CN106967004B - IDO1 and IDO2 double inhibitor, its preparation method and its medical usage containing urea groups - Google Patents

Abstract

The present invention relates to the field of medicinal chemistry, in particular to a class of ureido-containing IDO1 and IDO2 dual inhibitors (I) and a preparation method thereof. Pharmacodynamic tests have proved that the compounds of the present invention have dual inhibitory effects on IDO1 and IDO2 and can be used for the treatment of Diseases such as malignant tumors.

Description

IDO1 and IDO2 dual inhibitor containing urea group, its preparation method and its medical application

technical field

The invention relates to the field of medicinal chemistry, in particular to a class of ureido-containing IDO1 and IDO2 dual inhibitors, their preparation methods, pharmaceutical compositions containing these compounds and their use in treating malignant tumors.

Background technique

At present, cancer has become one of the main causes of human death. In 2016, more than 1.6 million patients were diagnosed with cancer in the United States, and nearly 600,000 patients died of cancer. In 2015, the number of new cancer patients in my country reached 4.3 million, and the number of cancer deaths reached 2.8 million. It is estimated that by 2030, the number of deaths from cancer worldwide will climb to 13.1 million. Therefore, cancer has become a public health problem and even a social problem that must be attached great importance to. The human immune system is responsible for safeguarding the health of the body, among which T cells play a vital role in the monitoring and killing of tumors. However, tumor cells can inhibit the anti-tumor activity of T cells through a variety of mechanisms, thereby evading the attack of the immune system and resulting in immune escape. With the continuous development of oncology and immunology, the treatment of tumors based on the human immune system has gradually been accepted by the scientific research community and the industry, and has become a hot field for new drug research and development.

Indoleamine 2,3-dioxygenase-1 (IDO1), an important enzyme that decomposes tryptophan, is expressed in a variety of tumor cells and mediates tumor-induced immunosuppression. This enzyme locally degrades tryptophan, inactivating the tumor surveillance role of the immune system and preventing tumor rejection. IDO1 affects the immunosuppressive molecules related to the body's immune system through various ways, eventually leading to immune escape of tumor cells and further transformation into cancer. Inhibiting the activity of IDO1 has a significant promotion effect on the treatment of tumors, and specific inhibitors of this enzyme have the potential to treat various types of tumors. At present, IDO1 has become the most important small molecule regulatory target for anti-tumor immunotherapy.

Indoleamine-2,3-dioxygenase-2 (IDO2) is a homologous protein of IDO1, containing 420 amino acids, with 43% homology with IDO1 in amino acid sequence, but with tryptophan 2,3- Oxygenase (TDO) proteins have no similar structure. Human and mouse IDO1 and IDO2 coding genes are arranged in head-to-tail tandem on chromosome 8. The expression of IDO2 protein is not as extensive as that of IDO1, but it is continuously expressed in antigen-presenting cells and human primary tumors including gastric cancer, colon cancer, and kidney. In human pancreatic cancer, IDO2 is expressed not only in tumor tissues but also in tumor-draining lymph nodes. Like IDO1, it is also the rate-limiting enzyme that catalyzes the metabolism of tryptophan through the kynurenine pathway. Studies have found that IDO1 and IDO2 may have a synergistic effect in tumor immunotherapy.

Contents of the invention

The purpose of the present invention is to search for anti-tumor candidate compounds with novel structure, high activity and less side effects. These compounds are used in combination with other anti-tumor drugs to improve the curative effect of the existing anti-tumor drugs and reduce the dosage and toxicity.

The present invention discloses a compound of general formula (I) or a pharmaceutically acceptable salt thereof.

Wherein R ¹ and R ² each independently represent: H, F, Cl, Br, CF ₃ , CH ₃ , OCH ₃ , CN or NO ₂ . R ¹ and R ² preferably each independently represent F, Cl or Br.

