CN109988173A - Ligand Compounds of Fluorenone α7 Nicotinic Acetylcholine Receptor and Its Application - Google Patents

Abstract

The embodiment of the present invention provides a ligand compound of the fluorenone α7 nicotinic acetylcholine receptor represented by the following formula (I), wherein X is a 6-10-membered nitrogen-containing heterocyclic group; the nitrogen-containing heterocyclic group Unsubstituted, or substituted by C _1-4 alkyl; R ₁ is selected from amino, nitro, halogen or a radioisotope of halogen, R ₂ is hydrogen; or R ₁ is hydrogen, R ₂ is selected from amino, nitro, halogen or a radioisotope of halogen; and when X is , R ₁ and R ₂ are not selected from amino group, fluorine and radioactive isotope of fluorine. The ligand compounds provided by the present invention have high affinity with α7 nicotinic acetylcholine receptors, and are excellent ligand compounds for α7 nicotinic acetylcholine receptors. After the ligand compound of the basic acetylcholine receptor is radiochemically labeled, it can be used as a PET imaging agent or a SPECT imaging agent.

Description

Ligand Compounds of Fluorenone α7 Nicotinic Acetylcholine Receptor and Its Application

technical field

The present invention relates to the technical field of medicine, in particular to a ligand compound of fluorenone α7 nicotinic acetylcholine receptor and application thereof.

Background technique

Nicotinic acetylcholine receptors (nAChRs) are a class of gated-transmitter ion channels that are ubiquitous in the central nervous system (CNS) and peripheral nervous system (PNS) and are associated with a variety of physiological functions. There are different subtypes of nAChR, which are generally composed of α subunits (eg α2-α10) and β subunits (β2-β4). Among them, α7 nicotinic acetylcholine receptor (α7nAChR) is a homopentamer composed of 5 identical α subunits, which mainly exists in the hippocampus, thalamus and cerebral cortex, which are important for memory, learning, etc. area. Recent clinical studies have found that α7nAChR protein density is reduced in the brains of patients with neurodegenerative diseases such as Alzheimer's Disease (AD) and Parkinson's disease. Ligand and other studies have shown that targeted α7nAChR ligands can improve cognitive ability and auditory gating defects, such as PNU-282987, PHA-543613 and A-582941 and other highly selective α7nAChR agonists improve sensory- Cognitive function in models of gating deficits, short-term working memory, and memory retention.

With the application of SPECT ((Single-Photon Emission Computed Tomography, single photon emission computed tomography) technology and autopsy studies on AD patients, it has been shown that the density of α7nAChR in the brains of healthy and patients is different. Therefore, α7nAChR It can be used as a potential drug target for early diagnosis and treatment of AD, and non-invasive quantification of α7nAChR in humans using radioligands will promote a better understanding of its role in various CNS diseases , thereby simplifying the development of drugs for the treatment of these diseases.

In view of this, the synthesis of α7 nicotinic acetylcholine receptor agonists and radioligands has attracted more and more attention.

SUMMARY OF THE INVENTION

The purpose of the embodiments of the present invention is to provide ligand compounds of fluorenone α7 nicotinic acetylcholine receptors and applications thereof. The specific technical solutions are as follows:

The present invention first provides the ligand compound of fluorenone α7 nicotinic acetylcholine receptor represented by the following formula (I),

Wherein, X is a 6-10-membered nitrogen-containing heterocyclic group; the nitrogen-containing heterocyclic group is unsubstituted or substituted by a C _1-4 alkyl group;

R ₁ is selected from amino, nitro, halogen or a radioisotope of halogen, and R ₂ is hydrogen; or R ₁ is hydrogen, and R ₂ is selected from amino, nitro, halogen or a radioisotope of halogen;

and when X is , R ₁ and R ₂ are not selected from amino group, fluorine and radioactive isotope of fluorine.

As used herein, "halogen" includes fluorine, chlorine, bromine or iodine.

Herein, the "C _1-4 alkyl" refers to a straight-chain or branched alkyl group derived from a hydrocarbon moiety containing 1-4 carbon atoms by removing one hydrogen atom, such as methyl, ethyl, n-propyl , isopropyl, n-butyl, etc.

In some embodiments of the present invention, the 6-10 membered nitrogen-containing heterocyclic group is selected from In the specific implementation process, the heterocyclic group can select the chiral structure of the above group, for example

In some embodiments of the invention, R ₁ is selected from fluorine, iodine or their radioisotopes; R ₂ is hydrogen; or R ₁ is hydrogen and R ₂ is selected from fluorine, iodine or their radioisotopes;

and when X is , R ₁ and R ₂ are not selected from fluorine and its radioactive isotopes.

In some embodiments of the invention, when X is , R ₁ is only iodine or its radioactive isotope, and R ₂ is hydrogen; or R ₁ is hydrogen and R ₂ is only iodine or its radioactive isotope.

In some embodiments of the invention, the radioisotope is selected ^from18F , ^123I , ^{125I or131I} .

In some embodiments of the present invention, the aforementioned fluorenone α7 nicotinic acetylcholine receptor ligand compound is selected from the compounds of the following structures:

In some specific embodiments, the ligand compounds of α7 nicotinic acetylcholine receptors provided by the present invention can act as agonists of α7 nicotinic acetylcholine receptors.

In some specific embodiments, the ligand compounds of α7 nicotinic acetylcholine receptors provided by the present invention can act as partial agonists of α7 nicotinic acetylcholine receptors.

As used herein, an "agonist" is understood to be given its broadest meaning, that is, as any molecule that partially or fully activates at least one biological activity of a target material (eg, an alpha7 nicotinic acetylcholine receptor) . For example, the ligand compounds of α7 nicotinic acetylcholine receptors provided by the present invention can specifically bind to the extracellular domain of α7 nicotinic acetylcholine receptors to induce intracellular signaling, thereby proving that they are useful in preventing or treating cognition. Disorders and efficacy in neurological rehabilitation.

[alpha]7 nicotinic acetylcholine receptors are known to be important in enhancing cognitive functions such as learning, memory and attention. For example, the alpha7 nicotinic acetylcholine receptor is associated with the following disorders: mild cognitive impairment, Alzheimer's disease, age-related and other cognitive disorders, schizophrenia, attention deficit disorder, attention deficit hyperactivity disorder Disorders (ADHD), dementia due to injections or metabolic disorders, dementia with Lewy bodies, seizures such as epilepsy, multiple cerebral infarction, mood disorders, compulsive and addictive behaviors, inflammatory disorders, and control of pain caused by these disorders related diseases and conditions. The activity of alpha7 nicotinic acetylcholine receptors can be altered or modulated by administration of alpha7 receptor ligands, non-limiting examples of which are: antagonists, agonists, partial agonists and inverse agonists . Alpha7 receptor ligands are useful in the treatment and prevention of these and various types of cognitive impairment and other conditions and diseases, while agonists and partial agonists are known to improve cognition in rodents, non-human primates and humans function and attention.

Based on this, the present invention also provides the use of the aforementioned fluorenone α7 nicotinic acetylcholine receptor ligand compound in the preparation of a medicament for preventing or treating cognitive impairment.

As used herein, the term "cognitive impairment" refers to a wide range of degradation in cognitive function or cognitive domains in animals, for example, in working memory, attention and alertness, language learning and memory, visual learning and memory, reasoning and problem-solving, especially, for example, in executive capacity, task processing speed and/or social cognition. Cognitive impairment is known to manifest as attention deficits, disorganized thinking, unresponsive thinking, difficulty understanding, poor concentration, loss of problem-solving skills, inaccurate memory, difficulty expressing and/or synthesizing thoughts, and feeling and behaving or Difficulty in eliminating irrational thinking.

As used herein, the term "treating" has its general meaning, and in particular herein refers to the treatment of a mammalian subject (preferably a human) already suffering from a cognitive impairment disorder described in the present invention with a medicament of the present invention, with a view to treating a The disease produces the effects of treating, curing, relieving, alleviating and the like. Similarly, as used herein, the term "prevention" has its general meaning, and in particular refers herein to mammals who may be afflicted with or are at risk of developing a cognitive impairment disorder according to the present invention. Individuals are treated with the medicament of the present invention in order to prevent, prevent, prevent, block, etc. the disease.

