CN114369060A

CN114369060A - Novel indoleamine 2, 3-dioxygenase inhibitor and application thereof in preparing antitumor drugs

Info

Publication number: CN114369060A
Application number: CN202011100808.9A
Authority: CN
Inventors: 谭瀛轩; 向道凤; 颜道静; 谭相宝
Original assignee: Hangzhou Xing'ao Biological Technology Co ltd
Current assignee: Hangzhou Xing'ao Biological Technology Co ltd
Priority date: 2020-10-15
Filing date: 2020-10-15
Publication date: 2022-04-19
Anticipated expiration: 2040-10-15
Also published as: WO2022077791A1; CN114369060B

Abstract

The invention belongs to the technical field of biological medicines, and provides an innovative inhibitor of indoleamine 2, 3-dioxygenase IDO1, namely a nicotinic acid derivative (a derivative of 3-pyridylpropionic acid). The derivatives of the 3-pyridylpropionic acid can effectively inhibit the metabolism of tryptophan catalyzed by indoleamine 2, 3-dioxygenase IDO1, can obviously inhibit the activity of high-expression IDO1 enzyme in human-derived cancer cells, and has low cytotoxicity. The nicotinic acid derivatives can be used as an IDO1 enzyme inhibitor to obviously inhibit the growth of transplanted tumors of mice when being used alone, and the nicotinic acid derivatives are used together with an innate immune pathway STING agonist or an immune checkpoint inhibitor, so that the effect of inhibiting the growth of the tumors is better, and the safety is also good. Therefore, the nicotinic acid derivative has wide application prospect in preparing antitumor drugs.

Description

Novel indoleamine 2, 3-dioxygenase inhibitor and application thereof in preparing antitumor drugs

Technical Field

The invention belongs to the technical field of biological medicines, and particularly relates to an inhibitor of nicotinic acid derivatives on indoleamine 2, 3-dioxygenase IDO1 and application thereof in preparation of antitumor drugs for immunity

Background

Tumors are one of the major diseases seriously harming human life and health, and are manifested by abnormal cell hyperproliferation and differentiation. According to WHO statistics, annual cancer cases may increase to 2400 million worldwide. At the same time, cancer imposes a huge burden on the global economy. Tumor immunotherapy is an emerging tumor treatment mode following surgery, radiotherapy and chemotherapy. It is a cancer treatment method for preventing, controlling and eliminating cancer by stimulating or enhancing the strength of the human body's own immune system. Compared with the traditional tumor treatment method, the immunotherapy has the unique advantages, can improve the curative effect of the traditional treatment and reduce the adverse reaction brought by the traditional treatment. Innate immune system agonists, immunodetection point blockers and cellular immunotherapy are currently the main areas of research for immunotherapy. The tumor immunotherapy strategy based on metabolic regulation can improve the effectiveness of immunotherapy and benefit more patients, and the regulation of tumor metabolism opens up a new direction for improving tumor immunotherapy.

The human immune system is responsible for recognizing self and non-self, thereby protecting the human body from exogenous and endogenous diseases. The human immune system, which consists of white blood cells and lymphoid organs and tissues including thymus, spleen, tonsil, lymph nodes, lymphatic vessels and bone marrow, maintains a sustained homeostasis of the body by recognizing and eliminating a variety of threats. Cancer immunotherapy refers to the stimulation of the immune system against cancer cells by the introduction of vaccines, cytokines, antibodies, or the metastatic immune cells themselves. The success of immunotherapy in tumor treatment, such as immune checkpoint inhibitors and CAR-T cell therapy, has established its role in cancer treatment. Cancer uses multiple mechanisms to select for host-tumor immune interactions, leading to immune escape. During the past few years, our research on host-tumor interactions has continued to advance, resulting in a variety of promising immunotherapeutic approaches.

