WO2019072231A1 - Magnetic resonance imaging compound, magnetic resonance imaging agent and application thereof as well as magnetic resonance imaging method - Google Patents

Abstract

The invention relates to a stapled peptide compound or a pharmaceutically acceptable salt thereof, a magnetic resonance imaging agent and application thereof as well as a magnetic resonance imaging method. The stapled peptide compound has a structure of formula (I), wherein R1, R2, R3, Ln and Xaa are as defined in the specification. The stapled peptide magnetic resonance imaging compound can penetrate cell membrane, enter cell nucleus, and bind to the estrogen receptor located in the cell nucleus to specifically and target-selectively performing intracellular imaging.

Description

Nuclear magnetic resonance imaging compound, nuclear magnetic resonance imaging agent and application, and magnetic resonance imaging method Technical field

The present invention relates to nuclear magnetic resonance imaging compounds, nuclear magnetic resonance imaging agents and applications, and nuclear magnetic resonance imaging methods.

Background technique

Estrogen can regulate the normal tissues and cell growth, replication, development, differentiation and other physiological processes, and also participate in the occurrence and development of a variety of tumors. Estrogen acts on cells mainly through two ER subtypes, estrogen receptor (ER) alpha and beta. Abnormal estrogen signaling can promote the development and progression of a variety of tumors. Estrogen and estrogen receptors are important factors affecting the biological behavior of various hormone-sensitive tumors such as breast cancer, endometrial cancer and pituitary adenoma, and are important drug targets for various tumors. Pituitary adenomas account for 10-15% of all brain tumors and are the second largest tumor in the brain. Non-functional adenomas (NFPA) are the most common subtype, lacking significant hormone-related clinical syndromes, often found in large adenomas or giant adenomas. The clinical manifestations are mostly tumor compression symptoms, which mainly impair the visual and endocrine functions of patients, seriously affecting the quality of life of patients, and causing a major disease burden to families and society. The current treatment is mainly surgery, and a considerable number of patients have poor surgical results. There are residual and recurrence after surgery, which is a problem that neurology is difficult to break through. Early use of expression profiling chips and proteomics confirmed that estrogen receptor-related pathways are critical pathways in nonfunctioning pituitary adenomas, and large-sample experiments using tissue microarray confirmed that more than 70% of pituitary adenoma patients have high expression of estrogen receptors. . Therefore, estrogen receptors are important targets for potential drug therapy for nonfunctioning pituitary adenomas. However, due to the special location of brain tumors, it is impossible to detect the expression of receptors by biopsy in other parts of the body to guide targeted drug therapy. Therefore, how to perform molecular imaging imaging in real time without noninvasiveness, and to understand the brain lesions in advance. The expression level of estrogen receptor is a key clinical problem that needs to be solved in the treatment of pituitary adenoma. However, in the prior art, it is not yet possible to accurately and non-invasively detect the distribution density of estrogen receptors in the brain.

CN106366075A discloses a benzamide methylpyrrolidine nuclear magnetic resonance imaging compound, and reports that the compound can specifically bind to the dopamine D ₂ receptor, and can specifically recognize the dopamine D ₂ receptor, and the dopamine D ₂ receptor The body has high expression in PRL type pituitary adenoma and can be applied to magnetic resonance detection or nuclear magnetic resonance detection of pituitary adenoma. However, such benzamide methylpyrrolidine nuclear magnetic resonance imaging compounds cannot penetrate the cell membrane and enter the nucleus for imaging.

Summary of the invention

The invention provides a novel book-binding peptide nuclear magnetic resonance imaging compound capable of penetrating cell membrane, entering the nucleus, and binding to an estrogen receptor localized in the nucleus, specifically targeting selective intracellular display image.

In one aspect, the invention provides a compound of formula (I), or a pharmaceutically acceptable salt thereof:

among them,

R _{1 is} selected from: an alkylene group;

R _{2 is} selected from the group consisting of hydrogen, an amino group, a group represented by the following formula (II), a group represented by the following formula (III), and a group represented by the following formula (IV);

Wherein X _{1 is} selected from the group consisting of: an alkylene group;

R _{3 is} selected from the group consisting of amino, alkanoylamino, carboxy, monoalkyl or dialkyl substituted amino groups (e.g., acylmethylamine, acylethylamine, acyl propylamine, isopropyl isopropylamine, acyl n-butylamine, acyl isobutylamine) , acyl tert-butylamine, acyl butylamine, etc.); optionally amino, alkanoylamino, carboxy, amidoamine, acylethylamine;

Here, R ₃ is an amino group, and represents an amino group which is condensed with a carboxyl group in a terminal serine residue to form an amide bond (ie, -(C=O)NH ₂ , wherein the -(C=O) moiety is derived from a terminal serine residue) R ₃ is an alkanoylamino group, and an alkanoylamino group in which an amino group is condensed with a carboxyl group in a terminal serine residue to form an amide bond, for example, when R ₃ is an acetylamino group, it represents -(C=O)NH(C= O) CH ₃ , wherein the left -(C=O) is derived from the carboxyl group of the terminal serine; R ₃ is a carboxyl group, which represents a carboxyl group contained in the terminal serine residue; and R ₃ is a monoalkyl or dialkyl substituted amino group, a monoalkyl or dialkyl substituted amino group in which an amino group is condensed with a carboxyl group in a terminal serine residue to form an amide bond, that is, -(C=O)NH-alkyl, or -(C=O)N a dialkyl group, wherein -(C=O) is derived from a terminal serine residue; R ₃ is a amide methylamine, ie (-(C=O)NHCH ₃ , wherein -(C=O) is derived from a terminal serine residue, R ₃ is acylethylamine, acyl n-propylamine, isopropyl isopropylamine, acyl n-butylamine, acylisobutylamine, acyl tert-butylamine, acyl butylamine, and the like, and so on.

Xaa is selected from the group consisting of: a glycine residue, an alanine residue, a β-aminopropionic acid residue, a γ-aminobutyric acid residue, a 6-aminocaproic acid residue, or a deletion;

Ln is selected from: Gd or Eu;

Glu is a glutamic acid residue, Lys is a lysine residue, His is a histidine residue, IIe is an isoleucine residue, Leu is a leucine residue, Arg is an arginine residue, Asp Is an aspartic acid residue, Ser is a serine residue, and S ₅ is a (S)-α-pentenylalanine residue;

The "alkylene group" is preferably a C ₁ -C ₁₀ linear or branched alkylene group, preferably a C ₁ -C ₈ linear or branched alkylene group, preferably selected from a methylene group. , ethylene, propylene, butylene, pentylene, hexylene; preferably, selected from the group consisting of methylene, ethylene, n-propyl, isopropylidene, n-butylene, and isobutylene a base, a tert-butyl group, a sec-butylene group, a n-n-pentyl group, an isoamyl group, a renepentyl group, a n-pentylene group, an n-hexylene group; preferably, an ethylene group, an n-propylene group, An n-butylene group, a sub-n-pentyl group, or an n-hexylene group.

Here, the number of carbon atoms in the alkanoyl group may be from 1 to 10, or from 1 to 6, or from 1 to 4. For example, the alkanoylamino group may be a formylamino group, an acetylamino group, a n-propionylamino group, a propionylamino group, a n-butyrylamino group, an isobutyrylamino group, a t-butyrylamino group, a sec-butyrylamino group, a n-pentanoylamino group or the like.

The alkyl group in the monoalkyl or dialkyl substituted amino group may have from 1 to 10 carbon atoms, or from 1 to 6, or from 1 to 4 carbon atoms. Examples of the alkyl group include a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, a hexyl group and the like; or a methyl group, an ethyl group, a n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group or a tertiary group. Butyl, sec-butyl, n-pentyl, isopentyl, neopentyl, n-pentyl, n-hexyl and the like.