In some embodiments of the invention, the cognitive impairment is selected from the group consisting of early-onset Alzheimer's disease, senile dementia, micro-infarct dementia, AIDS-related dementia, HIV dementia, Lewy bodies related dementia, Down syndrome-related dementia, mild cognitive impairment, age-related memory impairment, recent short-term memory impairment, age-related cognitive impairment, drug-related cognitive impairment, immunodeficiency syndrome-related Cognitive impairment, cognitive impairment associated with vascular disease, schizophrenia, attention deficit disorder, attention deficit hyperactivity disorder, and learning deficit disorder. The senile dementia may be early senile dementia or Alzheimer's type dementia. Lewy body-related dementia may be Lewy body dementia.

The present invention also provides the use of the fluorenone α7 nicotinic acetylcholine receptor ligand compound represented by the following formula (I) in the preparation of a reagent for a SPECT imaging agent,

Wherein, X is a 6-10-membered nitrogen-containing heterocyclic group; the nitrogen-containing heterocyclic group is unsubstituted or substituted by a C _1-4 alkyl group;

R ₁ is the single-photon radionuclide ¹²⁵ I; R ₂ is hydrogen; or R ₁ is hydrogen and R ₂ is the single-photon radionuclide ¹²⁵ I.

In some embodiments of the present invention, the 6-10 membered nitrogen-containing heterocyclic group is selected from

In some embodiments of the present invention, the structure of the ligand compound of the fluorenone α7 nicotinic acetylcholine receptor is shown in the following formula:

The invention also provides the use of the ligand compound of the fluorenone α7 nicotinic acetylcholine receptor represented by the following formula in the preparation of a reagent for a PET imaging agent,

abbreviation

Abbreviations referred to herein are as follows, and for abbreviations referred to but not listed, they have their usual meanings in the art.

DMSO Dimethyl sulfoxide

DPPA Diphenyl Phosphate Azide

Pd ₂ (dba) ₃ tris(dibenzylideneacetone)dipalladium

BINAP (±)-2,2'-bis-(diphenylphosphino)-1,1'-binaphthyl

The ligand compounds provided by the present invention have high affinity with α7 nicotinic acetylcholine receptors, and are excellent ligand compounds for α7 nicotinic acetylcholine receptors. After the ligand compound of the basic acetylcholine receptor is radiochemically labeled, it can be used as a PET imaging agent or a SPECT imaging agent, and has good affinity, strong specificity, high selectivity, brain uptake and metabolic rate Moderate characteristics, with clinical application value.

Description of drawings

In order to explain the embodiments of the present invention or the technical solutions in the prior art more clearly, the following briefly introduces the accompanying drawings that need to be used in the description of the embodiments or the prior art. Obviously, the accompanying drawings in the following description are only These are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained according to these drawings without creative efforts.

Fig. 1 is [ ¹²⁵ I]TM-14 and TM-14 co-injection HPLC chromatogram;

Fig. 2 is [ ¹²⁵ I]TM-4 and TM-4 co-injected HPLC chromatogram;

Fig. 3 is the specific binding curve of [ ¹²⁵ I]ɑ-bgt binding to ɑ7nAChRs membrane protein;

Figure 4 is a Hill line of the binding of [ ¹²⁵ I]ɑ-bgt to receptor membrane proteins.

Figure 5 is the Scatchard line;

Figure 6 is an HPLC chart of stability analysis of the radioligand [ ¹²⁵ I]TM-14 after incubation in fetal bovine serum for 2 hours;

Fig. 7 is the HPLC chart of the stability analysis of the radioligand [ ¹²⁵ I]TM-14 after incubation in normal saline for 2 hours;

Figure 8 is a SPECT image of the radioligand [ ¹²⁵ I]TM-14 in mice.

Detailed ways

The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, but not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts shall fall within the protection scope of the present invention.

Preparation Example 1 Synthesis of Intermediate Compounds 3-8

The synthetic route is as follows:

Step a Synthesize compound 3-6:

Dissolve chromium trioxide (138.0 g, 1.38 mol) in a mixed solution of 120 mL of water and 80 mL of acetic acid, stir to dissolve all of it, and stand by; add 40.0 g of fluoranthene (3-5) (0.2 mol) to the reaction flask and 500mL of acetic acid, and heated to 80-85 ℃, then the chromium trioxide solution was added dropwise to it, the temperature of the reaction system in the reaction flask was controlled at 80-85 ℃, and the temperature of the reaction system in the reaction flask was heated up to 110-120 ℃, after 2 hours of reaction, cooled to room temperature, and poured into 3L of water, a large amount of yellow solid was precipitated, after suction filtration, the obtained solid was dissolved in 600mL of 2M NaOH solution, suction filtered to remove insoluble impurities, and 500mL of water was used. Wash the filter cake; take the filtrate, adjust its pH to about 1.0 with concentrated hydrochloric acid, the yellow solid is precipitated again, and after suction filtration, it is vacuum-dried at 60 ° C to obtain the target product 3-6(9-fluorenone-1-carboxyl acid) (29.5 g, 66.0%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.18 (d, J=7.52 Hz, 1H), 7.74-7.53 (m, 5H), 7.36 (t, J=7.16 Hz, 1H); MS (M+H ⁺ ) :m/z=225.10.

Step b synthesizes compounds 3-7:

9-Fluorenone-1-carboxylic acid (3-6) (15.0 g, 0.067 mol) and 300 mL of water were added to the reaction flask, heated to 80-85 °C, and then 10 mL of Br ₂ (0.23 mol) was added dropwise to it , after the dropwise addition was completed, the reaction was continued for 16 hours, TLC detected (CH ₂ Cl ₂ : CH ₃ OH=10:1) after the reaction was complete, 300 mL of a 10% aqueous sodium bisulfite solution was added, stirred for 30 minutes, and filtered to obtain The yellow solid was dried under vacuum at 50°C to obtain the final product 3-7 (19.5 g, 96.5%). ¹ H NMR (400MHz, CDCl ₃ ) δ 8.22 (d, J=7.04Hz, 1H), 7.85 (s, 1H), 7.73-7.66 (m, 3H), 7.43 (d, J=7.84Hz, 1H) .

Step c: Synthesis of Compounds 3-8:

Under stirring, compound 3-7 (6 g, 19.87 mmol), toluene (60 mL), triethylamine (4.1 mL, 29.8 mmol), DPPA (6.4 mL, 29.8 mmol) and tert-butyl were successively added to the reaction flask. alcohol (10 mL), then heated to 110°C for reflux reaction, TLC detected the reaction after completion (developing solvent, petroleum ether: ethyl acetate=5:1), removed the solvent, and purified with silica gel column (petroleum ether: ethyl acetate=30 : 1) The yellow solid obtained was the product 3-8 (5.5 g, 74%). ¹ H NMR (400MHz, CDCl ₃ ) δ 8.15 (d, J=8.56Hz, 1H), 7.71 (d, J=1.72Hz, 1H), 7.59 (dd, J=7.88Hz, 1.8Hz, 1H), 7.42(dd,J＝7.44Hz,8.36Hz,1H),7.37(d,J＝7.92Hz,1H),7.09(d,J＝7.16Hz,1H),1.55(s,9H).

Preparation Example 2 Synthesis of Intermediate Compounds 3-9

The synthetic route is as follows:

Compound 3-8 (808 mg, 2.16 mmol) was dissolved in 40 mL of acetonitrile, 22 mL of 1M hydrochloric acid (21.6 mmol) was added to it, and the reaction was heated at 82°C for 4 hours. After the reaction was detected by TLC, it was cooled to room temperature and added to it. An appropriate amount of 1M NaOH solution was added to adjust the pH to strong alkalinity, then extracted with ethyl acetate, the organic phase was collected and dried with anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to remove the solvent to obtain a yellow solid that was the product 3-9 (500 mg, 84.5%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.71 (d, J=1.4 Hz, 1H), 7.55 (dd, J=7.8 Hz, 1H), 7.35 (d, J=7.9 Hz, 1H), 7.22 (t , J=7.6Hz, 1H), 6.81(d, J=7.1Hz, 1H), 6.52(d, J=8.4Hz, 1H), 5.54(s, 2H); MS(M+H ⁺ ): m/ z=273.99.

