Preparation method and application of theophylline derivative with targeting IDO1

Abstract

The invention discloses a preparation method and application of a theophylline derivative with targeting IDO1, and belongs to the technical field of medicine synthesis. According to the technical scheme, the theophylline derivative is characterized in that the molecular structure of the theophylline derivative is # imgabs0 #, wherein R is an aryl derivative or triazole. The invention designs and synthesizes a theophylline derivative with a novel structure, and the compound can target IDO1 protein as an IDO1 inhibitor.

Description

Technical Field

[0001] The invention belongs to the technical field of pharmaceutical chemical synthesis, and particularly relates to a preparation method and application of a theophylline derivative targeting IDO1. Background Art

[0002] Theophylline, chemical name: 1,3-dimethyl-3,7-dihydro-1H-purine-2,6-dione, belongs to xanthine compounds. Theophylline can reduce smooth muscle tension and dilate the airways; it can promote the release of endogenous adrenaline and norepinephrine, relax airway smooth muscles; inhibit the release of calcium ions from the endoplasmic reticulum of smooth muscles, reduce the intracellular calcium ion concentration and produce airway dilation, and has a strong relaxant effect on smooth muscles. It is used for bronchial asthma, acute bronchitis, asthmatic bronchitis, obstructive emphysema, etc. to relieve wheezing symptoms. It is also suitable for the symptoms of bronchospasm associated with chronic bronchitis and emphysema. In addition to being used to treat bronchitis and asthma, theophylline and its derivatives also have a variety of biological activities, such as anti-tumor activity, anti-inflammatory, central nervous system stimulant, immunomodulatory and hypoglycemic activity. For example, the combination of theophylline with carmustine or cyclophosphamide can produce a synergistic antitumor effect by increasing cyclophosphamide. Theophylline and caffeine can enhance the toxicity of doxorubicin to tumor cells. When theophylline is used in combination with gemcitabine or cisplatin, theophylline is found to induce programmed cell death in a variety of human cancer cell lines and malignantly transformed granulosa cell lines. The mechanisms of theophylline's pro-apoptotic effect have been found to include reducing the level of intracellular anti-apoptotic mediator Bcl-2, or acting as a potent aldehyde dehydrogenase 1 family member A1 (ALDH1A1) protein inhibitor to inhibit the proliferation of a variety of malignant tumors and cancer stem cells. Pentoxifylline and the proteasome inhibitor MG132 showed anti-leukemic activity in an experimental model of U937 human leukemia cells, and an additive effect was observed when the two compounds were used together.

[0003] Indoleamine-2,3-dioxygenase 1 (IDO1) is an important immunomodulatory enzyme widely present in various tissues and cells. It plays a key role in the metabolism of tryptophan along the kynurenine pathway and has become an attractive therapeutic target for cancer and other diseases (e.g., Alzheimer's disease and arthritis). Overexpression of IDO1 in the inflammatory and tumor microenvironment promotes the consumption of tryptophan and the increase of kynurenine, which in turn induces a combination of cell cycle arrest and apoptosis of effector T cells, and participates in the activation of regulatory T cells and myeloid-derived suppressor cells (MDSCs), inhibits the function of natural killer cells (NK), and plays an important immunosuppressive function. Therefore, in addition to checkpoint blockade or adoptive transfer of chimeric antigen receptor T cells, targeted IDO1 inhibition also provides therapeutic opportunities for cancer immunotherapy. On the other hand, in healthy postpartum individuals, IDO1 promotes tolerance by suppressing immune responses, while during pregnancy, IDO1 helps protect the fetus from maternal T lymphocytes. In addition, IDO1 plays a key role in promoting tumor angiogenesis by regulating the expression of interferon-γ (IFN-γ) and interleukin-6 (IL-6). IDO1 is overexpressed in the vast majority of cancers. Various strategies targeting IDO1 have been evaluated in multiple clinical trials and have produced encouraging results. Blocking IDO1 activity can reduce tumor proliferation in a T lymphocyte-mediated manner and enhance the efficacy of chemotherapy, radiotherapy, targeted therapy, and immunotherapy.