R ³ represents: C ₁ -C ₈ straight chain alkyl or C ₃ -C ₆ cycloalkyl; R ³ is preferably C ₂ -C ₅ straight chain alkyl or C ₃ -C ₅ cycloalkyl.

The compound of general formula (I) can form acid addition salt with pharmaceutically acceptable acid, and described acid comprises: hydrogen chloride, hydrogen bromide sulfuric acid, carbonic acid, oxalic acid, citric acid, succinic acid, tartaric acid, phosphoric acid, lactic acid, acetone acid, acetic acid, maleic acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid or ferulic acid.

The preferred partial compounds of the present invention are as follows:

General formula (I) compound of the present invention and its pharmaceutically acceptable acid addition salt (I.A) can be prepared by the following methods:

More specific methods include:

in:

The process of preparing compound III from compound II, the reaction reagents are sodium nitrite and 2mol/L hydrochloric acid, and the solvent is water.

The process of preparing compound IV from compound III through diazotization, the reaction reagents are sodium nitrite, 6mol/L hydrochloric acid and sodium chloride, and the solvent is glacial acetic acid.

The process of preparing compound VI by amination of compound IV and substituted aniline (V), the acid removal agent is sodium bicarbonate, and the solvent is ethanol and water.

The process of preparing compound VII from compound VI, the reactant is N,N'-carbonyldiimidazole (CDI), and the solvent is ethyl acetate.

The process of preparing compound I from compound VII first reacts compound VII with isocyanate in an anhydrous solvent under the assistance of a strong base, and the resulting product is directly hydrolyzed by adding water without separation to obtain the target compound I; the strong base is sodium hydride , potassium tert-butoxide or sodium tert-butoxide, preferably sodium hydride; the solvent is tetrahydrofuran, 1,4-dioxane, toluene, N,N-dimethylformamide, preferably tetrahydrofuran.

The process of preparing compound I A from compound I through salt formation, reactant (A) is hydrogen chloride, hydrogen bromide, sulfuric acid, phosphoric acid, carbonic acid, oxalic acid, citric acid, succinic acid, tartaric acid, lactic acid, pyruvic acid, acetic acid, horse Toric acid, methanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid or ferulic acid; the solvent is preferably methanol, ethanol, methylene chloride, acetone, ethyl acetate, toluene or tetrahydrofuran, or any mixed solvent.

Another object of the present invention is to provide a pharmaceutical composition, which comprises a pharmaceutically effective dose of the compound of the present invention or a salt thereof and a pharmaceutically acceptable carrier.

The compounds of the present invention can be prepared into common pharmaceutical preparations by adding pharmaceutically acceptable carriers, such as tablets, capsules, powders, syrups, liquids, suspensions, injections, spices, sweeteners, liquids or Common pharmaceutical excipients such as solid fillers or diluents.

The clinical administration of the compound of the present invention can be oral administration, injection and the like.

Generally, when the compound of the present invention is used for treatment, the dosage for human is in the range of 1 mg to 1000 mg/day. Depending on the dosage form and the severity of the disease, the dosage may exceed this range.

Pharmacological experiments and results of some compounds of the present invention are as follows:

1. Detect the activity of some target compounds in inhibiting IDO1/2 at the enzyme level

experimental method

A molecular screening model for IDO1/2 enzyme activity inhibition was established, and the inhibitory IC ₅₀ value and inhibition rate of the positive control drug Epacadostat were tested on this model. The results were similar to those reported in many literatures, indicating that the screening model was successfully constructed. IDO enzyme can catalyze the pyrrole epoxidation cleavage on tryptophan to produce N'-formylkynurenine (N'-formylkynurenine). At room temperature, mix 40nM IDO enzyme and 900uM L-tryptophan, and add reaction buffer (20mM ascorbate, 3.5uMmethylene blue and 0.2mg/mL catalase in 50mM potassium phosphate buffer pH6.5), at room temperature After reacting for 3 hours, ultraviolet measurement was carried out on a microplate reader, and the detection wavelength was 321nm. Percentage of enzyme activity (%) = (OD value administration well - OD value background) / (OD value control well - OD value background) × 100%, and then use PrismGraphPad sofeware software to fit and calculate IC ₅₀ value.