Alternatively, the compound represented by the formula (I) is a compound represented by the following formula (IA):

Alternatively, the group represented by the formula (II) is a group represented by the following formula (IIA)

Alternatively, the compound represented by the formula (I) has a structure represented by the formula (IA).

Optionally,

R _{1 is} selected from the group consisting of ethylene, propylene, butylene, pentylene, and hexylene;

R _{2 is} selected from the group consisting of hydrogen, an amino group, a group represented by the following formula (IIA), a group represented by the following formula (III), and a group represented by the following formula (IV).

Wherein X _{1 is} selected from the group consisting of ethylene, propylene, butylene, pentylene, and hexylene;

R _{3 is} selected from the group consisting of amino groups and carboxyl groups;

Xaa is selected from the group consisting of: a glycine residue, an alanine residue, a β-aminopropionic acid residue, a γ-aminobutyric acid residue, a 6-aminocaproic acid residue, or a deletion;

Ln is selected from Gd or Eu.

Preferably, the compound of the above formula (I), or a pharmaceutically acceptable salt thereof, is selected from the group consisting of the following compound 1 - compound 7::

Preferably, the pharmaceutically acceptable salt comprises an anionic salt and a cationic salt of a compound of formula I;

Preferably, the pharmaceutically acceptable salt comprises an alkali metal salt, an alkaline earth metal salt, an ammonium salt of a compound of formula I; preferably, the alkali metal comprises sodium, potassium, lithium, cesium, the alkaline earth metal comprising Magnesium, calcium, strontium;

Preferably, the pharmaceutically acceptable salt comprises a salt of a compound of formula I with an organic base; preferably, the organic base comprises a trialkylamine, pyridine, quinoline, piperidine, imidazole, picoline, two Methylaminopyridine, dimethylaniline, N-alkylmorpholine, 1,5-diazabicyclo[4.3.0]nonene-5 (DBN), 1,8-diazabicyclo[5.4.0] Undecen-7 (DBU), 1,4-diazabicyclo[2.2.2]octane (DABCO); preferably, the trialkylamine comprises trimethylamine, triethylamine, N-ethyl Diisopropylamine; preferably, the N-alkylmorpholine comprises N-methylmorpholine;

Preferably, the pharmaceutically acceptable salt comprises a salt of a compound of formula I with an acid; preferably, the acid comprises an inorganic acid, an organic acid; preferably, the inorganic acid comprises hydrochloric acid, hydrobromic acid, hydrogen iodine Acid, sulfuric acid, nitric acid, phosphoric acid, carbonic acid; preferably, the organic acid includes formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, citric acid, Tannic acid, tartaric acid, carbonic acid, picric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, glutamic acid, pamoic acid.

In another aspect, the invention provides a nuclear magnetic resonance imaging agent comprising the above compound of formula (I) or a pharmaceutically acceptable salt thereof;

Optionally, the nuclear magnetic resonance imaging agent is used for detection of an estrogen receptor;

Optionally, the nuclear magnetic resonance imaging agent is used for the diagnosis of a disease associated with estrogen receptor expression; alternatively, the disease is a pituitary adenoma.

In another aspect, the present invention provides a compound of the formula (I) or a pharmaceutically acceptable salt thereof, or the use of the above nuclear magnetic resonance imaging agent for nuclear magnetic resonance imaging, detection;

Optionally, the nuclear magnetic resonance detection comprises estrogen receptor imaging, estrogen receptor detection, or pituitary adenoma detection.

In another aspect, the invention provides a compound of formula (I), or a pharmaceutically acceptable salt thereof, or the use of a nuclear magnetic resonance imaging agent as described above for the diagnosis of a disease;

In another aspect, the invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, or the use of a nuclear magnetic resonance imaging agent as described above for the preparation of a disease diagnostic reagent; optionally, the disease is estrogen-accepting The body expresses a related disease, and optionally, the disease is a pituitary adenoma.

In another aspect of the present invention, there is provided a nuclear magnetic resonance imaging method, wherein the compound of the above formula (I) or a pharmaceutically acceptable salt thereof is used as a nuclear magnetic resonance imaging agent.

In another aspect of the present invention, there is provided a nuclear magnetic resonance detecting method of an estrogen receptor, wherein the compound of the above formula (I) or a pharmaceutically acceptable salt thereof is used as a nuclear magnetic resonance imaging agent.

The compound of the formula (I) according to the present invention, or a pharmaceutically acceptable salt thereof, has a ruthenium and osmium chelating structure which binds to an estrogen receptor and is formed by a chelating group in the molecule, and is specific for nuclear magnetic resonance. Enhancing the contrast of the estrogen receptor high expression tissue on the image in imaging is conducive to the diagnosis of the type of lesion.

In the present invention, when the amino acid is abbreviated in Chinese or English, the amino acid name and the abbreviated form commonly used in the field are used. If not explicitly stated, the individual amino acid name and amino acid abbreviation represent L-form amino acid, such as threonine or threonine. The acyl group (Thr) indicates that it is L-type threonine or L-type threonyl; in the case of a three-character abbreviated amino acid, the present invention adopts the amino acid name and the abbreviated form commonly used in the art, and the right side thereof is " -OH" means that the amino acid is in the form of a free carboxylic acid. When the left side is "H-", the amino acid is in the form of a free amino group. For example, "H-Thr-OH" means that the amino group and the carboxyl group are in a free form. Type threonine.

In the present invention, when a peptide chain formed of a plurality of amino acids in a three-character abbreviation is used, the amino acid name and the abbreviated form commonly used in the art are used, and when the right side is "-OH", the polypeptide is in the form of a free carboxylic acid. When the left side is "H-", the polypeptide is in the form of a free amino group. For example, "H-Gly-Trp-OH" means that the amino group and the carboxyl group are in a free form of a glycyl-tryptophan dipeptide.

In the general structure or specific compound of the present invention, unless otherwise indicated, the -NH- or -NH ₂ group on the left side of each amino acid represents a group in the amino acid structure, for example, -NH in "-NH-Glu-" - expressed as an amino group in glutamic acid, the amino group on the right side of the amino acid represents an amino group linked to the carboxyl group of the amino acid structure by an amide bond, for example, -NH ₂ in -Ser-NH ₂ is an amide moiety in the silk amino acid amide Amino group.

All documents cited in the present invention are hereby incorporated by reference in their entirety, and if the meanings expressed by these documents are inconsistent with the present invention, the expression of the present invention shall prevail. In addition, the various terms and phrases used in the present invention have the ordinary meanings well known to those skilled in the art, and even though the present invention has been described and explained with respect to the terms and phrases involved, and the present invention is inconsistent with the known meanings. The meaning of the statement shall prevail. The following is a definition of the terms of the present invention and is applicable to the entire specification unless otherwise specified in the specific case.

DRAWINGS

Figure 1 is a graph showing the dose-effect relationship between different concentrations of Compound 1-5 and cell viability in an in vitro cytotoxicity assay.

Figure 2 is a graph showing the fluorescence values of imaging agents after administration of different concentrations of Compound 1 and Gd-DTPA contrast agents to MDA-MB-231 and MCF-7 cells with different estrogen receptor expression levels.

Figure 3 is a graph of in vivo fluorescence imaging of different concentrations of Compound 1 administered to MDA-MB-231 and MCF-7 cells with different estrogen receptor expression levels.

Figure 4 is a T1-weighted image of different concentrations of Compound 1 administered to MDA-MB-231 and MCF-7 cells with different levels of estrogen receptor expression.

Figure 5 is a graph showing the localization of dopamine receptor imaging agent 1H and Example Compound 1 in cells.

Detailed ways

Specific embodiments of the present invention will be described in detail below. It is understood that the specific embodiments described herein are illustrative of the invention and are not intended to limit the invention.