Preparation Example 3 Synthesis of Intermediate Compound 3-26

The synthetic route is as follows:

10 mL of acetonitrile and 1 mL of concentrated hydrochloric acid were added to compound 3-9 (568.2 mg, 2.4 mmol), and the temperature was lowered to -5°C. Under stirring, an aqueous solution of 5 mL of NaNO ₂ (248.4 mg) was added dropwise, and the reaction was carried out for 30 min. The pre-cooled n-dibutylamine (619.2 mg, 4.8 mmol) and potassium carbonate (1.66 g, 12 mmol) in acetonitrile/water (6 mL) were added , 1:1) dropwise into the mixture. The system was warmed to room temperature, and the reaction was continued for 60 minutes, the solvent was concentrated under reduced pressure, and purified by silica gel column chromatography to obtain the product 3-26 (615.5 mg, 62.1%). ¹ H NMR (CDCl3, 600M) δ7.73 (d, J ＝1.8Hz,1H),7.55(dd,J＝1.8,7.9Hz,2H),7.38(d,J＝2.1Hz,1H),7.36(d,J＝1.8Hz,1H),7.34(d,J =1.3Hz,1H),3.83(t,J=7.6Hz,2H),3.76(t,J=7.3Hz,2H),1.75(dp,J=7.5,37.8Hz,4H),1.41(dq,J =7.5,30.7Hz,4H),0.98(dt,J=7.4,19.0Hz,6H); MS(ESI): m/z calcd for C13H8BrNO, 272.98; found, 273.99, 275.98(M+H ⁺ ).

Synthesis of Example 1 Compound TM-9

The synthetic route is as follows:

Step (1) Synthesis of compound BocTM-9

To the reaction flask was added intermediate compound 3-8 (9.0g), n-methylhomopiperazine (4.5g), Pd2(dba _{)3 (} 1.0g), BINAP (1.3g), cesium carbonate (12.5g) ), and toluene (270 mL), the reaction was replaced with nitrogen three times and then heated to 80-85 °C for 16 hours. The progress of the reaction was followed by TLC (dichloromethane:methanol=10:1) until the raw materials were no longer converted, 135 mL of dichloromethane was added to dilute the reaction system, the insolubles were removed by filtration, the solvent was concentrated under reduced pressure, and purified by silica gel column chromatography (DCM/DCM/ MeOH=50/1-10/1), concentrated under reduced pressure to obtain red product TM-9 (5.5g), yield 52.1%. ¹ H NMR (400 MHz, CDCl ₃ ) δ 9.26 (s, 1H), 7.95 (d, J=7.4 Hz, 1H), 7.30 (d, J=7.1 Hz, 1H), 7.28 (d, J=7.6 Hz ,1H),7.12(s,1H),6.90(m,J=8.4Hz,1H),6.85(m,J=7.1Hz,1H),3.23(s,4H),2.54(d,J=7.4, 4H), 2.32 (s, 3H), 1.53 (s, 9H); MS (M+H ⁺ ): m/z=408.33.

Step (2) Synthesis of Compound TM-9

Compound BocTM-9 (808 mg) was dissolved in 40 mL of acetonitrile, and 22 mL of 1M hydrochloric acid was added to it, and the reaction was heated at 82 °C for 4 h. After the reaction was detected by TLC, it was cooled to room temperature, and an appropriate amount of 1M NaOH solution was added to adjust it. pH to strong basicity, then extracted with ethyl acetate, the collected organic phase was dried with anhydrous sodium sulfate, filtered, concentrated under reduced pressure to remove the solvent, and the product TM-9 (500 mg, 84.5%) was obtained as a yellow solid. ¹ H NMR(400MHz, CDCl3)δ7.29(s,1H),7.27(s,1H),7.12(t,J=7.6Hz,1H),6.99(d,J=7.1Hz,1H),6.65( d, J＝8.4Hz, 1H), 6.33(d, J＝7.2Hz 1H), 5.42(s, 2H), 3.61(m, J＝7.44Hz, 2H), 3.54(t, J＝7.1Hz, 2H) ), 2.72(m, J=7.4Hz, 2H), 2.57(m, J=7.6Hz, 2H), 2.38(s, 3H), 2.02(m, J=8.4Hz, 2H); MS(M+H) ⁺ ): m/z=308.33.

The synthesis of embodiment 2 compound TM-2

The synthetic route is as follows:

Step (1) Synthesis of compound BocTM-2:

According to the synthesis method of step (1) in Example 1, the reaction substrate n-methylhomopiperazine is replaced with cis-2-methylhexahydropyrrolo[3,4-C]pyrrole, and the reaction is carried out to obtain the compound BocTM-2.

The synthesis of step (2) compound TM-2:

Compound BocTM-2 was reacted according to step (2) in Example 1 to obtain compound TM-2. MS (M+H ⁺ ): m/z=320.17.

The synthesis of embodiment 3 compound TM-7

According to the synthetic method of Example 2, the substrate cis-2-methylhexahydropyrrolo[3,4-C]pyrrole was replaced by cis-3,7-diazabicyclo[3.3.0]octane. After the reaction, the compound TM-7 is obtained. MS(M+H ⁺ ): m/z=306.15

Example 4 Synthesis of compound TM-13

According to the synthetic method of Example 1, the substrate n-methyl homopiperazine was replaced with homopiperazine for reaction to obtain compound TM-13. MS (M+H ⁺ ): m/z=294.15.

Example 5 Synthesis of compound TM-17

According to the synthesis method of Example 1, the substrate n-methylhomopiperazine was replaced with n-methylpiperazine for reaction to obtain compound TM-17. MS (M+H ⁺ ): m/z=294.15.

Example 6 Synthesis of compound TM-19

According to the synthetic method of Example 5, the substrate n-methylpiperazine was replaced with piperazine for reaction to obtain compound TM-17. MS (M+H ⁺ ): m/z=280.14.

Example 7 Synthesis of compound TM-16

The synthetic route is as follows:

Step (1) Synthesis of Intermediate Compound 3-20

To the intermediate compound 3-9 (100 mg, 0.36 mmol) was added 10 mL of tetrafluoroboric acid, and the temperature was lowered to 0°C. 5 mL of an aqueous solution of NaNO ₂ (0.1 g) was added dropwise with stirring, and the reaction was carried out for 30 min. After the reaction was completed, suction filtration, and the filter cake was washed with 20 mL of ethanol and 20 mL of methyl tert-butyl ether in turn, and then the filter cake was directly heated to 120° C. for 30 min. The remaining solid was dissolved in 20 mL of ethyl acetate and purified by silica gel column to obtain 3-20 as a pale yellow solid. (35 mg, 36.5%). ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.72 (m, J=8.4 Hz, 1H), 7.46 (s, 1H), 7.20 (d, J=7.6 Hz, 1H), 7.09 (s, 1H), 6.91 (d, J=7.4 Hz), 6.66 (d, J=7.3 Hz, 1H); ¹ 19 NMR (400 MHz, CDCl ₃ ) δ-112.40 (s); MS (M+K+): m/z=318.50.

Step (2) Synthesis of Compound TM-16

Compound 3-10 (100 mg, 0.358 mmol), n-methylpiperazine (98 mg, 0.989 mmol) and cesium carbonate (426 mg, 1.31 mmol) were dissolved in 2 mL of redistilled anhydrous toluene for use; under argon Under protection, Pd ₂ (dba) ₃ (30 mg, 0.033 mmol) and (±)-BINAP (61 mg, 0.099 mmol) were dissolved in 2 mL of redistilled anhydrous toluene, and stirred at 90 °C for 15 min (the reaction mixture was The dark purple turbid liquid turned into an orange-yellow clear liquid), and cooled to room temperature; then the aforementioned solution was added to the reaction system, protected by argon, and stirred at 60°C for 14 h (the reaction mixture changed from orange-yellow clear liquid to brown-red). liquid), cooled to room temperature, added 10 mL of water to quench the reaction, extracted with dichloromethane, collected the organic phase, dried over anhydrous sodium sulfate, filtered, evaporated under reduced pressure to remove the solvent, and purified by silica gel column chromatography (CH ₂ Cl ₂ : CH ₃ OH=20: 1), a purple-black solid TM-16 (36 mg, 36.8%) was obtained. ¹ H NMR (400 MHz, CDCl ₃ ) δ 7.40 (s, 1H), 7.37 (s, 1H), 7.24 (s, 1H), 7.17 (d, J=7.6 Hz, 1H), 6.95 (d, J= 7.4Hz, 1H), 6.81(s, J=7.3Hz, 1H), 3.34(s, 4H), 2.65(s, 4H), 2.41(s, 3H); MS(M+H ⁺ ): m/z = 297.33.

