CN109438265B - Compound with affinity with brown adipose tissue and preparation method and application thereof - Google Patents

Compound with affinity with brown adipose tissue and preparation method and application thereof Download PDF

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CN109438265B
CN109438265B CN201811485989.4A CN201811485989A CN109438265B CN 109438265 B CN109438265 B CN 109438265B CN 201811485989 A CN201811485989 A CN 201811485989A CN 109438265 B CN109438265 B CN 109438265B
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程妍
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Abstract

The invention provides a compound with affinity with brown adipose tissues, belonging to the field of specific molecular recognition diagnostic reagents. The invention provides a compound with affinity with brown adipose tissue, which has the structure shown in formula I, has the capability of targeting the brown adipose tissue in vivo, can be directly used as a fluorescent probe for detecting the brown adipose tissue in vivo or in a tissue sample, and can be applied to the early diagnosis, the curative effect monitoring and the research of treatment drugs of metabolic diseases such as obesity and the like.
Figure DDA0001894498840000011

Description

Compound with affinity with brown adipose tissue and preparation method and application thereof
Technical Field
The invention belongs to the technical field of specific molecular recognition diagnostic reagents, and particularly relates to a compound with affinity with brown adipose tissues, and a preparation method and application thereof.
Background
With the development of economy and the improvement of living standard, obesity and obesity-related metabolic diseases are increased correspondingly, including type 2 diabetes, hypertension, lipid metabolism disorder and the like, and the cardiovascular and cerebrovascular diseases are increased remarkably, so that the life quality of patients is seriously affected, and the heavy economic burden is brought to families and society of the patients. Therefore, the development of effective detection means and diagnostic drugs is extremely important.
In addition to the common white fat (WAT) used for energy storage, fat exists in the normal body in the form of Brown fat (BAT) that promotes energy expenditure. Brown fat is found in most newborns and infants and gradually fades away with age. In recent years, researches prove that a small amount of brown adipose tissues still exist in adults, are scattered and distributed in the areas of head and neck, clavicle, thoracic duct, thoracic mediastinum and the like, can be converted into brown adipose tissues from white adipose tissues under the conditions of body cooling and the like, and play an important role in the energy metabolism balance of organisms. Therefore, the increase of the amount of brown fat or the activity thereof can reduce white fat accumulation, increase energy consumption, reduce body weight, and improve metabolism, thereby effectively improving metabolic disorders such as obesity, hyperlipidemia, and insulin resistance. Therefore, brown adipose tissue has become a new target for metabolic diseases such as obesity and metabolic disorders of the body. The targeted probe and the molecular imaging technology are utilized to carry out the imaging of the brown adipose tissue, so that noninvasive and real-time in-vivo tracing and detection of the brown adipose tissue can be realized, and great convenience is further provided for the research of early diagnosis, curative effect monitoring, therapeutic drugs and the like.
At present, the clinical application is mainly18F-FDG for PET/CT for scanning of brown fat, however18F-FDG is a non-specific imaging agent. Therefore, the development of an imaging agent (molecular probe) targeting brown adipose tissues has very important scientific significance and practical value.
Disclosure of Invention
In view of the above, the present invention aims to provide a compound having affinity with brown adipose tissue, and a preparation method and applications thereof. The compound provided by the invention has affinity with brown adipose tissues, and can be directly used as a fluorescent probe for detecting the brown adipose tissues in vivo or in a tissue sample.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a compound with affinity with brown adipose tissues, which has a structure shown in a formula I:
Figure BDA0001894498820000021
in the formula I, X is
Figure BDA0001894498820000022
Or
Figure BDA0001894498820000023
Y is
Figure BDA0001894498820000024
Or
Figure BDA0001894498820000025
Z is
Figure BDA0001894498820000026
Or
Figure BDA0001894498820000027
1, 3, 2, 5 and 6 in said X represent a linking site, and 1 ', 6', 2 'and 5' in Y and Z represent a linking site;
m in the formula I is 1, 2 or 3; n is 1, 2 or 3;
in the formula I, R1And R2Independently H,18F、19F、123I、131I、OH、OCH3、N(CH3)2、NH2、(OCH2CH2)p 19F、(OCH2CH2)p 18F、NH-NODAGA-99mTc、NH-DOTA-68Ga、NH-NODAGA-68Ga、NH-DOTA-64Cu or NH-NODAGA-64Cu and p are 1, 2, 3, 4 or 5.
