Dihydroquinoline compounds, preparation methods thereof, pharmaceutical compositions and uses thereof

Abstract

The present invention discloses a dihydroquinoline compound, a preparation method thereof, a pharmaceutical composition and an application thereof. The structure of the compound is as shown in Formula I, and it also includes its pharmaceutically acceptable salts. This kind of compound has good affinity and selectivity for Aβ42 oligomers; at the same time, it has appropriate liposolubility, can penetrate the blood-brain barrier in vivo, has stable metabolism and can identify the pathological characteristics of AD; in addition, it also has the imaging characteristics of a near-infrared fluorescent molecular probe and can be used for the early diagnosis of Alzheimer's disease.

Description

Technical Field

[0001] The present invention relates to a dihydroquinoline compound, a preparation method thereof, a pharmaceutical composition and an application, and in particular to a dihydroquinoline compound that can be prepared into a near-infrared fluorescence probe, a preparation method thereof, a pharmaceutical composition and an application. Background Art

[0002] Patients with Alzheimer's disease (AD) have decreased cognitive function, reduced activities of daily living, and are accompanied by various neuropsychiatric symptoms and behavioral disorders. In the brains of patients with Alzheimer's disease, two toxic proteins are generally found, β-amyloid protein (which forms plaques) and tau protein (which forms tangles). The development of AD can be divided into three stages: preclinical stage, mild cognitive impairment, and dementia stage. In the preclinical stage, the lesions of brain neurons are relatively mild and patients generally have no obvious symptoms. By the time of mild cognitive impairment and dementia stage, when clinical symptoms appear, many neurons have already apoptosed and it is difficult to treat the disease. Therefore, diagnosing AD patients in the preclinical stage in a timely manner will win a time window for treatment and is extremely crucial for slowing down or reversing the development of the disease.

[0003] Currently, the main methods used clinically to diagnose AD patients include: neuropsychological clinical feature evaluation, magnetic resonance imaging (MRI), computed tomography (CT), positron emission tomography (PET), and toxic protein tracers. For neuropsychological clinical feature evaluation, the cognitive part of the AD assessment scale (ADAS-cog), the clinical dementia rating scale (CDR-SB), the mini-mental state examination (MMSE), etc. are used to evaluate the impairment of patients' memory, thinking skills, and abnormal behaviors and personalities. Magnetic resonance imaging (MRI) and computed tomography (CT) obtain cross-sectional images of the brain and analyze the degree of lesions based on the atrophy changes of the brain volume. Positron emission tomography (PET) uses fluorodeoxyglucose (FDG) to identify brain regions with reduced glucose metabolism. Tracers are used to track β-amyloid protein and tau protein, and a decrease in the monomer 42 of β-amyloid protein (Aβ42) and an increase in the concentration of tau protein in cerebrospinal fluid are detected as diagnostic indicators for AD.

[0004] However, the vast majority of the above diagnostic methods are applicable to diagnosing patients in the mild cognitive impairment and dementia stages, but there is a lack of effective detection means for preclinical patients. Neuropsychological feature diagnosis is the core evidence for clinically determining dementia. However, when dementia, cognitive, and mental symptoms are detected, the patient is already in the middle and late stages of the disease. Similarly, when imaging diagnoses structural brain lesions, the volumes of the hippocampus, cerebral cortex, and amygdala in the brain have shrunk and the number of neurons has been greatly reduced, limiting the efficacy of the disease. Diagnostic techniques that can detect AD patients in the preclinical stage are still an urgent clinical need. Summary of the Invention

[0005] Object of the Invention: The first object of the present invention is to provide a dihydroquinoline compound, the second object is to provide a preparation method of the compound, the third object is to provide a pharmaceutical composition containing the compound, and the fourth object is to provide an application of the compound and its pharmaceutical composition in the preparation of a near-infrared fluorescence probe.

