An indole compound, a preparation method and application thereof

Abstract

The present application relates to the technical field of organic synthesis, and particularly provides an indole compound, a preparation method and application thereof. The chemical structural formula of the indole compound is shown as formula I. The provided indole compound can inhibit the growth of Jurkat leukemia cells in combination with dexamethasone at different concentrations, and the indole compound in combination with dexamethasone can activate the phosphorylation level of glucocorticoid, inhibit the PI3K / AKT and JAK2 / STAT3 signal pathways related to glucocorticoid resistance to overcome the drug resistance of glucocorticoid, and make the leukemia cells die.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of organic synthesis, and in particular to an indole compound and a preparation method and application thereof. BACKGROUND

[0002] Acute lymphoblastic leukemia (ALL) is a kind of leukemia, which is a malignant tumor disease originated from lymphocytes. The incidence and recurrence rate of acute lymphoblastic leukemia are high, and the main treatment methods thereof include inhibition of hematopoietic stem cells, intervention of chemotherapeutic drugs and small molecule targeted immunotherapy. Glucocorticoids such as dexamethasone (DEX) and prednisone play an important role in the treatment of acute lymphoblastic leukemia. Glucocorticoids mainly bind to intracellular glucocorticoid receptors (GR), induce glucocorticoid receptor activation, promote transcription of target genes, and induce cycle arrest and apoptosis of leukemia cells. Dexamethasone is widely used in the treatment of leukemia, especially childhood acute lymphoblastic leukemia, because it reduces the risk of bone marrow and central nervous system recurrence. However, adverse reactions such as osteonecrosis, infection and mental problems frequently occur when glucocorticoids are administered in large doses for a long time. Primary or secondary drug resistance of patients to glucocorticoids makes the effect of glucocorticoids in the treatment of leukemia unsatisfactory.

[0003] Indole compounds play an important role in the development of anticancer drugs, and have broad prospects in solving the problem of drug resistance of anticancer drugs.

[0004] Therefore, it is of great significance to develop a highly efficient and low-toxicity therapeutic drug to make leukemia cells sensitive to glucocorticoids again and make the use of glucocorticoids in clinic more efficient. SUMMARY

[0005] Therefore, the present application provides an indole compound and a preparation method and application thereof. The indole compound of the present application can make leukemia cells sensitive to glucocorticoids again as a highly efficient and low-toxicity drug.

[0006] In order to achieve the above-mentioned application purposes, the present application provides the following technical solutions.

[0007] The present application provides an indole compound, the chemical structure of which is shown in formula I:

[0008]

[0009] The present application provides a preparation method of the indole compound, which comprises the following steps:

[0010] Mixing 3,5-dichloro-2-oxoindoline-3-carboxylic acid methyl ester, N-benzyl-2-(1H-indol-3-yl)ethan-1-amine, an organic solvent and an organic base to carry out a substitution reaction to obtain an indole compound.

[0011] Preferably, the mass ratio of the 3,5-dichloro-2-oxoindoline-3-carboxylic acid methyl ester and the N-benzyl-2-(1H-indol-3-yl)ethan-1-amine is 1:0.5-1.5.

[0012] Preferably, the organic solvent comprises one or more of acetonitrile, methanol and acetone.

[0013] The organic base comprises one or more of N,N-diisopropylethylamine, dimethylamine, aniline and pyridine.

[0014] Preferably, the temperature of the substitution reaction is 22-25℃ and the time is 1-3h.

[0015] Preferably, the substitution reaction further comprises post-treatment, which preferably comprises: concentrating the obtained substitution reaction solution and then purifying by column chromatography.

[0016] Preferably, the eluent of the column chromatography is a mixture of dichloromethane and methanol, and the volume ratio of the dichloromethane and the methanol is 150:1-300:1.

[0017] The present application provides the use of the indole compound in the above technical solution or the indole compound prepared by the preparation method in the above technical solution in the preparation of a drug for treating leukemia.

[0018] Preferably, the active ingredient of the drug for treating leukemia further comprises dexamethasone.