[0004] Theophylline structure has a very important function, which is that it has very good hydrophilicity. Because of its good hydrophilicity, theophylline is extracted by water extraction of tea leaves. Therefore, based on the advantage of the hydrophilicity of theophylline structure, we can develop it into an IDO1 inhibitor as an active group that acts on the hydrophilic pocket (Pocket B), and then introduce a 1,2,3-triazole structure that can act on heme, and a molecular structure that can act on the hydrophobic pocket (Pocket B). Specific operation: Theophylline acetic acid reacts with 4-aminophenylacetylene to introduce the terminal alkyne, and then reacts with azide compounds with different hydrophobic substituents to obtain the target compound. The 14 hydrophobic groups we selected have benzyl structures and phenyl structures, and have the same substituents for activity comparison. Summary of the invention

[0005] The method for preparing a theophylline derivative of the present invention is characterized in that the molecular structure of the theophylline derivative is: Wherein R is an aryl derivative or a triazole.

[0006] The method for preparing a theophylline derivative of the present invention is characterized by:

[0007] (1) At room temperature, add theophylline acetic acid (5 g, 0.02 mol), 4-aminophenylacetylene (3.69 g, 0.03 mol), HATU (12.96 g, 0.03 mol) and DIPEA (8.13 g, 0.06 mol) and solvent DMF into a 500 mL reaction bottle and stir under nitrogen for 24 hours. Monitor the reaction process by thin layer chromatography (TLC). After the reaction is completed, remove DMF with an oil pump, extract the product with dichloromethane, wash with saturated sodium chloride aqueous solution, dry with anhydrous sodium sulfate, and concentrate under reduced pressure. Recrystallize in methanol and filter to obtain a terminal acetylene derivative.

[0008] (2) In a reaction flask, add a terminal acetylene derivative, an azide compound, tert-butyl alcohol, water, tetrahydrofuran, anhydrous copper sulfate and sodium ascorbate in sequence, react at 70°C until the raw materials react completely, add dichloromethane, filter the reaction solution to obtain a yellow liquid, separate the organic phase, extract the aqueous phase twice with dichloromethane, combine the organic phases, dry over anhydrous magnesium sulfate, and concentrate under reduced pressure to obtain a solid, which is separated using a chromatography column to obtain the target compound.

[0009] (3) Take out the viable human cervical cancer Hela cell culture dish from the CO2 incubator and perform the following operations: perform aseptic operation next to an alcohol lamp, open the dish cover, aspirate the culture medium into a waste liquid tank, wash the culture medium in the culture bottle twice with 2 mL of PBS, digest with 0.25% trypsin, and terminate the digestion when the intercellular gaps increase and the cells become small circles. Use a pipette to blow the bottom of the culture bottle to make the cells fall off, transfer the obtained cell suspension to a sterile centrifuge tube, set the centrifuge to 1000 r / min, centrifuge for 3 min, then slowly pour out the supernatant in the centrifuge tube, add 2 to 5 mL of culture medium, and count the cells under an inverted microscope. According to the counting results, the viable human cervical cancer Hela cells in the logarithmic phase were plated in a 96-well cell culture plate with 50,000 cells per well, cultured with RPMI1640 containing 10% fetal bovine serum for 5 to 6 hours, and 100 μL of the test compound (concentration of 1.0 μM) and recombinant human interferon γ (final concentration of 100 ng / mg) diluted with culture medium were added to activate the IDO1 expression in Hela cells. After the operation, the 96-well cell culture plate was placed in a 37°C cell culture incubator rich in 5% carbon dioxide for 18 hours, and the reaction was terminated with a certain amount of 3.05N trichloroacetic acid, and then incubated at 50°C for 30 minutes. After the cell culture fluid was precipitated, the supernatant was taken and developed with (N,N-dimethyl) benzaldehyde, and the absorbance at 480nm was detected by a multifunctional microplate reader. The group treated with only IFNγ medium without drugs was taken as 100% (At), and the group treated with only 0.1% DMSO medium was taken as blank control 0% (Ab); the absorbance under different treatment conditions was calculated according to the following formula: Absorbance % = (A-Ab) / (At-Ab), A: drug treatment + 100 ng / mL IFNγ, Ab: blank control, At: no drug, only 100 ng / mL IFNγ.

[0010] The theophylline derivatives of the present invention have the following technical advantages: the triazole structure can form a π-π conjugation with PHE163 on heme, and the nitrogen-containing substituent connected to the benzene ring can coordinate with the ferrous ion in heme. Through the dual action, they can be used as IDO1 inhibitors for tumor immunotherapy, prevent and treat tumor cell immune escape, and reduce the levels of tumor cell-related inflammatory factors. DETAILED DESCRIPTION

[0011] The above contents of the present invention are further described in detail below through examples, but this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples, and all technologies implemented based on the above contents of the present invention belong to the scope of the present invention.