In the specific embodiment, the following instruments and reagents are used:

Thermo Exactive Plus (ESI/Obi-Trap) LC/MS;

German Sartorius-BSA type electronic balance;

CEM Discovery SPS Microwave Peptide Synthesizer;

Eyela rotary evaporator;

Vacumbrand diaphragm vacuum pump;

Purification of the polypeptide compound was performed using a Gilson GX-281 preparative chromatography system.

The anhydrous solvent was prepared by removing the water from a Pure Solv. solvent purification system using commercially available analytical reagents, and all other reagents were commercially available analytically pure.

Semi-preparative chromatography General conditions: Kromasil 21.2 × 250 mm C18 5μ reverse phase semi-preparative column (mobile phase A: 0.1% TFA in water, B: 0.1% TFA in acetonitrile, flow rate 15 ml / min, detection wavelength 220 nm);

The relevant abbreviations represent the following meanings:

S ₅ represents (S)-α-pentenylalanine, and the corresponding Fmoc-S ₅ -OH is (S)-N-fluorenylmethoxycarbonyl-α-pentenylalanine.

Aca is represented by 6-aminocaproic acid, Abu is represented by 4-aminobutyric acid, and Lys(N ₃ ) is represented by δ-azidolysine.

The HCTU is O-1-hydroxy-6-chlorobenzotriazole-tetramethylurea hexafluorophosphate.

Cl-HOBt is 1-hydroxybenzotriazole.

TFA is trifluoroacetic acid.

DMF is N,N-dimethylformamide.

THF is tetrahydrofuran.

TESi is triethylsilane.

DCM is dichloromethane.

DIPEA is N,N-diisopropylethylamine.

Cy5-OH is the following structure

The Grubbs I generation catalyst is: Benzylidene-bis (tricyclohexylphosphino)-dichloro-ruthenium.

Preparation method used in the detailed embodiment:

1. Washing method. The following "washing" of the solid phase resin means that the reaction liquid is filtered off, and the resin is washed three times with DMF × 2 times, DCM × 2 times, and DMF × 2 times, each washing for 2 to 3 minutes.

2. Condensation method. The following general method for the condensation of the solid phase resin is as follows: a 3-fold excess of the protected amino acid, a 3-fold excess of the condensing agent (HCTU except for the separately described), and a 3-fold excess of Cl-HOBt are dissolved in an appropriate amount of DMF. To a 0.25 mmol/ml solution, a 6-fold excess of DIPEA was added to the solution, and after stirring for 1 minute, it was added to the N-end-protected resin, and the mixture was stirred at room temperature for 2 hours and washed by "washing method".

3. Deprotection method. The following general method for removing the N-Fmoc protecting group from the solid phase resin is: adding an appropriate amount of 20% piperidine/DMF solution to the resin, shaking the mixture for 10 minutes at room temperature, filtering the solution, and adding 20% piperidine to the resin. /DMF solution, the mixture was shaken again for 10 minutes, and then the solution was filtered off and washed by "washing method".

4. RCM ring joint method. The following general method for performing olefin metathesis cyclization using Grubbs I catalyst is: adding an appropriate amount of anhydrous dichloroethane to the dried resin intermediate, and removing nitrogen or argon through the lower end of the solid phase reaction tube to remove the dissolved To the mixture, a Grubbs I generation catalyst equivalent to 20% by mole of the resin intermediate was added to the mixture, and the catalyst concentration was adjusted to 5 mg/ml by adjusting the amount of the solvent, and the reaction was carried out by bubbling nitrogen or argon for 2 hours. After the reaction solution was washed twice with dichloroethane, the resin was again bubbled with the same concentration and molar amount of Grubbs I catalyst for 2 hours, and then washed twice with dichloroethane to obtain a small amount of resin ( About 2 mg) 1 ml of a lysate containing TFA:TESi:H ₂ O=95:2.5:2.5 (volume ratio) was added, and the reaction was stirred for 1 hour, and then the resin was removed by filtration, and then nitrogen-dried and dissolved in methanol to carry out liquid-liquid analysis, and it was confirmed that there was no raw material ( After the unconjugated polypeptide), the remaining resin was washed in a "washing pass".

5. Peptide condensation cycle. The following synthetic step of peptide chain synthesis for each additional amino acid residue is a peptide condensation cycle which involves condensation of a N-terminal Fmoc protecting group according to the "decondensation method" after condensing the N-Fmoc protected amino acid according to the "condensation method". Unless otherwise stated, only the mass and molar amount of each of the amino acids involved in the condensation are listed below, and the specific procedures are not repeated.

6. Cracking method. The following general method for cracking the resin is: after the peptide resin intermediate is dried, TFA:TESi:H ₂ O=95:2.5:2.5 (volume) is added in a ratio of resin weight (g): lysate volume (ml) of 1:10. The lysate of the mixture was stirred for 3 hours, and the reaction solution was filtered off and the resin was washed twice with an appropriate amount of TFA. The filtrate was combined and concentrated by rotary evaporation to give an oily substance. 50-100 volume of ice-cooled water was added to the oil. The polypeptide was solidified and precipitated by diethyl ether. After centrifugation (speed: 4000 rpm), the supernatant was decanted, and the lower solid was triturated with anhydrous diethyl ether and centrifuged, and the supernatant was discarded, and the solid was vacuum dried to obtain a crude peptide product.

Synthesis of each example compound

Preparation 1. Preparation of N-tert-Butoxycarbonyl (O-benzyl) seryldiethylamine (1a)

11.814 g (40 mmol) of N-tert-butoxycarbonyl (O-benzyl)serine Boc-Ser(Bzl)-OH, 5.06 g of N-hydroxysuccinimide (HOSu) and 6.715 g of dicyclohexylcarbodiimide ( DCC) was dissolved in 100 ml of anhydrous DCM, and added to DIPEA under ice water for 30 minutes. After stirring for 30 minutes at room temperature, the reaction was stirred for 8 hours at room temperature. The filtrate was filtered with 1 mol/L hydrochloric acid, saturated aqueous NaHCO ₃ and saturated NaCl. The aqueous solution was washed 3 times each time, and the organic layer was dried over Na ₂ SO ₄ and filtered. The filtrate was concentrated, dried in vacuo, vacuum dried at 45 ° C, and the obtained product was dissolved in 200 ml of anhydrous DCM, and dropped into 18.4 ml of ethylenediamine solution, ice. The reaction was stirred in an external water bath, and after the completion of the dropwise addition, the ice bath was removed and reacted at room temperature for 4 hours. After adding 100 ml of water, the reaction was stirred for 20 minutes, transferred to a separatory funnel, and the organic phase was kept. The aqueous phase was extracted with DCM three times, then the organic phase was combined, dried over Na ₂ SO ₄ and filtered, and the filtrate was concentrated to silica gel column column chromatography. Separation, mobile phase DCM: MeOH (methanol) = 50:1 (volume ratio), the target fractions were collected and concentrated to give a brown oil of 10.048 g yield 74.5%.

Analysis of the _{product: 1H NMR (400MHz, CDCl 3} ) δ7.32 (m, 5H), 6.79 (s, 1H), 6.24 (s, 1H), 5.13 (t, J = 7.0Hz, 1H), 4.91 (d, J = 12.4 Hz, 1H), 4.45 (d, J = 12.3 Hz, 1H), 4.19 (dd, J = 12.5, 7.0 Hz, 1H), 3.78 (td, J = 12.2, 3.1 Hz, 1H), 3.60 ( Dd, J = 12.4, 6.9 Hz, 1H), 3.05 (td, J = 12.1, 3.1 Hz, 1H), 2.91 (m, 2H), 1.42 (d, J = 18.4 Hz, 11H).; ESI-MS: Calculated 338.20 [M+H]+, found: 338.20.

Preparation 2. Preparation of (O-benzyl) seryldiethylamine hydrochloride (1b)

9.418 g (27.9 mmol) of the compound 1a prepared in Preparation Example 1 was added to 10 ml of methanol, and a 3 mol/L saturated HCl solution of ethyl acetate was added thereto, and the reaction mixture was stirred for 3 hours in an ice water external bath, and the reaction liquid was concentrated and dried in a vacuum oven at 45 ° C. After 12 hours, 8.18 g of a pale yellow solid was obtained, yield 93.7%.