Indoleamine 2, 3-dioxygenase (IDO1) is a monomeric heme protein with a molecular weight of 45 KD. IDO1 catalyzes molecules with an indole ring and is therefore called indoleamine 2, 3-dioxygenase. IDO1 is a key rate-limiting metabolic enzyme in the tryptophan metabolic pathway. Numerous studies have shown that IDO1 is highly expressed in many types of human cancers, subsequently leading to accumulation of tryptophan metabolites, resulting in immune tolerance of the body to tumor antigens, ultimately leading to immune escape from the tumor. A number of preclinical and clinical trial studies have shown that IDO1 inhibitors are an effective tool against a variety of cancers. Currently, clinical studies on the treatment of tumors by IDO1 inhibitors are ongoing, and these inhibitors can effectively activate tumor-infiltrating T cells and/or reduce tumor-resident immunosuppressive regulatory T cells, thereby enhancing human immunity and achieving the goal of tumor resistance. Because of the important role of the IDO1 in tumor immunotherapy and no IDO1 inhibitor for tumor therapy is on the market at present, the method has important significance in the screening of a new IDO1 inhibitor and the research of an anti-tumor medicament based on the IDO1 enzyme inhibitor.

Cyclic dinucleotide synthetase (cGAS) is an important cytoplasmic DNA receptor in the innate immune pathway. cGAS catalyzes ATP and GTP in vivo to synthesize cGAMP, and the cGAMP is used as a secondary messenger molecule to induce the generation of interferon IFN-beta and other cytokines through a STING protein pathway on endoplasmic reticulum membrane, regulate the expression of downstream proteins, induce cell growth arrest and apoptosis and generate antiviral effect. The STING pathway can regulate innate immune recognition of immunogenic tumors and promote anti-tumor effects of interferons. IFN-gamma plays an anti-tumor role through TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) in vivo and promotes tumor cell apoptosis. cGAMP is a key stimulator of innate immune response, is an endogenous activator of STING, and has immune anti-tumor effects.

The research invents an innovative class of inhibitors of indoleamine 2, 3-dioxygenase IDO1, namely nicotinic acid derivatives (derivatives of 3-pyridylpropionic acid). The derivatives of the 3-pyridylpropionic acid can effectively inhibit the indole amine 2, 3-dioxygenase IDO1 from catalyzing tryptophan metabolism, can obviously inhibit the activity of high-expression IDO1 enzyme in human-derived cancer cells, and has low cytotoxicity. The nicotinic acid analogues can be used as an IDO1 enzyme inhibitor to remarkably inhibit the growth of transplanted tumors of mice when being used alone, and are combined with an innate immune pathway STING agonist or an immune checkpoint inhibitor and/or an anti-tumor chemical drug, so that the efficacy of inhibiting the growth of the tumors is better, and the safety is also good. Therefore, the nicotinic acid derivative has wide application prospect in preparing antitumor drugs.

Disclosure of Invention

The invention provides an innovative inhibitor of indoleamine 2, 3-dioxygenase IDO1, namely a nicotinic acid derivative (a derivative of 3-pyridylpropionic acid). The derivatives of the 3-pyridylpropionic acid can effectively inhibit the indole amine 2, 3-dioxygenase IDO1 from catalyzing tryptophan metabolism, can obviously inhibit the activity of high-expression IDO1 enzyme in human-derived cancer cells, and has low cytotoxicity. The nicotinic acid derivatives can be used as an IDO1 enzyme inhibitor to obviously inhibit the growth of transplanted tumors of mice when being used alone, and the nicotinic acid derivatives are combined with an innate immune pathway STING agonist or an immune checkpoint inhibitor to have better drug effect and good safety in inhibiting the growth of tumors. Therefore, the nicotinic acid derivative has wide application prospect in preparing antitumor drugs.