Example 8 Synthesis of compound TM-1

According to the synthetic method of step (2) in Example 7, the intermediate compound 3-20 was reacted with the substrate cis-2-methylhexahydropyrrolo[3,4-C]pyrrole to obtain compound TM- 1. MS (M+H ⁺ ): m/z=323.15.

Example 9 Synthesis of compound TM-5

According to the synthetic method of step (2) in Example 7, the intermediate compound 3-20 was combined with the substrate cis-3,7-diazabicyclo[3.3.0]octano[3,4-C]pyrrole The reaction was carried out to obtain compound TM-5. MS (M+H ⁺ ): m/z=309.13.

Example 10 Synthesis of compound TM-8

According to the synthesis method of step (2) in Example 7, the intermediate compound 3-20 was reacted with the substrate n-methylhomopiperazine to obtain compound TM-8. MS (M+H ⁺ ): m/z=311.15.

Example 11 Synthesis of compound TM-11

According to the synthesis method of step (2) in Example 7, the intermediate compound 3-20 is reacted with the substrate homopiperazine to obtain compound TM-11. MS (M+H ⁺ ): m/z=283.12.

Example 12 Synthesis of compound TM-18

According to the synthesis method of step (2) in Example 7, the intermediate compound 3-20 was reacted with the substrate piperazine to obtain compound TM-18. MS (M+H ⁺ ): m/z=297.13.

Example 13 Synthesis of compound TM-4

The synthetic route is as follows:

Step (1) Synthesis of Intermediate Compound 4-2

Compound 4-1 (100 mg, 0.296 mmol), 1,4-diazabicyclo[3.2.2]nonane (46.9 mg, 0.384 mmol) and cesium carbonate (426 mg, 1.31 mmol) were dissolved in 2 mL of redistilled In aqueous toluene, for use; under argon protection, Pd ₂ (dba) ₃ (30 mg, 0.033 mmol) and (±)-BINAP (61 mg, 0.099 mmol) were dissolved in 2 mL of redistilled anhydrous toluene, 90 After stirring for 15 min under the condition of ℃ (the reaction mixture changed from dark purple turbid liquid to orange-yellow clear liquid), it was cooled to room temperature; then the aforementioned solution was added to the reaction system under argon protection, and after stirring at 60 ℃ for 14 h (reaction The mixture changed from orange-yellow clear liquid to brown-red liquid), cooled to room temperature, added 10 mL of water to quench the reaction, extracted with dichloromethane, collected the organic phase, dried over anhydrous sodium sulfate, filtered, and the solvent was removed by rotary evaporation under reduced pressure, After purification by silica gel column chromatography (CH ₂ Cl ₂ :CH ₃ OH=20:1), 4-2 was obtained as a purple-black solid (37 mg, 36.8%). ¹ H NMR (CDCl3, 400M) δ 7.62 (d, J=2.0 Hz, 1H), 7.47 (dd, J=7.9, 1.9 Hz, 1H), 7.27 (d, J=8.3 Hz, 1H), 7.17 ( d, J=7.8Hz, 1H), 7.05 (d, J=2.6Hz, 1H), 6.75 (dd, J=8.4, 2.6Hz, 1H), 4.07 (dt, J=4.6, 2.3Hz, 1H), 3.56 (t, J=5.7Hz, 2H), 3.31–3.05 (m, 4H), 2.99 (tdd, J=12.8, 6.4, 3.6Hz, 2H), 2.43–2.03 (m, 2H), 1.75 (ddt, J=14.7,9.7,4.7Hz,2H).MS(M+H ⁺ ): m/z=383.07.

Step (1) Synthesis of Intermediate Compound 4-3

Compound 4-2 (100 mg) was added to 50 mL of anhydrous toluene, dissolved and then added with 30 mg of tetrakistriphenylphosphine palladium and 455.5 mg of n-hexabutylditin, heated to 90° C. and refluxed for 18 h. After the reaction was detected by TCL, the product 4-3 (35 mg, 41.5%) was obtained by silica gel column purification. ¹ H NMR(CDCl3, 600M)δ7.64,7.46(dd,J=7.2,0.8Hz,1H),7.28(d,J=5.5Hz,1H),7.27(d,J=4.2Hz,1H), 7.07(d,J＝2.7Hz,1H),6.75(dd,J＝8.3,2.7Hz,1H),4.06(dt,J＝4.5,2.3Hz,1H),3.58–3.51(m,2H),3.20 –3.06(m,4H),2.98(dddd,J=15.1,10.0,5.2,2.4Hz,2H),2.10(tdd,J=12.3,5.1,2.5Hz,2H),1.72(ddt,J=14.7, 9.9, 4.7Hz, 2H), 1.59–1.48 (m, 6H), 1.32 (h, J=7.3Hz, 6H), 1.07–1.03 (m, 6H), 0.88 (t, J=7.4Hz, 9H). 13C NMR(151MHz,CDCl3)δ195.88,150.42,143.10,141.08,133.47,132.47,121.33,118.64,117.50,109.44,77.29,77.08,76.87,57.16,51.92,46.64,44.75,29.77,27.41,27.02,13.73.MS (ESI): m/z calcd for C32H46N2OSn, 594.26; found, 595.2713 (M+H ⁺ ).

Step (3) Synthesis of Compound TM-4

4-3 (100 mg) was dissolved in 10 mL of dichloromethane, and 10 mL of saturated dichloromethane solution of ₁ was added dropwise with stirring at room temperature. After the reaction for 30 min, an appropriate amount of saturated NaHSO ₃ solution was added to quench, and the organic phase was separated and filtered through silica gel. TM-4 (10 mg, 54.2%) was obtained after column purification. ¹ H NMR (CDCl3, 600M) δ 7.83 (d, J=1.7 Hz, 1H), 7.68 (dd, J=7.9, 1.7 Hz, 1H), 7.28 (d, J=8.4 Hz, 1H), 7.07 ( d, J=7.9Hz, 1H), 7.06 (d, J=2.9Hz, 1H), 6.76 (dd, J=8.4, 2.6Hz, 1H), 4.06 (q, J=2.2Hz, 1H), 3.59– 3.52 (m, 2H), 3.15–3.04 (m, 4H), 2.98 (dddd, J=17.3, 7.2, 5.2, 2.4Hz, 2H), 2.13–2.05 (m, 2H), 1.73 (ddt, J=14.6 ,9.6,4.6Hz,2H).13CNMR(151MHz,CDCl3)δ193.54,150.61,145.21,143.19,136.04,135.30,131.20,120.77,117.63,109.40,77.29,77.01,76.87,51.79,46.6.6. .MS(ESI): m/zcalcd for C20H19IN2O, 430.05; found, 431.0620(M+H ⁺ ).

Example 14 Synthesis of compound TM-15

According to the synthesis method of Example 13, the substrate 1,4-diazabicyclo[3.2.2]nonane was replaced with cis-2-methylhexahydropyrrolo[3,4-C]pyrrole and the reaction was carried out , the compound TM-15 was obtained. MS (M+H ⁺ ): m/z=431.05.

