A 2,3,4,9-tetrahydro-1H-pyrido[3,4-b]indole compound, a preparation method and application thereof

Abstract

The application discloses a 2,3,4,9-tetrahydro-1H-pyridine [3,4-b] indole compound and a preparation method and application thereof. The 2,3,4,9-tetrahydro-1H-pyridine [3,4-b] indole compound is prepared from 3-hydroxy-4-methoxybenzaldehyde and 1-(chloromethyl)-2,6-difluorobenzene as starting materials through two-step reactions. The 1-(3-((2,6-difluorophenyl)oxy)-4-methoxyphenyl)-2,3,4,9-tetrahydro-1H-pyridine [3,4-b] indole provided by the application has a significant inhibitory effect on RANKL-induced osteoclast differentiation and has no obvious cytotoxicity, and can be used as a novel candidate drug for inhibiting osteoclast differentiation and for targeted osteoclast treatment of bone mass loss related diseases.

Description

Technical Field

[0001] This invention belongs to the field of compound preparation technology, specifically relating to a 2,3,4,9-tetrahydro-1H-pyridine[3,4-b]indole compound and its preparation method, as well as its application in the preparation of osteoclast differentiation inhibitors. Background Technology

[0002] Osteoclasts are highly differentiated multinucleated giant cells originating from monocyte / macrophage hematopoietic stem cell lines. They are primarily responsible for bone resorption and participate in bone remodeling. Osteoclasts play a crucial role in bone development, growth, repair, and remodeling by secreting acidic substances and enzymes to degrade the bone matrix. Abnormal osteoclast activity is associated with a variety of diseases, including osteoporosis, fracture healing, osteoarthritis, and primary / metastatic bone tumors.

[0003] Globally, the prevalence of osteoporosis in women over 50 years of age can reach 33%, with an overall prevalence of osteoporotic fractures of approximately 15%. Among these, the one-year mortality rate for hip fractures is 20%, and the disability rate is as high as 50%. Patients with osteoporosis-related fractures have poor prognoses, leading to a significant decline in quality of life and often experiencing complications such as pneumonia and embolic diseases. Currently, the main treatments for osteoporosis include the use of anti-resorption drugs (such as bisphosphonates and calcitonin) and bone-forming drugs (such as parathyroid hormone). However, drug treatment is not ideal and carries complications associated with surgical intervention; for example, long-term use of bisphosphonates may cause side effects such as osteonecrosis of the mandible. Therefore, long-term use of these drugs is limited, making the development of safe and effective new drugs targeting osteoclasts an urgent priority. Summary of the Invention

[0004] In view of this, embodiments of this application provide a 2,3,4,9-tetrahydro-1H-pyridine[3,4-b]indole compound, its preparation method, and its application.

[0005] According to embodiments of this application, a 2,3,4,9-tetrahydro-1H-pyridine[3,4-b]indole compound is provided, the structural formula of which is shown in formula (1):

[0006]

[0007] The chemical formula of this compound is 1-(3-((2,6-difluorophenyl)oxo)-4-methoxyphenyl)-2,3,4,9-tetrahydro-1H-pyridine[3,4-b]indole.

[0008] The preparation method of the above-mentioned 2,3,4,9-tetrahydro-1H-pyridine[3,4-b]indole compounds includes the following steps:

[0009] (1) 3-hydroxy-4-methoxybenzaldehyde reacts with 1-(chloromethyl)-2,6-difluorobenzene under alkaline conditions to produce 3-((2,6-difluorophenyl)oxo)-4-methoxybenzaldehyde.

[0010] (2) 3-((2,6-difluorophenyl)oxo)-4-methoxybenzaldehyde reacts with tryptophan at room temperature under acidic conditions to cyclize and give 1-(3-((2,6-difluorophenyl)oxo)-4-methoxyphenyl)-2,3,4,9-tetrahydro-1H-pyridine[3,4-b]indole.