The specific invention content characteristics are:

1. nicotinic acid derivatives (derivatives of 3-pyridylpropionic acid), are novel inhibitors of the indoleamine 2, 3-dioxygenase IDO 1. The nicotinic acid derivative is characterized in that: nicotinic acid is used as a structural mother nucleus, atoms which are easy to form hydrogen bonds such as S, N, O, F and the like are introduced by utilizing a carboxylic acid isostere mode, the binding force of the inhibitors and IDO1 enzyme protein molecules is enhanced, a compound formed by the inhibitors and IDO1 protein molecules is stabilized, and the effect of inhibiting the activity of IDO1 enzyme is increased. The specific nicotinic acid derivative is shown in figure 1 (structural formula and name of nicotinic acid derivative (1-10)).

2. Application of nicotinic acid derivative (3-pyridine propionic acid derivative) in preparing antitumor drugs is provided. The anti-tumor drug is applied to treating various tumors, including but not limited to colorectal cancer, breast cancer, testicular cancer, ovarian cancer, prostate cancer, lung cancer, nasopharyngeal cancer, esophageal cancer, malignant lymphoma, head and neck cancer, thyroid cancer, osteogenic sarcoma, bladder cancer, cervical cancer, germ cell tumor and the like.

3. The application of nicotinic acid derivative (3-pyridine propionic acid derivative) and immune pathway STING agonist in preparing antitumor medicine. STING agonists include, but are not limited to, cGAMP and derivatives thereof. The anti-tumor medicament is applied to treating various tumors.

4. An application of a nicotinic acid derivative (a derivative of 3-pyridine propionic acid) and an immune checkpoint inhibitor in preparing an anti-tumor medicament in a combined mode. The immune checkpoint inhibitor comprises but is not limited to monoclonal antibodies such as anti-PD 1/PD-L1, anti-CD-47, anti-VEGF, anti-CTLA-4 and the like and nanobodies of the monoclonal antibodies; the anti-tumor medicament is applied to treating various tumors.

5. The application of the combination of nicotinic acid derivatives (derivatives of 3-pyridylpropionic acid) and chemotherapeutic antitumor drugs in preparing antitumor drugs. The chemotherapy drugs include but are not limited to platinum metal drugs, 5-fluorouracil, rhodol and other antitumor drugs, and are applied to treating various types of tumors.

6. The application of nicotinic acid derivatives (derivatives of 3-pyridine propionic acid) in preparing antitumor drugs comprises unit preparations with different specifications and pharmaceutically acceptable pharmaceutical preparations, including but not limited to one or more of intravenous injection, intramuscular injection, subcutaneous injection, intravenous drip, nasal drip, oral administration and the like, and the related tumors are treated.

Detailed Description

The present invention will be described in detail with reference to examples. In the present invention, the following examples are given to better illustrate the present invention and are not intended to limit the scope of the present invention.

Example 1: research on inhibition effect of nicotinic acid derivative on IDO1 metalloenzyme

All biochemical reagents were analytical grade, purchased from Sigma-Aldrach. The nicotinic acid derivative 1-10 has a structural formula and an English name shown in figure 1.

1. Indoleamine 2, 3-dioxygenase 1(IDO1) was prepared according to literature procedures

pGEX-6P-1 is selected as a vector for constructing a human IDO1 expression plasmid, IDO1 protein with a GST fusion tag can be expressed in escherichia coli, IDO1 fusion protein is adsorbed and purified by using a GST affinity column and has extremely high protein purity, recombinant HRV 3C protease is adopted for enzyme digestion of the GST tag protein, enzyme is carried out overnight at the temperature of 16 ℃, the enzyme-digested IDO1 protein is purified by using a glutaminone Sepharose affinity column for the second time, and electrophoresis pure protein is obtained and identified by SDS-Page and mass spectrometry. SDS-PAGE shows that the purity of IDO1 protein is higher than 95%, MALDI-TOF mass spectrum further confirms that the molecular weight of IDO1 is 45KD, which is consistent with the theoretical molecular weight; the ultraviolet visible spectrum characterization shows that the characteristic absorption peak is 404nm, and the electrochemical reduction potential is-0.37V. Experimental results prove that the human IDO1 protein with high purity is successfully obtained, and a foundation is laid for the subsequent screening of IDO1 inhibitors and the smooth performance of enzyme activity related experiments.