Example 15 Synthesis of compound TM-14

The synthetic route is as follows:

Step (1) Synthesis of Compound 3-27

Intermediate compound 3-26 (413.3 mg, 1.0 mmol), 1,4-diazabicyclo[3.2.2]nonane (146.4 mg, 1.2 mmol) and cesium carbonate (651.6 mg, 2.0 mmol) were dissolved in 30 mL In re-distilled anhydrous toluene, set aside; under argon, Pd ₂ (dba) ₃ (45.8 mg, 0.05 mmol) and (±)-BINAP (61 mg, 0.099 mmol) were dissolved in 10 mL of re-distilled anhydrous In water toluene, after stirring for 15min at 90°C (the reaction mixture changed from dark purple turbid liquid to orange-yellow clear liquid), cool to room temperature; then add the aforementioned solution to the reaction system, under argon protection, under 80°C After stirring for 14 hours (the reaction mixture changed from orange-yellow clear liquid to brown-red liquid), it was cooled to room temperature, 10 mL of water was added to quench the reaction, extracted with dichloromethane, the organic phase was collected, dried over anhydrous sodium sulfate, filtered, and reduced in pressure. The solvent was removed by rotary evaporation and purified by silica gel column chromatography (CH ₂ Cl ₂ :CH ₃ OH=20:1) to obtain 3-27 as a red solid (300.1 mg, 65.3%). ¹ H NMR (CDCl3, 600M) δ 7.34–7.26 (m, 1H), 7.27 (dd, J=8.3, 7.2Hz, 1H), 7.13 (dd, J=8.3, 1.0Hz, 1H), 7.11–7.02 (m, 1H), 6.75 (dd, J=8.3, 2.7Hz, 1H), 4.11–4.01 (m, 1H), 3.78 (d, J=38.6Hz, 4H), 3.60–3.50 (m, 2H), 3.16–3.02 (m, 4H), 2.98 (dddd, J=15.1, 10.0, 5.2, 2.3Hz, 2H), 2.10 (dddd, J=12.5, 10.2, 5.0, 2.5Hz, 2H), 1.71 (td, J =10.1,5.1Hz,4H),1.61(t,J=7.6Hz,3H),1.50–1.33(m,3H),0.97(t,J=8.2Hz,6H).13C NMR(151MHz,CDCl3)δ150 .36,150.09,136.75,134.97,131.82,125.11,120.99,117.02,116.98,116.98,115.06,108.90,77.30,77.08,76.87,51.94,46.65,44.71,27.00.MS(ESI):m/z calcd for C28H37N5O,459.30 ;found,460.2950(M+H+).

Step (2) Synthesis of Compound TM-14

3-27 (459.3 mg, 1.0 mmol) was dissolved in 30 mL of acetonitrile, 10 mL of saturated aqueous sodium iodide solution and trifluoroacetic acid (0.5 mL, 6.7 mmol) were added to the system, and the reaction system was heated to 80 ° C and kept at 30 minute. Sodium hydroxide solution was added to quench the reaction, the solvent was concentrated under reduced pressure, extracted with dichloromethane, and purified by silica gel column chromatography. ¹ H NMR (CDCl3, 600M) δ 7.51 (d, J=7.9Hz, 1H), 7.30 (d, J=2.7Hz, 1H), 7.29 (d, J=2.0Hz, 1H), 7.07 (d, J=2.7Hz, 1H), 7.06–6.97 (m, 1H), 6.79 (dd, J=8.4, 2.6Hz, 1H), 4.07 (p, J=2.2Hz, 1H), 3.57 (t, J=5.7 Hz, 2H), 3.10 (dt, J=11.1, 5.0Hz, 4H), 2.98 (dddd, J=15.1, 10.0, 5.2, 2.4Hz, 2H), 2.09 (dtd, J=10.5, 5.2, 2.8Hz, 2H), 1.74 (tt, J=9.7, 4.9Hz, 2H). 13C NMR (151MHz, CDCl3) δ192.68, 150.61, 148.72, 138.28, 135.86, 135.23, 133.95, 129.49, 121.19, 118.62, 117.76, 109.35, 917.76, 109.35 77.30, 77.09, 76.88, 57.09, 51.77, 46.57, 46.55, 44.62, 26.86. MS(ESI): m/z calcd for C20H19IN2O, 430.05; found, 431.0611(M+H+).

Example 16 Synthesis of compound TM-3

According to the synthetic method of Example 15, the substrate 1,4-diazabicyclo[3.2.2]nonane was replaced by cis-2-methylhexahydropyrrolo[3,4-C]pyrrole, and then mixed with The intermediate compound 3-26 is reacted to finally obtain compound TM-3. MS (M+H ⁺ ): m/z=431.05.

Example 17 Synthesis of compound TM-6

According to the synthetic method of Example 15, the substrate 1,4-diazabicyclo[3.2.2]nonane was replaced by cis-3,7-diazabicyclo[3.3.0]octano[3, After 4-C]pyrrole, react with the intermediate compound 3-26, and finally obtain compound TM-6. MS (M+H ⁺ ): m/z=417.04.

Example 18 Synthesis of compound TM-10

According to the synthesis method of Example 15, the substrate 1,4-diazabicyclo[3.2.2]nonane was replaced by n-methyl homopiperazine, and then reacted with the intermediate compound 3-26 to finally obtain compound TM -10. MS (M+H ⁺ ): m/z=419.05.

Example 19 Synthesis of compound TM-12

According to the synthesis method of Example 15, after replacing the substrate 1,4-diazabicyclo[3.2.2]nonane with homopiperazine, it reacts with the intermediate compound 3-26 to finally obtain the compound TM-12. MS (M+H ⁺ ): m/z=405.04.

Example 20 Synthesis of compound [ ¹²⁵ I]TM-14

Compound 3-27 (0.1 mg) was dissolved in 100 μL of acetonitrile, 1 mCi Na ¹²⁵ I and 50 μL of TFA (trifluoroacetic acid) were added respectively, 10 μL of H ₂ O was added, the temperature was raised to 80 °C and reacted for 30 minutes. The saturated sodium hydrogen carbonate solution was quenched, the pH of the saturated sodium hydrogen carbonate solution was adjusted to neutral, and the reaction solution was separated by radio-HPLC (acetonitrile: water = 28:72, flow rate 4 mL/min, detection wavelength 280 nm, inertsil ODS-3 type semi-preparation) Chromatographic column, 5μm, 10mm*250mm). After separation and purification by radio-HPLC, the radiochemical purity of the radioligand was greater than 98%, and the radiolabeling rate was about 63.1% (without decay correction). The purified [ ¹²⁵ I]TM-14 and unlabeled stable Compound TM-14 was co-injected for HPLC analysis, the mobile phase composition was acetonitrile: water (containing 0.2% trifluoroacetic acid) = 70:30, the flow rate was 2 mL/min, the wavelength was 280 nm, and the analytical column was Agela Technologies, Venusil XBP C18 (L ), 5μm, 4.6×250mm; the analysis results are shown in Figure 1, the retention times of [ ¹²⁵ I]TM-14 and TM-14 are 7.257min and 6.931min, respectively, and the retention times of the two match, confirming the accuracy of the radioligand .

Example 21 Synthesis of compound [ ¹²⁵ I]TM-4

To a solution of tributyltin precursor (4-3) (0.2 mg, 0.00034 mmol) in ethanol (200 μl) at room temperature was added 1 mCi Na ¹²⁵ I in 0.1 N NaOH followed by 1 N HCl (50 μl) at room temperature and hydrogen peroxide solution (50 μl). The reaction mixture was kept at room temperature for 30 minutes with intermittent shaking and quenched with saturated sodium bisulfite solution. It was then diluted with a _CH3CN / _H2O mixture (7/3 200 μl) and separated by high performance liquid chromatography (Agela Technologies, 5mm, 4.6×250mm). [ ¹²⁵ I]TM-4 peaked at 50.8 minutes, collected and loaded on a Waters Sep-Pak C18 solid phase extraction cartridge. The column was washed with purified water (10ml), the product was eluted with methanol (10ml), and the solvent was removed in vacuo. The final product was analyzed by high performance liquid chromatography using a 280 nm UV detector and a radioactive detector to determine the radiochemical purity of the synthesized compounds (Figure 2). The total synthesis time was approximately 100 minutes, the radiochemical yield was 84.5% (uncorrected for decay), and the radiochemical purity was greater than 98%.