</ RTI> <RTI ID=0.0></RTI> </RTI> <RTIgt;

Preparation 3. Preparation of (R)-2-amino-3-((2-aminoethyl)-amino)-1-propanol) (1c)

6.724 g (21.67 mmol) of the compound 1b prepared in Preparation Example 2 was suspended in 50 ml of anhydrous THF, and 11 ml of a borane dimethyl sulfide solution having a concentration of 9.89 mmol/ml was added dropwise, and the reaction was refluxed at 65 ° C for 36 hours after the completion of the dropwise addition. 11 ml of 6 mol/L HCl aqueous solution was added dropwise, and the mixture was heated under reflux for 30 minutes, and then evaporated to dryness. The product was purified by ion-exchange resin Dowax 50WX2 to obtain a brown oil. The oil was added to 100 ml of 6N aqueous HCl solution and sealed at 90 ° C. After the reaction, the reaction mixture was concentrated and dried in a vacuum oven to yield 3.34 g of brown oil.

Analysis of the product: 1H NMR (400 MHz, D ₂ O) δ 3.86 (ddd, J = 12.3, 7.0, 5.1 Hz, 1H), 3.54 (ddd, J = 12.3, 7.0, 5.1 Hz, 1H), 2.98 (m, 2H), 2.85 (m, 2H), 2.72 (m, 2H), 2.61 (dd, J = 12.3, 7.0 Hz, 1H).

Preparation 4. N,N,N',N',N",N"-penta-butoxycarbonylmethyl-(R)-2-amino-3-((2-aminoethyl)-amino)- Preparation of 1-propanol (1d)

3.34 g (13.77 mmol) of the compound 1c prepared in Preparation Example 3 was dissolved in 40 ml of anhydrous DMF, and 36 ml of DIPEA was added, and the reaction was stirred at room temperature under nitrogen atmosphere, and 15 ml of t-butyl bromoacetate was added dropwise, and the reaction was carried out at room temperature for 16 hours. the reaction mixture was concentrated and saturated aqueous NaCl, and extracted 3 times with ethyl acetate, the organic layer with distilled water, saturated aqueous NaHCO _3, washed with saturated aqueous NaCl 3 times each, dried Na ₂ SO _4, filtered and concentrated the residue was dried in a vacuum Drying in a box gave 6.555 g of a brown oil, yield 67.7%.

</ RTI> <RTI ID=0.0></RTI> </RTI>

Preparation 5. N, N, N', N', N", N"-penta-butoxycarbonylmethyl-(R)-2-amino-3-((2-aminoethyl)-amino)- Preparation of 1-(O-propargyl)propanol (1e)

2.9 g (4.1 mmol) of the compound 1d prepared in Preparation Example 4 was dissolved in 10 ml of anhydrous DMF, and 60% NaH 0.656 g was added under argon atmosphere. After stirring for 15 minutes, bromopropyne 0.962 ml was added dropwise, and the room temperature was completed. for 16 hours, washed with saturated aqueous NaCl 100mlDCM three times, the organic layer was dried over Na ₂ SO ₄ filtered, and the filtrate was concentrated to give a red brown oil, medium pressure silica gel preparative chromatography, the mobile phase DCM:MeOH = 20:1 ( The volume fraction was collected, and the fraction was collected and concentrated to give a brown oily substance (yield: 29.9 g). ,

</ RTI> <RTI ID=0.0></RTI> </RTI> <RTI ID=0.0>

Preparation 6. Preparation of N-fluorenylmethoxycarbonyl-α-(4-azidobutyl)glycine (Fmoc-Lys(N ₃ )-OH)

2.34 g (5 mmol) of Fmoc-Lys(Boc)-OH was dissolved in 10 ml of 80% trifluoroacetic acid in dichloromethane, and the reaction mixture was stirred for 1 hour in an ice water external bath, and the reaction mixture was evaporated to dryness. The white solid was suction filtered and dried under an infrared lamp to yield 2.278 g of white solid. The solid was dissolved in 15 ml of methanol, 1.129 g of imidazosulfonyl azide hydrochloride was added, and 12 mg of copper sulfate pentahydrate, 1.175 g of NaHCO ₃ , stirred under argon for 5 hours, and then adjusted to pH with 2 mol/L aqueous hydrochloric acid. 1~2, the aqueous layer was extracted with ethyl acetate 3 times, the organic layer was washed 3 times with 1 mol/L hydrochloric acid aqueous solution, washed with saturated aqueous NaCl solution 3 times, dried over Na ₂ SO ₄ , filtered, concentrated, and solidified to white solid in the refrigerator. 1.41 g, yield 73%.

Product analysis: ESI-MS: calc. 395.17 [M+H]+, found: 395.17. 1H-NMR: (400 MHz, Chloroform-d) δ 8.83 (s, 1H), 7.81 (d, J = 7.6 Hz , 2H), 7.75-7.53 (m, 2H), 7.45 (t, J = 7.5 Hz, 2H), 7.36 (t, J = 7.5 Hz, 2H), 5.38 (d, J = 8.3 Hz, 1H), 4.47 (t, J = 6.0 Hz, 2H), 4.27 (t, J = 6.9 Hz, 1H), 3.29 (dt, J = 29.3, 6.5 Hz, 2H), 2.05-1.89 (m, 1H), 1.88-1.22 ( m,6H). ¹³ C NMR (101MHz, CDCl ₃ ) δ 176.89, 156.12, 143.80, 143.65, 141.35, 127.79, 127.11, 125.06, 120.06, 77.38, 77.26, 77.06, 76.75, 67.17, 53.53, 51.08, 47.16, 31.86, 28.37, 22.47.

Preparation 7. Preparation of N-fluorenylmethoxycarbonyl-α-(4-azidopropyl)glycine (Fmoc-Orn(N ₃ )-OH)

2.272 g (5 mmol) of Fmoc-Orn(Boc)-OH was dissolved in 10 ml of 80% trifluoroacetic acid in dichloromethane, and the reaction was stirred for 1 hour in an ice water external bath, and the reaction mixture was evaporated to dryness. The white solid was filtered under suction and dried with EtOAc. The solid was dissolved in 15 ml of methanol, 1.125 g of imidazosulfonyl azide hydrochloride was added, and 12 mg of copper sulfate pentahydrate, 1.175 g of NaHCO ₃ , stirred under argon for 5 hours, and then adjusted to pH with 2 mol/L aqueous hydrochloric acid. To 1-2, the aqueous layer was extracted with ethyl acetate three times, the organic layer was washed three times with 1 mol/L hydrochloric acid aqueous solution, and washed with saturated aqueous NaCl solution three times, dried over Na ₂ SO ₄ , filtered, concentrated and evaporated to white The solid was 1.481 g and the yield was 82%.

</ RTI><RTIID=0.0></RTI></RTI></RTI><RTIID=0.0></RTI></RTI>^<RTIgt; , 2H), 7.72-7.55 (m, 2H), 7.45 (t, J = 7.5 Hz, 2H), 7.33 (t, J = 7.5 Hz, 2H), 5.24 (d, J = 8.3 Hz, 1H), 4.45 (t, J = 6.0 Hz, 2H), 4.35 (t, J = 6.9 Hz, 1H), 3.29 (dt, J = 29.3, 6.5 Hz, 2H), 2.11-1.95 (m, 1H), 1.92-1.32 ( m,4H). ¹³ C NMR (101MHz, CDCl ₃ ) δ 174.89, 156.15, 143.56, 143.46, 141.18, 140.38, 127.07, 126.38, 125.48, 121.08, 66.72, 54.34, 51.98, 47.08, 29.55, 25.36.

