阅读说明：本技术 一种与Aβ寡聚体具有亲和力的荧光化合物及制备方法与应用 (Fluorescent compound with affinity with Abeta oligomer as well as preparation method and application thereof ) 是由 李玉艳 曾繁天 徐云根 杨剑 刘正世 李小芳 任陇飞 于 2019-05-24 设计创作，主要内容包括：本发明公开了一种与Aβ寡聚体具有亲和力的荧光化合物,并公开了其制备方法以及应用。本发明获得的荧光化合物最大激发和荧光发射光谱值分别在550～560nm和600～700nm之间,大部分的化合物的发射光谱值>650nm；适合用于阿尔茨海默氏病可溶性Aβ寡聚体成像的近红外荧光分子探针的特性,适合应用于制备NIRF探针,特别是靶向可溶性Aβ寡聚体的NIRF探针,该探针可用于阿尔兹海默症的早期诊断。(The invention discloses a fluorescent compound with affinity with Abeta oligomer, and discloses a preparation method and application thereof. The maximum excitation and fluorescence emission spectrum values of the fluorescent compound obtained by the invention are respectively between 550-560 nm and 600-700 nm, and the emission spectrum values of most compounds are more than 650 nm; the near infrared fluorescent molecular probe is suitable for imaging the soluble Abeta oligomers of Alzheimer's disease, is suitable for preparing NIRF probes, particularly the NIRF probes targeting the soluble Abeta oligomers, and can be used for early diagnosis of Alzheimer's disease.)

1. A compound shown as a formula I is provided,

wherein R1 is-NRaRb, Ra and Rb are independently selected from hydrogen, C1-6 alkyl or C1-6 hydroxyalkyl, but Ra and Rb are not hydrogen at the same time;

R₂is phenyl, C3-6 cycloalkyl or C_1-4An alkyl group;

r3 is hydrogen, C1-6 alkyl, wherein R3 represents mono-or di-substitution;

x is a C atom or a N atom;

n＝1、2、3、4。

2. a compound of claim 1, wherein R is_a、R_bAnd R₃C as described in (1)_1-6Alkyl is methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl or tert-butyl.

3. The compound of claim 1, wherein said C3-6 cycloalkyl of R2 is cyclopropyl, cyclobutyl, cyclopentyl, or cyclohexyl.

4. A compound according to claim 1, wherein R1 is selected from N, N-dimethylamino, N-diethylamino, N-methylhydroxyethylamino.

5. The compound of claim 1, wherein the compound is selected from any one of the following compounds:

6. a process for the preparation of a compound as claimed in any one of claims 1 to 5, comprising the steps of: in an organic solvent, under the catalysis of alkali and in the presence of a dehydrating agent, carrying out a condensation reaction on a compound shown as a formula 3 and a compound shown as a formula 4, wherein the reaction formula is as follows:

7. the process according to claim 6, wherein the organic solvent is a benzene-based solvent, the base is tetrahydroisoquinoline, and the dehydrating agent is glacial acetic acid.

8. The method according to claim 6, wherein the molar ratio of the base to the compound represented by the formula 3 is 0.1 to 0.9: 1, and the molar ratio of the dehydrating agent to the compound represented by the formula 3 is 0.1 to 0.9: 1.

9. The method according to claim 6, wherein the compound represented by formula 3 is prepared by:

10. use of a compound according to any one of claims 1 to 5 in the preparation of a NIRF probe.

Technical Field

The invention belongs to the field of specific molecular recognition diagnostic reagents, and particularly relates to a fluorescent compound with affinity with Abeta oligomers, and preparation and application thereof.

Background

Alzheimer's Disease (AD) is the most common degenerative disease of the nervous system, with clinical manifestations of decreased cognitive and memory function, diminished daily living capacity, and various neuropsychiatric symptoms and behavioral disorders. About 4700 million of AD patients worldwide are expected to reach 1.31 hundred million by 2050 due to aging population. The etiology has not been clear to date. Clinically, it is mainly diagnosed by evaluating the cognitive impairment of the patient, and at this time, the diagnosed patient often has entered the middle or late stage of the disease course and treatment is delayed. The lack of effective detection means has become a significant obstacle to the early diagnosis and treatment of AD. Therefore, an effective and reliable diagnosis means is developed, the survival rate of patients is improved by early diagnosis and early treatment of AD, and the method has higher clinical application value.