[0019] The present application also provides a drug for treating leukemia, comprising an active ingredient and a pharmaceutically acceptable adjuvant; the active ingredient comprises an indole compound or a mixture of an indole compound and dexamethasone; the indole compound is the indole compound in the above technical solution or the indole compound prepared by the preparation method in the above technical solution.

[0020] The indole compound provided by the present application can inhibit the growth of Jurkat leukemia cells in combination with dexamethasone at different concentrations, and the indole compound in combination with dexamethasone can activate the phosphorylation level of glucocorticoid, inhibit the PI3K / AKT and JAK2 / STAT3 signal pathways related to glucocorticoid resistance to overcome the resistance of glucocorticoid and cause the death of leukemia cells.

[0021] The present application also provides a medicine for treating leukemia, wherein the medicine comprises the indole compound described in the foregoing scheme, and the medicine can effectively treat glucocorticoid-resistant leukemia. BRIEF DESCRIPTION OF DRAWINGS

[0022] Figure 1 The structural characterization diagram of the indole compound prepared in Example 1 is shown from top to bottom as the hydrogen spectrum, the carbon spectrum and the mass spectrum;

[0023] Figure 2 The result diagram of the influence of Example 2 on Jurkat cell proliferation is shown;

[0024] Figure 3 The result diagram of the cell cycle distribution of different groups of Jurkat in Example 3 is shown;

[0025] Figure 4 The distribution statistical diagram of Jurkat cell cycle in Example 3 is shown;

[0026] Figure 5 The result diagram of the distribution of Jurkat cell apoptosis in Example 4 is shown;

[0027] Figure 6 The statistical diagram of the apoptosis rate of Jurkat cells in Example 4 is shown;

[0028] Figure 7 The diagram of the influence of the indole compound combined with DEX on G1 phase related proteins in Example 5 is shown;

[0029] Figure 8 The diagram of the influence of the indole compound combined with DEX on apoptosis related proteins in Example 5 is shown

[0030] Figure 9 The result diagram of the influence of Example 6 on glucocorticoid resistance related protein expression of Jurkat cells is shown, wherein A is the expression of glucocorticoid receptor protein GR caused by DEX, the indole compound and DEX combined with the indole compound, B is the expression diagram of JAK2 / STAT3 signal protein induced by DEX, the indole compound and DEX combined with the indole compound, and C is the expression diagram of PI3K / AKT signal protein induced by DEX, the indole compound and DEX combined with the indole compound. DETAILED DESCRIPTION

[0031] The present application provides an indole compound, and the chemical structural formula is shown as formula I:

[0032]

[0033] The present application also provides a preparation method of the indole compound described in the foregoing scheme, comprising the following steps:

[0034] Mixing 3,5-dichloro-2-oxoindoline-3-carboxylic acid methyl ester, N-benzyl-2-(1H-indol-3-yl)ethan-1-amine, an organic solvent and an organic base to perform a substitution reaction to obtain an indole compound.

[0035] In the present application, the required materials are all commercially available and well known to those skilled in the art, unless otherwise specified.

[0036] In the present application, the synthetic route of the indole compound is as follows:

[0037]

[0038] In the present application, the mass ratio of the 3,5-dichloro-2-oxoindoline-3-carboxylic acid methyl ester and the N-benzyl-2-(1H-indol-3-yl)ethan-1-amine is preferably 1:0.5-1.5, more preferably 1:0.8-1.2, and further preferably 1:1.

[0039] In the present application, the organic solvent preferably includes one or more of acetonitrile, methanol and acetone, and preferably acetonitrile. In the present application, the mass-to-volume ratio of the 3,5-dichloro-2-oxoindoline-3-carboxylic acid methyl ester and acetonitrile is preferably 1 g:5-15 mL, more preferably 1 g:8-12 mL, and further preferably 1 g:10 mL.

[0040] In the present application, the organic base includes one or more of N,N-diisopropylethylamine, dimethylamine, aniline and pyridine. In the present application, the mass ratio of the 3,5-dichloro-2-oxoindoline-3-carboxylic acid methyl ester and the organic base is preferably 1:0.5-2, more preferably 1:1.2-1.5, and further preferably 1:1.5.