[0011] Specifically, according to a specific embodiment of the present invention, the preparation method may include the following steps:

[0012] (1) 3-hydroxy-4-methoxybenzaldehyde (compound 1) and 1-(chloromethyl)-2,6-difluorobenzene (compound 2) were placed in a flask, sodium iodide and potassium carbonate were added, dissolved in acetone, and the mixture was heated overnight. After the reaction was completed, the reaction solution was concentrated under reduced pressure. The resulting mixture was diluted with water, extracted with ethyl acetate, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The white solid 3-((2,6-difluorophenyl)oxo)-4-methoxybenzaldehyde (compound 3) was purified by silica gel column chromatography.

[0013] (2) 3-((2,6-difluorophenyl)oxo)-4-methoxybenzaldehyde (compound 3) and tryptophan were dissolved in dichloromethane, and trifluoroacetic acid was added. The reaction was carried out overnight at room temperature. After the reaction was completed, sodium bicarbonate aqueous solution was added to quench the reaction. After extraction, the mixture was washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by column chromatography to obtain 1-(3-((2,6-difluorophenyl)oxo)-4-methoxyphenyl)-2,3,4,9-tetrahydro-1H-pyridine[3,4-b]indole (compound 4).

[0014] The synthesis route is as follows:

[0015]

[0016] In the above scheme, further, the heating reaction temperature in (1) is 40-80℃;

[0017] Furthermore, the room temperature in (2) is 15-45℃;

[0018] Furthermore, in (2), the molar ratio of 3-((2,6-difluorophenyl)oxo)-4-methoxybenzaldehyde to tryptamine is 1:(1~2).

[0019] The application of the above-mentioned 2,3,4,9-tetrahydro-1H-pyridine[3,4-b]indole compounds in the preparation of osteoclast differentiation inhibitors.

[0020] The compound is used in the preparation of medicaments for treating postmenopausal osteoporosis, bone destruction due to tumor metastasis, or inflammatory bone destruction.

[0021] The present invention also provides a medicament for treating postmenopausal osteoporosis, bone destruction due to tumor metastasis, or inflammatory bone destruction, comprising the 2,3,4,9-tetrahydro-1H-pyridine[3,4-b]indole compound as an active ingredient, and further comprising pharmaceutically acceptable excipients.

[0022] The drug dosage form is tablet, pill, powder, capsule, injection, oral liquid, ointment or cream.

[0023] The administration method can be oral, injection, or topical.

[0024] This invention utilizes a RANKL-induced mouse bone marrow macrophage osteoclast differentiation model. Experiments have demonstrated that the 2,3,4,9-tetrahydro-1H-pyridine[3,4-b]indole compounds of this invention significantly inhibit RANKL-induced osteoclast differentiation without significant cytotoxicity. This provides a novel candidate drug for inhibiting osteoclast differentiation, potentially enabling targeted treatment of bone loss-related diseases. Attached Figure Description

[0025] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments consistent with this application and, together with the specification, serve to explain the principles of this application.

[0026] Figure 1 The example shows the 1H NMR spectrum of 1-(3-((2,6-difluorophenyl)oxo)-4-methoxyphenyl)-2,3,4,9-tetrahydro-1H-pyridine[3,4-b]indole.

[0027] Figure 2 The carbon NMR spectrum of 1-(3-((2,6-difluorophenyl)oxo)-4-methoxyphenyl)-2,3,4,9-tetrahydro-1H-pyridine[3,4-b]indole is given in the examples.

[0028] Figure 3 These are experimental results obtained by using the CCK-8 assay to detect the effects of substituted 2,3,4,9-tetrahydro-1H-pyridine[3,4-b]indole compounds on the cell viability of mouse bone marrow-derived macrophages (BMMs).

[0029] Figure 4These are the results of TRAP staining to detect the effect of substituted 2,3,4,9-tetrahydro-1H-pyridine[3,4-b]indole compounds on RANKL-induced osteoclast differentiation of BMMs. Scale bar: 200 μm.

[0030] Figure 5 These are experimental results obtained by qRT-PCR to detect the effects of substituted 2,3,4,9-tetrahydro-1H-pyridine[3,4-b]indole compounds on the expression of osteoclast differentiation marker genes. Data are expressed as mean ± standard deviation. ## p <0.01 vs Ctrl / 0 μM group, * p <0.05, ** p <0.01 vs RANKL / 0 μM group. Detailed Implementation

[0031] The present invention will be further described below with reference to embodiments and accompanying drawings. The following embodiments are merely illustrative and not intended to limit the invention in any way.