Preferably, the compound has a structure represented by formulas (1) to (4):
Figure BDA0001894498820000028
preferably, the compound has a structure represented by formulas (5) to (10):
Figure BDA0001894498820000029
Figure BDA0001894498820000031
the invention also provides a preparation method of the compound in the technical scheme,
when said R is1And R2Independently H,18F、19F、123I、131I、OH、OCH3、N(CH3)2Or NH2The preparation method of the compound comprises the following steps:
mixing ketone substances, a compound with a structure shown in a formula II, a compound with a structure shown in a formula III, ethanol and a potassium hydroxide aqueous solution for condensation reaction to obtain a compound with a structure shown in a formula I;
Figure BDA0001894498820000032
the ketone substance is acetone, cyclopentanone or cyclohexanone;
wherein Y is
Figure BDA0001894498820000041
Or
Figure BDA0001894498820000042
Z is
Figure BDA0001894498820000043
Or
Figure BDA0001894498820000044
1 ', 6', 2 'and 5' in said Y and Z represent attachment sites;
m is 1, 2 or 3; n is 1, 2 or 3;
when the compound has the structure of formula (5), the preparation method comprises the following steps:
mixing a compound with a structure shown in a formula IV, a compound with a structure shown in a formula V, N-dimethylformamide and anhydrous potassium carbonate to carry out a first substitution reaction to obtain a compound with a structure shown in a formula VI;
Figure BDA0001894498820000045
wherein m is 1, 2 or 3, n is 1, 2 or 3, and q is 0, 1, 2, 3 or 4;
mixing a compound with a structure shown in a formula VI, 4-tosyl chloride and pyridine for a second substitution reaction to obtain a compound with a structure shown in a formula VII;
Figure BDA0001894498820000046
wherein m is 1, 2 or 3, n is 1, 2 or 3, and p is 1, 2, 3, 4 or 5;
mixing a compound with a structure shown as a formula VII, anhydrous acetonitrile and K222/K2CO3Is/are as follows18F-Carrying out fluoridation marking on the marker to obtain a compound with a structure shown in a formula (5);
when the compound has the structure from formula (6) to formula (10), the preparation method comprises the following steps:
carrying out chelation reaction on a compound with a structure shown in a formula VIII, a bifunctional ligand and acetonitrile to obtain a compound with a structure shown in a formula IX, wherein the bifunctional ligand is DOTA-NHS or NODAGA-NHS;
Figure BDA0001894498820000047
the structure of the compound having the structure shown in formula IX is shown in formula IX-1 or IX-2:
Figure BDA0001894498820000051
subjecting a compound having a structure represented by formula IX, an organic solvent, and a labeling substance containing a radioactive ion, which is a compound having a structure represented by formula (6) to formula (10), to radioactive ion labeling68Ga、99mTc or64Cu。
Preferably, the molar ratio of the ketone substance to the compound having the structure shown in formula II to the compound having the structure shown in formula III is 1:1: 1.
Preferably, the temperature of the condensation reaction is 20-100 ℃, and the time of the condensation reaction is 10 min-18 h.
Preferably, the temperature of the fluoridation marking is 90-150 ℃, and the time of the radioactive ion marking is 5 min-3 h.
Preferably, the temperature of the radioactive ion label is 20-120 ℃, and the time of the radioactive ion label is 5 min-5 h.
The invention also provides application of the compound in the technical scheme or the compound prepared by the preparation method in the technical scheme in preparation of a medicine for diagnosing metabolic diseases, wherein the medicine for diagnosing metabolic diseases is a nuclear medicine imaging agent or an optical imaging agent for imaging brown adipose tissues.
The invention also provides a composition for imaging brown adipose tissues, which comprises an effective ingredient and a pharmaceutically acceptable carrier, wherein the effective ingredient is the compound in the technical scheme or the compound prepared by the preparation method in the technical scheme.