[0041] The present application does not have special limitations on the mixing method, and the raw materials can be mixed uniformly, for example, by stirring. The mixing order is preferably: first mixing the 3,5-dichloro-2-oxoindoline-3-carboxylic acid methyl ester, the N-benzyl-2-(1H-indol-3-yl)ethan-1-amine and the acetonitrile, and second mixing the obtained first mixture with the organic base. The temperature of the first mixing is preferably room temperature. The temperature of the second mixing is preferably 0-4°C, and more preferably 0°C. The present application does not have special limitations on the time of the first mixing and the second mixing, and the raw materials can be mixed uniformly.

[0042] In the present application, the temperature of the substitution reaction is preferably 22-25°C, and more preferably 25°C. The time of the substitution reaction is preferably 1-3 h, and more preferably 2 h.

[0043] After the substitution reaction is completed, the present application also preferably comprises post-treatment, which preferably comprises: after the obtained substitution reaction solution is concentrated, column chromatography purification is performed to obtain the indole compound. The present application does not have special limitations for the concentration, and a concentration method well known to those skilled in the art can be used, such as reduced pressure concentration. In the present application, the eluent used in the column chromatography purification is preferably a mixture of dichloromethane and methanol, and the volume ratio of dichloromethane to methanol in the eluent is preferably 150:1 to 300:1.

[0044] The present application also provides the use of the indole compound described in the foregoing scheme or the indole compound prepared by the foregoing scheme in the preparation of a drug for treating leukemia. In the present application, the active ingredient in the drug for treating leukemia preferably further comprises dexamethasone (DEX) in addition to the indole compound; when the active ingredient is a mixture of the indole compound and dexamethasone, the indole compound can be combined with dexamethasone to treat leukemia, induce G1 phase arrest of Jurkat cells, down-regulate the expression of the cell cycle-related oncogene C-Myc, down-regulate the expression of G1 phase-related cell cycle proteins CDK2 and cyclin E1, and inhibit the growth of Jurkat cells; and the indole compound of the present application can also be combined with dexamethasone to significantly increase the apoptosis rate of Jurkat cells, and can execute the apoptosis signal through the caspase pathway by up-regulating the cleaved caspase 9, cleaved caspase 3, and cleaved PARPA protein, while the indole compound of the present application can also regulate the expression of genes related to the mitochondrial apoptosis pathway, up-regulate the expression of the pro-apoptotic gene Bim and down-regulate the expression of the anti-apoptotic gene Bcl-2; in addition, the indole compound of the present application combined with dexamethasone can overcome the drug resistance of Jurkat cells to DEX by regulating the phosphorylation expression of GR through the PI3K / AKT and JAK2 / STAT3 signaling pathways, and the indole compound combined with dexamethasone can up-regulate the phosphorylation level of the glucocorticoid ser211 site of Jurkat cells and down-regulate the levels of phosphorylated PI3K, phosphorylated AKT, phosphorylated JAK2, and phosphorylated STAT3 after administration, thereby overcoming the glucocorticoid resistance of Jurkat cells and playing an anti-leukemia role.

[0045] The present application also provides a medicine for treating leukemia, which comprises an active ingredient and pharmaceutically acceptable adjuvants; the active ingredient comprises an indole compound or a mixture of an indole compound and dexamethasone; the indole compound is the indole compound described in the foregoing scheme or the indole compound prepared in the foregoing scheme. The present application is not particularly limited to the pharmaceutically acceptable adjuvants, and the pharmaceutically acceptable adjuvants well known to those skilled in the art can be used. The present application is not particularly limited to the dosage form and administration method of the medicine for treating leukemia, and the dosage form and administration method well known to those skilled in the art can be used.

[0046] The technical solutions in the present application will be described clearly and completely below in combination with the embodiments in the present application. Obviously, the described embodiments are only some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all the other embodiments obtained by those skilled in the art without creative efforts belong to the scope of protection of the present application.