2. IC of nicotinic acid derivative for inhibiting IDO1 enzyme activity₅₀Value determination

The experimental method comprises the following steps:

by KH₂PO₄(100mM, pH 6.5) buffer solution, 1. mu.M protein at 404nm UV absorbance calculated from the molar extinction coefficient of IDO1 to prepare protein samples. Preparing a 1.5mL EP tube, and marking each concentration gradient of the compound; then 1mL of 1 mu M protein is taken to be put in an EP tube to be used as the volume of a reaction system; methylene blue, catalase, L-ascorbic acid (pH adjusted to 6.5 with NaOH) were then added thereto at final reagent concentrations of 10. mu.M, 200. mu.g/mL, and 10mM, respectively; adding compounds with corresponding concentration gradients into each tube, arranging blank holes without the compounds, turning upside down, mixing uniformly, and incubating in a 37 ℃ water bath for 10 min; the substrate L-tryptophan was then added at 100. mu.M prior to kinetic testing, followed immediately by monitoring at 320nmThe absorption value of the reaction product N' -formylkynurenine is 60 s. Experimental selection inhibition at Tryptophan concentration 100. mu.M IC of inhibitor was calculated₅₀The value is obtained.

Data processing:

in data processing, firstly, linear fitting is carried out on the kinetic experiment data of each reaction to obtain an initial speed V; then calculating the corresponding inhibition rate under different compound concentrations according to a formula, and plotting the inhibition rate to the Log10 value of the concentration of the nicotinic acid derivative inhibitor according to the IC₅₀Fitting data by formula to obtain IC of each compound₅₀The value is obtained.

The experimental results are as follows: IC (integrated circuit)₅₀The value measurement shows that 10 nicotinic acid derivatives have inhibition effect on IDO 1. On

Compounds

6, 7, 8, 9, 10, their IC₅₀The values were all around 10. mu.M. All 8 compounds showed 4-PI (IC) as compared to the positive control compound₅₀49.03 +/-6.67 μ M), wherein the

compounds

9 and 10 have the best effect, the inhibition rate of the compounds is the highest at the maximum concentration of 200 μ M, and the inhibition rate of the compound 9 can reach 90 percent.

TABLE 1 IC inhibition of IDO1 metalloenzyme by nicotinic acid derivatives₅₀And K_iValue of

3. Inhibition of IDO1 by nicotinic acid derivatives constant K_iMeasurement of

Experimental methods and data processing:

the same nicotinic acid derivative inhibitor was subjected to parallel kinetics of inhibitory activity at three substrate concentrations, 50. mu.M, 100. mu.M and 150. mu.M, respectively. Firstly, carrying out linear fitting on kinetic experimental data of each reaction to obtain an initial rate V, then adopting a Cornish-Bowden mapping method, taking the concentration of an inhibitor as an abscissa and the ratio (S/V) of the concentration of a substrate to the initial rate V as an ordinate in the same axis, and finally carrying out linear fitting on three groups of data, wherein the absolute value of the intersection point of a fitting straight line is an inhibition constant Ki, and the inhibition type of the inhibitor is judged by the position of the coordinate axis where the intersection point Ki is located. The derived experimental data were plotted using Origin 7.5.

The experimental results are as follows:

ki values were determined by selecting compounds with an inhibition of 60% or more at a concentration of 100. mu.M, respectively compounds 2, 4, 5, 8, 9, 10. The experiment is carried out in parallel by using different inhibitor concentrations under the three nicotinic acid derivative substrate concentrations of 50 mu M, 100 mu M and 150 mu M respectively, S/V is used for plotting the concentration of the nicotinic acid derivative inhibitor, and the absolute value of the intersection point of the straight lines linearly fitted by the three groups of data is the inhibition constant Ki.