The invention provides a compound with affinity with brown adipose tissue, which has the structure shown in formula I, has the capability of targeting the brown adipose tissue in vivo, can be directly used as a fluorescent probe for detecting the brown adipose tissue in vivo or in a tissue sample, and can be applied to the early diagnosis, the curative effect monitoring and the research of treatment drugs of metabolic diseases such as obesity and the like.
Drawings
FIG. 1 is a photograph obtained in example 818HPLC profile of F labeled ligand;
FIG. 2 is a graph showing the appearance of IVIS Lumina Series IIIimaging system in mice sacrificed 1 hour after injection of the compound of example 2;
FIG. 3 is a photograph showing the IVIS lumineSeries III imaging system after digestion of the scapular brown fat of the compound-injected mice of example 2 at 37 ℃ for 2 h;
FIG. 4 is a photograph showing the IVIS Lumina Series IIIimaging system after the compound of example 2 is injected into mice via tail vein;
FIG. 5 is a photograph showing the IVIS Lumina Series III imaging system at the scapular region of a mouse sacrificed 1 hour after injection of the compound of example 2.
Detailed Description
The invention provides a compound with affinity with brown adipose tissues, which has a structure shown in a formula I:
Figure BDA0001894498820000061
in the formula I, X is
Figure BDA0001894498820000062
Or
Figure BDA0001894498820000063
Y is
Figure BDA0001894498820000064
Or
Figure BDA0001894498820000065
Z is
Figure BDA0001894498820000066
Or
Figure BDA0001894498820000067
1, 3, 2, 5 and 6 in said X represent a linking site, and 1 ', 6', 2 'and 5' in Y and Z represent a linking site;
m in the formula I is 1, 2 or 3; n is 1, 2 or 3;
in the formula I, R1And R2Independently H,18F、19F、123I、131I、OH、OCH3、N(CH3)2、NH2、(OCH2CH2)p 19F、(OCH2CH2)p 18F、NH-NODAGA-99mTc、NH-DOTA-68Ga、NH-NODAGA-68Ga、NH-DOTA-64Cu or NH-NODAGA-64Cu and p are 1, 2, 3, 4 or 5.
In the present invention, the structure of the compound is preferably as shown in formulas (1) to (4):
Figure BDA0001894498820000068
Figure BDA0001894498820000071
in the present invention, the structure of the compound is preferably represented by formulas (5) to (10):
Figure BDA0001894498820000072
the invention also provides a preparation method of the compound in the technical scheme,
when said R is1And R2Independently H,18F、19F、123I、131I、OH、OCH3、N(CH3)2Or NH2The preparation method of the compound comprises the following steps:
mixing ketone substances, a compound with a structure shown in a formula II, a compound with a structure shown in a formula III, ethanol and a potassium hydroxide aqueous solution for condensation reaction to obtain a compound with a structure shown in a formula I;
Figure BDA0001894498820000081
the ketone substance is acetone, cyclopentanone or cyclohexanone;
wherein Y is
Figure BDA0001894498820000082
Or
Figure BDA0001894498820000083
Z is
Figure BDA0001894498820000084
Or
Figure BDA0001894498820000085
1 ', 6', 2 'and 5' in said Y and Z represent attachment sites;
m is 1, 2 or 3; n is 1, 2 or 3;
when the compound has the structure of formula (5), the preparation method comprises the following steps:
mixing a compound with a structure shown in a formula IV, a compound with a structure shown in a formula V, N-dimethylformamide and anhydrous potassium carbonate to carry out a first substitution reaction to obtain a compound with a structure shown in a formula VI;
Figure BDA0001894498820000086
wherein m is 1, 2 or 3, n is 1, 2 or 3, and q is 0, 1, 2, 3 or 4;
mixing a compound with a structure shown in a formula VI, 4-tosyl chloride and pyridine for a second substitution reaction to obtain a compound with a structure shown in a formula VII;
Figure BDA0001894498820000087
wherein m is 1, 2 or 3, n is 1, 2 or 3, and p is 1, 2, 3, 4 or 5;
mixing a compound with a structure shown as a formula VII, anhydrous acetonitrile and K222/K2CO3Is/are as follows18F-Carrying out fluoridation marking on the marker to obtain a compound with a structure shown in a formula (5);
when the compound has the structure from formula (6) to formula (10), the preparation method comprises the following steps:
carrying out chelation reaction on a compound with a structure shown in a formula VIII, a bifunctional ligand and acetonitrile to obtain a compound with a structure shown in a formula IX, wherein the bifunctional ligand is DOTA-NHS or NODAGA-NHS;