[0047] Embodiment 1

[0048] Preparation of the indole compound

[0049] Methyl 3,5-dichloro-2-oxoindoline-3-carboxylate (1.0 g) and N-benzyl-2-(1H-indol-3-yl)ethan-1-amine (0.96 g) were dissolved in 10 mL of MeCN, DIPEA (2.01 mL) was slowly added at 0℃, and the reaction mixture was stirred at room temperature for 2 h, concentrated under reduced pressure, and purified by column chromatography (DCM:MeOH, volume ratio = 300:1 to 150:1) to obtain the indole compound (white solid, 1.15 g, yield 63%, purity >98%).

[0050] Figure 1 The structure characterization chart of the indole compound is shown below, from top to bottom, the hydrogen spectrum chart, the carbon spectrum chart and the mass spectrum chart. Figure 1 It can be seen that the indole compound prepared by the present application is: methyl 3-((2-(1H-indol-3-yl)ethyl ethyl)(benzyl)amino)-5-chloro-2-oxoindoline-3-carboxylate and methyl 3-((1-indol-3-yl-3-yl)methyl benzyl)-5-chloroindole-3-carboxylate; the characterization data of the indole compound are as follows: m.p: 153.2-154.5.1H 1H NMR (600 MHz, CDC13) δ 8.56 (br s., 1 H.), 7.35 (d, J = 7 Hz, 2 H), 7.21 - 7.29 (m, 3 H), 7.11 (t d, J = 7.35, 1.51 Hz, 1 H), 6.94 - 7.00 (m, 2 H), 4.13 (s, 2 H), 3.75 (s, 3 H), 2.96 - 3.00 (m, 1 H), 2.86 - 2.87 (m, 3 H), 2.58 (td, J = 9.70, 5.72 Hz, 2 H);13C NMR (150 MHz, CDC13) δ 176.9, 169.3, 140.2, 140.1, 136.1, 130.3, 128.7, 128.4, 128.3, 128.2, 127.3, 127.2, 126.0, 121.8, 121.7, 119.0, 118.5, 113.5, 111.8, 111.1, 76.6, 55.7, 53.4, 52.5, 25.3; HRMS (ESI) m / z calcd for C 13 C 27 H 24 O3N3Cl[M + Na] + : 496.13984, found: 496.13843.

[0051] Example 2

[0052] The effect of different concentrations of indole compounds combined with DEX on the proliferation of Jurkat cells was determined by MTT method.

[0053] Acute lymphoblastic leukemia cells Jurkat were planted in 96-well plates at a density of 1 x 10 4 μM, 4 μM, 8 μM, 12 μM and 16 μM of the indole compound prepared in Example 1 were added, and the combined drug group was administered with the above different concentrations of the indole compound and 100 μM of DEX. In addition, a DMSO control group was set up, 10% MTT solution (5 mg / mL) was added to each well after 24 h, 48 h and 72 h of culture, 100 μL of triplex solution (SDS 10 g, isobutyl alcohol 5 mL, 10 M HCl 0.1 mL) was added to dissolve the formazan crystals until complete dissolution, and the OD value of each group was detected at 570 nm using an enzyme marker, the survival rate of the cells was calculated, and the results are shown in Table 1. Figure 2

[0054] Survival rate calculation method: survival rate = (treatment group OD value / control group OD value) x 100%.

[0055] From Figure 2 ​It can be seen that the indole compound prepared in Example 1 combined with DEX has concentration and time-dependent inhibition of Jurkat cell proliferation.

[0056] Example 3

[0057] Effect of the indole compound prepared in Example 1 combined with DEX on the cell cycle of Jurkat cells.

[0058] Jurkat cells were seeded at 3x10 5 cells per well in a 6-well plate, and DMSO, DEX, the indole compound prepared in Example 1 (4 μM), the indole compound prepared in Example 1 (4 μM) + DEX (100 μM), the indole compound prepared in Example 1 (8 μM), the indole compound prepared in Example 1 (8 μM) + DEX (100 μM), LWX-473 (12 μM), and LWX-473 (12 μM) + DEX (100 μM) were added, respectively, and the cells were incubated for 36 h. After washing twice with PBS, the cells were fixed with 500 μL of 70% ethanol overnight, and then centrifuged to remove the supernatant. After washing twice with PBS, the cells were resuspended with 500 μL of PBS, and then Annexin V-FITC and PI were added. After incubation at 37°C for 30 min, the cells were centrifuged to remove the dye, resuspended with PBS, and then detected by flow cytometry.