The experimental results are shown in Table 1

Table 1 IC ₅₀ of some target compounds on IDO1 and IDO2 at the enzyme level

2. Detection of the inhibitory activity of some target compounds on human colon cancer cell line HT29

experimental method

Using the sulforhodamine B (SRB) colorimetric method, the HT29 human colon cancer cells in the logarithmic growth phase were blown into a single-cell suspension, seeded in a 96 ^- well culture plate (5x10 cells/well), and cultured in each well. Base 200 μL, incubate overnight in 37°C, 5% CO ₂ incubator; after cells adhere to the wall, add appropriate concentration of test compound and positive control drug and incubate in incubator for another 72 hours. After adding 10% trichloroacetic acid, fix at 4°C for 1 hour. Wash 5 times with double distilled water, after drying, add 70 microliters of SRB solution (4 mg/mL) to each well, stain at room temperature for 20 minutes, wash 5 times with 1% acetic acid, and dry. Add 100 μL of 10 mM Tris-Base solution to each well to dissolve SRB. Detect the OD value of each well with a microplate reader (detection wavelength: 515nm); record the results; calculate the inhibition rate according to the following formula: inhibition rate (%) = (OD control - OD administration) / OD control × 100%, and calculate IC ₅₀ .

Table 2 Part of the target compound's inhibitory activity IC ₅₀ on human colon cancer cell line HT29

Detailed ways

Example 1

Preparation of N-(3-bromo-4-fluorophenyl)-4-(3-ethylureido)-N′-hydroxy-1,2,5-oxadiazole-3-carboxamidine (I-1)

Preparation of 3-Amino-4-Aminoximidofurazan(III)

Add NaNO ₂ (62.67g, 0.91mol) into a 1L three-necked flask filled with H ₂ O (120mL), stir to dissolve at room temperature (25°C), slowly add malononitrile (II) (30g, 0.45mol) dropwise to dissolve In 450mL of 2mol/L hydrochloric acid solution, stir overnight at room temperature, cool to 0°C, add dropwise a solution of hydroxylamine hydrochloride (69.74g, 1.0mol) dissolved in 80mL of water, stir for 30 minutes after the addition, and use 10mol/L under 20°C Adjust the pH to 10 with sodium hydroxide solution, raise the temperature to 35° C. for 2 hours, and then heat to reflux for 2 hours. Cooled to room temperature, the reaction solution was extracted with ethyl acetate (100mL×3), washed twice with saturated NaCl (30mL×2), dried over anhydrous Na ₂ SO ₄ , and the solvent was evaporated under reduced pressure to obtain 46.8 g of white powder, yield 72%, mp 192-193°C. ¹ H-NMR(300MHz,DMSO-d ₆ ),δ(ppm):10.45(s,1H),6.25(s,2H),6.17(s,2H); MS(ESI(-)70V)m/z :142[MH] ^- .

Preparation of 4-amino-N-hydroxy-1,2,5-oxadiazole-3-carboxamidine (IV)

Add compound III (20.75g, 145mmol), H ₂ O (300mL), glacial acetic acid (150mL) and 6mol/L HCl (75mL, 435mmol) into a 1L three-necked flask, raise the temperature to 45°C, and stir until compound III is completely Dissolve, add NaCl (25.42g, 435mmol), stir until dissolved, cool down to 0°C, slowly add NaNO ₂ (10g, 145mmol) in 35mL of water dropwise, stir for 4 hours below 0°C, rise to room temperature, pump Filter, wash the filter cake with water, recrystallize from water/ethanol to obtain 12.1 g of white powder, yield 51.3%, mp 188-189°C. ¹ H-NMR (300MHz, DMSO-d ₆ ), δ (ppm): 13.40 (s, 1H), 6.30 (s, 2H); MS (ESI (-) 70V) m/z: 161 [MH] ^- .