[0012] Example 1

[0013]

[0014] At room temperature, theophylline acetic acid (5g, 0.02mol), 4-aminophenylacetylene (3.69g, 0.03mol), HATU (12.96g, 0.03mol) and DIPEA (8.13g, 0.06mol) were added to a 500mL reaction bottle together with solvent DMF and stirred for 24 hours under nitrogen protection. The reaction process was monitored by thin layer chromatography (TLC). After the reaction was completed, DMF was removed by an oil pump, the product was extracted with dichloromethane, washed with saturated sodium chloride aqueous solution, dried with anhydrous sodium sulfate, and concentrated under reduced pressure. Recrystallized in methanol and filtered to obtain a terminal acetylene derivative

[0015] Example 2

[0016]

[0017] In a reaction flask, 1 g of a terminal acetylene derivative, 1 g of 2-chlorobenzyl azide, 20 mL of tert-butanol, 20 mL of water, 20 mL of tetrahydrofuran, 0.5 g of anhydrous copper sulfate and 1 g of sodium ascorbate were added in sequence. The reaction was carried out at 70 °C for 6 h. After the raw materials reacted completely, 50 mL of dichloromethane was added, and the reaction solution was filtered to obtain a yellow liquid. The organic phase was separated, and the aqueous phase was extracted twice with 20 mL of dichloromethane. The combined organic phases were dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain a solid, which was separated using a chromatography column to obtain compound 1.

[0018] Example 3

[0019]

[0020] In a reaction flask, 1 g of a terminal acetylene derivative, 1 g of 3-methoxyazide, 20 mL of tert-butanol, 20 mL of water, 20 mL of tetrahydrofuran, 0.5 g of anhydrous copper sulfate and 1 g of sodium ascorbate were added in sequence. The reaction was carried out at 70°C for 6 h. After the raw materials were completely reacted, 50 mL of dichloromethane was added, and the reaction solution was filtered to obtain a yellow liquid. The organic phase was separated, and the aqueous phase was extracted twice with 20 mL of dichloromethane. The combined organic phases were dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain a solid, which was separated using a chromatography column to obtain compound 2.

[0021] Example 4

[0022]

[0023] In a reaction flask, 1 g of a terminal acetylene derivative, 1 g of 4-chlorobenzyl azide, 20 mL of tert-butanol, 20 mL of water, 20 mL of tetrahydrofuran, 0.5 g of anhydrous copper sulfate and 1 g of sodium ascorbate were added in sequence and reacted at 70 °C for 6 h. After the raw materials reacted completely, 50 mL of dichloromethane was added and the reaction solution was filtered to obtain a yellow liquid. The organic phase was separated and the aqueous phase was extracted twice with 20 mL of dichloromethane. The combined organic phases were dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain a solid, which was separated using a chromatography column to obtain compound 3.

[0024] Example 5

[0025]

[0026] In a reaction flask, 1 g of a terminal acetylene derivative, 1 g of 2-trifluoromethylbenzyl azide, 20 mL of tert-butanol, 20 mL of water, 20 mL of tetrahydrofuran, 0.5 g of anhydrous copper sulfate and 1 g of sodium ascorbate were added in sequence. The reaction was carried out at 70 °C for 6 h. After the raw materials reacted completely, 50 mL of dichloromethane was added, and the reaction solution was filtered to obtain a yellow liquid. The organic phase was separated, and the aqueous phase was extracted twice with 20 mL of dichloromethane. The combined organic phases were dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain a solid, which was separated using a chromatography column to obtain compound 4.

[0027] Example 6

[0028]

[0029] In a reaction flask, 1 g of a terminal acetylene derivative, 1 g of benzyl azide, 20 mL of tert-butanol, 20 mL of water, 20 mL of tetrahydrofuran, 0.5 g of anhydrous copper sulfate and 1 g of sodium ascorbate were added in sequence. The mixture was reacted at 70 °C for 6 h. After the reaction of the raw materials was complete, 50 mL of dichloromethane was added. The reaction solution was filtered to obtain a yellow liquid. The organic phase was separated, and the aqueous phase was extracted twice with 20 mL of dichloromethane. The combined organic phases were dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain a solid, which was separated using a chromatography column to obtain compound 5.

