03 and C02 (1:0.5-1:3 eq.) were dissolved in DMSO, 1-5 equivalents of N, N-diisopropylethylamine was added, reacted at 0-50? C. for 8 h, and the desired product PL-005 was obtained after purification by column chromatography; The compounds obtained in Examples 6-13 in Table 2 below were prepared according to the method of Embodiment 2 after replacing the reaction intermediates.

TABLE-US-00003 TABLE 2 Embodiments Compounds Purity Yield 6 [00038] 92.1% 43.1% 7 [00039] 88.5% 55.3% 8 [00040] 92.2% 60.9% 9 [00041] 82.1% 45.3% 10 [00042] 86.7% 65.2% 11 [00043] 92.8% 43.7% 12 [00044] 88.3% 49.8% [00045]

[0080] After preparation of PL-005, PL-008, and PL-013, it was found that this compound was easily soluble in highly polar organic solvents (methanol, N, N-dimethylformamide, dimethyl sulfoxide, etc.), and difficult to soluble in water (<0.1 mg/mL).

Embodiment 13

[0081] The longitudinal relaxation rate (r1) of the contrast agent was determined in a 0.35T small nuclear magnetic resonance imaging system using an IR reaction sequence. The relaxation rates of the contrast agent in pure water and in bovine serum protein solution (1%, w/w) in different solvents are shown in Table 3 below.

TABLE-US-00004 TABLE 3 r.sub.1 in BSA r.sub.1 in Water solution(1%) (mM.sup.?1s.sup.?1) (mM.sup.?1s.sup.?1) PL-002 11.8 16.2 PL-003 15.4 18.9 PL-004 14.7 17.6 PL-006 14.9 17.5 PL-007 15.2 18.6 PL-009 35.5 19.1 PL-010 34.7 17.9 PL-011 14.3 18.5 PL-012 10.9 14.6

Embodiment 14

[0082] The aqueous solution of the contrast agent (0.1 mg/mL) was left at room temperature and its stability was measured by calibrating its relative content by the HPLC method at different time points, and the stability of the contrast agent is shown in Table 4.

TABLE-US-00005 TABLE 4 PL-001 PL-002 PL-003 PL-004 PL-006 decomposition rate % 0 h 0 0 0 0 0 2 h 5.9 <LOQ <LOQ <LOQ <LOQ 24 h 40.0 <LOQ <LOQ <LOQ <LOQ 48 h 65.4 <LOQ <LOQ <LOQ <LOQ PL-007 PL-009 PL-0010 PL-0011 PL-0012 decomposition rate % 0 h 0 0 0 0 0 2 h <LOQ <LOQ <LOQ <LOQ <LOQ 24 h <LOQ <LOQ 1.5 4.7 <LOQ 48 h <LOQ <LOQ 5.4 1.0.5 <LOQ

Embodiment 15

[0083] The maximum tolerated dose (MTD) of the contrast agent was preliminarily determined by administration to mice by tail intravenous injection. Four ICR female mice (8 weeks old) per group were selected for tail intravenous injection at doses of 25, 50, 75, 100, 125, and 150 mg/kg and were continuously observed for a week, and the results are shown in Table 5.

TABLE-US-00006 TABLE 5 MTD mg/kg ICG 60* PL-001 75 PL-002 125 PL-003 150 PL-004 125 PL-006 100 PL-007 150 PL-009 100 PL-010 125 PL-011 75 PL-012 150

Embodiment 16

[0084] The cell entry rate of the contrast agent at the cell level was determined by flow cytometers. Logarithmically grown murine hepatocellular carcinoma cells H22 were collected and spread evenly in 12-well plates, with an approximate cell number of 1.5?105 per well. After wall attachment was complete for 24 h, PBS, PL-002, PL-003, PL-004, and PL-006 were added to each well, respectively, and the cells were trypsin-digested after a certain time of incubation and washed twice with PBS, and then finally resuspended in 0.5 mL of PBS solution, and their cell entry was detected by flow cytometry. The results are shown in FIG. 2, PL-003 has the fastest cellular entry.

Embodiment 17

[0085] Magnetic resonance imaging of liver in situ tumors with a contrast agent was performed using a GE 3.0T magnetic resonance imaging system. H22 cells were inoculated in Balb/c mice through liver in-situ injection, and liver conditions were observed through magnetic resonance. After the tumor was formed, 1 mg/mL contrast agent solution (5 mL/kg) was injected through the tail intravenous, and the liver was subjected to magnetic resonance T1-enhanced imaging at different time points after the administration of the agent. The results are shown in FIG. 3. The imaging effect of the tumor site can be significantly enhanced after the injection of the contrast agent, and PL-003 has a better enhancement effect.

Embodiment 18

[0086] Liver in situ tumor mice tissue fluorescence was observed by a small animal live imaging analyzer (Perkin Elmer). After magnetic resonance imaging, the major organs of the mice were removed and observed by live imaging, and the results are shown in FIG. 4. All of the contrast agents have more obvious fluorescence in the liver, and the main fluorescence range coincides with the magnetic resonance imaging results, in which the PL-003 tumor is distinguished from the normal tissue and has better fluorescence intensity. This figure indicates that the contrast agent of the present invention can effectively improve the distinction between normal tissue and tumor tissue in imaging, which is helpful in assisting preoperative planning and intraoperative fluorescence navigation.