[0032] Example 1 Preparation of 1-(3-((2,6-difluorophenyl)oxo)-4-methoxyphenyl)-2,3,4,9-tetrahydro-1H-pyridine[3,4-b]indole

[0033]

[0034] (1) 3-((2,6-difluorophenyl)oxo)-4-methoxybenzaldehyde

[0035] Take a 100 mL flask and add 10 mmol (1.52 g) of 3-hydroxy-4-methoxybenzaldehyde, 10 mmol (1.62 g) of 1-(chloromethyl)-2,6-difluorobenzene, 15 mmol (2.25 g) of sodium iodide, and 20 mmol (2.76 g) of potassium carbonate to 50 mL of acetone in that order. Heat the mixture at 60 °C overnight. After the reaction is complete, concentrate the reaction mixture under reduced pressure. Dilute the resulting mixture with water, extract with ethyl acetate, wash with saturated brine, dry to anhydrous sodium sulfate, and concentrate under reduced pressure. Purify the concentrate by silica gel column chromatography to give a white solid 3-((2,6-difluorophenyl)oxo)-4-methoxybenzaldehyde in 55% yield.

[0036] 1 H NMR (400 MHz, CDCl3) δ 9.86 (s, 1H), 7.46 (dd, J = 8.0, 1.6 Hz, 1H), 7.42 (d, J= 2.0 Hz, 1H), 7.38 – 7.31 (m, 1H), 7.15 (d, J = 8.0 Hz, 1H), 6.94 (t, J = 8.0 Hz, 2H), 5.26 (s, 2H), 3.90 (s, 3H). ESI-MS: m / z = 279.1 [M+H] + .

[0037] (2) Preparation of 1-(3-((2,6-difluorophenyl)oxo)-4-methoxyphenyl)-2,3,4,9-tetrahydro-1H-pyridine[3,4-b]indole

[0038] Compound 3-((2,6-difluorophenyl)oxo)-4-methoxybenzaldehyde (2 mmol, 560 mg) and tryptophan (2 mmol, 320 mg) were dissolved in 10 mL of dichloromethane, and trifluoroacetic acid (2 mmol, 228 mg) was added. The mixture was reacted overnight at room temperature. After the reaction was completed, 10% sodium bicarbonate aqueous solution was added to quench the reaction. The mixture was extracted three times with DCM, washed with saturated brine, dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain 0.63 g of white solid (i.e., formula (1)), with a yield of 75%. Its 1H NMR and 1C NMR spectra are shown below. Figure 1 , Figure 2 As shown.

[0039] 1 H NMR (400 MHz, CDCl3) δ 7.59 – 7.53 (m, 2H), 7.34 – 7.28 (m, 1H),7.24 (d, J = 8.0 Hz, 1H), 7.13 (dt, J = 13.2, 6.4 Hz, 2H), 6.98 (d, J = 8.0 Hz,1H), 6.92 – 6.82 (m, 4H), 5.16 (s, 2H), 5.11 (s, 1H), 3.76 (s, 3H), 3.44 –3.39 (m, 1H), 3.18 – 3.11 (m, 1H), 2.97 – 2.90 (m, 1H), 2.81 (d, J = 15.2 Hz, 1H). 13C NMR (101 MHz, CDCl3) δ 163.3, 160.8, 152.0, 150.5, 148.0, 135.8,135.5, 134.7, 130.7, 127.5, 121.7, 120.7, 119.4, 118.2, 114.7, 112.2, 111.6,111.3, 110.9, 110.0, 59.4, 58.2, 56.1, 43.3, 22.5. ESI-MS: m / z =421.17[M+H] + .

[0040] Example 2 evaluates the cytotoxicity of 1-(3-((2,6-difluorophenyl)oxo)-4-methoxyphenyl)-2,3,4,9-tetrahydro-1H-pyridine[3,4-b]indole and its inhibitory effect on RANKL-induced osteoclast differentiation.

[0041] The specific experiment is as follows:

[0042] Experiment 1: The CCK8 assay was used to detect the cytotoxicity of substituted 2,3,4,9-tetrahydro-1H-pyridine[3,4-b]indole compounds on mouse bone marrow-derived macrophages (BMMs).