As shown in Table 1, the Ki values for the three strong IDO1 nicotinic acid derivative inhibitors 8 (15.56. mu.M), 9 (8.68. mu.M) and 10 (9.93. mu.M) are in the 15. mu.M range. The lower the Ki value, the stronger the inhibitory effect, and hence nicotinic acid derivative inhibitor 9 had the strongest inhibitory effect on IDO 1. The above results indicate that the two fluorine-containing

compounds

9 and 10 have the strongest inhibitory activity against IDO1 as a compound having 3-pyridylpropionic acid as a parent nucleus. Lowest IC of the two₅₀And Ki of about 10 μ M. The inhibitory effect of inhibitor 9 was most pronounced primarily for two reasons, on the one hand, only 9 had oxygen and three fluorine atoms, and the introduction of F enhanced the hydrogen bonding force between IDO1 and the inhibitor, as compared to other inhibitors. In particular, 9 contains a trifluoroethyl group, which can significantly improve the binding capacity of the inhibitor. On the other hand, the carboxylic acid in 9 is substituted by fluorine, which increases the hydrophobicity of the inhibitor and favors 9 occupying the hydrophobic cavity of IDO 1. In summary, compounds containing elements that readily form hydrogen bonds, particularly those containing trifluoroethyl groups, have the potential advantage of inhibiting IDO 1.

Example 2: research on inhibition effect of nicotinic acid derivative on IDO1 enzyme of cancer cells

1. Experimental reagent consumable

0.25 Xpancreatin-EDTA, diabody (penicillin/streptomycin) from Gibco; 1640 medium, FBS fetal bovine serum from weissent biotechnology (nanjing) limited; isopropanol, DMSO, glacial acetic acid, absolute ethyl alcohol and copper sulfate are purchased from chemical reagents of national drug group, Inc.; the Nalgene program cooling box was purchased from beijing soja technologies ltd; sterile PBS buffer solution and sterile 0.22 mu m aqueous phase filter membrane are purchased from Biotechnology engineering (Shanghai) GmbH; the CCK-8 kit is purchased from Biyuntian biotechnology limited; 60mm and 100mm cell culture dishes, cell cryopreservation tubes were purchased from Corning; 96-well plates were purchased from NEST; human cervical cancer cells (Hela), human liver cancer cells (HepG-2), human breast cancer cells (MCF-7) and human normal liver cells (L02) were purchased from Shanghai academy of sciences cell banks; IFN gamma from Sigma-Aldrich; p-dimethylaminobenzaldehyde, trichloroacetic acid, 4-phenylimidazole are all available from Aladdin reagents (Shanghai) Inc.

2. Laboratory apparatus

-80 ℃ refrigerator (commercial electric appliances of Citsubishi, encyclopedia, Anhui); an electric heating constant temperature water bath (Shanghai Ke Heng industry development Co., Ltd.); a vertical pressure steam sterilizer (Shanghai Boxun practice Co., Ltd.); a double-person single-side vertical air supply clean bench (Shanghai Tianheng medical instruments Co., Ltd.); a carbon dioxide incubator (model: BPN-50CH, Shanghai-Hengyue instruments Co., Ltd.); microscope (Shanghai optical instruments, Inc.); a vertical refrigerator (Hefei Meiling Ltd.); centrifuge (Sorvall Legend Micro 17R/21R, Thermo Fisher Scientific); microplate reader (BioTek staining TM 3).

3. Experiment content and experiment method

This experiment uses IFN γ to induce cancer cells to produce IDO1 for the detection of IDO1 inhibitor activity at the viable cell level.

Day one of experiment: HeLa, HepG-2 and MCF-7 cancer cells cultured to a certain cell density with 100mm were digested with trypsin, observed under a microscope, after the cell morphology became round, digestion was stopped with the whole medium, the cells were gently blown off with a pipette, and pancreatin was removed by centrifugation. At 5X 10⁴Cell numbers per mL were seeded into 96-well plates and blanks were set. The cells were cultured in a carbon dioxide incubator containing 5% CO2 at 37 ℃ for 12 hours.

The next day of the experiment: cells in 96-well plates were changed and 200. mu.L of medium was added, followed by co-culture for 48h in each culture space with IFN γ at 10ng/mL final concentration, tryptophan at 100. mu.M and inhibitor at different concentration gradients. The final concentrations of the inhibitor were 0. mu.M, 0.5. mu.M, 1. mu.M, 5. mu.M, 10. mu.M, 20. mu.M, 40. mu.M, 60. mu.M, 80. mu.M, 100. mu.M, 150. mu.M, 200. mu.M, respectively. The final concentrations of the positive control 4-PI were 0. mu.M, 10. mu.M, 20. mu.M, 40. mu.M, 6. mu.M, 100. mu.M, 150. mu.M, and 200. mu.M, respectively. Duplicate wells for each sample concentration 3.

The fourth day of the experiment: after the cells are cultured by adding drugs for 48h, 140 mu L of supernatant is taken from each culture hole to a 1.5mL centrifuge tube, 10 mu L of 30% (w/v) trichloroacetic acid solution is added, and the mixture is incubated for 15min under the condition of water bath at 65 ℃ to stop the catalytic reaction of IDO1 on tryptophan and convert N' -formyl kynurenine into kynurenine; centrifuging at 10000rpm for 10min, adding 100 μ L of supernatant solution into a new 96-well plate, adding 2% (w/v) p-dimethylaminobenzaldehyde acetic acid solution with the same volume, mixing, and performing display reaction; finally, the absorbance at 492nm was measured by a microplate reader, and the reaction rate of the IDO1 enzyme was calculated from the amount of the product. Data were exported in excel format and then plotted using GraphPad 7.00. Cell experiments were performed in duplicate technical replicates. Inhibition rate on IDO1 live cells was calculated as: inhibition% ([ 1- (a/B) ] × 100% (1) (a represents the absorption value in the case of containing an inhibitor, and B represents the absorption value in the case of not containing an inhibitor.)

4. Results of the experiment

To evaluate the inhibitory activity of nicotinic acid derivatives against IDO1 enzyme in living cells, six inhibitors with an inhibition rate of greater than 60% selected in the enzyme inhibition assay of example 1 were analyzed in a cell assay, using three human cancer cell lines. Firstly, the inhibition rate of the drug on IDO1 in three cancer cells under the same inhibitor concentration of 20 mu M is determined, and the experimental results are shown in Table 2, the inhibition rate of 9 and 10 on IDO1 is the highest in Hela cell line and is 14.70% and 20.06%, respectively, in HepG-2 cell line, 9 and 10 also produce the best inhibition effect, the inhibition rate is 45.61% and 29.52%, respectively, while the inhibition effect of the inhibitor on MCF-7 cell line is weaker, the inhibition rate of the inhibitor is slightly more significant and is 13.54%, and the inhibition rate of other 5 inhibitors is about 10%. We used 9 and 10 inhibitors to determine their EC for IDO1 on HepG-2 cells₅₀. The results show that EC of 9 and 10₅₀The values were 11.04. mu.M and 14.06. mu.M, respectively. EC of other inhibitors₅₀The values were all over 100. mu.M (Table 2).

TABLE 2 parameters for the inhibition of IDO1 inhibitory activity of nicotinic acid derivatives in human cancer cells

Example 3: detection of anti-tumor effect of nicotinic acid derivative by tumor-bearing mouse model

Animal(s) production

BALB/C normal mice, male, 20-22g in weight, 7-8 weeks old, SPF grade, purchased from shanghai slek laboratory animals llc [ laboratory animal quality certification no: SCXK (Shanghai) 2007 + 0005 ].

Feeding conditions

All mice were left free to feed and drink water and were kept at room temperature (25 + -2) ° c. The feed and water are sterilized by high pressure, and the whole experimental feeding process is SPF grade.

Dose setting

(1) Nicotinic acid derivative (compound 9) was set at 20mg/kg in 1 dose group;

(2) combination of nicotinic acid derivatives with cGAMP: nicotinic acid derivative 20mg/kg + cGAMP20 mg/kg;

(3) combination of nicotinic acid derivatives with anti-PD-L1: nicotinic acid derivative 20 mg/kg/day + anti-PD-L1 monoclonal antibody, 200 mug/time, once a week;

test control

Negative control: physiological saline solution

Positive control:

(1)cGAMP，20mg/kg；

(2) anti-PD-L1 monoclonal antibody, 200 μ g/time, once a week;

method of administration

The administration route is as follows: tail vein injection

Administration volume: 100 microliter/piece; the administration times are as follows: the administration was continued for 21 days, 1 time every other day.

Number of animals per group: 10 pieces of

Mouse colorectal cancer cell line CT26 and mouse breast cancer cell line 4T1 were purchased from cell banks of Chinese academy of sciences.

The main steps of the test

Establishment and intervention of tumor model mouse

Cell culture, passage, cell collection at log phase, concentration of 1.0X 10 per ml⁷) The right anterior axillary region of the mouse was injected with 0.2ml of the cell suspension (cell number 2.0X 10)⁶One/one), the tumorigenicity is successful about 8 days. According to two mouse tumor model experiment categories, 6 groups are respectively and randomly divided into a model control group, a cGAMP positive drug control group, an anti-PD-L1 positive drug control group, a nicotinic acid derivative 9 and cGAMP combined drug group, and a nicotinic acid derivative 9 and anti-PD-L1 monoclonal antibody combined drug group. After 21 days, the mice were sacrificed and tumor body weights were taken and tumor inhibition rates were calculated.

Respectively preparing a mouse colorectal cancer cell line CT26, transplanting the cell line CT26 to a BalB/C common mouse and a mouse breast cancer cell line 4T1, transplanting the cell line CT26 to a BalB/C common mouse, and observing the anti-tumor effect of different drugs.

Statistical analysis

Data are expressed in x ± s, treated with SPSS10.0 software, and the significance of tumor weight differences of each group was compared using one-way ANOVA (one-way ANOVA) test, with a significance level a of 0.05.

Results of the experiment

The mice are inoculated with tumor cells subcutaneously to prepare a subcutaneous transplantation tumor model successfully, the nicotinic acid derivative (compound 9) can obviously inhibit the growth of tumors, and the tumor recurrence after 21 days of administration is obviously lower than that of a negative control model group (P <0.05, P < 0.01); the combined drug combination of the nicotinic acid derivative (compound 9) and the cGAMP, and the nicotinic acid derivative (compound 9) and the anti-PD-L1 monoclonal antibody show the effect of inhibiting the tumor drug effect which is obviously improved compared with the effect of single component of each drug. The specific results are shown in tables 2-3.

TABLE 2 inhibition of mouse colorectal cancer cell CT26 subcutaneous transplantable tumor by nicotinic acid derivatives and combinations thereof

(n＝10，mean±SD)

P <0.01vs negative control group.

TABLE 3 inhibition of 4T1 subcutaneous mouse breast cancer by nicotinic acid derivatives and combinations thereof

(n＝10，mean±SD)

P <0.01vs negative control group.

Example 4 Niacin derivative acute toxicity study in mice

The experimental method comprises the following steps:

20 ICR mice (purchased from Shanghaisleke laboratory animals, Limited liability company [ laboratory animal quality certification number: SCXK (Shanghai) 2007-0005]), each half of male and female, the weight of the mice is 18-22 g, and the mice are fed with pellet feed and can freely eat and drink water.

The nicotinic acid derivative (Compound 9) was formulated into a solution at a concentration of 200mg/mL with physiological saline.

ICR mice were dosed with a single intraperitoneal injection of 2g/kg of the novel compound per body weight, and mice were observed for toxicity and death within 14 days after dosing. As a result, the mice were found to be normally active after a single intraperitoneal injection administration. Within 14 days after administration, the mice did not die, and on day 15, all mice were sacrificed, dissected, and examined by naked eyes for each organ, and no obvious lesion was observed.

The experimental results are as follows:

the results of the acute toxicity experiments show that the maximum tolerated dose MTD of the intraperitoneal administration is not less than 2g/Kg, which indicates that the acute toxicity of the nicotinic acid derivative 9 is low.

Figure 1 shows the structural formula and the name of nicotinic acid derivative (1-10).

Claims

1. Nicotinic acid derivatives, i.e. derivatives of 3-pyridylpropionic acid, are novel inhibitors of the indoleamine 2, 3-dioxygenase IDO 1. The nicotinic acid derivative is characterized in that: nicotinic acid is used as a structural mother nucleus, atoms which are easy to form hydrogen bonds such as S, N, O, F and the like are introduced by utilizing a carboxylic acid isostere mode, the binding force of the inhibitors and IDO1 enzyme protein molecules is enhanced, a compound formed by the inhibitors and IDO1 protein molecules is stabilized, and the effect of inhibiting the activity of IDO1 enzyme is increased. Nicotinic acid derivatives include, but are not limited to, the compounds listed in figure 1.

2. The use of nicotinic acid derivatives as claimed in claim 1 for the preparation of antitumor medicaments. The anti-tumor drug is applied to treating various tumors, including but not limited to colorectal cancer, breast cancer, testicular cancer, ovarian cancer, prostate cancer, lung cancer, nasopharyngeal cancer, esophageal cancer, malignant lymphoma, head and neck cancer, thyroid cancer, osteogenic sarcoma, bladder cancer, cervical cancer, germ cell tumor and the like.

3. Use of a nicotinic acid derivative in combination (including a complex) with an immune pathway STING agonist for the preparation of an anti-neoplastic medicament, according to claim 1. STING agonists include, but are not limited to, cGAMP and derivatives thereof. The anti-tumor drug is applied to treating various tumors, including but not limited to colorectal cancer, breast cancer, testicular cancer, ovarian cancer, prostate cancer, lung cancer, nasopharyngeal cancer, esophageal cancer, malignant lymphoma, head and neck cancer, thyroid cancer, osteogenic sarcoma, bladder cancer, cervical cancer, germ cell tumor and the like.

4. The use of a nicotinic acid derivative in combination with an immune checkpoint inhibitor for the preparation of an anti-tumor medicament according to claim 1. The immune checkpoint inhibitor comprises but is not limited to monoclonal antibodies such as anti-PD 1/PD-L1, anti-CD-47, anti-VEGF, anti-CTLA-4 and the like and nanobodies of the monoclonal antibodies; the anti-tumor drug is applied to treating various tumors, including but not limited to colorectal cancer, breast cancer, testicular cancer, ovarian cancer, prostate cancer, lung cancer, nasopharyngeal cancer, esophageal cancer, malignant lymphoma, head and neck cancer, thyroid cancer, osteogenic sarcoma, bladder cancer, cervical cancer, germ cell tumor and the like.

5. The use of a nicotinic acid derivative (derivative of 3-pyridylpropionic acid) in combination with a chemotherapeutic antitumoral drug for the preparation of an antitumoral drug, according to claim 1. The chemotherapy drugs include but are not limited to platinum metal drugs, 5-fluorouracil, rhodol and other antitumor drugs, and are applied to treating various types of tumors. The anti-tumor drug is applied to treating various tumors, including but not limited to colorectal cancer, breast cancer, testicular cancer, ovarian cancer, prostate cancer, lung cancer, nasopharyngeal cancer, esophageal cancer, malignant lymphoma, head and neck cancer, thyroid cancer, osteogenic sarcoma, bladder cancer, cervical cancer, germ cell tumor and the like.

6. The application of the nicotinic acid derivatives in preparing the antitumor drugs according to claim 1, which comprises unit preparations with different specifications and pharmaceutically acceptable pharmaceutical preparations, including but not limited to one or more of intravenous injection, intramuscular injection, subcutaneous injection, intravenous drip, nasal drip, oral administration and the like, for treating the related tumors.