[0030] Example 7

[0031]

[0032] In a reaction flask, 1 g of a terminal acetylene derivative, 1 g of 2-bromobenzyl azide, 20 mL of tert-butanol, 20 mL of water, 20 mL of tetrahydrofuran, 0.5 g of anhydrous copper sulfate and 1 g of sodium ascorbate were added in sequence. The mixture was reacted at 70 °C for 6 h. After the raw materials were completely reacted, 50 mL of dichloromethane was added and the reaction solution was filtered to obtain a yellow liquid. The organic phase was separated and the aqueous phase was extracted twice with 20 mL of dichloromethane. The combined organic phases were dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain a solid, which was separated using a chromatography column to obtain compound 6.

[0033] Example 8

[0034]

[0035] In a reaction flask, 1 g of a terminal acetylene derivative, 1 g of 4-trifluoromethylbenzyl azide, 20 mL of tert-butanol, 20 mL of water, 20 mL of tetrahydrofuran, 0.5 g of anhydrous copper sulfate and 1 g of sodium ascorbate were added in sequence. The reaction was carried out at 70°C for 6 h. After the raw materials were completely reacted, 50 mL of dichloromethane was added, and the reaction solution was filtered to obtain a yellow liquid. The organic phase was separated, and the aqueous phase was extracted twice with 20 mL of dichloromethane. The combined organic phases were dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain a solid, which was separated using a chromatography column to obtain compound 7.

[0036] Example 9

[0037]

[0038] In a reaction flask, 1 g of a terminal acetylene derivative, 1 g of 2-chlorophenyl azide, 20 mL of tert-butanol, 20 mL of water, 20 mL of tetrahydrofuran, 0.5 g of anhydrous copper sulfate and 1 g of sodium ascorbate were added in sequence and reacted at 70 °C for 6 h. After the raw materials reacted completely, 50 mL of dichloromethane was added and the reaction solution was filtered to obtain a yellow liquid. The organic phase was separated and the aqueous phase was extracted twice with 20 mL of dichloromethane. The combined organic phases were dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain a solid, which was separated using a chromatography column to obtain compound 8.

[0039] Example 10

[0040]

[0041] In a reaction flask, 1 g of a terminal acetylene derivative, 1 g of 3-methoxyphenyl azide, 20 mL of tert-butanol, 20 mL of water, 20 mL of tetrahydrofuran, 0.5 g of anhydrous copper sulfate and 1 g of sodium ascorbate were added in sequence. The reaction was carried out at 70°C for 6 h. After the raw materials were completely reacted, 50 mL of dichloromethane was added, and the reaction solution was filtered to obtain a yellow liquid. The organic phase was separated, and the aqueous phase was extracted twice with 20 mL of dichloromethane. The combined organic phases were dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain a solid, which was separated using a chromatography column to obtain compound 9.

[0042] Embodiment 11

[0043]

[0044] In a reaction flask, 1 g of a terminal acetylene derivative, 1 g of 4-chlorophenyl azide, 20 mL of tert-butanol, 20 mL of water, 20 mL of tetrahydrofuran, 0.5 g of anhydrous copper sulfate and 1 g of sodium ascorbate were added in sequence and reacted at 70 °C for 6 h. After the raw materials reacted completely, 50 mL of dichloromethane was added and the reaction solution was filtered to obtain a yellow liquid. The organic phase was separated and the aqueous phase was extracted twice with 20 mL of dichloromethane. The combined organic phases were dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain a solid, which was separated using a chromatography column to obtain compound 10.

[0045] Example 12

[0046]

[0047] In a reaction flask, 1 g of a terminal acetylene derivative, 1 g of 2-trifluoromethylphenyl azide, 20 mL of tert-butanol, 20 mL of water, 20 mL of tetrahydrofuran, 0.5 g of anhydrous copper sulfate and 1 g of sodium ascorbate were added in sequence. The reaction was carried out at 70 °C for 6 h. After the raw materials were completely reacted, 50 mL of dichloromethane was added, and the reaction solution was filtered to obtain a yellow liquid. The organic phase was separated, and the aqueous phase was extracted twice with 20 mL of dichloromethane. The combined organic phases were dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain a solid, which was separated using a chromatography column to obtain compound 11.