[0006] Technical Solution: The dihydroquinoline compound of the present invention has the structure of Formula I, and also includes its pharmaceutically acceptable salts:

[0007]

[0008] Wherein:

[0009] R is selected from unsubstituted or substituted C1-C6 alkyl, 3-6 membered cycloalkyl, phenyl, and the substituents are selected from halogen, hydroxyl, nitro, cyano, amino, mercapto, C1-C6 alkoxy, C1-C6 haloalkoxy, C1-C6 haloalkyl.

[0010] Preferably, in the structure:

[0011] R is selected from unsubstituted or substituted C1-C4 alkyl, 3-5 membered cycloalkyl, and the substituents are selected from halogen, hydroxyl.

[0012] More preferably, in the structure:

[0013] R is selected from unsubstituted or substituted methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, cyclopropyl, cyclobutyl, and the substituents are selected from hydroxyl.

[0014] Even more preferably, in the structure:

[0015] R is selected from unsubstituted or substituted ethyl, n-propyl, cyclopropyl, cyclobutyl, and the substituents are selected from hydroxyl.

[0016] Preferably, the number of the substituents is selected from 1, 2, 3.

[0017] Specifically, the dihydroquinoline compound of the present invention is selected from any of the following compounds:

[0018]

[0019] Wherein, the pharmaceutically acceptable salt is a salt formed by the compound and an acid selected from any of the following: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, carbonic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, malic acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid, ferulic acid.

[0020] The maximum emission wavelength of near-infrared (NIR) fluorescence imaging probes is in the range of 600 - 1700 nm. In this wavelength range, the fluorescence background of biological tissues is weak, and the probes have high sensitivity. After the NIR probes bind to the target proteins, significant changes occur in their fluorescence properties (such as fluorescence intensity, emission wavelength, and quantum yield). Without the need to connect a reporter group, the interaction with the target proteins can be directly detected. Therefore, NIR small molecule probes have become a very efficient method for studying AD diagnosis.

[0021] Years before the appearance of mild cognitive impairment symptoms in cognitive impairment, the balance of the generation of β-amyloid (Aβ) monomers is disrupted. Aβ42 and Aβ40 misfold and aggregate into neurotoxic Aβ oligomers (AβO) in the brains of AD patients, leading to synaptic damage and neuronal apoptosis. AβO is related to the severity of the disease. Therefore, choosing AβO as a biomarker, developing sensitive and specific active small molecules, and then developing new diagnostic reagents will be of great significance for the timely detection and treatment of AD patients.

[0022] The present invention designs a class of novel structure near-infrared fluorescence probes and their derivatives, which produce enhanced near-infrared fluorescence by targeting amyloid oligomers that appear in the early stage of Alzheimer's disease and are used for the early screening of diseases in vitro and in vivo.

[0023] The preparation method of the dihydroquinoline compounds described in the present invention comprises the following steps:

[0024] Under the catalysis of a base, in an organic solvent, the compound of formula II and the compound of formula III are subjected to a condensation reaction to obtain the compound of formula I;

[0025]

[0026] Wherein, the definition of R is as described above;

[0027] The corresponding acid is salted with the compound I prepared by the above method to obtain the pharmaceutically acceptable salt.

[0028] Specifically, the organic solvent can be a conventional organic solvent in this type of reaction in the art, preferably acetonitrile, ethanol, benzene solvents; preferably, the volume molar ratio of the compound of formula II to the compound of formula III is 1:1.

[0029] The base can be a conventional base in this type of reaction in the art, preferably tetrahydroisoquinoline (i.e., 1,2,3,4-tetrahydroisoquinoline); the amount of the base used is generally a catalytic amount, preferably the molar ratio of the base to the compounds of formula II and formula III is (0.1 - 1):1, more preferably (0.1 - 0.2):1.

[0030] The condensation reaction temperature can be the conventional temperature for such reactions in this field, 0 - 100 °C, preferably 20 - 70 °C; the reaction progress can be monitored by the conventional detection methods for organic synthesis reactions in this field, such as TLC, GC, HPLC or NMR, etc.; the reaction time is preferably 2 - 24 hours, for example, 10 hours.

