CN106749184B - 8-aminoquinoline-hydroxycarbazole heterozygotes, process for their preparation and pharmaceutical compositions containing them - Google Patents

Abstract

The invention relates to synthesis of a series of 8-aminoquinoline-hydroxycarbazole heterozygotes. Can selectively chelate copper ions and simultaneously play a good role in protecting neurons, so that the copper ions become candidate compounds for developing medicaments for treating AD.

Description

8-Aminoquinoline-hydroxycarbazole hybrid, its preparation method and pharmaceutical composition

technical field

The present invention relates to the synthesis of a series of 8-aminoquinoline-hydroxycarbazole hybrids. The structural formula is shown in formula (I) in Figure 1. They can selectively chelate copper ions while exhibiting good neuroprotective effects, making them candidate compounds for drug development in AD.

Background technique

Alzheimer's disease (AD), namely senile dementia, is a chronic progressive central nervous system disease, which seriously affects the cognitive function, memory function, visuospatial function and social life ability of patients. With the rapid growth of the world's elderly population, the number of AD patients has also increased year by year. Currently, there are about 46.8 million people suffering from AD, and it is expected that the number of AD patients will increase to 130 million by 2050. The main pathological features of AD are diffuse atrophy of the cerebral cortex, massive reduction of neurons, senile plaques (SPs) formed by β-amyloid, and neurofibrillary tangles (NFTs) formed by hyperphosphorylation of tau protein. At present, the pathogenesis of AD is not very clear. It is generally believed that AD is a multi-causal, multi-gene association and multi-link disease. The existing hypotheses include Aβ cascade hypothesis, tau protein hypothesis, cholinergic defect, metal ion disorder hypothesis, genetic theory and other etiological hypotheses, but none of them can provide a comprehensive explanation for the pathogenesis of AD.

Recent findings report that the dynamics of multiple metal ions, including copper, zinc, iron, and aluminum, play important roles in AD. Copper metal is an essential trace element for our body and an important transition metal for various metabolic pathways. It has been reported in the literature that copper ions can inhibit the excessive activation of excitatory neurotransmitter receptor N-Methyl-D-aspartic acid (NMDA), and can promote Aβ aggregation and amyloid plaque formation. It also has the effect of anti-oxidative damage. 5-Chloro-7-iodo-8-hydroxyquinoline (clioquinol) can reduce brain Aβ deposition in AD mouse model, improve animal cognitive function and improve overall health status, becoming the first anti-AD metal to enter clinical research Ion chelating agent. The 8-hydroxyquinoline derivative PBT-2 can significantly reduce the level of Aβ42 in the cerebrospinal fluid (CSF) of AD _animals . The 8-aminoquinoline derivative PA1673 has high selectivity for copper ions, and has almost no chelating effect on zinc ions. It was able to completely reverse memory loss in mice injected or orally with Aβ. Using 8-hydroxyquinoline and 8-aminoquinoline as the core framework to develop metal ion modulators for AD treatment is a feasible strategy for the development of new AD drugs.

Oxidative stress is an early manifestation in the occurrence and development of AD. Studies have found that oxidative stress is caused by the irregular production of reactive oxygen species in the body, which in turn leads to the death and loss of neurons. Therefore, the search for exogenous antioxidants has become one of the directions in the research and development of AD therapeutic drugs. It has been reported that carbazole derivatives can directly scavenge reactive oxygen species in the body and have strong antioxidant activity. Recent studies have shown that carbazole derivatives can also inhibit Aβ aggregation and inhibit cholinesterase. Carvedilol and P7C3, the carbazole derivatives, have good neuroprotective effects. Taking the anti-oxidative stress as the assumption, the introduction of carbazole as the active site of anti-oxidation has become an important direction of anti-AD drug research and development.

SUMMARY OF THE INVENTION

Metal ions play a key role in the occurrence and development of AD pathology, and metal ion chelators, especially copper ion chelators, are one of the important strategies for developing AD drugs. Therefore, the design of selective copper ion chelators as drugs for the treatment of AD is the focus of the present invention. Based on our research basis, 8-aminoquinoline is a good selective copper ion chelator, and in vivo, 8-aminoquinoline derivatives can reduce divalent copper ions to monovalent copper ions, thereby making divalent copper ions The ions lose their ability to bind to Aβ, and Aβ aggregation decreases. On the other hand, existing studies have shown that carbazole has a good neuronal protective effect and has various properties related to the treatment of AD. Therefore, we designed a hybrid of 8-aminoquinoline and hydroxycarbazole, and estimated the therapeutic properties of the two to achieve a "multi-target, multi-functional" synergistic effect of one drug in AD treatment.

The compounds of the present invention are characterized by the presence of two main units: a carbazole moiety and an 8-aminoquinoline moiety. On the one hand, they can selectively chelate copper ions; on the other hand, they can play a good neuronal protective effect and improve the symptoms of AD, making them candidates for drug development.

The present invention relates to compounds of formula (I) of Figure 1 or tautomers, pharmaceutically acceptable salts, prodrugs or solvates thereof.

Wherein, R ₁ =H, F, Cl, Br; R ₂ =H, OH, OCH ₃ ; R ₁ is at 5-position, 6-position, 7-position or 8-position of carbazole; R ₂ is at quinoline The 5'-position, 6'-position or 7'-position of -O-CH ₂ - is at the 1-position, 2-position, 3-position or 4-position of carbazole.

Unless otherwise indicated, the compounds of the present invention are also intended to include compounds that differ only by the presence of one or more isotopically enriched atoms. For example, compounds of this structure other than the replacement of hydrogen with deuterium or tritium, or the replacement of carbon atoms ^{with13C or14C} -enriched carbon atoms, ^or15N -enriched nitrogen are within the scope of this invention.

Figure 2 Reaction Scheme exemplifies methods for preparing the compounds of the present invention.

The specific preparation method is:

(1) dissolve compound 1 in acetonitrile in Fig. 2, add catalyst potassium carbonate and epichlorohydrin, heat under reflux until the reaction of raw materials is complete, spin dry solvent, extract, and obtain intermediate product 2 after column chromatography purification;

(2) The intermediate product 2 was dissolved in methanol, compound 3 was added, heated to 50° C. until the reaction of the raw materials was complete, the solvent was evaporated to dryness, and the crude product was purified by column chromatography to obtain the final product.

Description of drawings

Figure 1 8-aminoquinoline-hydroxycarbazole hybrid structure of general formula (I).

Fig. 2 The synthetic route of 8-aminoquinoline-hydroxycarbazole hybrid.

Figure 3 The specific synthetic route of P-Z-001.

Figure 4 Protective effect of compounds on L-glutamate-induced HT22 cell death.

Figure 5 UV-Vis absorption spectrum of the interaction between compound P-Z-001 and common metal ions (copper, iron, zinc) in living organisms.

Example

The following examples are provided to further illustrate the invention and should not be construed to limit the scope of the invention.

Example 1: Synthesis of compound P-Z-001

The reaction scheme of Figure 3 lists the method for synthesizing P-Z-001.

4-Hydroxycarbazole (1 g, 5.5 mmol) was dissolved in 20 mL of acetonitrile, catalyst potassium carbonate (1.52 g, 11 mmol) and epichlorohydrin (5.0 mL, 6.4 mmol) were added, and the reaction was heated to reflux until the reaction of the raw materials was complete. The reaction time About 24h. The solvent was evaporated under reduced pressure, the residue was dissolved in 50 mL of ethyl acetate, and washed three times with 20 mL of water each time. The organic layer was separated and washed once with 20 mL of saturated brine, then dried over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain a crude product. The crude product was purified by column chromatography (dichloromethane:petroleum ether=1:1) to give product a (0.86 g, 65%) as a white solid. ¹ H NMR (400 MHz, CDCl ₃ ) δ 8.36 (d, J=7.9, 0.9 Hz, 1H), 8.10 (s, 1H), 7.46-7.39 (m, 2H), 7.35 (t, J=8.0 Hz, 1H), 7.27 (dd, J=5.8, 2.2Hz, 1H), 4.49 (dd, J=11.0, 3.3Hz, 1H), 4.30 (dd, J=11.0, 5.4Hz, 1H), 3.59 (dddd, J =5.6, 4.1, 3.4, 2.6 Hz, 1H), 3.03 (dd, J=5.0, 4.1 Hz, 1H), 2.92 (dd, J=5.0, 2.6 Hz, 1H). ¹³ C NMR (100 MHz, CDCl ₃ ) δ 155.02, 141.01, 138.76, 126.62, 125.14, 123.24, 122.55, 119.78, 112.92, 110.00, 104.09, 101.39, 68.84, 50.40, 44.92.MS ESI: 239.84.

The intermediate product a (300 mg, 1.26 mmol) was dissolved in 15 mL of methanol, 8-aminoquinoline (222 mg, 1.5 mmol) was added, and the mixture was heated to 50° C. until the reaction of the raw materials was complete. The reaction time was about 16 h. The solvent was evaporated under reduced pressure to obtain a crude product, which was purified by column chromatography (petroleum ether:ethyl acetate=10:3) to obtain a pale yellow solid PZ-001 (298 mg, 62%). ¹ H NMR (400MHz, DMSO) δ 11.26 (s, 1H), 8.73 (d, J=3.7Hz, 1H), 8.28 (d, J=7.8Hz, 1H), 8.20 (d, J=8.1Hz, 1H), 7.49 (dd, J=8.2, 4.2Hz, 1H), 7.44 (d, J=8.1Hz, 1H), 7.30 (dq, J=16.0, 8.0, 7.5Hz, 3H), 7.12 (t, J =7.4Hz, 1H), 7.06(dd, J=8.0, 4.0Hz, 2H), 6.76(d, J=7.7Hz, 1H), 6.70(d, J=7.9Hz, 1H), 5.56(s, 1H) ), 4.43-4.34 (m, 1H), 4.26 (d, J=5.1Hz, 2H), 3.68 (d, J=10.2Hz, 1H), 3.48 (dd, J=12.8, 7.0Hz, 1H). ¹³ C NMR(100MHz，DMSO)δ155.31，147.42，145.11，141.60，139.40，138.03，136.45，128.78，128.22，126.95，125.03，122.99，122.20，122.17，119.06，113.85，112.05，110.84，104.90，104.45， 100.95, 70.79, 68.05, 55.38, 46.57.

HRMS ESI(+)m/z calculated for _C24H21N3O2 [ _{M +} H] ⁺ : _384.1713 . Found: 384.1707.

HPLC purity: 98.4%.

Example 2: Biological Assessment

Protective effect of L-glutamate-induced HT22 cell death

The mouse hippocampal neuron cell line HT22 was routinely cultured in DMEM complete medium containing 10% fetal bovine serum at 37°C, saturated humidity, and a carbon dioxide incubator containing 5% CO ₂ and 95% air. Take the cells in the logarithmic growth phase, digest with 0.25% trypsin, resuspend in complete medium, count the cells under a microscope and adjust the cell concentration to 10×10 ⁴ cells/ml, inoculate a 96-well cell culture plate, 100 μL/well , and cultured overnight to allow cells to adhere. The medium in the 96-well plate was aspirated, the compounds to be tested were dissolved in DMSO, diluted with complete medium, and added to the 96-well plate, 100 μL/well. After 30 min of pre-incubation, 2 μL of 100 mM L-glutamate was added. In the model group, 2μL of 100mM L-glutamate was directly added without the test compound. After 24 hours of incubation, 10 μL of 5 mg/mL MTT was added to each well, incubated for 2 hours, the supernatant was discarded, 100 μL of DMSO was added to each well, and the product formazan was fully dissolved by shaking. The survival rate of cells promoted by the formula compound (%)=100%*(A _{test compound} -A _{model group} )/(A _{model group} -A _blank ) was used to calculate the cell survival rate.

Example 3: Biological Assessment

Interaction with common metal ions in living organisms

The total volume of the reaction system was 1 ml, in which the final concentration of the compound to be tested was 20 μM, and the final concentration of the metal ion solution was 20 μM. At the same time, a separate test compound group and a blank group were set up, and the components were mixed and incubated at room temperature for 30 min. Scan the UV absorption spectra of each experimental group in the range of 200-700 nM on a UV spectrophotometer, with one absorption point per 0.5 nM. First, add 1 mL of absolute ethanol to the quartz cuvette to scan the baseline, then scan the absolute ethanol as the solvent control group, and then scan the compound solution to be tested and the compound plus metal ion solution group. At least three independent experiments were performed for each concentration.

Example 4: Biological Assessment

Inhibitory effect of copper ion-induced Aβ aggregation

Aβ _1-42 was dissolved in a 1% ammonia solution to obtain a stock solution, which was stored in a -80°C refrigerator after being subpackaged. Aβ stock solution was diluted with 20 μM HEPES (containing 150 μM NaCl), 10 μL Aβ (25 μM final concentration) was added to 10 μL copper ion (25 μM final concentration) and 10 μL test compound (50 μM final concentration) mixture, and incubated at 37°C 24h. Take 20 μL of sample solution, dilute it to 200 μL with 50 mM glycine-NaOH buffer and mix well, and use the fluorescence intensity scan for 300 seconds ((λ _exc = 450 nm; λ _em = 485 nm). Percent inhibition rate = (1-IF _i / IF _c )×100, IF _i and IF _c represent the fluorescence intensity with and without the inhibitor after subtracting background, respectively.

Table 1. Th-T fluorescence assay to determine the inhibitory effect of compounds on copper ion-induced Aβ aggregation

Claims (4)

1.8-aminoquinoline-hydroxycarbazole heterozygote having the structural formula shown in formula (I):

wherein R is₁＝H，F，Cl，Br；R₂＝H，OH，OCH3；R₁At the 5-, 6-, 7-or 8-position of the carbazole; r₂At the 5 ' -position, 6 ' -position or 7 ' -position of quinoline; the oxygen end of-O-CH 2-is at the 1-, 2-, 3-or 4-position of the carbazole.

2. The method of preparing the 8-aminoquinoline-hydroxycarbazole hybrid of claim 1, comprising the steps of:

(1) will be provided with

Dissolving in acetonitrile, adding catalyst potassium carbonate and epoxy chloropropane, heating and refluxing until the raw materials react completely, spin-drying solvent, extracting, and purifying by column chromatography to obtain intermediate product

(2) Will be provided with

Dissolving in methanol, adding

Heating to 50 deg.C until the raw materials react completely, evaporating solvent, and purifying the crude product by column chromatography to obtain final product

3. The use of the heterobody of claim 1 for the preparation of a medicament for the treatment of alzheimer's disease.

4. The hybrid body of claim 1, wherein the dosage form of the hybrid body for preparing the medicament for treating the Alzheimer disease is tablets, pills, capsules, injections, suspensions or emulsions.

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