[0043] Isolation and culture of primary mouse bone marrow-derived macrophages (BMMs)

[0044] One-month-old C57BL / 6 mice were sacrificed and disinfected with 75% medical alcohol. The hind limbs of the mice were dissected in a biosafety cabinet, and the femur and tibia were separated. The upper end of the femur and the lower end of the tibia were cut open with sterile scissors. The bone marrow cavity was flushed with 1 ml of α-MEM complete culture medium (containing 10% fetal bovine serum and 1% penicillin-streptomycin antibiotics). The cells were centrifuged at 1000 rpm for 5 min, and α-MEM complete culture medium containing 40 ng / ml M-CSF was added. The cells were gently mixed by pipetting and transferred to 6 cm cell culture dishes and incubated at 37°C in a 5% CO2 incubator. The cells were cultured for approximately 3-4 days until they reached 80%-90% confluence. These cells are the primary bone marrow-derived macrophages (BMMs).

[0045] (2) Detection of cytotoxicity of 1-(3-((2,6-difluorophenyl)oxo)-4-methoxyphenyl)-2,3,4,9-tetrahydro-1H-pyridine[3,4-b]indole to BMMs by CCK-8 assay

[0046] BMMs are divided into 5×10 3Cells were seeded at a density of 96-well plates, with 7 groups per plate: one control group (containing only the drug solvent dimethyl sulfoxide) and 6 drug-treated groups (containing 0.3125, 0.625, 1.25, 2.5, 5, and 10 μM of the drug, respectively), with 6 replicates per group. α-MEM complete culture medium containing 40 ng / ml M-CSF was added, and the cells were cultured for 24, 48, and 72 hours, with the medium changed every other day. At the corresponding time points, 10 μl of CCK-8 reagent was added to each well, and after incubation in the dark for 2 hours, the absorbance at 450 nm was measured. The results showed that drug concentrations of 10 μM and below had no significant cytotoxicity to BMMs (see [link to relevant documentation]). Figure 3 ).

[0047] Experiment 2: The effect of substituted 2,3,4,9-tetrahydro-1H-pyridine[3,4-b]indole compounds on RANKL-induced osteoclast differentiation of BMMs was assessed using tartrate-resistant acid phosphatase (TRAP) staining.

[0048] Five groups were set up: a RANKL-induced group and five groups treated with different concentrations of 1-(3-((2,6-difluorophenyl)oxo)-4-methoxyphenyl)-2,3,4,9-tetrahydro-1H-pyridine[3,4-b]indole, with three replicates for each group. BMMs were arranged in a 5 × 10⁻⁶ pattern. 3Cells were seeded at a density of 40 ng / ml M-CSF in 96-well plates and cultured overnight in α-MEM complete medium. Subsequently, the cells were treated as follows: the RANKL group was treated with osteoclastogenic culture medium containing the same amount of dimethyl sulfoxide as the drug-treated groups (α-MEM complete medium containing 40 ng / ml M-CSF and 50 ng / ml RANKL); the 0.3125, 0.625, 1.25, 2.5, and 5 μM drug-treated groups were treated with osteoclastogenic culture medium containing 0.3125, 0.625, 1.25, 2.5, and 5 μM of 1-(3-((2,6-difluorophenyl)oxo)-4-methoxyphenyl)-2,3,4,9-tetrahydro-1H-pyridine[3,4-b]indole, respectively. The culture medium was changed every other day. After culturing for 5-7 days, the culture medium was discarded, and the cells were washed with PBS and fixed with 4% paraformaldehyde for 10 min. Tartrate-resistant acid phosphatase staining was performed using a kit. Microscopic images were taken and the number of mature osteoclasts (TRAP-positive cells with 3 or more nuclei) was counted to assess the effect of 1-(3-((2,6-difluorophenyl)oxo)-4-methoxyphenyl)-2,3,4,9-tetrahydro-1H-pyridine[3,4-b]indole on osteoclast differentiation. TRAP staining results showed that substituted 2,3,4,9-tetrahydro-1H-pyridine[3,4-b]indole compounds dose-dependently inhibited RANKL-induced osteoclast differentiation, especially with very few mature osteoclasts in the 2.5 and 5 μM drug treatment groups. Quantitative results further showed that the number of osteoclasts in the 0.3125 μM drug treatment group was not significantly different from that in the RANKL group, the number of osteoclasts in the 0.625 μM drug treatment group was somewhat reduced, and the number of osteoclasts in the 1.25 μM, 2.5 μM and 5 μM drug treatment groups was significantly reduced compared with the RANKL group. Figure 4 ).