The present invention can be better understood from the following biological experimental examples. However, those skilled in the art can easily understand that the contents described in the experimental examples are only used to illustrate the present invention, and should not and will not limit the present invention.

Biological Experiment Example 1 Ligand Compound Receptor Affinity Test

1. Preparation of receptor protein and determination of its concentration

The receptor proteins used in the experiment were all isolated and extracted from the brains of female SD rats (180-200 g). After the female SD rats (180-200g) were sacrificed by cervical dislocation, their brains were quickly taken out and placed on ice cubes. After rinsing the blood capillaries with ice-cold saline, the cerebral cortex was dissected out (this area is enriched with ɑ7nAChRs receptor protein), and then placed on ice. in 10 volumes of ice-cold 50 mM Tris-HCl buffer solution (50 mM Tris, 120 mM NaCl, 5 mM KCl, 2 mM CaCl ₂ , 1 mM MgCl ₂ , pH=7.4, 4 °C), then place the beaker in an ice-water bath with a hand-held The mixture was homogenized for 30 s with a tissue homogenizer (set to No. 6). Divide the homogenized membrane solution into three equal parts into 50mL centrifuge tubes, centrifuge with a low-temperature high-speed centrifuge for 20min (4°C, 48000g), discard the supernatant after centrifugation, and dissolve the lower sediment in 10 times the volume of ice-cold The mixture was homogenized, centrifuged and washed in the same manner in 50 mM Tris-HCl buffer solution. After repeating this step for 3 times, the lower precipitate obtained is the receptor membrane protein, which is dissolved in 10-fold volume of ice-cold 50 mM Tris-HCl buffer solution, and homogenized to make it well mixed. Take out 10 μL of the uniformly mixed receptor membrane protein solution, measure the protein concentration by the Lowry method, divide the remaining membrane solution into 2 mL centrifuge tubes, and store them in a -80°C refrigerator for later use.

2. Saturation Binding Experiment of Receptor Membrane Proteins

Saturation binding experiments were performed by measuring the binding of the radioligand [ ¹²⁵ I]ɑ-bungaratoxin ([ ¹²⁵ I]ɑ-bungarotoxin, abbreviated: [ ¹²⁵ I]ɑ-bgt) to mouse brain membrane proteins. In the experiment, 8 different concentration points (0.005-5nM) were set for [ ¹²⁵ I]ɑ-bgt, and each concentration point was in parallel with 3 groups. Take out the receptor membrane protein stored in the -80°C refrigerator, freeze-thaw at 4°C, and add an appropriate volume of ice-cold 50mM Tris-HCl buffer solution (50mM Tris, 120mM NaCl, 5mM after freezing and thawing) according to the determined protein concentration. KCl, 2 mM CaCl ₂ , 1 mM MgCl ₂ , pH=7.4, 4° C.) for dilution. The total volume of the reaction mixture in the total binding tube was 500 μL, including 100 μL of membrane protein solution (final amount of protein in each tube was 1.5 mg), 10 μL of various concentrations of the radioligand [ ¹²⁵ I]ɑ-bgt and 390 μL of ice-cold 50 mM Tris-HCl buffer solution, the order of loading is: membrane protein, Tris-HCl buffer solution and [ ¹²⁵ I]ɑ-bgt (Table 1). Nonspecific binding was determined by 2 μM unlabeled ɑ-bgt, the reaction mixture in test tubes consisted of 100 μL of membrane protein solution (final amount of protein in each test tube was 1.5 mg), 10 μL of various concentrations of radioligand [ ¹²⁵ I ]ɑ-bgt, 100 μL of 2 μM of ɑ-bgt and 290 μL of ice-cold 50 mM Tris-HCl buffer solution for a total volume of 500 μL in the order of: membrane protein, Tris-HCl buffer solution, ɑ-bgt and [ ^125I ]ɑ -bgt (Table 2). After adding the sample, seal the test tube with parafilm, vortex for a few seconds to fully mix, and then incubate in a constant temperature incubator at 37°C for 2.5 hours. After incubation, take out the test tube and place it in an ice-water bath. To stop the binding of receptor protein and ligand, the mixture was filtered with a 48-well cell harvester onto Whatman GF/B filter paper (soaked in 0.5% polyacetimide solution for 2.5h in advance). Rinse three times with 50mM Tris-HCl buffer solution, remove the filter paper, cut the filter paper piece and place it in the measuring PE tube, and measure the count with γ-counter. The difference between total binding (TB) and non-specific binding (NSB) is specific binding (SB), ie SB(cpm)=TB(cpm)-NSB(cpm).

Table 1 Saturated Binding Experiment Total Binding Tube Loading Table

Table 2 Saturated Binding Experiment Non-specific Binding Tube Loading Table

3. Competitive Binding Experiment

To quantitatively determine the affinity of ligand compounds to ɑ7nAChRs, we performed in vitro competitive binding experiments using [ ¹²⁵ I]ɑ-bgt as a radioactive standard. In the experiment, membrane protein solution (the amount of protein in each reaction tube was 1.5 mg), 0.4 nM [ ¹²⁵ I]ɑ-bgt solution and a series of different concentrations (3 groups of each concentration were measured in parallel) of unlabeled ligands The solution was co-incubated in a constant temperature incubator at 37°C for 2.5 hours. After the incubation was completed, it was processed in the same manner as in the aforementioned saturation binding experiment and counted by γ-counter. At the same time, in order to ensure the accuracy and reliability of this experimental system, MLA (JMward, VB Cockcroft, GGLunt, FSSmillie, S. Wonnacott, Methyllycaconitine: aselective probe for neuronal alpha-bungatoxin binding sites, FEBS LETT, 270 (1990) 45- 8.) (known high-selectivity, high-affinity ligands for ɑ7nAChRs) was used as a reference ligand, and its affinity with ɑ7nAChRs in mouse brain was determined. The preparation method and loading method of the ligand compound are shown in Table 3 and Table 4 below:

The preparation method of table 3 ligand compound

Table 4 Sample addition table for competition binding experiment

Note: The order of loading is: protein, Tris-HCl buffer solution, drug (ligand compound or MLA), [ ¹²⁵ I]ɑ-bgt.

4. Experimental results:

In the saturation binding experiment, the radioligand [ ¹²⁵ I]ɑ-bgt was established at 8 different concentrations (0.005-5 nM), and each concentration was measured in parallel in 3 groups. The experimental results showed that the specific binding of [ ¹²⁵ I]ɑ-bgt to the receptor membrane protein rapidly reached saturation within the determined concentration range, and its specific binding curve was shown in Figure 3; according to the specific binding curve and The formula log[B/(B _max -B)]=n _H log[L]-logK _d plots log[B/(B _max -B)] against log[L] to obtain the Hill straight line (as shown in Figure 4) : y=1.05828x+0.08731 (R=0.99031), the slope is the Hill coefficient n _H =1.058, indicating that the binding of [ ¹²⁵ I]ɑ-bgt to receptor membrane protein is a simple single-site interaction system.

According to the Scatchard equation of the single-site action system: B/F=-B/K _d +B _max /K _d , the specific binding amount B is plotted against the corresponding B/F (as shown in Figure 5), and the linear regression equation y is obtained =46.42857-1.2987x(R=1), according to this equation, the equilibrium dissociation constant of [ ¹²⁵ I]ɑ-bgt under the experimental conditions is K _d =0.77±0.088nM (95% confidence interval is 0.36-1.172 nM), the maximum binding amount was _Bmax = 35.75±4.64 fmol/mg protein (95% confidence interval 29.88-41.62 fmol/mg protein). The experimental results are consistent with the data reported in the literature (K _d = 1.5 ± 0.7 nM, B _max = 63 ± 17 pmol/mg protein), indicating that the assay method used in this paper is credible and can be used in the future Assays to measure the biological activity of compounds.