Figure BDA0001894498820000091
the structure of the compound having the structure shown in formula IX is shown in formula IX-1 or IX-2:
Figure BDA0001894498820000092
subjecting a compound having a structure represented by formula IX, an organic solvent, and a labeling substance containing a radioactive ion, which is a compound having a structure represented by formula (6) to formula (10), to radioactive ion labeling68Ga、99mTc or64Cu。
In the present invention, when said R is1And R2Independently H,18F、19F、123I、131I、OH、OCH3、N(CH3)2Or NH2The preparation method of the compound comprises the following steps:
mixing ketone substances, a compound with a structure shown in a formula II, a compound with a structure shown in a formula III, ethanol and a potassium hydroxide aqueous solution for condensation reaction to obtain a compound with a structure shown in a formula I;
Figure BDA0001894498820000093
the ketone substance is acetone, cyclopentanone or cyclohexanone;
wherein Y is
Figure BDA0001894498820000094
Or
Figure BDA0001894498820000095
Z is
Figure BDA0001894498820000096
Or
Figure BDA0001894498820000097
1 ', 6', 2 'and 5' in said Y and Z represent attachment sites;
m is 1, 2 or 3; n is 1, 2 or 3.
In the present invention, the molar ratio of the ketone, the compound having the structure represented by formula II, and the compound having the structure represented by formula III is preferably 1:1: 1.
In the invention, the temperature of the condensation reaction is preferably 20-100 ℃, and the time of the condensation reaction is preferably 10 min-18 h.
The amount of the ethanol or the aqueous solution of potassium hydroxide to be used and the concentration of the aqueous solution of potassium hydroxide are not particularly limited.
The source of the compound having the structure shown in formula II and the compound having the structure shown in formula III is not particularly limited in the present invention, and the compounds can be prepared by using commercially available products well known to those skilled in the art or by using preparation methods well known to those skilled in the art.
In the present invention, when the compound has the structure of formula (5), the preparation method comprises the steps of:
mixing a compound with a structure shown in a formula IV, a compound with a structure shown in a formula V, N-dimethylformamide and anhydrous potassium carbonate to carry out a first substitution reaction to obtain a compound with a structure shown in a formula VI;
Figure BDA0001894498820000101
wherein m is 1, 2 or 3, n is 1, 2 or 3, and q is 0, 1, 2, 3 or 4;
mixing the compound with the structure shown in the formula VI, 4-tosyl chloride and pyridine for a second substitution reaction to obtain a compound with the structure shown in the formula VII;
Figure BDA0001894498820000102
wherein m is 1, 2 or 3, n is 1, 2 or 3, and p is 1, 2, 3, 4 or 5;
the compound with the structure shown in the formula VII, anhydrous acetonitrile and K-containing222/K2CO3Is/are as follows18F-The label is subjected to fluoridation to obtain the compound with the structure shown in the formula (5).
Mixing a compound with a structure shown in a formula IV, a compound with a structure shown in a formula V, N-dimethylformamide and anhydrous potassium carbonate to carry out a first substitution reaction to obtain a compound with a structure shown in a formula VI;
Figure BDA0001894498820000111
wherein m is 1, 2 or 3, n is 1, 2 or 3, and q is 0, 1, 2, 3 or 4.
In the invention, the compound with the structure shown in the formula IV is obtained by mixing a ketone substance, a compound with the structure shown in the formula II, a compound with the structure shown in the formula III, ethanol and a potassium hydroxide aqueous solution for condensation reaction. The restriction of the condensation reaction, ethanol and potassium hydroxide aqueous solution in the invention is consistent with the technical scheme, and the details are not repeated.
The amounts of the N, N-dimethylformamide and anhydrous potassium carbonate used in the present invention are not particularly limited.
In the present invention, the temperature of the first substitution reaction is preferably 90 to 120 ℃.