[0049] Experiment 3: The effect of substituted 2,3,4,9-tetrahydro-1H-pyridine[3,4-b]indole compounds on the expression of osteoclast differentiation marker genes was detected by qRT-PCR.

[0050] Six drug groups with different concentrations of substituted 2,3,4,9-tetrahydro-1H-pyridine[3,4-b]indole compounds and a RANKL-induced group were simultaneously treated with different drug concentrations, with three replicates per group. BMMs were distributed at 2 × 10⁻⁶. 5Cells were seeded at a density of 0.5 μM / well in 24-well plates and cultured overnight in α-MEM complete medium containing 40 ng / ml M-CSF. Subsequently, cells were treated with 0, 0.3125, 0.625, 1.25, 2.5, and 5 μM of the drug, respectively, while the RANKL induction group was induced simultaneously with 50 ng / ml RANKL. The medium was changed every other day, and after 3 days of culture, the culture medium was discarded, and the cells were washed with PBS. RNA was extracted using an RNA extraction kit and then subjected to qRT-PCR to detect osteoclast differentiation marker genes. NFATc1 , Trap , DC-Stamp and Ctsk The expression of RANKL-induced osteoclast differentiation marker genes was inhibited by qRT-PCR. The results showed that 1-(3-((2,6-difluorophenyl)oxo)-4-methoxyphenyl)-2,3,4,9-tetrahydro-1H-pyridine[3,4-b]indole in a dose-dependent manner. Figure 5 ).

[0051] The above descriptions are merely some preferred embodiments of the present invention, used to help illustrate the invention. The scope of protection of the present invention is not limited to the above embodiments; all technical solutions falling within the scope of the present invention's concept are within its protection.

Claims

1. A 2,3,4,9-tetrahydro-1H-pyridine[3,4-b]indole compound, characterized in that, Its structural formula is shown in equation (1): ； The chemical name of this compound is 1-(3-((2,6-difluorobenzyl)oxy)-4-methoxyphenyl)-2,3,4,9-tetrahydro-1H-pyridine[3,4-b]indole.

2. The method for preparing the 2,3,4,9-tetrahydro-1H-pyridine[3,4-b]indole compound according to claim 1, characterized in that, Includes the following steps: (1) 3-hydroxy-4-methoxybenzaldehyde reacts with 1-(chloromethyl)-2,6-difluorobenzene under alkaline conditions to produce 3-((2,6-difluorobenzyl)oxy)-4-methoxybenzaldehyde; (2) 3-((2,6-difluorobenzyl)oxy)-4-methoxybenzaldehyde reacts with tryptophan at room temperature under acidic conditions to cyclize and give 1-(3-((2,6-difluorobenzyl)oxy)-4-methoxyphenyl)-2,3,4,9-tetrahydro-1H-pyridine[3,4-b]indole.

3. The method for preparing the 2,3,4,9-tetrahydro-1H-pyridine[3,4-b]indole compound according to claim 2, characterized in that, The heating reaction temperature in step (1) is 40-80℃.

4. The method for preparing the 2,3,4,9-tetrahydro-1H-pyridine[3,4-b]indole compound according to claim 2, characterized in that, The room temperature in step (2) is 15-45℃.

5. The method for preparing the 2,3,4,9-tetrahydro-1H-pyridine[3,4-b]indole compound according to claim 2, characterized in that, In step (2), the molar ratio of 3-((2,6-difluorobenzyl)oxy)-4-methoxybenzaldehyde to tryptamine is 1:(1~2).

6. A drug for treating osteoporosis, bone metastasis of tumors, or inflammatory bone destruction, characterized in that, The drug comprises the compound as described in claim 1 as an active ingredient and medically acceptable pharmaceutical excipients.

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