[0048] Example 13

[0049]

[0050] In a reaction flask, 1 g of a terminal acetylene derivative, 1 g of phenyl azide, 20 mL of tert-butanol, 20 mL of water, 20 mL of tetrahydrofuran, 0.5 g of anhydrous copper sulfate and 1 g of sodium ascorbate were added in sequence. The mixture was reacted at 70 °C for 6 h. After the raw materials were completely reacted, 50 mL of dichloromethane was added and the reaction solution was filtered to obtain a yellow liquid. The organic phase was separated and the aqueous phase was extracted twice with 20 mL of dichloromethane. The combined organic phases were dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain a solid, which was separated using a chromatography column to obtain compound 12.

[0051] Embodiment 14

[0052]

[0053] In a reaction flask, 1 g of a terminal acetylene derivative, 1 g of 2-bromophenyl azide, 20 mL of tert-butanol, 20 mL of water, 20 mL of tetrahydrofuran, 0.5 g of anhydrous copper sulfate and 1 g of sodium ascorbate were added in sequence. The reaction was carried out at 70 °C for 6 h. After the raw materials were completely reacted, 50 mL of dichloromethane was added, and the reaction solution was filtered to obtain a yellow liquid. The organic phase was separated, and the aqueous phase was extracted twice with 20 mL of dichloromethane. The combined organic phases were dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain a solid, which was separated using a chromatography column to obtain compound 13.

[0054] Embodiment 15

[0055]

[0056] In a reaction flask, 1 g of a terminal acetylene derivative, 1 g of 4-trifluoromethylphenyl azide, 20 mL of tert-butanol, 20 mL of water, 20 mL of tetrahydrofuran, 0.5 g of anhydrous copper sulfate and 1 g of sodium ascorbate were added in sequence. The reaction was carried out at 70°C for 6 h. After the raw materials were completely reacted, 50 mL of dichloromethane was added, and the reaction solution was filtered to obtain a yellow liquid. The organic phase was separated, and the aqueous phase was extracted twice with 20 mL of dichloromethane. The combined organic phases were dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain a solid, which was separated using a chromatography column to obtain compound 14.

[0057] Example 16

[0058]

[0059] In a reaction flask, 1 g of a terminal acetylene derivative, 1 g of 4-azido-1H-indole, 20 mL of tert-butanol, 20 mL of water, 20 mL of tetrahydrofuran, 0.5 g of anhydrous copper sulfate and 1 g of sodium ascorbate were added in sequence. The mixture was reacted at 70 °C for 6 h. After the raw materials were completely reacted, 50 mL of dichloromethane was added. The reaction solution was filtered to obtain a yellow liquid. The organic phase was separated, and the aqueous phase was extracted twice with 20 mL of dichloromethane. The combined organic phases were dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain a solid, which was separated using a chromatography column to obtain compound 15.

[0060] Embodiment 17

[0061]

[0062] In a reaction flask, 1 g of a terminal acetylene derivative, 1 g of 5-azido-1H-indole, 20 mL of tert-butanol, 20 mL of water, 20 mL of tetrahydrofuran, 0.5 g of anhydrous copper sulfate and 1 g of sodium ascorbate were added in sequence. The mixture was reacted at 70 °C for 6 h. After the raw materials were completely reacted, 50 mL of dichloromethane was added and the reaction solution was filtered to obtain a yellow liquid. The organic phase was separated and the aqueous phase was extracted twice with 20 mL of dichloromethane. The combined organic phases were dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain a solid, which was separated using a chromatography column to obtain compound 16.

[0063] Embodiment 18

[0064]

[0065] In a reaction flask, 1 g of a terminal acetylene derivative, 1 g of 6-azido-1H-indole, 20 mL of tert-butanol, 20 mL of water, 20 mL of tetrahydrofuran, 0.5 g of anhydrous copper sulfate and 1 g of sodium ascorbate were added in sequence. The mixture was reacted at 70°C for 6 h. After the raw materials were completely reacted, 50 mL of dichloromethane was added and the reaction solution was filtered to obtain a yellow liquid. The organic phase was separated and the aqueous phase was extracted twice with 20 mL of dichloromethane. The combined organic phases were dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain a solid, which was separated using a chromatography column to obtain compound 17.

[0066] Embodiment 19

[0067]

[0068] In a reaction flask, 1 g of a terminal acetylene derivative, 1 g of 7-azido-1H-indole, 20 mL of tert-butanol, 20 mL of water, 20 mL of tetrahydrofuran, 0.5 g of anhydrous copper sulfate and 1 g of sodium ascorbate were added in sequence. The mixture was reacted at 70 °C for 6 h. After the raw materials were completely reacted, 50 mL of dichloromethane was added. The reaction solution was filtered to obtain a yellow liquid. The organic phase was separated, and the aqueous phase was extracted twice with 20 mL of dichloromethane. The combined organic phases were dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain a solid, which was separated using a chromatography column to obtain compound 18.

[0069] Embodiment 20

[0070]

[0071] In a reaction flask, 1 g of a terminal acetylene derivative, 1 g of 5-azido-1H-benzo[D]-imidazole, 20 mL of tert-butanol, 20 mL of water, 20 mL of tetrahydrofuran, 0.5 g of anhydrous copper sulfate and 1 g of sodium ascorbate were added in sequence. The mixture was reacted at 70°C for 6 h. After the raw materials were completely reacted, 50 mL of dichloromethane was added and the reaction solution was filtered to obtain a yellow liquid. The organic phase was separated and the aqueous phase was extracted twice with 20 mL of dichloromethane. The combined organic phases were dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain a solid, which was separated using a chromatography column to obtain compound 19.

[0072] Embodiment 21

[0073]

[0074] In a reaction flask, 1 g of a terminal acetylene derivative, 1 g of 6-azidoquinoxaline, 20 mL of tert-butanol, 20 mL of water, 20 mL of tetrahydrofuran, 0.5 g of anhydrous copper sulfate and 1 g of sodium ascorbate were added in sequence. The mixture was reacted at 70 °C for 6 h. After the raw materials were completely reacted, 50 mL of dichloromethane was added and the reaction solution was filtered to obtain a yellow liquid. The organic phase was separated and the aqueous phase was extracted twice with 20 mL of dichloromethane. The combined organic phases were dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain a solid, which was separated using a chromatography column to obtain compound 20.

[0075] Embodiment 22

[0076]

[0077] In a reaction flask, 1 g of a terminal acetylene derivative, 1 g of 5-azidobenzimidazolone, 20 mL of tert-butanol, 20 mL of water, 20 mL of tetrahydrofuran, 0.5 g of anhydrous copper sulfate and 1 g of sodium ascorbate were added in sequence. The mixture was reacted at 70 °C for 6 h. After the raw materials were completely reacted, 50 mL of dichloromethane was added and the reaction solution was filtered to obtain a yellow liquid. The organic phase was separated and the aqueous phase was extracted twice with 20 mL of dichloromethane. The combined organic phases were dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain a solid, which was separated using a chromatography column to obtain compound 21.

[0078] Embodiment 23

[0079]

[0080] In a reaction flask, 1 g of a terminal acetylene derivative, 1 g of TMS-azide, 20 mL of tert-butanol, 20 mL of water, 20 mL of tetrahydrofuran, 0.5 g of anhydrous copper sulfate and 1 g of sodium ascorbate were added in sequence. The mixture was reacted at 70 °C for 6 h. After the raw materials were completely reacted, 50 mL of dichloromethane was added and the reaction solution was filtered to obtain a yellow liquid. The organic phase was separated and the aqueous phase was extracted twice with 20 mL of dichloromethane. The combined organic phases were dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain a solid, which was separated using a chromatography column to obtain compound 22.

[0081] Embodiment 24

[0082]

[0083] In a reaction flask, 1 g of a terminal acetylene derivative, 1 g of 5-azidobenzotriazole, 20 mL of tert-butanol, 20 mL of water, 20 mL of tetrahydrofuran, 0.5 g of anhydrous copper sulfate and 1 g of sodium ascorbate were added in sequence. The mixture was reacted at 70°C for 6 h. After the raw materials were completely reacted, 50 mL of dichloromethane was added and the reaction solution was filtered to obtain a yellow liquid. The organic phase was separated and the aqueous phase was extracted twice with 20 mL of dichloromethane. The combined organic phases were dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain a solid, which was separated using a chromatography column to obtain compound 23.

[0084] Embodiment 25

[0085]

[0086] In a reaction flask, 1 g of a terminal acetylene derivative, 1 g of 5-azidobenzofuran, 20 mL of tert-butanol, 20 mL of water, 20 mL of tetrahydrofuran, 0.5 g of anhydrous copper sulfate and 1 g of sodium ascorbate were added in sequence. The mixture was reacted at 70°C for 6 h. After the raw materials were completely reacted, 50 mL of dichloromethane was added and the reaction solution was filtered to obtain a yellow liquid. The organic phase was separated and the aqueous phase was extracted twice with 20 mL of dichloromethane. The combined organic phases were dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain a solid, which was separated using a chromatography column to obtain compound 24.

[0087] Embodiment 26

[0088]

[0089] In a reaction flask, 1 g of a terminal acetylene derivative, 1 g of 5-azidoindazolane, 20 mL of tert-butanol, 20 mL of water, 20 mL of tetrahydrofuran, 0.5 g of anhydrous copper sulfate and 1 g of sodium ascorbate were added in sequence. The mixture was reacted at 70°C for 6 h. After the raw materials were completely reacted, 50 mL of dichloromethane was added and the reaction solution was filtered to obtain a yellow liquid. The organic phase was separated and the aqueous phase was extracted twice with 20 mL of dichloromethane. The combined organic phases were dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain a solid, which was separated using a chromatography column to obtain compound 25.

[0090] Embodiment 27

[0091]

[0092] In a reaction flask, 1 g of terminal acetylene derivative, 1 g of azide benzene, 20 mL of tert-butanol, 20 mL of water, 20 mL of tetrahydrofuran, 0.5 g of anhydrous copper sulfate and 1 g of sodium ascorbate were added in sequence and reacted at 70°C for 6 h. After the raw materials reacted completely, 50 mL of dichloromethane was added and the reaction liquid was filtered to obtain a yellow liquid. The organic phase was separated and the aqueous phase was extracted twice with 20 mL of dichloromethane. The combined organic phases were dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain a solid, which was separated using a chromatography column to obtain compound 26.

[0093] Embodiment 28

[0094]

[0095] In a reaction flask, 1 g of terminal acetylene derivative, 1 g of azidobiphenyl, 20 mL of tert-butanol, 20 mL of water, 20 mL of tetrahydrofuran, 0.5 g of anhydrous copper sulfate and 1 g of sodium ascorbate were added in sequence and reacted at 70°C for 6 h. After the raw materials reacted completely, 50 mL of dichloromethane was added and the reaction liquid was filtered to obtain a yellow liquid. The organic phase was separated and the aqueous phase was extracted twice with 20 mL of dichloromethane. The combined organic phases were dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain a solid, which was separated using a chromatography column to obtain compound 27.

[0096] Embodiment 29

[0097]

[0098] In a reaction flask, 1 g of a terminal acetylene derivative, 1 g of 5-azidobenzothiazole, 20 mL of tert-butanol, 20 mL of water, 20 mL of tetrahydrofuran, 0.5 g of anhydrous copper sulfate and 1 g of sodium ascorbate were added in sequence. The mixture was reacted at 70 °C for 6 h. After the raw materials were completely reacted, 50 mL of dichloromethane was added. The reaction solution was filtered to obtain a yellow liquid. The organic phase was separated, and the aqueous phase was extracted twice with 20 mL of dichloromethane. The combined organic phases were dried over anhydrous magnesium sulfate and concentrated under reduced pressure to obtain a solid, which was separated using a chromatography column to obtain compound 28.

[0099] Embodiment 30

[0100] Take out a viable human cervical cancer Hela cell culture dish from the CO2 incubator and perform the following operations: perform aseptic operations next to an alcohol lamp, open the dish cover, aspirate the culture medium into a waste tank, wash the culture medium in the culture bottle twice with 2 mL of PBS, digest with 0.25% trypsin, and terminate the digestion when it is observed that the cell gap increases and the cells become small circles. Use a pipette to blow the bottom of the culture bottle to make the cells fall off, transfer the obtained cell suspension to a sterile centrifuge tube, set the centrifuge to 1000 r / min, centrifuge for 3 minutes, then slowly pour out the supernatant in the centrifuge tube, add 2 to 5 mL of culture medium, and count the cells under an inverted microscope. According to the counting results, the viable human cervical cancer Hela cells in the logarithmic phase were plated in a 96-well cell culture plate with 50,000 cells per well, cultured with RPMI1640 containing 10% fetal bovine serum for 5 to 6 hours, and 100 μL of the test compound (concentration of 1.0 μM) and recombinant human interferon γ (final concentration of 100 ng / mg) diluted with culture medium were added to activate the IDO1 expression in Hela cells. After the operation, the 96-well cell culture plate was placed in a 37°C cell culture incubator rich in 5% carbon dioxide for 18 hours, and the reaction was terminated with a certain amount of 3.05N trichloroacetic acid, and then incubated at 50°C for 30 minutes. After the cell culture fluid was precipitated, the supernatant was taken and developed with (N,N-dimethyl) benzaldehyde, and the absorbance at 480nm was detected by a multifunctional microplate reader. The group treated with only IFNγ medium without drugs was taken as 100% (At), and the group treated with only 0.1% DMSO medium was taken as blank control 0% (Ab); the absorbance under different treatment conditions was calculated according to the following formula: Absorbance % = (A-Ab) / (At-Ab), A: drug treatment + 100 ng / mL IFNγ, Ab: blank control, At: no drug, only 100 ng / mL IFNγ.

[0101]

[0102]

[0103] The above embodiments describe the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are only for illustrating the principles of the present invention. Without departing from the scope of the principles of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of protection of the present invention.

Claims

1. A preparation method and application of a theophylline derivative targeting IDO1, characterized in that the molecular structure of the theophylline derivative is: Wherein R is an aryl derivative or a triazole.

2. The method for preparing a theophylline derivative according to claim 1, characterized in that The specific process is as follows: at room temperature, theophylline acetic acid, 4-aminophenylacetylene, HATU and DIPEA and solvent DMF are added to a reaction bottle, and stirred for 24 hours under nitrogen protection; the reaction process is monitored by thin layer chromatography (TLC); after the reaction is completed, DMF is removed by an oil pump, the product is extracted with dichloromethane, washed with a saturated sodium chloride aqueous solution, dried with anhydrous sodium sulfate, and concentrated under reduced pressure; recrystallized in methanol, filtered, and a terminal acetylene derivative is obtained.

3. The method for preparing a theophylline derivative according to claim 1, characterized in that The specific process is: in a reaction bottle, add a terminal acetylene derivative, an azide compound, tert-butanol, water, tetrahydrofuran, anhydrous copper sulfate and sodium ascorbate in sequence, react at 70°C until the raw materials react completely, add dichloromethane, filter the reaction solution to obtain a yellow liquid, separate the organic phase, extract the aqueous phase twice with dichloromethane, combine the organic phases, dry over anhydrous magnesium sulfate, and concentrate under reduced pressure to obtain a solid, which is separated by a chromatography column to obtain the target compound.

4. The use of a theophylline derivative according to claim 1, characterized in that The specific process is as follows:

1. Take out the viable human cervical cancer Hela cell culture dish from the CO2 incubator, and perform the following operations respectively: perform aseptic operation next to the alcohol lamp, open the dish cover, aspirate the culture solution into the waste liquid tank, wash the culture solution in the culture bottle with 2mL of PBS twice, digest with 0.25% trypsin, and terminate the digestion when the cell gap is observed to increase and the cells become small circles, use a pipette to blow the bottom of the culture bottle to make the cells fall off, transfer the obtained cell suspension to a sterile centrifuge tube, set the centrifuge to 1000r / min, 3min, centrifuge, then slowly pour out the supernatant in the centrifuge tube, add 2-5mL of culture solution, and count the cells under an inverted microscope; according to the counting results, the viable human cervical cancer Hela cells in logarithmic growth are plated in a 96-well cell culture plate at a number of 50,000 cells per well, and RPMI1640 containing 10% fetal bovine serum is used. After 5-6 hours of culture, 100 μL of the test compound (concentration of 1.0 μM) and recombinant human interferon γ (final concentration of 100 ng / mg) diluted with culture medium were added to activate IDO1 expression in Hela cells; after the operation, the 96-well cell culture plate was placed in a 37°C cell culture incubator rich in 5% carbon dioxide and cultured for 18 hours, and a certain amount of 3.05N trichloroacetic acid was used to terminate the reaction, and then incubated at 50°C for 30 minutes; after the cell culture fluid was precipitated, the supernatant was taken and developed with (N,N-dimethyl)benzaldehyde, and the absorbance at 480 nm was detected by a multifunctional microplate reader; the group treated with IFNγ culture medium without drug was taken as 100% (At), and the group treated with 0.1% DMSO culture medium was taken as blank control 0% (Ab); the absorbance under different treatment conditions was calculated according to the following formula: Absorbance % = (A-Ab) / (At-Ab), A: drug treatment + 100 ng / mL IFNγ, Ab: blank control, At: no drug but only 100 ng / mL IFNγ.

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