In the present invention, the first substitution reaction is preferably carried out under stirring conditions.
After the first substitution reaction is completed, the first substitution reaction product is preferably subjected to column chromatography purification to obtain the compound with the structure shown in the formula VI. The present invention preferably employs TLC to detect the end of the first substitution reaction. The specific method for the column chromatography purification is not particularly limited in the present invention.
After the compound with the structure shown in the formula VI is obtained, the compound with the structure shown in the formula VI, 4-tosyl chloride and pyridine are mixed for a second substitution reaction to obtain a compound with the structure shown in the formula VII;
Figure BDA0001894498820000112
wherein m is 1, 2 or 3, n is 1, 2 or 3, and p is 1, 2, 3, 4 or 5.
In the invention, the molar ratio of the compound with the structure shown in the formula VI to the 4-tosyl chloride is preferably 1: 1-2. The amount of the pyridine is not particularly limited, and the compound having the structure shown in formula VI and the 4-tosyl chloride can be uniformly mixed.
In the present invention, the temperature of the second substitution reaction is preferably room temperature to 30 ℃.
In the present invention, the second substitution reaction is preferably carried out under stirring conditions.
After the second substitution reaction is completed, the second substitution reaction product is preferably subjected to column chromatography purification to obtain the compound with the structure shown in the formula VII. The present invention preferably employs TLC to detect the end of the secondary substitution reaction.
After the compound with the structure shown in the formula VII is obtained, the compound with the structure shown in the formula VII, anhydrous acetonitrile and K-containing222/K2CO3Is/are as follows18F-The label is subjected to fluoridation to obtain the compound with the structure shown in the formula (5).
In the invention, the temperature of the fluoridation label is preferably 90-150 ℃, more preferably 100-120 ℃, and the time of the radioactive ion label is preferably 5-30 min, more preferably 10-20 min.
In the present invention, the compound having the structure represented by the formula VII and18F-in the marker K222/K2CO3The mass ratio of (A) to (B) is preferably 1-5: 5 to 30. The invention is directed to said K222/K2CO3The source of (A) is not particularly limited, and commercially available products known to those skilled in the art may be used.
In the present invention, the18F-In the marker18F-The concentration of (B) is preferably 5 to 50 mCi.
After the fluorinated labeling is completed, the present invention preferably passes the fluorinated labeled reaction product through C18The reverse phase column is washed with water to remove the rest18F-And then eluting with acetonitrile, drying by nitrogen, and separating by HPLC to obtain the compound with the structure shown in the formula (5).
In the present invention, when the compound has the structures of formulae (6) to (10), the preparation method comprises the following steps:
carrying out chelation reaction on a compound with a structure shown in a formula VIII, a bifunctional ligand and acetonitrile to obtain a compound with a structure shown in a formula IX, wherein the bifunctional ligand is DOTA-NHS or NODAGA-NHS;
Figure BDA0001894498820000121
the structure of the compound having the structure shown in formula IX is shown in formula IX-1 or IX-2:
Figure BDA0001894498820000122
subjecting a compound having a structure represented by formula IX, an organic solvent, and a labeling substance containing a radioactive ion, which is a compound having a structure represented by formula (6) to formula (10), to radioactive ion labeling68Ga、99mTc or64Cu。
In the present invention, the organic solvent is preferably acetonitrile, ethanol, DMSO, or methanol.
Carrying out a chelation reaction on a compound with a structure shown in a formula VIII, a bifunctional ligand and acetonitrile to obtain a compound with a structure shown in a formula IX, wherein the bifunctional ligand is DOTA-NHS or NODAGA-NHS;
Figure BDA0001894498820000131
the structure of the compound having the structure shown in formula IX is shown in formula IX-1 or IX-2:
Figure BDA0001894498820000132
the source of the bifunctional ligand is not particularly limited in the present invention, and commercially available products well known to those skilled in the art may be used.
In the present invention, the molar ratio of the compound having the structure represented by formula VIII to the bifunctional ligand is preferably 1: 5. The amount of acetonitrile used in the present invention is not particularly limited, and the compound having the structure represented by formula VIII and the bifunctional ligand can be dissolved.