In the brains of AD patients, the deposition of β -amyloid plaques in the brain is one of the most prominent pathological hallmarks of AD. However, more and more studies have shown that the size of a β plaques is less correlated with the severity of AD. Plaques in the brain do not necessarily progress to AD. Compared with insoluble precipitated plaques, soluble A beta oligomer has stronger neurotoxicity than plaques, on one hand, the A beta oligomer with smaller volume has large contact area with neurons, can enter synaptic cleft and gather on cell membranes and lipid valves to cause membrane division and damage; on the other hand, a β oligomers bind to receptors on cell membranes, induce endocytosis into cells, affect synaptic signaling, eventually leading to synaptic loss and impaired memory. In addition, a β oligomers can also cause abnormal depolarization of mitochondrial cell membranes, releasing the apoptosis factor cytochrome C, which has been shown to be closely associated with synaptic dysfunction and impaired memory.

Over the past decades, more molecular probes with radioactivity directed to a β plaques have entered clinical trials and related PET imaging agents are on the market (Florbetapir, Florbetaben, Flutemetamol), however, the application of the radioactive imaging method is also limited by factors such as the radiation emitted by radioactive elements causing some radiation damage to the human body, requiring expensive equipment and specialized personnel. In contrast, optical imaging has many advantages such as safety, no radioactivity, short data acquisition time, and low cost, and has been widely regarded in medical diagnosis and the like in recent years. Especially near infrared fluorescence imaging technology, its advantage lies in: (1) the sensitivity is high, and the detection of weak signals can be realized; (2) the detection is safe and does not contact radioactive elements; (3) time is not needed, and real-time imaging is realized in the data acquisition process; (4) the cost is moderate, and expensive equipment and high-tech personnel are not needed; (5) when the NIRF probe is combined with the target protein, the fluorescence characteristics (such as fluorescence intensity, emission wavelength and quantum yield) are changed remarkably, and the interaction between the probe and the target protein can be directly detected without connecting a reporter group. At present, probes targeting insoluble plates have been studied more successfully, such as CRNAD-2, AOI987, NIAD-16, DANIR-2C, DBA-SLOH and the like. However, designing NIRF probes that specifically target soluble Α β oligomers presents significant difficulties.

Disclosure of Invention

The purpose of the invention is as follows: in view of the above technical problems in the prior art, the present application provides a fluorescent compound that can selectively bind to soluble a β oligomers, and provides a preparation method and applications thereof.

The technical scheme is as follows: the invention discloses a compound shown as a formula I,

wherein R1 is-NRaRb, wherein Ra and Rb are independently selected from hydrogen, C1-6 alkyl or C1-6 hydroxyalkyl, but Ra and Rb are not hydrogen at the same time;

R₂is phenyl, C3-6 cycloalkyl or C_1-4An alkyl group;

r3 is hydrogen, C1-6 alkyl, wherein R3 represents mono-or di-substitution (e.g., di-substitution at the 3, 5-position);

x represents a C atom or a N atom;

n＝1、2、3、4。

further, in the above-mentioned case,R_a、R_band R₃C as described in (1)_1-6Alkyl is preferably C_1-4The alkyl group, further, said C1-4 alkyl group is preferably a methyl group, an ethyl group, an n-propyl group, an isopropyl group, an n-butyl group, an isobutyl group or a tert-butyl group.

Further, the C3-6 cycloalkyl group as described in R2 is preferably a cyclopropyl group, a cyclobutyl group, a cyclopentyl group or a cyclohexyl group. Further, R2 is preferably hydrogen, 3-ethyl or 2, 5-dimethyl.

R1 is preferably N, N-dimethylamino, N-diethylamino or N, N-methylhydroxyethylamino.