[0031] More preferably, the preparation method of the compound of formula III is as follows:

[0032]

[0033] The pharmaceutical composition of the present invention comprises the dihydroquinoline compound and a pharmaceutically acceptable carrier, and is specifically made into common pharmaceutical preparations such as pills, ointments, tablets, capsules, syrups, suspensions, oral liquid preparations or injections (subcutaneous injections, intravenous injections, etc.) by adding common pharmaceutical excipients such as fragrances, sweeteners, liquid / solid fillers, diluents, etc.

[0034] The dihydroquinoline compound and its pharmaceutical composition of the present invention are applied to the preparation of a near-infrared fluorescence probe, specifically an active molecular probe targeting amyloid oligomers, as an early detection tracer for Alzheimer's disease.

[0035] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages:

[0036] 1. The compound has good affinity and selectivity for neurotoxic Aβ42 oligomers. Compared with the affinity for non-toxic Aβ monomers, the fluorescence intensity is more than 10 times higher after binding; the logP value is 2.91, having appropriate lipophilicity and being able to penetrate the blood-brain barrier in vivo; the maximum excitation wavelength is 540 nm and the maximum emission wavelength is 610 nm, possessing the characteristics of a near-infrared fluorescence molecular probe for imaging; it is metabolically stable in vivo, capable of performing near-infrared fluorescence imaging and differentiating the pathological features of AD;

[0037] 2. As a near-infrared fluorescence probe, especially for imaging by specifically targeting and binding to Aβ oligomers closely related to the occurrence and development of AD, it can be used for the early diagnosis of Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] Figure 1 It is the fluorescence emission spectra of compound P1 with Aβ42 monomer, oligomer, protein fiber, and PBS;

[0039] Figure 2 It is the binding constant of compound P1 with Aβ42 oligomer;

[0040] Figure 3 It is the near-infrared imaging map of compound P1 in mice;

[0041] Figure 4 It is the synthetic route diagram of compound P1;

[0042] Figure 5 It is for compound P1's 1 1H NMR spectrum. Specific embodiments

[0043] The technical solutions of the present invention will be further described below in conjunction with the embodiments.

[0044] Example 1: Preparation of 2-(3,4-dihydroquinolin-1(2H)-yl)ethanol

[0045]

[0046] Add tetrahydroquinoline (1.59 g / 1.5 mL, 12 mmol), 2-bromoethanol (2.25 g / 1.28 mL, 18 mmol), and sodium bicarbonate (1.3 g, 15.6 mmol) into a 50 mL eggplant-shaped flask. Connect the condenser and heat up to 60 °C. Monitor the reaction by TLC after reacting for 26 h. Post-treatment: Cool the reaction solution to room temperature, filter by suction, wash the filter cake with DCM, and collect the filtrate. Extract with water and collect the organic phase. Wash with saturated brine, dry with anhydrous sodium sulfate, and concentrate under reduced pressure. Purify by column chromatography, and the eluents are PE:EA = 15:1, 10:1, 8:1 in sequence. Finally, 2 g of a pale yellow oily liquid is obtained, and the yield is 95%. 1 1H NMR (300 MHz, Chloroform-d) δ 7.13–7.03 (m, 1H), 6.99 (dd, J = 7.4, 1.6 Hz, 1H), 6.72 (d, J = 8.2 Hz, 1H), 6.64 (td, J = 7.3, 1.1 Hz, 1H), 3.84 (t, J = 5.8 Hz, 2H), 3.47 (t, J = 5.8 Hz, 2H), 3.41–3.31 (m, 2H), 2.81 (t, J = 6.4 Hz, 2H), 2.04–1.92 (m, 2H), 1.87 (s, 1H).

[0047] Example 2: Preparation of ethyl 2-(3,4-dihydroquinolin-1(2H)-yl)acetate

[0048]

[0049] Dissolve 3-1 (1.9 g, 10.71 mmol) in 120 mL of DCM, add EDCI (6.16 g, 32.2 mmol), DMAP (654 mg, 5.36 mmol), and acetic acid (1.61 g / 1.53 mL, 26.8 mmol), react at room temperature for 4 h, and monitor the reaction by TLC thin-layer plate. The developing solvent is PE:EA = 6:1. Post-treatment: Wash the reaction solution successively with water and saturated brine, dry over anhydrous sodium sulfate, concentrate under reduced pressure, and purify by column chromatography. The eluent is PE:EA = 20:1 to obtain 2.27 g of a colorless oily liquid, yield: 97%. 1 H NMR (300 MHz, Chloroform-d) δ 7.09–6.99 (m, 1H), 6.94 (d, J = 7.3 Hz, 1H), 6.60 (dd, J = 16.0, 8.6 Hz, 2H), 4.26 (t, J = 6.3 Hz, 2H), 3.52 (t, J = 6.3 Hz, 2H), 3.43–3.24 (m, 2H), 2.75 (t, J = 6.4 Hz, 2H), 2.04 (s, 3H), 1.94 (p, J = 6.3 Hz, 2H).

[0050] Example 3: Preparation of ethyl 2-(6-formyl-3,4-dihydroquinolin-1(2H)-yl)acetate

[0051]

[0052] After purging the three-necked flask with nitrogen, add 0.75 mL of DMF with a syringe, pre-cool in an ice bath, and add phosphorus oxychloride (0.17 mL, 1.82 mmol) dropwise. Stir at room temperature for 30 min; pre-cool the reaction solution in an ice bath again, and add 3-2 (200 mg, 0.91 mmol) dissolved in 1.5 mL of DMF dropwise. Stir at room temperature for 2 h. Post-treatment: Pour the reaction solution into crushed ice, adjust the pH to alkaline with 2N NaOH, extract with EA, combine the organic phases, wash with saturated brine, dry over anhydrous sodium sulfate, concentrate under reduced pressure, and purify by column chromatography. The eluent is PE:EA = 3:1 to obtain 190 mg of a milky white liquid, yield: 84.4%. 1 H NMR (300 MHz, Chloroform-d) δ 9.71 (s, 1H), 7.59 (dd, J = 8.6, 2.1 Hz, 1H), 7.51 (s, 1H), 6.71 (d, J = 8.6 Hz, 1H), 4.33 (t, J = 6.1 Hz, 2H), 3.67 (t, J = 6.1 Hz, 2H), 3.56–3.40 (m, 2H), 2.83 (t, J = 6.3 Hz, 2H), 2.07 (s, 3H), 2.05–1.96 (m, 2H).

[0053] Example 4: Preparation of 1-(2-Hydroxyethyl)-1,2,3,4-tetrahydroquinoline-6-carbaldehyde

[0054]

[0055] Dissolve W-3 (190 mg, 0.77 mmol) in 8 mL of methanol, and then dropwise add NaOH (50 mg, 1.25 mmol) dissolved in 1 mL of water. Stir at room temperature for 30 min. Post-treatment: Wash the reaction solution with water and saturated brine, dry over anhydrous sodium sulfate, concentrate under reduced pressure, and purify by column chromatography. The eluent is PE:EA = 3:1 to obtain 120 mg of a pale yellow oily liquid. Yield: 92%. 1 HNMR(300MHz,Chloroform-d)δ9.69(s,1H),7.57(dd,J=8.6,2.1Hz,1H),7.50(s,1H),6.75(s,1H),3.94(t,J=5.8Hz,2H),3.61(t,J=5.8Hz,2H),3.57–3.48(m,2H),2.85(t,J=6.3Hz,2H),2.05–1.98(m,2H).