In the invention, the temperature of the chelation reaction is preferably room temperature, no additional heating or cooling is needed, and the time of the chelation reaction is preferably 1-5 h.
After the chelation reaction is finished, the invention preferably performs column chromatography purification on the chelation reaction product to obtain the compound with the structure shown in the formula VIII.
After obtaining the compound having the structure represented by the formula IX, the present invention provides compounds having the structures represented by the formulae (6) to (10) by subjecting a compound having the structure represented by the formula IX, acetonitrile and a label containing a radioactive ion of 68Ga, 99mTc or 64Cu to radioactive ion labeling.
In the invention, the temperature of the radioactive ion label is preferably 20-120 ℃, and the time of the radioactive ion label is preferably 5 min-5 h.
In the present invention, the concentration of the label containing radioactive ions is preferably 1 to 10 mCi.
After the radioactive ion labeling is finished, the present invention preferably passes the radioactive ion labeling reaction product through a C18 reversed phase column, water is used for washing to remove the residual radioactive ions, then ethanol is used for rinsing, after nitrogen drying, a C18 solid phase small column or HPLC is used for separating to obtain the compounds with the structures shown in the formulas (6) to (10).
The invention also provides application of the compound in the technical scheme or the compound prepared by the preparation method in the technical scheme in preparation of a medicine for diagnosing metabolic diseases, wherein the medicine for diagnosing metabolic diseases is a nuclear medicine imaging agent or an optical imaging agent for imaging brown adipose tissues.
In the present invention, the metabolic disease to which the diagnostic metabolic disease drug is directed is preferably diabetes, hyperlipidemia, metabolic disorder, or obesity.
The invention also provides a compound in the technical scheme or a derivative of the compound prepared by the preparation method in the technical scheme. In the present invention, the derivative is preferably a pharmaceutically acceptable salt, ester, amide compound or prodrug.
The invention also provides a composition for imaging brown adipose tissues, which comprises an effective ingredient and a pharmaceutically acceptable carrier, wherein the effective ingredient is the compound in the technical scheme or the compound prepared by the preparation method in the technical scheme.
In the present invention, the pharmaceutically acceptable carrier preferably includes an excipient and a diluent.
In the present invention, the formulation form of the pharmaceutically acceptable carrier includes a liquid. In the present invention, the liquid includes water, physiological saline, glycerol or ethanol.
The following examples are provided to illustrate the compounds having affinity for brown adipose tissue and the preparation and use thereof in detail, but they should not be construed as limiting the scope of the present invention.
Example 1
Dissolving p-dimethylaminocinnamaldehyde (350mg, 2mmol) and acetone (58mg, 1mmol) in ethanol, adding 10% KOH (6mL) dropwise, heating and refluxing for condensation reaction, monitoring the progress of the reaction by TLC, cooling to precipitate crystals, and filtering to obtain a product (150mg, 40.2%) with the structure shown as the following formula:
Figure BDA0001894498820000141
1H NMR(CDCl3,600MHz)：δ7.504-7.441(m,2H),7.390-7.328(m,4H),6.918-6.879(m,2H),6.799-6.763(m,2H),6.679-6.659(m,4H),6.480-6.443(m,2H),3.007(s,12H).13CNMR(CDCl3,150MHz):δ189.0,143.8,142.0,127.6,127.0,122.7,112.2,40.2.HRMS(ESI)Calcd for C21H24N2O[M+H]+:373.2280；Found 373.2277.
example 2
Dissolving p-dimethylaminocinnamaldehyde (350mg, 2mmol) and cyclopentanone (98mg, 1mmol) in ethanol (5mL), adding 10% KOH (6mL) dropwise, heating and refluxing for condensation reaction, monitoring the reaction progress by TLC, and performing column chromatography on the obtained initial product by reduced pressure rotary evaporation to obtain a product (70mg, 17.6%) with the structure shown in the following formula:
Figure BDA0001894498820000151
1H NMR(CDCl3,600MHz)：δ7.406-7.392(m,4H),7.251-7.232(d,2H,J＝11.4Hz),6.913-6.887(d,2H,J＝15.6Hz),6.795-6.750(dd,2H,J＝11.4Hz,J＝11.4Hz),6.686-6.672(m,4H),3.013(s,12H),2.861(s,4H).13C NMR(CDCl3,150MHz):δ194.6,150.8,141.7,137.8,133.4,128.7,124.9,120.6,112.0,40.2,24.0.HRMS(ES+)m/z:[M+H]+Calcd forC27H30N2O 399.2436；Found 399.2436.
example 3
Dissolving p-dimethylamino cinnamaldehyde (350mg, 2mmol) and cyclohexanone (98mg, 1mmol) in ethanol (5mL), dropwise adding 10% KOH (6mL), and heating and refluxing for condensation reaction; the progress of the reaction was monitored by TLC and the crude product obtained by rotary evaporation under reduced pressure was purified by column chromatography to give the product (150mg, 36.4%) which has the following structure:
Figure BDA0001894498820000152
1H NMR(CDCl3,600MHz)：δ7.490-7.474(m,2H),7.397-7.381(m,4H),6.890(s,4H),6.672-6.652(m,4H),2.997(s,12H),2.750(s,4H),1.845(s,2H).13C NMR(CDCl3,150MHz):δ188.6,150.8,141.3,137.2,133.1,128.6,125.84,119.6,112.1,40.2,26.6,22.2.HRMS(ES+)m/z:[M+H]+Calcd for C28H32N2O 413.2593；Found 413.2594.
example 4
Dissolving p-dimethylaminocinnamaldehyde (175mg, 1mmol) and cyclopentanone (98mg, 1mmol) in ethanol (5mL), adding 10% KOH (3mL) dropwise, and heating under reflux for condensation reaction; the progress of the reaction was monitored by TLC and the crude product obtained by rotary evaporation under reduced pressure was purified by column chromatography to give the product (48.7mg, 20.2%), MS [ M + H ]242, of the formula:
Figure BDA0001894498820000161
example 5
P-hydroxycinnamaldehyde (74mg, 0.5mmol) and the compound (121mg, 0.5mmol) obtained in example 4 were dissolved in ethanol (5mL), 10% KOH (2mL) was added dropwise, and condensation reaction was performed by heating under reflux; the progress of the reaction was monitored by TLC and the crude product obtained by rotary evaporation under reduced pressure was purified by column chromatography to give the product (94.8mg, 51.2%), MS [ M + H ]372, of the formula:
Figure BDA0001894498820000162
example 6
2- [2- (2-chloroethoxy) ethoxy ] ethanol (84mg, 0.5mmol) and the compound obtained in example 5 (185mg, 0.5mmol) were dissolved in DMF (3mL), and anhydrous sodium carbonate (273mg, 1.98mmol) was added and the reaction was heated at 100 ℃ to react; the progress of the reaction was monitored by TLC and the crude product obtained by rotary evaporation under reduced pressure was purified by column chromatography to give the product (126mg, 49.6%), MS [ M + H ]504, of the formula:
Figure BDA0001894498820000163
example 7
P-toluenesulfonyl chloride (95mg, 0.5mmol) and the compound obtained in example 6 (250mg, 0.5mmol) were dissolved in pyridine (3mL) and reacted at room temperature; the progress of the reaction was monitored by TLC and the crude product obtained by rotary evaporation under reduced pressure was purified by column chromatography to give the product (83.6mg, 25.4%), MS [ M + H ]659, of the formula:
Figure BDA0001894498820000164
example 818Preparation of F-labelled ligand
10mCi of18F]F-Adding the solution into a glass reaction tube, adding 10 mg of Kryptofix2.2.2 and 2mg of potassium carbonate, continuously introducing nitrogen at 120 ℃ for drying, adding 1ml of acetonitrile for drying in three times, and ensuring that a reaction system does not existWater; 2mg of a labeling precursor (a compound having a structure represented by formula IV, wherein m ═ 1, n ═ 1) was dissolved in 500 μ l of anhydrous acetonitrile, and the solution was transferred to a solution containing [, [ solution ] of a compound having a structure represented by formula IV, and then the solution was dissolved in 500 μ l of anhydrous acetonitrile18F]F-The glass reaction tube (2) is heated and reacted for 5 minutes at the temperature of 100 ℃, the solution passes through a Sep-Pak solid phase extraction column after cooling, and is rinsed by 10mL of deionized water to remove the reacted solution18F]F-Sequentially eluting the labeled ligand and the labeled precursor adsorbed on the column by using anhydrous acetonitrile, concentrating the eluent, separating and purifying by HPLC, collecting the effluent of the target radioligand, and removing the solvent by rotary evaporation to obtain the compound with the structural formula shown in the following formula:
Figure BDA0001894498820000171
HPLC assay is shown in FIG. 1: column:5C 18-AR-II (4.6X 150mm), mobile phase acetonitrile: water (60:40)1mL/min,254nm, room temperature, radioligand peak at 16 min. As can be seen from the figure, the structural formula of the product prepared by the embodiment is shown as the formula.