Preparation Example 8. Preparation of the peptide resin a

0.91 g of Rink amide resin (degree of substitution 0.55 mmol/g) was taken, and the Fmoc protecting group was removed by deprotection. According to the "peptide condensation cycle" method, 1.5 mmol of each, Fmoc-Ser(tBu)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-S ₅ -OH, Fmoc-Leu-OH, Fmoc- are sequentially and sequentially linked. Leu-OH, Fmoc-Arg(Pbf)-OH, Fmoc-S ₅ -OH, Fmoc-Leu-OH, Fmoc-Ile-OH, Fmoc-Lys(Boc)-OH, Fmoc-His(Trt)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Glu(OtBu)-OH, followed by cyclization according to the "RCM loop-through method", followed by removal of the Fmoc protecting group according to the "deprotection method" to obtain a peptide resin a.

Example 1. Preparation of Compound 1:

The book peptide resin a prepared in Preparation Example 8 was sequentially condensed by Fmoc-Aca, Fmoc-Lys(N ₃ )-OH according to the "peptide condensation cycle" method, and then the Fmoc protecting group was removed according to the "deprotection method", and then The resin was cleaved according to the "cracking method" to obtain 0.625 g of a crude peptide product. The crude peptide was separated by semi- preparative chromatography and HPLC to obtain 134 mg of purified peptide. 0.58 g of the purified peptide and the compound 1e obtained in Preparation Example 5 were dissolved in a solution of t-butanol and water (2:1 by volume), 1.78 mg of copper sulfate and 1.26 mg of ascorbic acid were added, and the reaction was stirred for 12 hours to prepare a half-preparative chromatogram. The separation was carried out, and the product was collected in a ratio of 35% B for 15 minutes. The product obtained from the target peak was collected, and the obtained product was lyophilized, dissolved in 15 ml of TFA (trifluoroacetic acid), stirred at room temperature for 2 hours, and then concentrated to give a brown oil. The mixture was triturated with anhydrous diethyl ether to give a pale yellow powder (yield: 169). NaHCO ₃ was added thereto, the pH was adjusted to 6.5, and 1 ml of an aqueous solution of 18 mg of GdCl ₃ was added dropwise with stirring in an ice water external bath. After the dropwise addition, the ice bath was removed, pH=5.5, and after 3 hours of reaction, it was separated by preparative HPLC, and then frozen. Dry, 187 mg of a white solid, yield 86.3%.

Product analysis: ESI-MS: calculated value 1241.61 [M+2H] ²⁺ , found: 1241.61, molecular ion peaks are shown as yttrium isotope characteristic signals.

Example 2. Preparation of Compound 2:

The staple peptide resin a prepared in Preparation Example 8 was condensed 6-azido-1-hexanoic acid according to the "peptide condensation cycle" method, and then the resin was cleaved according to "cracking method" to obtain 0.325 g of a crude peptide product. The crude peptide was separated using semi-preparative HPLC to obtain 86 mg of purified peptide. 0.58 g of the purified peptide and the compound 1e obtained in Preparation Example 5 were dissolved in a solution of t-butanol and water (2:1 by volume), 1.2 mg of copper sulfate and 0.9 mg of ascorbic acid were added, and the reaction was stirred for 12 hours to prepare a semi-preparative HPLC. The product was isolated, and the obtained product was lyophilized, dissolved in 15 ml of TFA (trifluoroacetic acid), and the mixture was stirred at room temperature for 2 hours, then the reaction mixture was concentrated to give a brown oil. %. NaHCO ₃ was added thereto, the pH was adjusted to 6.5, and 1 ml of an aqueous solution of 18 mg of GdCl ₃ was added dropwise with stirring in an ice water external bath. After the dropwise addition, the ice bath was removed, pH=5.5, and after 3 hours of reaction, it was separated by preparative HPLC, and then frozen. Dry to give a white solid (92 mg, yield: 82.1%).

Product analysis: ESI-MS: calculated value 1177.56 [M+2H] ²⁺ , found: 1177.56, molecular ion peaks are shown as yttrium isotope characteristic signals.

Example 3. Preparation of Compound 3

The staple peptide resin a prepared in Preparation Example 8 was sequentially condensed according to the "peptide condensation cycle" method, Fmoc-Aca, Fmoc-Lys(N ₃ )-OH, 6-azido-1-hexanoic acid, and then "cracked" The general procedure "cracked the resin to give the crude peptide product 0.325 g. 47 mg of the purified peptide and 0.264 g of the compound 1e obtained in Preparation Example 5 were dissolved in a solution of t-butanol and water (2:1 by volume), 8 mg of copper sulfate and 1.8 mg of ascorbic acid were added, and the reaction was stirred for 12 hours to prepare a semi-preparative HPLC. The product was separated, and the obtained product was lyophilized, dissolved in 15 ml of TFA (trifluoroacetic acid), and the mixture was stirred at room temperature for 2 hours. The reaction mixture was concentrated to give a brown oil. %. NaHCO ₃ was added thereto, the pH was adjusted to 6.5, and 1 ml of an aqueous solution of 83 mg of GdCl ₃ was added dropwise with stirring in an ice water external bath. After the dropwise addition, the ice bath was removed, pH=5.5, and after 3 hours of reaction, it was separated by preparative HPLC, and then frozen. Dry to give a white solid 26mg, yield 56.2%.

Product analysis: ESI-MS: calculated value 1078.78 [M+3H] ³⁺ , found: 1078.79 molecular ion peaks are shown as yttrium isotope characteristic signals.

Example 4. Preparation of Compound 4

The book peptide resin a prepared in Preparation Example 8 was sequentially condensed Fmoc-Aca, Fmoc-Orn(N ₃ )-OH according to the "peptide condensation cycle" method, and then the Fmoc protecting group was removed according to the "deprotection method", and then The resin was cleaved according to "cracking method" to give the crude peptide product 0.384 g. The crude peptide was separated using semi-preparative HPLC to obtain 89 mg of the purified peptide. The purified peptide and 0.039 g of the compound 1e obtained in Preparation Example 5 were dissolved in a solution of t-butanol and water (2:1 by volume), 1.24 mg of copper sulfate and 0.88 mg of ascorbic acid were added, and the reaction was stirred for 12 hours to prepare a semi-preparative HPLC. The product was isolated, and the obtained product was lyophilized, dissolved in 10 ml of TFA (trifluoroacetic acid), and the mixture was stirred at room temperature for 2 hr. %. NaHCO ₃ was added thereto, the pH was adjusted to 6.5, and 1 ml of an aqueous solution of 13.3 mg of GdCl ₃ was added dropwise with stirring in an ice water external bath. After the completion of the dropwise addition, the ice bath was removed, pH=5.5, and after 3 hours of reaction, separation was carried out by preparative HPLC, and then Lyophilized to give 88 mg of a white solid, yield 78.5%.

Product analysis: ESI-MS: Calculated value 1235.10 [M+2H] ²⁺ , found: 1235.11, molecular ion peak showed 钆 isotope characteristic signal.

Example 5. Preparation of Compound 5:

The book peptide resin a prepared in Preparation Example 8 was sequentially condensed in accordance with the "peptide condensation cycle" method, Fmoc-Abu-OH, Fmoc-Orn(N ₃ )-OH, and then the Fmoc protecting group was removed according to the "deprotection method". Then, the resin was cleaved according to "cracking method" to obtain 0.296 g of a crude peptide product. The crude peptide was separated using semi-preparative HPLC to obtain 87 mg of the purified peptide. The purified peptide and 0.037 g of the compound 1e obtained in Preparation Example 5 were dissolved in a solution of t-butanol and water (2:1 by volume), 1.23 mg of copper sulfate and 0.86 mg of ascorbic acid were added, and the reaction was stirred for 12 hours to prepare a semi-preparative HPLC. The product was isolated, and the obtained product was lyophilized, dissolved in 10 ml of TFA (trifluoroacetic acid), and the mixture was stirred at room temperature for 2 hr. %. NaHCO ₃ was added thereto, the pH was adjusted to 6.5, and 1 ml of an aqueous solution of 12.5 mg of GdCl ₃ was added dropwise with stirring in an ice water external bath. After the dropwise addition, the ice bath was removed, pH=5.5, and after 3 hours of reaction, separation was carried out by preparative HPLC, and then Lyophilized to give a white solid, 80 mg, yield 76.5%.