[0056] Example 5: (E)-2-(6-(2-(6-Ethyl-5-(4-ethylphenyl)-2,2-difluoro-2H-1λ3,3,2λ4-dioxaborolane-4-yl)vinyl)-3,4-dihydroquinolin-1(2H)-yl)ethan-1-ol (P1)

[0057]

[0058] Using Y-2 (260 mg, 0.974 mmol) and W-4 (200 mg, 0.974 mmol) as reaction raw materials, the preparation and experimental method of Y-2 refer to CN110615808A. The polarity of the developing agent used during the reaction monitoring is PE:EA = 2:1. Purify by column chromatography, and the polarity of the mobile phase is PE:EA:DCM = 6:1:0.5 (adding a small amount of DCM is to improve solubility and prevent the product from precipitating and adhering to the silica gel column during column chromatography). Collect the product spot, concentrate by rotary evaporation to remove the solvent, and then slurry with petroleum ether to obtain 77 mg of a dark red solid. The yield is 17%.

[0059] 11H NMR (300 MHz, Chloroform-d) δ 8.05 (d, J = 15.1 Hz, 1H), 7.35 (s, 2H), 7.23–7.15 (m, 3H), 7.07 (s, 1H), 6.66 (d, J = 8.8 Hz, 1H), 6.15 (d, J = 15.1 Hz, 1H), 3.91 (t, J = 5.6 Hz, 2H), 3.58 (t, J = 5.7 Hz, 2H), 3.52 (t, J = 5.7 Hz, 2H), 2.78 (dt, J = 12.1, 6.6 Hz, 4H), 2.37 (q, J = 7.5 Hz, 2H), 1.99 (t, J = 5.9 Hz, 2H), 1.37 (t, J = 7.6 Hz, 3H), 1.18 (t, J = 7.5 Hz, 3H).

[0060] HRMS (ESI) C 26 H 30 BF2NO3, [M+Na] + calculated = 476.2185; found = 476.2175.

[0061] Example 6: Determination of the Wavelength of Compound P1

[0062] Determination of the excitation wavelength: A DMSO solution of 10 μM compound P1. When using an ultraviolet spectrophotometer (scanning wavelength range of 300 - 900 nm), first add the solvent DMSO into the cuvette to balance the baseline. After the baseline is stable, add the solution to be measured into the cuvette for full-wavelength scanning measurement. If the peak shape of the obtained picture is normal and there is no plateau, it is the optimal measurement concentration. Select the maximum absorption wavelength of the ultraviolet spectrophotometer as the excitation wavelength of the fluorescence spectrophotometer, which is 540 nm.

[0063] Measurement of the maximum emission wavelength: Place the four-side light-transmitting cuvette filled with the solution to be measured into the fluorescence spectrophotometer to measure the emission wavelength, which is 610 nm.

[0064] Example 7: Fluorescence Study on the Binding of (E)-2-(6-(2-(6-Ethyl-5-(4-ethylphenyl)-2,2-difluoro-2H-1λ3,3,2λ4-dioxaborolan-4-yl)vinyl)-3,4-dihydroquinolin-1(2H)-yl)ethan-1-ol to Amyloid Fibrils, Oligomers and Monomers

[0065] 1. Experimental Method

[0066] Probe concentration in the detection solution: protein concentration = 250 nM:750 nM; Add 960 μL of PBS to a four-sided light-transmitting cuvette, mix well, measure the emission spectrum as a blank control; then add 10 μL of a DMSO solution of 25 μM compound P1, stir well, and measure the emission spectrum; add 30 μL of a HFIP solution of 25 μM Aβ42, stir gently, and measure the emission spectrum.

[0067] The fluorescence detection methods for the binding of compound P1 to Aβ42 oligomers and fibrils are the same as those for monomers.

[0068] 2. Experimental results

[0069] Table 1. Wavelength of compound P1 and selectivity for Aβ protein

[0070]

[0071] Note: I is the fluorescence intensity of P1 itself, I M is the fluorescence intensity of P1 binding to Aβ monomer, I A is the fluorescence intensity of P1 binding to Aβ polymer, I O is the fluorescence intensity of P1 binding to Aβ oligomer, ΔI O / ΔI M is the fluorescence intensity ratio of oligomer to monomer binding, ΔI O / ΔI A is the fluorescence intensity ratio of oligomer to polymer binding.

[0072] As can be seen from Table 1 and Figure 1 Compound P1 has good affinity and selectivity for neurotoxic Aβ42 oligomers, and the fluorescence intensity after binding is more than 10 times higher than that for non-toxic Aβ monomers.