[0059] Figure 3 The cell cycle distribution results of Jurkat cells in different groups are shown in Table 1, Figure 4 The distribution of the cell cycle of Jurkat cells is shown in Figure 1. From Figures 3-4 It can be seen that the indole compound combined with DEX at different concentrations can induce Jurkat cell cycle arrest at the G1 phase.

[0060] Example 4

[0061] Effect of the indole compound prepared in Example 1 combined with DEX on the apoptosis of Jurkat cells.

[0062] After incubation for 36 h, the cells were collected, washed twice with PBS, resuspended with 50 μL of 1x Binding buffer, and then stained with 2.5 μL of Annexin V-FITC and 2.5 μL of PI for 15 min. After centrifugation to remove the dye, the cells were resuspended with 200 μL of pre-cooled PBS, and then detected by flow cytometry.

[0063] Figure 5 The apoptosis distribution results of Jurkat cells are shown in Table 2, Figure 6The statistical chart of Jurkat cell apoptosis rate. From Figures 5-6 It is known that the indole compound of different concentrations combined with DEX can induce Jurkat cell apoptosis, which has a concentration-dependent effect.

[0064] Example 5

[0065] The effect of the indole compound prepared in Example 1 combined with DEX on the expression of Jurkat cell cycle and apoptosis-related proteins.

[0066] Jurkat cells were planted in 100mm dishes at a density of 1x10 7 DMSO (0.1%), DEX (100 μM), the indole compound prepared in Example 1, and the indole compound prepared in Example 1 + DEX (100 μM) were added, respectively, and the cells were incubated for 36 h. Then the cells were collected, washed twice with PBS, and the cell pellets were obtained. After adding appropriate cell lysis solution PIPA (containing 1% PMSF), the cells were lysed on ice for 30 min. Then the cells were centrifuged at 12000 rpm and 4°C for 15 min, and the protein supernatant was collected. The protein was quantified according to the instructions of the BCA kit, and the loading volume was calculated according to the protein concentration. After mixing the protein with 5xloading buffer (volume ratio = 5:1), the mixture was heated at 100°C for 3-5 min, and then aliquoted and stored at -80°C.

[0067] According to the loading amount and the target band, the appropriate concentration of separation gel was selected. Generally, 8-12% separation gel and 5% concentration gel were used. After the gel was prepared, the sample was loaded according to the loading volume of each group, and the protein sample was separated at 80V for 30 min and 120V for 60 min. The protein was transferred to the PVDF membrane by "sandwich" structure wet transfer method at 220mmA constant current, and the membrane was washed with 1xTBST for 3 times, each for 5 min. After blocking with 3% BSA or 3% skimmed milk powder for 1-2 h, the membrane was washed with 1xTBST for 3 times, each for 5 min. The target protein primary antibody diluent was added, and the membrane was incubated on a shaker at 4°C for 12 h. After collecting the primary antibody, the membrane was washed with 1xTBST for 3 times, each for 5 min. Then 3% secondary antibody diluent was added, and the membrane was incubated at room temperature for 2 h. After recovering the secondary antibody, the membrane was washed with 1xTBST for 3 times, each for 5 min.

[0068] The washed protein membrane was developed by Odyssey imager, and the fluorescence values of the target bands of each group were normalized. β-actin was used as a quantitative control.

[0069] Figure 7 The effect of indole compound combined with DEX on G1 phase-related proteins, Figure 8 The effect of indole compound combined with DEX on apoptosis-related proteins. FromFigures 7-8 It can be seen that the combination of indole compounds and DEX can significantly down-regulate the expression of oncogene C-Myc, down-regulate CDK2 and Cyclin E1 cyclins; the combination of indole compounds and DEX can down-regulate the anti-apoptotic protein Bcl-2 and up-regulate the expression of pro-apoptotic protein Bim; and it can also increase the cleaved forms of caspase 9, caspase 3 and PARP, indicating that the combination indeed induces apoptosis of Jurkat cells.

[0070] Example 6

[0071] Effect of the combination of indole compounds prepared in Example 1 and DEX on glucocorticoid resistance related pathways of Jurkat cells

[0072] According to the plating administration and protein collection procedure of Example 5, Western-Blot detection was performed, and the results are shown in Figure 9 Figure A is the expression of glucocorticoid receptor protein GR caused by DEX, indole compounds and the combination of DEX and indole compounds, Figure B is the expression of JAK2 / STAT3 signaling protein induced by DEX, indole compounds and the combination of DEX and indole compounds, and Figure C is the expression of PI3K / AKT signaling protein induced by DEX, indole compounds and the combination of DEX and indole compounds. Figure 9 It can be seen that the expression of GR phosphorylation level closely related to glucocorticoid resistance is significantly increased after the administration of the combination of indole compounds and DEX, and the combination of indole compounds and DEX can also regulate the PI3K / AKT signaling pathway and down-regulate the phosphorylation levels of PI3K and AKT.

[0073] As can be seen from Examples 1-6, the indole compound synthesized in the present application can inhibit the proliferation of Jurkat leukemia cells, induce cell cycle arrest at the G1 phase, induce Jurkat cell apoptosis, regulate cell cycle and cell apoptosis protein inhibition and kill leukemia cells, and overcome the dexamethasone resistance of Jurkat cells by regulating the expression of GR and PI3K / AKT, JAK2 / STAT3 signaling pathways, thereby playing a role in treating leukemia.

[0074] The above only describes the preferred embodiments of the present application, and it should be noted that those skilled in the art can make several improvements and refinements without departing from the principles of the present application, and these improvements and refinements should also be considered within the protection scope of the present application.

Claims

1. An indole compound, characterized by, The chemical structural formula is shown as Formula I: Formula I.

2. The method of producing an indole compound according to claim 1, characterized by, The method comprises the following steps: The 3,5-dichloro-2-oxoindoline-3-carboxylic acid methyl ester and the N-benzyl-2-(1H-indol-3-yl)ethane-1-amine are mixed in a mass ratio of 1:0.5-1.

5.

3. The production method according to claim 2, characterized by, The organic solvent comprises one or more of acetonitrile, methanol and acetone.

4. The production method according to claim 2, characterized by, The organic base comprises one or more of N,N-diisopropyl ethylamine, dimethylamine, aniline and pyridine. The temperature of the substitution reaction is 22-25°C, and the time is 1-3 hours.

5. The production method according to claim 2, 3 or 4, characterized in that, The substitution reaction is followed by post-treatment.

6. The preparation method according to claim 2, characterized in that, The post-treatment comprises concentrating the obtained substitution reaction liquid and then purifying it by column chromatography.

7. The production method according to claim 6, characterized by, The eluent for the column chromatography is a mixture of dichloromethane and methanol, and the volume ratio of the dichloromethane to the methanol is 150:1-300:

1.

8. The method of claim 7, wherein, 9. Use of the indole compound of claim 1 or the indole compound prepared by the preparation method of any one of claims 2-8 in the preparation of a medicament for treating leukemia. The medicament for treating leukemia further comprises dexamethasone as an active ingredient.

10. Use according to claim 9, characterized in that, The medicament comprises an active ingredient and pharmaceutically acceptable adjuvants; the active ingredient comprises the indole compound; the indole compound is the indole compound of claim 1 or the indole compound prepared by the preparation method of any one of claims 2-8.

11. A medicament for treating leukemia, characterized by comprising the compound of claim 1. The medicament comprises an active ingredient and pharmaceutically acceptable adjuvants; the active ingredient comprises a mixture of the indole compound and dexamethasone; the indole compound is the indole compound of claim 1 or the indole compound prepared by the preparation method of any one of claims 2-8.

12. A medicament for treating leukemia, characterized by comprising the compound of claim 1. ​

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