Product analysis: ESI-MS: calculated value 1228.09 [M+2H] ²⁺ , found: 1228.10, the molecular ion peak is shown as the yttrium isotope characteristic signal.

Example 6. Preparation of Compound 6:

The book peptide resin a prepared in Preparation Example 8 was sequentially condensed in a "peptide condensation cycle" method by Fmoc-Abu-OH, Fmoc-Lys(N ₃ )-OH, Fmoc-Abu-OH, and then according to the "deprotection method". Remove the Fmoc protecting group, add 250 mg of 5-FAM (0.5 mmol), 90 mg of HOBt (0.6 mmol) and 1.2 mmol of DIC to the resin, react at room temperature for 12 hours, drain the resin and wash the resin by general method, then follow the "cracking" The resin was cleaved to give a crude peptide product of 0.096 g. The crude peptide was separated using semi-preparative HPLC to obtain 28 mg of the purified peptide. 10 mg of the purified peptide and the compound 1e obtained in Preparation Example 5 were dissolved in a solution of t-butanol and water (2:1 by volume), 0.3 mg of copper sulfate and 0.2 mg of ascorbic acid were added, and the reaction was stirred for 12 hours, and subjected to semi-preparative HPLC. After separation, the obtained product was lyophilized, dissolved in 10 ml of TFA (trifluoroacetic acid), and the mixture was stirred at room temperature for 2 hours, and then the mixture was concentrated to give a brown oil. NaHCO ₃ was added thereto, the pH was adjusted to 6.5, and 1 ml of an aqueous solution of 2.63 mg (10 μmol) of GdCl ₃ was added dropwise with stirring in an ice water external bath. After the dropwise addition, the ice bath was removed, pH=5.5, and after reacting for 3 hours, C18 was solidified. The extract was separated by phase extraction and then lyophilized to give 8 mg of a yellow solid.

Product analysis: ESI-MS: Calculated value 1463.15828 [M+2H] ²⁺ , measured value 1463.16643 [M+2H] ²⁺ , molecular ion peak showed 钆 isotope characteristic signal.

Example 7. Preparation of Compound 7:

The book peptide resin a prepared in Preparation Example 8 was sequentially condensed in a "peptide condensation cycle" method by Fmoc-Abu-OH, Fmoc-Lys(N ₃ )-OH, Fmoc-Abu-OH, and then according to the "deprotection method". Remove the Fmoc protecting group, add 80 mg (0.135 mmol) of Cy5-OH, 24 μL of DIEPA (137 μmol) to the resin, react at room temperature for 4 hours, drain the resin, and wash the resin by general method, then lyse according to the "cracking method". Resin gave 0.124 g of crude peptide product. The crude peptide was separated using semi-preparative HPLC to obtain 34 mg of the purified peptide. The purified peptide and the compound 1e obtained in Preparation Example 1 12.75 mg were dissolved in a solution of t-butanol and water (2:1 by volume), 0.4 mg of copper sulfate and 0.3 mg of ascorbic acid were added, and the reaction was stirred for 12 hours to prepare a semi-preparative HPLC. The obtained product was separated, and the obtained product was lyophilized, dissolved in 10 ml of TFA (trifluoroacetic acid), and the mixture was stirred at room temperature for 2 hours, and then the mixture was concentrated to give a brown oil. NaHCO ₃ was added thereto, the pH was adjusted to 6.5, and 1 ml of an aqueous solution of 2.63 mg (10 μmol) of GdCl ₃ was added dropwise with stirring in an ice water external bath. After the dropwise addition, the ice bath was removed, pH=5.5, and after reacting for 3 hours, C18 was solidified. The phases were separated by extraction and then lyophilized to give 9.6 mg of a blue solid.

Product analysis: HRMS: calculated value 1015.85817 [M + 3H] ^{3 +} , measured value 1015.75880 [M + 3H] ^{3 +} , molecular ion peaks are shown as strontium isotope signature signals.

Experimental Example 1. In vitro cytotoxicity test

1.1 Cells and reagents:

The rat pituitary tumor cell line GH3 is a product of the American ATCC company. DMEM medium and high quality fetal bovine serum were purchased from Gibco, Dimethylsulphoxide (DMSO), trypsin was purchased from Sigma, USA, MTT was purchased from Genview, USA, penicillin and streptomycin were purchased from North China Pharmaceutical Company. All other reagents were of commercially available analytical grade.

1.2 Experimental instruments and equipment:

HERAcell150 CO ₂ cell incubator (Hellet, Germany), IMT-2 inverted microscope (Olympus, Japan), 550 enzyme-linked immunosorbent assay (BIO-RAD, USA); consumables for Petri dishes, 96 cell well plates (Costar, USA).

1.3 routine culture and passage of pituitary adenoma cells GH3

The frozen rat pituitary tumor cell line GH3 cells were resuscitated, and the DMEM medium containing 100 mL/L fetal bovine serum and 100 U/mL penicillin and 100 U/mL streptomycin was used at 37 ° C, 5% CO ₂ and saturated humidity. The cells were cultured in a constant temperature incubator, periodically passaged according to the growth conditions, and experiments were carried out using the cells after the third passage.

1.4. Effect of compound 1-5 prepared in the examples on proliferation of pituitary adenoma GH3 cells

5 experimental groups: Compounds 1-5 were formulated into 5 different final concentrations in DMEM medium containing 10% fetal bovine serum: 1 μM/L, 5 μM/L, 10 μM/L, 50 μM/L, 500 μM/L. ;

1 group of blank control group (blank group): only DMEM medium containing 10% fetal bovine serum was added;

The third passage of pituitary adenoma GH3 cells was divided into 6 groups (5 groups of experimental groups and 1 group of control groups). The passage cells were inoculated into three 96-well cell culture plates at 4000/100 ul per well, and divided into 6 groups according to the above grouping scheme, and 8 replicates were set for each group. After the cells were attached to the cells for 24 hours, the cells were replaced with the corresponding concentrations of the compound 1-5, and cultured in a constant temperature incubator at 37 ° C, 5% CO ₂ and saturated humidity. After 72 hours, 20 μl of a 5 mg/ml MTT solution (3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrayl) was added to each well in the wells to be tested. The azole bromo salt, trade name is thiazolyl blue, the solvent is dimethyl sulfoxide), the incubation is continued at 37 ° C for 4 h, the culture is terminated, the culture supernatant in the well is aspirated, and 100 μl of dimethyl sulfoxide (DMSO) is added to each well. Shake for 10 min. The wavelength of 490 nm was selected, and the absorbance value OD _{490 of} each well was measured on an enzyme-linked immunosorbent detector.

The experimental results are shown in Figure 1. The test compound had no significant effect on the proliferation of pituitary adenoma cells at a concentration of 1-500 μM/L. This series of compounds has a low cytotoxic effect. From this, it can be seen that the compound of the formula (I) of the present invention as a contrast agent has low toxicity for nuclear magnetic resonance imaging of pituitary adenoma cells.

Experimental Example 2. Nuclear Magnetic Resonance Relaxation Rate Detection

Example Compounds 1-5 and gadopentetate (Gd-DTPA, diethylene pentamine acetate, clinical nuclear magnetic contrast agent) contrast agent were prepared at the same concentration, and Example Compounds 1-5 and Gd-DTPA were respectively used. PBS (phosphate buffer solution, concentration 0.01 mol/L, pH 7.2) was prepared at a final concentration of 0, 5 μM, 10 μM, 50 μM, 500 μM and 5 mM, and resuspended in a 0.5 mL microcentrifuge tube (which is an Eppendof tube, below) Also referred to as Eppendof tube). The Eppendof tube was placed on a Bruker small animal nuclear magnetic resonance machine (Bruker, Germany) for magnetic resonance scanning T1-weighted imaging.

The signal of compound 1-5 and Gd-DTPA contrast agent were measured, and the signal intensity (T1 relaxation time) was measured by selecting the ROI (return on interesting) in each sample size. The experiment was repeated 3 times and the measurement was taken. The average of the results. The signal intensities of the compound of Example 1-5 and the Gd-DTPA contrast agent were subjected to correlation regression analysis, and the slope was the relaxation rate (R) of the compound 1-5 and the Gd-DTPA contrast agent. The ratio of the relaxation rate of any of the compounds of Examples 1-5 to the Gd-DTPA contrast agent is the difference in signal. The results are shown in Table 1. The ratio of the compound/Gd-DTPA signal of any of the compounds of Examples 1-5 was more than 1, and the results showed that the relaxation rate of the compound of Example 1-5 was significantly better than that of Gd-DTPA. rate. The effect of contrast enhancement of NMR T1 imaging at the same concentration was shown for Example Compounds 1-5 compared to the contrast agent. That is, at room temperature, the compound of Example 1-5 was superior to the nuclear magnetic imaging effect of the Gd-DTPA contrast agent. It is shown that the compound of the formula (I) of the present invention has excellent contrast ability and can be used as a contrast agent for magnetic resonance imaging.

It should be noted that gadopentetate (Gd-DTPA) is a small molecule nuclear magnetic resonance or magnetic resonance contrast agent currently widely used, and is a non-selective non-specific contrast agent.

Table 1 Example compound 1H-5H and Gd-DTPA contrast agent relaxation ratio ratio

Experimental Example 3. In vitro targeted detection

Breast cancer MDA-MB-231 cells (derived from ATCC) and estrogen receptor-positive breast cancer MCF-7 cells (derived from ATCC) with negative estrogen receptor expression were used with 100 mL/L fetal calf The DMEM medium containing serum and 100 U/mL penicillin and 100 U/mL streptomycin was cultured in a constant temperature incubator at 37 ° C, 5% CO ₂ and saturated humidity, and periodically passaged according to the growth condition, and the cells after the third passage were used. conduct experiment.

The MDA-MB-231 and MCF-7 cells were seeded in a six-well plate at a cell density of 1*10 ⁶ /ml, respectively. After the cells were attached, different concentrations of the compound compounds of the example compound were coupled after the fluorophores were attached. 1 and Gd-DTPA contrast agent (1μM, 5μM, 10μM, 50μM, 100μM and 500μM, solvent concentration of 0.01mol/L, pH 7.2 PBS) were added to MDA-MB-231 and MCF-7 cell culture medium for 2h. Then, the cells were washed three times with the above PBS, the cells were completely digested with 0.25% trypsin, centrifuged at 1200 rpm for 4 min, and resuspended in a 0.5 mL Eppendof tube. The Eppendof tube was placed on a small animal living fluorescent machine (PE company, Germany) for in vivo fluorescence imaging. The Eppendof tube was placed on a Bruker small animal nuclear magnetic resonance machine (Bruker, Germany) for magnetic resonance scanning T1-weighted imaging.

Experimental results:

Figure 2 shows the imaging agent fluorescence values after administration of different concentrations of Compound 1 and Gd-DTPA contrast agents to MDA-MB-231 and MCF-7 cells with different estrogen receptor expression levels. As shown in Figure 2, there was no significant difference in nuclear magnetic signal intensity between Md-MB-231 and MCF-7 cells in which Gd-DTPA contrast agent differed in estrogen receptor expression, whereas Example Compound 1 contained estrogen receptor. The concentration of the gradient-dependent fluorescence signal was increased in MCF-7 cells, while there was no significant fluorescence signal intensity in MDA-MB-231 cells negative for estrogen receptor expression. At the same time, the fluorescence signal intensity in MCF-7 cells positive for estrogen receptor expression was significantly higher than that in MDA-MB-231 cells negative for estrogen receptor expression.

Figure 3 is a graph of in vivo fluorescence imaging of different concentrations of Compound 1 administered to MDA-MB-231 and MCF-7 cells with different estrogen receptor expression levels. As a result, as shown in Fig. 3, the fluorescence intensity of the compound of Example 1 at a concentration of 0-500 μM in the estrogen receptor-positive MCF-7 cells was significantly higher than that in the MDA-MB-231 cells negative for estrogen receptor expression. Fluorescence signal intensity. MCF-7 cells showed a fluorescent signal from the concentration of compound 1 at 10-7, while MDA-MB-231 cells showed no fluorescence signal. Moreover, as the concentration of Compound 1 increased, the fluorescence signal intensity in MCF-7 cells gradually increased.

Figure 4 is a T1-weighted image of different concentrations of Compound 1 administered to MDA-MB-231 and MCF-7 cells with different levels of estrogen receptor expression. As a result, as shown in Fig. 4, the concentration of nuclear magnetic signal of compound 1 in the concentration of 0-500 μM in MCF-7 cells positive for estrogen receptor expression was significantly higher than that in MDA-MB-231 cells negative for estrogen receptor expression. Signal strength. In the T1-weighted imaging image, the denser the red dots, the stronger the nuclear magnetic signal intensity. It can be seen from the figure that the nuclear magnetic signal intensity in the MCF-7 cells gradually increases with the increase of the concentration of the compound 1.

The above results indicate that Example Compound 1 has estrogen receptor targeting at the in vitro cell level compared to Gd-DTPA contrast agent, and can target nuclear magnetic display of estrogen receptor levels.

Experimental Example 4. Target-specific carrying imaging molecules enter the cell

Breast cancer MCF-7 cells (derived from ATCC) and dopamine receptor-positive PC12 cells with positive estrogen receptor expression were used with 100 mL/L fetal bovine serum and 100 U/mL penicillin, 100 U/mL streptomycin, respectively. The DMEM medium was cultured in a constant temperature incubator at 37 ° C, 5% CO ₂ and saturated humidity, and was periodically passaged according to the growth condition, and the cells were subjected to the third passage.

The MCF-7 and PC12 cells were separately subjected to cell scrawling, and after the cells were attached, 50 μM of the conjugated fluorophore-expressing Example Compound 1 and the dopamine receptor-targeted imaging agent 1H (developer disclosed in CN106366075A) Incubate with MCF-7 and PC12 cell culture medium for 2 h, rinse with PBS for 3 times, and fix 4% paraformaldehyde for 10 min. Rinse in PBS for 5 min and repeat three times. Incubate 3% Triton X-100 for 10 min at room temperature, rinse for 5 min in PBS, and repeat three times. The slide was observed under a fluorescence microscope.

Experimental results:

Figure 5 is a graph showing the localization of dopamine receptor imaging agent 1H and Example Compound 1 in cells.

As shown in Fig. 5A, red fluorescence represents dopamine D2 receptor, green fluorescence is FITC (referred to as fluorescein isothiocyanate) coupled dopamine receptor imaging agent 1H, and blue fluorescence is DAPI (refers to 4', 6 - Dimercapto-2-phenylindole fluorescent dye) localized nuclei. The green fluorescent particles in Fig. 5A are all concentrated outside the cell membrane. It indicates that the dopamine receptor imaging agent 1H is difficult to enter the cell interior, and the dopamine receptor imaging agent 1H can only bind to the dopamine D2 receptor on the cell membrane surface.