In vitro fluorescence imaging
The compound of example 2 (1.0mg/kg, 45% DMSO and 55% propylene glycol) was injected via the tail vein into normal C57BL/6 mice (8 weeks, male, n ═ 3), and the mice were sacrificed 1 hour after injection, fat was removed from the scapular region, brown adipose tissue was isolated and placed in the IVIS luminea Series III imaging system for imaging, and the experimental results are shown in fig. 2, with fluorescence mainly concentrated in brown adipose tissue and no significant fluorescence continuum in white adipose tissue. After being placed in an Eppendorf tube and minced, the mixture was digested at 37 ℃ for 2 hours with the addition of Hanks' Balanced Salt Solution containing collagenic enzyme (2 mg/mL). Centrifuging at 400rpm for 15 min after digestion, and developing by placing in IVIS Lumina Series III imaging system; the results are shown in FIG. 3, in which A in FIG. 3 shows that BAT is digested with collagenase, brown cells are on the upper layer, and blood cells are on the bottom of the tube; b represents that the fluorescence is mainly concentrated on the upper layer (brown cells) of the test tube, while the fluorescence of the lower layer (blood cell layer) is weak, which indicates that the compound provided by the invention targets brown fat; c is a quantitative analysis of fluorescence of the layers, showing that fluorescence accumulation in brown cells is much higher than in blood cells.
Fluorescence imaging of living bodies
Norepinephrine (NE) can activate body brown adipose tissue. 1 hour after intraperitoneal injection of NE at different concentrations into C57BL/6 mice, the compound of example 2 (1.0mg/kg, 45% DMSO and 55% propylene glycol) was injected into normal C57BL/6 mice via tail vein and placed in an IVIS lumine Series III imaging system for imaging. Fluorescence intensity data were obtained by locating the region of interest at the scapula, and semi-quantitative data processing was performed using Living Image software. The experimental results are shown in fig. 4, in which a indicates that significant fluorescence enhancement is shown at the scapular region after stimulation with different concentrations of NE in fig. 4; b and C show that fluorescence quantitative analysis at the scapular region shows that the fluorescence intensity is in direct proportion to the quantity of NE, which indicates that the compound provided by the invention can be used for imaging and quantitatively monitoring brown adipose tissues in vivo.
Fluorescence imaging of isolated tissues and organs
The compound of example 2 (1.0mg/kg, 45% DMSO and 55% propylene glycol) was injected via tail vein into normal C57BL/6 mice (8 weeks, male, n ═ 3), and the mice were sacrificed 1 hour after injection, fat at the shoulder blades and various tissue organs were removed and placed in IVIS luminea Series III imaging system for imaging, data on fluorescence intensity were obtained, and semi-quantitative data processing was performed with Living Image software. The experimental result is shown in fig. 5, the fluorescence is mainly concentrated in the brown adipose tissue, and the white adipose tissue has no obvious secondary fluorescence, which indicates that the compound provided by the invention can be used for displaying the brown adipose tissue.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (1)

1. The application of a compound with affinity with brown adipose tissue in preparing a medicine for diagnosing metabolic diseases, wherein the medicine for diagnosing metabolic diseases is a nuclear medicine imaging agent or an optical imaging agent for imaging brown adipose tissue, and the structural formula of the compound with affinity with brown adipose tissue is shown as the following formula:
Figure DEST_PATH_IMAGE001
。
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