In the competition binding experiment, MLA was selected as the reference ligand, and the affinity of MLA and the designed test compound for ɑ7nAChRs was determined simultaneously under the same experimental conditions. The reference ligand MLA and a series of test compounds were established at 8 different concentrations (10 ^-3 -10 ^-10 mol/L), by inhibiting 0.4nM [ ¹²⁵ I]ɑ-bgt (K _d =0.77±0.088nM) on Binding of ɑ7 nAChRs was determined with _IC50 values, and the respective Ki values were calculated by the Cheng-Prusoff formula (K _i =IC ₅₀ /(1+[L]/K _d ) ₎ . The measurement results are shown in Table 5. Under this experimental condition, the inhibitory constant K _i of MLA = 2.88 ± 0.78 nM, which is consistent with literature (JMward, VB Cockcroft, GGLunt, FSSmillie, S. Wonnacott, Methyllycaconitine: aselective probe for neuronal alpha-bungarotoxin binding sites, FEBS LETT, 270 (1990) 45-8.) reported K _i values (1.09 ± 0.09 nM) that were substantially similar, indicating the feasibility of the experimental method employed in this paper.

Table 5. In vitro binding affinity (K _i , nM) of MLA and each tested ligand compound to ɑ7 nAChRs

[a] Mean ± standard deviation of three measurements

As can be seen from Table 5, each ligand compound showed affinity for the ɑ7nAChR membrane protein, and the inhibition constant (K _i ) was distributed in the range of 2.23-521.1 nM, among which compounds TM-14 and TM-4 showed an affinity for [ ¹²⁵ I]α-bungaratoxin showed a strong inhibitory effect with K _i values of 2.23±0.56nM and 9.26±0.37nM, respectively.

Biological experiment example 2 In vitro stability experiment of [ ¹²⁵ I]TM-14

The in vitro stability of radioligands is of great significance for further in vivo studies, usually in saline and animal serum. The specific method is as follows: take 10 μCi of HPLC-purified radioligand [ ¹²⁵ I]TM-14 and 100 μL fetal bovine serum and incubate them at 37°C for 1 h and 2 h respectively. Then centrifuge at 4°C for 5 min (7000 rpm), collect the supernatant, filter through a membrane, and take 100 ^μL for HPLC analysis; After incubation for 1 h and 2 h at room temperature, the analysis was performed directly by HPLC.

The experimental results of the in vitro stability of the radioligand [ ¹²⁵ I]TM-14 are shown in Figures 6 and 7. It can be seen from the figures that [ ¹²⁵ I]TM-14 showed good performance in both normal saline and fetal bovine serum. stability. Under the condition of 37°C, after incubation in fetal bovine serum for 2 hours (as shown in Figure 6), the radiochemical purity was greater than 98%; at room temperature, after incubation in normal saline for 2 hours (as shown in Figure 7), the radiochemical purity was still greater than 98%.

Biological experiment example 3 [ ¹²⁵ I]TM-14 in vivo distribution experiment

3.1 Biodistribution experiments

HPLC-purified [ ¹²⁵ I]TM-14 (1 μCi, dissolved in 0.1 mL of normal saline, containing 5% DMSO) was injected into normal Kunming mice (18-22 g, female, n=5) by tail vein injection. ), mice were sacrificed by decapitation at 5min, 15min, 30min, 60min, 90min, 120min respectively, and the blood, brain, heart, lung, liver, spleen, lung, kidney, muscle, bone, intestine, stomach and tail were dissected and taken out. , Weigh the wet weight of each organ and measure its count with γ-counter, the uptake of each tissue is finally expressed as %ID/g, %ID/g=ID/g÷1%, where ID/g=tissue radioactivity Counts (counts)÷tissue mass (mg), 1%=mean value of 1% ID in each phase-tail radioactive counts/100, the results of the in vivo distribution of the radioligand are shown in Table 6.

Table 6 [ ¹²⁵ I]TM-14 biodistribution results

The data in the table are the mean ± standard deviation of five measurements;

It can be seen from Table 6 that the ¹²⁵ I-labeled radioligand [ ¹²⁵ I]TM-14 has a very high initial brain uptake in the mouse brain, and its uptake value reaches 6.47% ID/g 5min after injection, 15min After administration, the highest brain uptake value was 9.49%ID/g; at the same time, the radioligand showed a suitable brain clearance rate, and the uptake value in the brain decreased to 6.21%ID/g after 60min and 120min of administration, respectively and 3.26%ID/g, which indicates that the compound has suitable intracerebral kinetic properties; in addition, the uptake value of [ ¹²⁵ I]TM-14 in blood is very low, showing a high brain/blood ratio, which is 8.11 and 8.87 at 15min and 60min, respectively.

Biological experiment example 4 [ ¹²⁵ I]TM-14 SPECT dynamic imaging experiment in mice

The radioligand [ ¹²⁵ I]TM-14 (0.2 mL, 60 μCi) was injected into female Kunming mice (18-22 g) by tail vein injection, and then the mice were anesthetized with 3% isoflurane until coma Afterwards, the mice were fixed on a small animal micro-SPECT/CT imaging device (TriFoil imaging Triumph SPECT/CT) in a prone position, and 1% isoflurane was used to maintain the mice under anesthesia during the scanning imaging. Images were collected 1min-60min after administration, divided into 12 frames, one frame every 5 minutes, to observe the distribution of [ ¹²⁵ I]TM-14 in the mouse brain.

Figure 8 is the coronal, sagittal and transverse micro-SPECT images of the brain of Kunming mice injected with [ ¹²⁵ I]TM-14 for 15 min, 30 min, 60 min and 90 min, respectively. It can be seen from Figure 8 that [ ¹²⁵ I]TM-14 has a high uptake in the mouse brain, and its distribution in the brain is basically consistent with the results of the biological experiment example 3 animal body distribution experiment, the uptake is the highest at 30min, With the prolongation of time, the concentration of radioligand gradually decreased, and at the same time, the retention in the brain was more suitable, and a certain concentration of enrichment could still be observed after 60 min of administration. According to the above-mentioned good imaging results, [ ¹²⁵ I]TM-14 can be used as a SPECT imaging agent for α7nAChR.

Biological experiment example 5 [ ¹²⁵ I]TM-4 in vivo distribution experiment

3.1 Biodistribution experiments

HPLC-purified [ ¹²⁵ I]TM-4 (1 μCi, dissolved in 0.1 mL of normal saline, containing 5% DMSO) was injected into normal Kunming mice (18-22 g, female, n=5) by tail vein injection. ), mice were sacrificed by decapitation at 5min, 15min, 30min, 60min, 90min, 120min respectively, and the blood, brain, heart, lung, liver, spleen, lung, kidney, muscle, bone, intestine, stomach and tail were dissected and taken out. , Weigh the wet weight of each organ and measure its count with γ-counter, the uptake of each tissue is finally expressed as %ID/g, %ID/g=ID/g÷1%, where ID/g=tissue radioactivity Counts (counts)÷tissue mass (mg), 1%=mean value of 1% ID in each phase-tail radioactive counts/100, the results of the in vivo distribution of the radioligand are shown in Table 7.

Table 7 [ ¹²⁵ I]TM-4 biodistribution results

The data in the table are the mean ± standard deviation of five measurements

It can be seen from Table 7 that the ¹²⁵ I-labeled radioligand [ ¹²⁵ I]TM-4 has a very high initial brain uptake in the mouse brain, and its uptake value reaches 4.20% ID/g 5min after injection, 15min After administration, the highest brain uptake value was 7.46%ID/g; at the same time, the radioligand showed a suitable brain clearance rate, and the uptake value in the brain decreased to 4.56%ID/g after 60min and 120min of administration, respectively. and 2.84% ID/g, which indicates that the compound has suitable intracerebral kinetic properties. In addition, the uptake value of [ ¹²⁵ I]TM-4 in blood was very low, showing a high brain/blood ratio of 6.27 and 5.30 at 15 min and 60 min, respectively.

Biological experiment example 6 [ ¹²⁵ I]TM-14 mouse intracranial distribution experiment

HPLC-purified [ ¹²⁵ I]TM-14 (1 μCi, dissolved in 0.1 mL of normal saline, containing 5% DMSO) was injected into normal Kunming mice (28-32 g, female, n=5) by tail vein injection. ), the mice were sacrificed by cervical dislocation at 5min, 15min, 30min, 60min, and 90min, respectively, the brain was quickly dissected out and placed on ice, the blood was removed with ice-cold saline, and then the cortex was dissected out in different regions. , striatum, hippocampus, superior and inferior colliculus, thalamus, cerebellum and cerebellum, the wet weight of each brain region was weighed and the radioactivity count was determined by γ-counter, and the radioligand uptake of each region was finally expressed as %ID/g , %ID/g=ID/g÷1%, where ID/g=tissue radioactivity counts (counts)÷tissue mass (mg), 1%=average of 1% ID per phase, the radioligand The distribution of brain regions is shown in Table 8.