[0073] Example 8: Study on the binding constant of compound P1 to Aβ42 oligomers

[0074] 1. Experimental method

[0075] Fix the protein concentration at 500 nM, change the concentration of the compound solution, so that the concentration of the compound in the cuvette solution at the final measurement is 10, 25, 50, 100, 200 nM respectively (the corresponding concentrations during compound preparation are 1, 2.5, 5, 10, 20 μM), and measure the maximum fluorescence intensity at different compound concentrations. Use the concentration of the compound as the abscissa and the fluorescence intensity obtained under different compound concentration conditions as the ordinate, and use GraphPad Prism 8.0.1 to process the data to calculate the Kd value.

[0076] 2. Experimental results

[0077] The binding constant of compound P1 with Aβ42 oligomers is 43.99 nM,( Figure 2 ).

[0078] Example 9: Study on the Near-Infrared Imaging Performance of Compound P1 in Organisms

[0079] 1. Experimental Method

[0080] Prepare a compound P1 solution (4 mg / kg): a mixed solution of 15% DMSO + 15% polyoxyl castor oil + 70% PBS. After stabilizing at 25 °C for 20 min, inject it into the tail vein. Use 3 six-month-old APP / PS1 mice and 3 littermate controls (WT mice). Inject 100 μL of the above solution into each mouse; all data are measured using an IVIS Spectrum in vivo imaging system (Caliper Life Sciences, Perkin Elmer, Hopkinton, MA). The fluorescence signal intensity analysis is obtained using software 4.2.1. For all mice, select the same-sized ROI (Region of Interest).

[0081] 2. Experimental Results

[0082] The in vivo imaging results of 6-month-old AD mice show that near-infrared fluorescence imaging can be performed, and 6-month-old AD mice can be distinguished from normal mice( Figure 3 ).

[0083] Figure 3 Among them, A is the fluorescence presentation map of WT mice, B is the fluorescence imaging map of APP / PS1 mice, and C is the fluorescence signal intensity curve graph.

Claims

1. A dihydroquinoline compound, characterized in that, Having the structure of formula I and also including its pharmaceutically acceptable salts: Wherein: R is selected from unsubstituted or substituted C1-C6 alkyl, and the substituents are selected from halogen, hydroxy, nitro, cyano, amino, mercapto.

2. The dihydroquinoline compound according to claim 1, wherein In the said structure: R is selected from unsubstituted or substituted C1-C4 alkyl, and the substituents are selected from halogen, hydroxy.

3. The dihydroquinoline compound according to claim 2, wherein In the said structure: R is selected from unsubstituted or substituted methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, and the substituents are selected from hydroxy.

4. The dihydroquinoline compound according to any one of claims 1-3, characterized in that, The number of the substituents is selected from 1, 2, 3.

5. The dihydroquinoline compound according to claim 1, wherein, Selected from any one of the following compounds:

6. The dihydroquinoline compound according to claim 1, wherein The pharmaceutically acceptable salts are salts formed by the said compound and an acid selected from any one of the following: hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, carbonic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, naphthalenesulfonic acid, citric acid, malic acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, succinic acid, fumaric acid, salicylic acid, phenylacetic acid, mandelic acid, ferulic acid.

7. A pharmaceutical composition, characterized in that, Comprising the dihydroquinoline compound as claimed in claim 1 and a pharmaceutically acceptable carrier.

8. The pharmaceutical composition according to claim 7, wherein, Its preparation form is selected from pills, ointments, tablets, capsules, syrups, suspensions, oral liquids or injections.

9. Use of the dihydroquinoline compound as claimed in claim 1 or the pharmaceutical composition as claimed in claim 7 in the preparation of a near-infrared fluorescence probe.

10. The application according to claim 9, wherein The probe is an active molecular probe targeting amyloid oligomers.

11. The application according to claim 9, wherein The said probe is a tracer for early detection of Alzheimer's disease.

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