As shown in Figure 5B, red fluorescence represents the estrogen receptor, green fluorescence is the FITC-coupled Example Compound 1, and blue fluorescence is the DAPI-localized nuclei. The green fluorescent particles in Fig. 5B are distributed on the surface of the cell membrane and inside the nucleus. It is shown that the compound of the example can bind to the estrogen receptor on the surface of the cell membrane, and can also enter the cell, penetrate the nuclear membrane, enter the nucleus, and bind to the estrogen receptor localized in the nucleus (Fig. 5B, green) Fluorescence coincides with red fluorescence, indicating that Example Compound 1 colocalizes with the estrogen receptor).

The above results indicate that the compound of the example 1 can make the macromolecular imaging group enter the interior of the cell and bind to the estrogen receptor in the nucleus, thus being able to specifically target the estrogen receptor, compared to the dopamine receptor imaging agent 1H. Targeted selective intracellular imaging.

According to the experimental results of Example 3 and Example 4, the compound of the formula (I) of the present invention and a pharmaceutically acceptable salt thereof can specifically bind to an estrogen receptor, that is, the compound of the formula (I) of the present invention A pharmaceutically acceptable salt that targets to recognize estrogen receptors. Further, the compound of the formula (I) of the present invention and a pharmaceutically acceptable salt thereof can be applied to estrogen receptor-targeted magnetic resonance detection or nuclear magnetic resonance detection.

While the present invention has been described with reference to the embodiments of the present invention, it will be understood by those skilled in the art that various changes can be made without departing from the scope of the invention. These variations are considered to be within the scope of protection of the present invention.

Claims (10)

1. a compound of the formula (I), or a pharmaceutically acceptable salt thereof:

   

   among them,

   R _{1 is} selected from: an alkylene group;

   R _{2 is} selected from the group consisting of hydrogen, an amino group, a group represented by the following formula (II), a group represented by the following formula (III), and a group represented by the following formula (IV);

   

   Wherein X _{1 is} selected from the group consisting of: an alkylene group;

   R _{3 is} selected from the group consisting of amino, alkanoylamino, carboxy, monoalkyl or dialkyl substituted amino groups (e.g., acylmethylamine, acylethylamine, acyl propylamine, isopropyl isopropylamine, acyl n-butylamine, acyl isobutylamine) , acyl tert-butylamine, acyl butylamine), optionally amino, alkanoylamino, carboxy, amidoamine, acylethylamine;

   Xaa is selected from the group consisting of: a glycine residue, an alanine residue, a β-aminopropionic acid residue, a γ-aminobutyric acid residue, a 6-aminocaproic acid residue, or a deletion;

   Ln is selected from: Gd or Eu;

   Glu is a glutamic acid residue, Lys is a lysine residue, His is a histidine residue, IIe is an isoleucine residue, Leu is a leucine residue, Arg is an arginine residue, Asp Is an aspartic acid residue, Ser is a serine residue, and S ₅ is a (S)-α-pentenylalanine residue;

   The "alkylene group" is preferably a C ₁ -C ₁₀ linear or branched alkylene group, preferably a C ₁ -C ₈ linear or branched alkylene group, preferably selected from a methylene group. , ethylene, propylene, butylene, pentylene, hexylene; preferably, selected from the group consisting of methylene, ethylene, n-propyl, isopropylidene, n-butylene, and isobutylene a base, a tert-butyl group, a sec-butylene group, a n-n-pentyl group, an isoamyl group, a renepentyl group, a n-pentylene group, an n-hexylene group; preferably, an ethylene group, an n-propylene group, An n-butylene group, a sub-n-pentyl group, or an n-hexylene group.

   Alternatively, the compound represented by the formula (I) is a compound represented by the following formula (IA):

   

   Alternatively, the group represented by the formula (II) is a group represented by the following formula (IIA)

   
2. The compound of the formula (I) or a pharmaceutically acceptable salt thereof according to claim 1, wherein the compound of the formula (I) has a structure represented by the formula (IA).

   

   Optionally,

   R _{1 is} selected from the group consisting of ethylene, propylene, butylene, pentylene, and hexylene;

   R _{2 is} selected from the group consisting of hydrogen, an amino group, a group represented by the following formula (IIA), a group represented by the following formula (III), and a group represented by the following formula (IV).

   

   Wherein X _{1 is} selected from the group consisting of ethylene, propylene, butylene, pentylene, and hexylene;

   R _{3 is} selected from the group consisting of amino groups and carboxyl groups;

   Xaa is selected from the group consisting of a glycine residue, an alanine residue, a β-aminopropionic acid residue, a γ-aminobutyric acid residue, a 6-aminocaproic acid residue, or a deletion; and Ln is selected from Gd or Eu.
3. A compound of formula (I), or a pharmaceutically acceptable salt thereof, according to claim 1 or 2, which is selected from the group consisting of the following compounds 1 - compound 7:

   

   

   
4. A compound of formula (I), or a pharmaceutically acceptable salt thereof, according to any one of claims 1 to 3, wherein

   The pharmaceutically acceptable salt comprises an anionic salt and a cationic salt of a compound of formula I;

   Preferably, the pharmaceutically acceptable salt comprises an alkali metal salt, an alkaline earth metal salt, an ammonium salt of a compound of formula I; preferably, the alkali metal comprises sodium, potassium, lithium, cesium, the alkaline earth metal comprising Magnesium, calcium, strontium;

   Preferably, the pharmaceutically acceptable salt comprises a salt of a compound of formula I with an organic base; preferably, the organic base comprises a trialkylamine, pyridine, quinoline, piperidine, imidazole, picoline, two Methylaminopyridine, dimethylaniline, N-alkylmorpholine, 1,5-diazabicyclo[4.3.0]nonene-5 (DBN), 1,8-diazabicyclo[5.4.0] Undecen-7 (DBU), 1,4-diazabicyclo[2.2.2]octane (DABCO); preferably, the trialkylamine comprises trimethylamine, triethylamine, N-ethyl Diisopropylamine; preferably, the N-alkylmorpholine comprises N-methylmorpholine;

   Preferably, the pharmaceutically acceptable salt comprises a salt of a compound of formula I with an acid; preferably, the acid comprises an inorganic acid, an organic acid; preferably, the inorganic acid comprises hydrochloric acid, hydrobromic acid, hydrogen iodine Acid, sulfuric acid, nitric acid, phosphoric acid, carbonic acid; preferably, the organic acid includes formic acid, acetic acid, propionic acid, oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, citric acid, Tannic acid, tartaric acid, carbonic acid, picric acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, glutamic acid, pamoic acid.
5. A nuclear magnetic resonance imaging agent comprising the compound of formula (I) according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof; optionally, the nuclear magnetic resonance imaging agent is used Detection of estrogen receptors;

   Optionally, the nuclear magnetic resonance imaging agent is used for the diagnosis of a disease associated with estrogen receptor expression; alternatively, the disease is a pituitary adenoma.
6. The use of the compound of the formula (I) according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, or the nuclear magnetic resonance imaging agent according to claim 5, for nuclear magnetic resonance imaging, detection;

   Optionally, the nuclear magnetic resonance detection comprises estrogen receptor imaging, estrogen receptor detection, or pituitary adenoma detection.
7. Use of a compound of formula (I) according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, or a nuclear magnetic resonance imaging agent according to claim 5 for the diagnosis of disease;

   Optionally, the disease is a disease associated with estrogen receptor expression, optionally the pituitary adenoma.
8. Use of a compound of formula (I), or a pharmaceutically acceptable salt thereof, according to any one of claims 1 to 4, or a nuclear magnetic resonance imaging agent according to claim 5, for the preparation of a disease diagnostic reagent;

   Optionally, the disease is a disease associated with estrogen receptor expression, optionally the pituitary adenoma.
9. A nuclear magnetic resonance imaging method using the compound of the formula (I) according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, as a nuclear magnetic resonance imaging agent.
10. A method of nuclear magnetic resonance detection of an estrogen receptor, wherein the compound of the formula (I) according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, is used as a nuclear magnetic resonance imaging agent.

Images