Table 8 [ ¹²⁵ I]TM-14 distribution results in the brain

The data in the table are the mean ± standard deviation of five measurements;

It can be seen from Table 8 that after injecting 1 μCi [ ¹²⁵ I]TM-14 into mice, the radioligand has higher absorption in the cortex and hippocampus where α7nAChR is most enriched, and reaches a peak 30 minutes after administration , were 7.22 ± 0.62% ID/g and 6.02 ± 0.35% ID/g, respectively, and the uptake values in these regions gradually decreased during the subsequent observation period; the regions with moderate uptake were the hypothalamus and thalamus, and the region with the lowest uptake was the cerebellum (region of the mouse brain with the least distribution of α7nAChR). The distribution characteristics of this region are consistent with the distribution of α7nAChR in vitro and in vivo. The uptake and clearance rates of radioactivity in the cerebellum were fast compared to other brain regions. The tissue/cerebellum ratio gradually increased throughout the experiment and reached a peak 90 minutes after administration, indicating that The radioligand had better specific binding to α7nAChR.

The brain region distribution of this radioligand is similar to that of [ ¹²⁵ I]ASEM, and its uptake value in α7nAChR-intensive regions has certain advantages compared with [ ¹²⁵ I]ASEM: [ ¹²⁵ I]ASEM showed some advantages at 20 min after administration. Peak absorption was reached with uptake values of 6.3% ID/g and 5.1% ID/g in the cortex and hippocampus, respectively.

Biological experiment example 7 [ ¹²⁵ I]TM-4 mouse intracranial distribution experiment

HPLC-purified [ ¹²⁵ I]TM-4 (1 μCi, dissolved in 0.1 mL of normal saline, containing 5% DMSO) was injected into normal Kunming mice (28-32 g, female, n=5) by tail vein injection. ), the mice were sacrificed by cervical dislocation at 5min, 15min, 30min, 60min, and 90min, respectively, the brain was quickly dissected out and placed on ice, the blood was removed with ice-cold saline, and then the cortex was dissected out in different regions. , striatum, hippocampus, superior and inferior colliculus, thalamus, cerebellum and cerebellum, the wet weight of each brain region was weighed and the radioactivity count was determined by γ-counter, and the radioligand uptake of each region was finally expressed as %ID/g , %ID/g=ID/g÷1%, where ID/g=tissue radioactivity counts (counts)÷tissue mass (mg), 1%=average of 1% ID per phase, the radioligand The distribution of brain regions is shown in Table 9.

Table 9 [ ¹²⁵ I]TM-4 distribution results in the brain

The data in the table are the mean ± standard deviation of five measurements;

It can be seen from Table 9 that after injecting 1 μCi [ ¹²⁵ I]TM-4 into mice, the radioligand has higher absorption in the cortex and hippocampus where α7nAChR is most enriched, and reaches a peak value 30 min after administration , were 6.55 ± 0.53% ID/g and 5.98 ± 0.35% ID/g, respectively, and the uptake values in these regions gradually decreased during the subsequent observation period; the regions with moderate uptake were the hypothalamus and thalamus, and the region with the lowest uptake was the cerebellum (region of the mouse brain with the least distribution of α7nAChR). The distribution characteristics of this region are consistent with the distribution of α7nAChR in vitro and in vivo. The uptake and clearance rates of radioactivity in the cerebellum were fast compared to other brain regions. The tissue/cerebellum ratio gradually increased throughout the experiment and reached a peak 90 minutes after administration, indicating that The radioligand had better specific binding to α7nAChR.

The brain region distribution characteristics of this radioligand are similar to [ ¹²⁵ I]ASEM, and its uptake value in α7nAChR-dense regions has certain advantages compared with [ ¹²⁵ I]ASEM: [ ¹²⁵ I]ASEM at 20 min after administration Reaching the highest value of absorption, the uptake values of cortex and hippocampus were 6.3% ID/g and 5.1% ID/g, respectively, and then gradually cleared, [ ¹²⁵ I]TM-4 showed better than [ ¹²⁵ I]ASEM The retention and brain dynamics properties of .

The ligand compounds of the fluorenone α-ketonicotinic acetylcholine receptors provided by the present invention and their applications have been introduced in detail above. The principles and implementations of the present invention are described herein by using specific embodiments, and the descriptions of the above embodiments are only used to help understand the method and the central idea of the present invention. It should be pointed out that for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can also be made to the present invention, and these improvements and modifications also fall into the protection of the claims of the present invention.

Claims (11)

1. A ligand compound of the fluorenone α7 nicotinic acetylcholine receptor represented by the following formula (I), Wherein, X is a 6-10-membered nitrogen-containing heterocyclic group; the nitrogen-containing heterocyclic group is unsubstituted or substituted by a C _1-4 alkyl group; R ₁ is selected from amino, nitro, halogen or a radioisotope of halogen, and R ₂ is hydrogen; or R ₁ is hydrogen, and R ₂ is selected from amino, nitro, halogen or a radioisotope of halogen; and when X is , R ₁ and R ₂ are not selected from amino group, fluorine and radioactive isotope of fluorine. 2. The ligand compound of fluorenone α7 nicotinic acetylcholine receptor according to claim 1, wherein the 6-10-membered nitrogen-containing heterocyclic group is selected from the group consisting of 3. The ligand compound of fluorenone class α7 nicotinic acetylcholine receptor according to claim 1, wherein, R ₁ is selected from fluorine, iodine or their radioisotopes; R ₂ is hydrogen; or R ₁ is hydrogen, and R ₂ is selected from fluorine, iodine or their radioisotopes; and when X is , R ₁ and R ₂ are not selected from fluorine and its radioactive isotopes. 4. The ligand compound of fluorenone α7 nicotinic acetylcholine receptor according to claim 1, wherein the radioisotope is selected from ¹⁸ F, ¹²³ I, ¹²⁵ I or ¹³¹ I. 5. the ligand compound of fluorenone class α7 nicotinic acetylcholine receptor as claimed in claim 1, it is selected from the compound of following structure: 6. Use of the fluorenone α7 nicotinic acetylcholine receptor ligand compound according to any one of claims 1-5 in the preparation of a medicament for preventing or treating cognitive impairment. 7. The use of claim 6, wherein the cognitive impairment is selected from the group consisting of early-onset Alzheimer's disease, senile dementia, microinfarct dementia, AIDS-related dementia, HIV dementia , Lewy body-related dementia, Down syndrome-related dementia, mild cognitive impairment, age-related memory impairment, recent short-term memory impairment, age-related cognitive impairment, drug-related cognitive impairment, immunodeficiency syndrome symptom-related cognitive impairment, vascular disease-related cognitive impairment, schizophrenia, attention deficit disorder, attention deficit hyperactivity disorder, and learning deficit disorder. 8. Use of a fluorenone α7 nicotinic acetylcholine receptor ligand compound represented by the following formula (I) in the preparation of a reagent for a SPECT imaging agent, Wherein, X is a 6-10-membered nitrogen-containing heterocyclic group; the nitrogen-containing heterocyclic group is unsubstituted or substituted by a C _1-4 alkyl group; R ₁ is the single-photon radionuclide ¹²⁵ I; R ₂ is hydrogen; or R ₁ is hydrogen and R ₂ is the single-photon radionuclide ¹²⁵ I. 9. purposes as claimed in claim 8, wherein said 6-10 membered nitrogen-containing heterocyclic group is selected from 10. The use according to claim 8 or 9, wherein the structure of the ligand compound of the fluorenone α7 nicotinic acetylcholine receptor is shown in the following formula: 11. Use of the ligand compound of the fluorenone α7 nicotinic acetylcholine receptor shown in the following formula in the preparation of a reagent for a PET imaging agent,