Preferably, the compound described herein is selected from any one of the following compounds:

the invention also discloses a preparation method of the compound shown in the formula I, which comprises the following steps: in an organic solvent, under the catalysis of alkali and in the presence of a dehydrating agent, carrying out a condensation reaction on a compound shown as a formula 3 and a compound shown as a formula 4, wherein the reaction formula is as follows:

the organic solvent may be an organic solvent conventional in the art for such reactions, preferably a benzene-based solvent, wherein the benzene-based solvent is preferably toluene. Preferably, the volume molar ratio of the compounds represented by the formulas 3 and 4 is 1: 1.

The base may be one conventional in such reactions in the art, preferably tetrahydroisoquinoline (i.e., 1, 2, 3, 4-tetrahydroisoquinoline). The amount of the base to be used is not particularly limited, and is generally a catalytic amount, and preferably a molar ratio thereof to the compound represented by the formula 3 is 0.1 to 0.9: 1, more preferably 0.1 to 0.3: 1.

The dehydrating agent may be one conventional in the art for such reactions, preferably glacial acetic acid. The amount of the dehydrating reagent to be used may be an amount conventionally used in such reactions in the art, and it is preferably in a molar ratio of 0.1 to 0.9: 1, more preferably 0.2 to 0.5: 1, to the compound represented by the formula 3.

The molar ratio of the compound represented by the formula 3 to the compound represented by the formula 4 is not particularly limited as long as the reaction is not affected, and is preferably 1: 1.

The temperature of the condensation reaction may be a temperature conventional in the art for such reactions, preferably room temperature (0-30 ℃). The progress of the condensation reaction can be monitored by detection methods (e.g., TLC, GC, HPLC, NMR, etc.) which are conventional in the art for organic synthesis reactions, and is usually determined as the end point of the reaction when the compound represented by formula 4 disappears. The time for the condensation reaction is preferably 2 to 6 hours (e.g., 3 hours).

Further preferably, the compound represented by the above formula 4 can be prepared by the following steps:

the invention also discloses application of the compound in preparing an NIRF probe.

Further, the use of the compounds for the preparation of NIRF probes targeting soluble A beta oligomers.

The probe can be used for early diagnosis of Alzheimer's disease.

Has the advantages that: the invention discloses a fluorescent compound with a novel structure and further discloses a preparation method thereof, the maximum excitation and fluorescence emission spectrum values of the fluorescent compound obtained by the invention are respectively between 550-560 nm and 600-700 nm, and the emission spectrum values of most compounds are more than 650 nm; the properties of near infrared fluorescent molecular probes suitable for imaging alzheimer's disease soluble Α β oligomers; the in vivo and in vitro experiments prove that the in vivo metabolism is stable, the affinity with soluble Abeta oligomer is good, and the in vivo imaging result shows that the near infrared fluorescence imaging can be performed on the amyloid protein in the brain of AD mice with the age of 4 months, and the AD mice with the age of 4 months can be distinguished from normal mice, so that the probe is suitable for preparing a NIRF probe, particularly a NIRF probe targeting the soluble Abeta oligomer, and the probe can be used for early diagnosis of Alzheimer's disease.

Drawings

FIG. 1 is a fluorescence emission spectrum of the most preferred fluorescent compounds of the present invention before and after mixing with A β 42 monomers, A β 40 monomers, A β 42 oligomers, polymers, BSA;

FIG. 2 is a graph of MTT cell viability assay for the most preferred fluorescent compounds of the present invention;

FIG. 3 is a graph showing the binding constants of the most preferred fluorescent compounds of the present invention to A β 42 oligomers;

FIG. 4 is a graph showing the stability of the most preferred fluorescent compounds of the present invention to mouse serum;

FIG. 5 is a near infrared image of a live small animal most preferably a fluorescent compound;

FIG. 6 is a transmission electron microscope image.

Detailed Description

The present invention will be described in detail with reference to specific examples.

The compound shown in the formula I can be prepared by the following steps: