Desloratadine derivative and application thereof in preparation of medicine for Alzheimer's disease

Abstract

The invention discloses a desloratadine derivative and application of the desloratadine derivative in preparation of a medicine for treating and / or preventing inflammatory neurodegenerative diseases. A BV2 cell model and a 3 * Tg-AD mouse verify that the desloratadine derivative can significantly inhibit the inflammation level of BV2 cells and improve cognitive impairment of the 3 * Tg-AD mouse, and has a significant effect on inflammatory neurodegenerative diseases, especially Alzheimer's disease.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and particularly relates to desloratadine derivatives and their use in preparing drugs for Alzheimer's disease. Background Art

[0002] Alzheimer's Disease (AD) is a progressive neurodegenerative disease with clinical manifestations of memory loss and cognitive dysfunction. According to statistics, there are more than 50 million dementia patients in the world today, spending more than 1 trillion US dollars each year, and about 50-60% of them are Alzheimer's patients. In addition, there is evidence that Alzheimer's disease mostly occurs in people over 50 years old. As the world's aging level increases, the number of Alzheimer's patients will continue to increase, which brings great pressure to individuals and society. At present, there is still no effective means to cure AD. There are very few drugs for treating AD in clinical practice, and they can only slow down the patient's symptoms, but cannot effectively block the progression of AD. Therefore, there is an urgent need to discover new anti-AD drugs that can block the pathological process of AD.

[0003] Studies have found that neuroinflammation is a major factor contributing to the development and progression of Alzheimer's disease. "Neuroinflammation" here refers to the secondary response of inflammation to the central nervous system, which in turn leads to damage to neurons. Inflammation is considered an early event in the AD disease process, and many chronic inflammatory reactions exist in the normal elderly population. Numerous studies have shown that high levels of inflammatory factors are present in the brains of AD patients and AD model animals.

[0004] Desloratadine is a non-sedating antihistamine that poorly crosses the blood-brain barrier. It is primarily used clinically to alleviate the systemic and localized symptoms of chronic idiopathic urticaria and allergic rhinitis. Recent reports suggest that desloratadine can improve memory and cognitive impairment in Alzheimer's disease (AD) model mice.

[0005] However, desloratadine has limited effects on Alzheimer's disease, and more drugs with better effects on this condition are needed. Summary of the Invention

[0006] In response to the deficiencies of the prior art, the present invention provides a series of desloratadine derivatives, which significantly improve the neuroinflammation inhibitory effect and significantly enhance the therapeutic effect of Alzheimer's disease compared to desloratadine.

[0007] In a first aspect, the present invention provides a compound represented by Formula A or a pharmaceutically acceptable salt thereof. The structure of Formula A is as follows:

[0008]

[0009] R is selected from dimethylamino, amino, methoxy, isopropyl and trifluoromethyl.

[0010] Preferably R is dimethylamino.

[0011] In a second aspect, the present invention provides a pharmaceutical composition comprising the compound represented by formula A described in the first aspect or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

[0012] Preferably, the pharmaceutical composition contains the compound represented by formula A or a pharmaceutically acceptable salt thereof as the sole active ingredient.

[0013] In a third aspect, the present invention provides the use of the compound represented by formula A described in the first aspect or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition described in the second aspect, in the preparation of drugs for treating and / or inflammatory neurodegenerative diseases.

[0014] Preferably, the neurodegenerative disease is an inflammatory neurodegenerative disease.

[0015] More preferably, the neurodegenerative disease is Alzheimer's disease or Parkinson's disease.

[0016] Most preferably, the neurodegenerative disease is Alzheimer's disease.

[0017] Compared with the prior art, the present invention has the following advantages:

[0018] This study, based on a series of structural modifications to desloratadine and using BV2 cells (immortalized mouse microglia) to create a neuroinflammatory cell model, demonstrated significant improvements in neuroinflammation inhibition compared to desloratadine. Furthermore, using a 3×Tg-AD mouse model as a research subject, it was found that these derivative compounds, such as Compound A1, exhibited significantly better long-term memory and spatial exploration abilities than desloratadine.

[0019] The above research results fully demonstrate the significant advantages of the series of compounds of the present invention over desloratadine in treating inflammatory neurological diseases such as Alzheimer's disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 The mRNA expression levels of NLRP3 and IL-1β in BV2 cells in each group. * p<0.05, ** p<0.01, *** p<0.001, ns means no significant difference. One-way ANOVA.

[0021] Figure 2 The time required for mice in each group to find the platform. *p<0.05, ** p<0.01, *** p<0.001, ns means no significant difference. One-way ANOVA.

[0022] Figure 3 The number of times mice in each group crossed the platform. * p<0.05, ** p<0.01, *** p<0.001, ns means no significant difference. One-way ANOVA. DETAILED DESCRIPTION

[0023] The following examples are intended to enable those skilled in the art to more fully understand the technical solutions and implementation effects of the present invention, but the scope of protection of the present invention is not limited thereto. This section further describes the present invention in detail in conjunction with specific implementation cases, and its technical features and advantages will be clearly reflected in the description. It should be pointed out that the embodiments are only exemplary descriptions and are not intended to limit the scope of the claims of the present invention. Any detailed adjustments, equivalent replacements, or adaptive improvements based on the core principles of the present invention fall within the substantive protection scope of the present invention.

[0024] Example 1 Synthesis of Compound A1

[0025]

[0026] Desloratadine (1 g, 1 equivalent) and dimethylaminoethanol (0.382 ml, 1.2 equivalents) were placed in a 50 ml reaction flask. 6 ml of anhydrous dichloromethane (DCM) and triethylamine (2.44 ml, 6 equivalents) were added. The mixture was stirred at 0°C, and a solution of triphosgene (BTC, 350 mg, 0.36 equivalents) in anhydrous dichloromethane (10 ml) was added dropwise. The mixture was allowed to react at room temperature for 20 minutes. The reaction solution was washed with water, dried, and concentrated. The residue was purified by column chromatography using a gradient elution ratio of petroleum ether (60-90°C) to ethyl acetate (5:1-2:1) to obtain 700 mg of a white semisolid, Compound A1, in a 51% yield. HPLC purity: 99.91%. 1H NMR(500MHz,D2O)δ8.49(dd,J=5.9,1.5Hz,1H),8.34(dd,J=8.1,1.5Hz,1H),7.83(dd,J =8.0,5.8Hz,1H),7.39(d,J=2.2Hz,1H),7.30(dd,J=8.2,2.2Hz,1H),7.19(d,J=8.2Hz, 1H),4.45(m,2H),3.93(m,2H),3.58–3.52(m,1H),7.51(t,J=4.9Hz,2H),3.49–3.42(m, 1H),3.22–3.04(m,3H),2.95(s,6H),2.93–2.89(m,1H),2.54–2.40(m,3H),2.35(m,1H); 13 C NMR(126MHz,D2O)δ155.4,148.7,148.4,144.9,139.7,139.1,138.3,135.2,133.7,130.2,128.9 ,126.6,126.0,125.5,59.9,56.3,46.7,44.0,43.8,43.1,30.5,30.0,29.5; HRMS(ESI):m / z[M+H] + calcdfor C 24 H 29 ClN3O2 + :426.1948,found:426.1963.

[0027] Example 2 Synthesis of Compound A2

[0028]

[0029] Following the procedure of Example 1, the reaction was repeated, substituting dimethylaminoethanol for N-(tert-butyloxycarbonyl)ethanolamine. The intermediate was purified by chromatography, and then a 4M HCl solution in 1,4-dioxane was added, stirred for 10 minutes, and the solvent was evaporated to afford 462 mg of compound A2 in a 36% overall yield. HPLC purity: 96.87%. 1H NMR(500MHz,D2O)δ8.45(d,J=5.6Hz,1H),8.17(m,1H),7.71(t,J=8.2Hz,1H) ,7.31(m,1H),7.23(m,1H),7.16(d,J=8.2Hz,1H),4.36(m,2H),3.93–3.75(m ,2H),3.51–3.47(m,1H),3.44–3.39(m,1H),3.35–3.33(m,2H),3.10(m,2H), 2.98(m,1H),2.87–2.83(m,1H),2.51–2.45(m,1H),2.40(m,2H),2.30(m,1H); 13 CNMR(126MHz,D2O)δ155.8,139.9,135.7,133.6,130.3,129.0,126.5,125.1,62.2,48.9,44.2,44.0,38.9,30.5,30.0,29.9; HRMS(ESI):m / z[M+H] + calcd for C 22 H 25 ClN3O2 + :398.1635,found:398.1641.

[0030] Example 3 Synthesis of Compound A3

[0031]

[0032] Following the steps of Example 1, compound A3 was synthesized by replacing dimethylaminoethanol with 2-methoxyethanol in a 64% yield. HPLC purity: 99.91%. 1 H NMR (500MHz, CDCl3) δ8.41 (dd, J=5.4, 1.2Hz, 1H), 7.45 (dd, J=7.7, 1.8Hz, 1H), 7.15(m,1H),7.12–7.11(m,2H),7.09–7.07(m,1H),4.25(t,J=4.6Hz,2H),3.83 (m,2H),3.60(t,J=4.7Hz,2H),3.41–3.37(m,1H),3.36(s,3H),3.48–3.30(m,1 H),3.21–3.15(m,2H),2.89–2.77(m,2H),2.53–2.47(m,1H),2.41–2.30(m,3H); 13CNMR(126MHz, CDCl3)δ157.1,155.4,146.7,139.6,137.7,137.6,137.4,134.3,133.4,133.0 ,130.6,129.1,126.2,122.4,71.0,64.6,59.0,45.0,44.9,31.8,31.5; HRMS(ESI):m / z[M+H] + calcd for C 23 H 26 ClN2O3 + :413.1632,found:413.1629.

[0033] Example 4 Synthesis of Compound A4

[0034]

[0035] Following the steps of Example 1, compound A4 was synthesized by replacing dimethylaminoethanol with isoamyl alcohol in a 68% yield. HPLC purity: 98.23%; 1 H NMR(500MHz, CDCl3)δ8.41(m,1H),7.45(dd,J=7.7,1.7Hz,1H),7.14(m,1H) ,7.11(m,2H),7.09–7.07(m,1H),4.09(t,J=6.8Hz,2H),3.78(m,2H),3.43-3 .32(m,2H),3.21–3.11(m,2H),2.86-2.75(m,2H),2.51-2.45(m,1H),2.35–2 .26(m,3H),1.71–1.63(m,1H),1.53(q,J=6.8Hz,2H),0.92(d,J=6.7Hz,6H); 13 C NMR (126MHz, CDCl3) δ157.2,155.7,146.8,139.6,137.8,137.7,134.3,133.5,133.1,130.7 ,129.1,126.3,122.4,64.2,44.9,44.9,37.9,31.8,31.6,25.3,22.6; HRMS(ESI):m / z[M+H] + calcdfor C 25 H 20 ClN2O2 + :425.1996,found:425.2007.

[0036] Example 5 Synthesis of Compound A5

[0037]

[0038] Compound A5 was synthesized using the same method as in Example 1, except that dimethylaminoethanol was replaced with 3,3,3-trifluoropropanol. The yield was 37%. HPLC purity: 96.18%. 1 H NMR(500MHz, CDCl3)δ8.39(m,1H),7.43(d,J=7.7Hz,1H),7.15(m,1H),7.13–7.11(m,2H),7.11–7.08(m,1H),4.31(t,J=6.1Hz, 2H),3.77(m,2H),3.43–3.28(m,2H),3.21–3.11(m,2H),2.89–2.74(m,2H),2.50–2.41(m,3H),2.50–2.41(m,3H),2.33(m,3H); 19 F NMR (471MHz, CDCl3) δ64.81, 64.83, 64.85; 13 C NMR (126MHz, CDCl3) δ157.1,154.7,146.8,139.6,137.7,137.2,134.5,133.5,133.1,130 .6,129.1,126.3,125.0,122.5,58.3,44.9,34.0,33.7,31.8,31.6; HRMS(ESI):m / z[M+H] + calcd for C 23 H 23 ClF3N2O2 + :451.1400,found:451.1396.

[0039] Test Example 1: Inhibitory effect of the series of compounds of the present invention on BV2 cells

[0040] 1.1 Experimental Principle

[0041] In this study, LPS (1 μg / mL) was used to pre-activate the NLRP3 inflammasome, followed by the addition of ATP (3 mM) to promote NLRP3 activation and assembly, as well as the release of the inflammatory cytokine IL-1β, thereby simulating an in vivo inflammatory state. RT-PCR was used to detect changes in NLRP3 and IL-1β gene levels in BV2 cells.

[0042] 1.2 Experimental Materials and Methods

[0043] 1) LPS and ATP used to induce cell damage were purchased from Sigma, cell culture reagents were purchased from Gibco, BV2 cells were from ATCC, and RNA extraction kits and reverse transcriptase were purchased from Novozymes.

[0044] 2) RT-PCR experiment: BV2 cells were cultured overnight at 2×10 5 Cells were seeded in 24-well plates. After stabilization and the appearance of intact morphology, they were pre-incubated with compound (10 μM) for 6 h, pre-activated with LPS (1 μg / mL) for 6 h, and stimulated with ATP (3 mM) for 30 min before being harvested. The supernatant was aspirated, the cells were washed three times with PBS, and lysed with 0.5 mL of TRIzol at room temperature for 5 min. 200 μL of chloroform was added, the sample was shaken vigorously for 15 seconds, allowed to stand for 5 min, and then centrifuged in a centrifuge (12,000 rpm / min, 4°C, 15 min). After centrifugation, the sample was separated into two layers, the top layer containing RNA. The RNA was transferred to a new EP tube (approximately 400 μL, avoiding the middle layer). 500 μL of isopropanol was added, the sample was gently shaken briefly, allowed to stand for 10 min, and then centrifuged in a centrifuge (12,000 rpm / min, 4°C, 10 min). After centrifugation, discard the supernatant. A small amount of precipitate will remain at the bottom of the tube. Wash with 0.5 mL of 75% ethanol and centrifuge again (12,000 rpm / min, 4°C, 10 min). Discard the supernatant and repeat the wash cycle. After washing, discard the 75% ethanol and air-dry the tube. Add the appropriate volume of DEPC water to each tube and place in a 55-60°C oven for 10 min to dissolve the precipitate. Measure the RNA concentration using a nucleic acid analyzer, with an A260 / A280 ratio between 1.8 and 2.0. Reverse transcriptase the mRNA to cDNA using a reverse transcription kit. A 20 μL system is prepared with 2 μg of RNA and 4 μL of reverse transcriptase, with the remainder filled with RNase-free ddH2O provided in the kit. Reverse transcription protocol: 37°C for 15 min; 85°C for 5 s; 10°C for ∞. The cDNA obtained by reverse transcription is analyzed using a kit containing the fluorescent substrate SYBR for real-time quantitative PCR in a 96-well plate. The specific RT-PCR program is as follows: (1) 94°C, 1 min; (2) 95°C, 30 s; (3) 63°C, 45 s; (4) 72°C, 45 s; (5) 80°C, 1 s; (6) plate reading; (7) repeat steps (2) to (6) for another 44 cycles; (8) 72°C, 10 min; (9) gradually increase the temperature from 50 to 95°C, reading the plate once every 0.5°C increase; (10) 10°C, 5 min, end the program.

[0045] 1.3 Experimental Results

[0046] The results are as follows Figure 1As shown in the results, compounds A1-A5 were able to significantly improve (p<0.001) the elevated NLRP3 and IL-1β levels induced by LPS / ATP in BV2 cells. In particular, compounds A1-A5 had a significantly enhanced inhibitory effect on IL-1β compared with desloratadine (p<0.001).

[0047] The above results show that compounds A1-A5 have obvious neuroinflammation inhibitory effects and have significantly enhanced neuroinflammation inhibitory effects compared with desloratadine.

[0048] Test Example 2: Compound A1 improves cognitive dysfunction in 3×Tg-AD mice

[0049] The present invention uses 7-month-old 3×Tg-AD mice and utilizes a water maze test to detect the effect of compound A1 on the long-term memory ability and spatial exploration ability of 3×Tg-AD mice. The experiment shows that A1 has the effect of significantly improving the long-term memory ability and spatial exploration ability of 3×Tg-AD mice.

[0050] 2.1 Experimental Principle

[0051] The 3×Tg-AD mice (34830-JAX) used in the experiment were purchased from the Jackson Laboratory in the United States. The three-allele homozygous mice (homozygous for the Psen1 mutation and homozygous for the co-injected APPSwe and tauP301L transgenes (Tg(APPSwe, tauP301L)1Lfa)) are all fertile and do not show severe physical or behavioral abnormalities. Overexpressed transgenic translation occurs in the central nervous system and shows plaque and tangle pathology associated with synaptic dysfunction, which is similar to the phenomenon observed in Alzheimer's patients. Therefore, we chose 3×Tg-AD mice as the AD model mice for this study.

[0052] The Morris water maze (MWM) test exploits the rodent's tendency to avoid water to assess mice's long-term memory and spatial exploration abilities. The water maze test apparatus consists of a circular pool with a diameter of 1.5 meters and a depth of 0.6 meters. White food coloring is added to the pool, concealing a potential platform approximately 2 cm below the water surface. The pool is divided into four quadrants: the first quadrant is farthest from the platform, the second and fourth quadrants are equidistant from the platform, and the platform is located in the third quadrant. The water maze consists of a training phase and a testing phase. During the training phase, mice are placed in the water from the first, second, and third quadrants, respectively, and allowed to freely explore the water for 1 minute while searching for the platform. If they fail to find the platform, they are lured to the platform and remain there for 10 seconds. This experiment is repeated over five days, representing the training phase. During the testing phase, the platform is removed from the pool, and mice are placed in the water from the first quadrant. The number of times they enter the fourth quadrant, where the platform is located, within 1 minute is measured. The long-term memory and spatial exploration abilities of mice were evaluated by the time required for them to find the platform during the training period and the number of times they crossed the platform within 1 minute during the testing period.

[0053] 2.2 Experimental Materials and Methods

[0054] 1) The 3×Tg-AD mice (34830-JAX) used in the experiment were purchased from the Jackson Laboratory in the United States.

[0055] 2) Animal grouping.

[0056] 7-month-old 3×Tg-AD male mice and 7-month-old C57 male mice were divided into 5 groups, with 8 mice in each group:

[0057] (1) WT + saline (VEH): C57 mice + saline group;

[0058] (2) 3×Tg-AD + saline (VEH): 3×Tg-AD mice + saline group;

[0059] (3) 3×Tg-AD+Compound A1(13.5): 3×Tg-AD mice+Compound A1(13.5mg / kg / day) group;

[0060] (4) 3×Tg-AD+Compound A1 (27): 3×Tg-AD mice+Compound A1 (27 mg / kg / day) group;

[0061] (5) 3×Tg-AD+DLT(20): 3×Tg-AD mice+DLT (20 mg / kg / day) group.

[0062] 3) Animal drug administration.

[0063] The positive compound DLT (ie, desloratadine) was dissolved in physiological saline containing 6% Tween-80, and A1 was dissolved in physiological saline. The compounds were administered by gavage once a day for 90 consecutive days to conduct a water maze test.

[0064] 4) Water maze experiment.

[0065] The Morris water maze (MWM) test exploits the rodent's tendency to avoid water to assess mice's long-term memory and spatial exploration abilities. The water maze test apparatus consists of a circular pool with a diameter of 1.5 meters and a depth of 0.6 meters. White food coloring is added to the pool, concealing a potential platform approximately 2 cm below the water surface. The pool is divided into four quadrants: the first quadrant is farthest from the platform, the second and fourth quadrants are equidistant from the platform, and the platform is located in the third quadrant. The water maze consists of a training phase and a testing phase. During the training phase, mice are placed in the water from the first, second, and third quadrants, respectively, and allowed to freely explore the water for 1 minute while searching for the platform. If they fail to find the platform, they are lured to the platform and remain there for 10 seconds. This experiment is repeated over five days, representing the training phase. During the testing phase, the platform is removed from the pool, and mice are placed in the water from the first quadrant. The number of times they enter the fourth quadrant, where the platform is located, within 1 minute is measured. The long-term memory and spatial exploration abilities of mice were evaluated by the time required for them to find the platform during the training period and the number of times they crossed the platform within 1 minute during the testing period.

[0066] 2.3 Experimental Results

[0067] according to Figure 2 It can be seen that all dose groups of Compound A1 and the desloratadine group can reduce the time required for 3×Tg-AD mice to find the platform, especially the high-dose group of Compound A1 had a more significant reducing effect than the desloratadine group (p<0.01).

[0068] according to Figure 3 It can be seen that all dose groups of Compound A1 and the desloratadine group can increase the number of times 3×Tg-AD mice find the platform, especially the high-dose group of Compound A1 has a more significant increase compared with the desloratadine group (p<0.05).

[0069] The above results show that compound A1 can significantly improve the long-term memory and spatial exploration ability of 3×Tg-AD mice, and its effect is significantly better than that of desloratadine.

[0070] Finally, it should be noted that the above embodiments are only used to illustrate rather than limit the technical solutions of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art should understand that the present invention can still be modified or replaced by equivalents. Any modification or partial replacement that does not depart from the spirit and scope of the present invention should be included in the scope of the claims of the present invention.

Claims

1. The compound represented by formula A or a pharmaceutically acceptable salt thereof, wherein the structure of formula A is as follows: R is selected from dimethylamino, amino, methoxy, isopropyl and trifluoromethyl. 2 . The compound according to claim 1 , or a pharmaceutically acceptable salt thereof, wherein R is a dimethylamino group.

3. A pharmaceutical composition comprising the compound according to claim 1 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient.

4. The pharmaceutical composition according to claim 3, characterized in that The pharmaceutical composition contains the compound or a pharmaceutically acceptable salt thereof as the sole active ingredient.

5. Use of the compound according to claim 1 or 2 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to claim 3, in the preparation of a drug for treating and / or preventing neurodegenerative diseases.

6. The use according to claim 5, characterized in that The neurodegenerative disease is an inflammatory neurodegenerative disease.

7. The use according to claim 5, characterized in that The neurodegenerative disease is Alzheimer's disease or Parkinson's disease.

8. The use according to claim 7, characterized in that The neurodegenerative disease is Alzheimer's disease.

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