Preparation of 4-amino-N-(3-bromo-4-fluorophenyl)-Nˊ-hydroxy-1,2,5-oxadiazole-3-carboxamidine (VI-1)

Dissolve compound IV (10g, 61.53mmol) in ethanol (300mL), add 3-bromo-4-fluoroaniline (V-1) (12.86g, 67.68mmol), then add NaHCO ₃ (12.92g, 153.83mmol) The solution dissolved in 150mL of water was heated to 60°C, and the reaction was detected by TLC, and the reaction was stopped after the compound IV was completely reacted. The solvent was evaporated under reduced pressure, the residue was dissolved in ethyl acetate (100 mL), washed three times with water (30 mL×3), washed twice with saturated NaCl (30 mL×3), dried over anhydrous Na ₂ SO ₄ overnight, and the solvent was evaporated under reduced pressure , recrystallized from ethyl acetate/n-hexane to obtain 8.03 g of light yellow solid, yield 41.3%, mp 179-180°C. ¹ H-NMR (300MHz, DMSO-d ₆ ), δ(ppm): 11.46(s, 1H), 8.89(s, 1H), 6.99(t, J=8.8Hz, 1H), 6.81(dd, J= 6.0, 2.7Hz, 1H), 6.56-6.51(m, 1H), 6.28(s, 2H); MS (ESI(-)70V) m/z: 314[MH] ^- .

3-(4-amino-1,2,5-oxadiazol-3-yl)-4-(3-bromo-4-fluorophenyl)-1,2,4-oxadiazol-5(4H) - Preparation of ketone (VII-1)

Compound VI-1 (10g, 31.64mmol) was dissolved in ethyl acetate (120mL), N,N'-carbonyldiimidazole (7.7g, 47.64mmol) was added, and reacted at 25°C. The reaction was followed by TLC until the reaction of the raw materials was complete. Wash with 1mol/L HCl (116mL×2), dry over anhydrous sodium sulfate, evaporate the solvent under reduced pressure, and recrystallize the residue with methanol to obtain 10.6g white solid, yield 98%, mp192-193°C. ¹ H-NMR (300MHz, DMSO-d ₆ ), δ (ppm): 8.10 (dd, J = 6.2, 2.4 Hz, 1H), 7.74 (m, 1H), 7.61 (t, J = 8.7Hz, 1H) , 6.63 (s, 2H); MS (ESI (-) 70V) m/z: 340 [MH] ^- .

Preparation of N-(3-bromo-4-fluorophenyl)-4-(3-ethylureido)-N′-hydroxy-1,2,5-oxadiazole-3-carboxamidine (I-1)

Mix 60% sodium hydride (140mg, 3.51mmol) with anhydrous tetrahydrofuran (3mL), stir and cool down to -78°C, slowly add compound VII-1 (0.4g, 1.17mmol) in 3mL anhydrous tetrahydrofuran After the dropwise addition, stir for 1 hour, add dropwise a solution of ethyl isocyanate (VIII-1) (99 mg, 1.4 mmol) dissolved in 1 mL of anhydrous tetrahydrofuran to the reaction solution, react for 2 hours, and heat up to -68 The reaction was continued at ℃ for 2 hours, and the temperature was raised to -58 ℃ again for overnight reaction. The reaction solution was slowly heated to -8°C (10°C/hour), and 1 mL of H ₂ O was added. After reacting at this temperature for 3 hours, it was filtered with suction, and the filtrate was evaporated under reduced pressure to remove tetrahydrofuran, and ethyl acetate (20 mL) was added to dissolve it. It was washed with saturated ammonium chloride (10 mL×2) and saturated NaCl (10 mL×2), dried and stored over anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure to obtain a brown-yellow oil. Separation by column chromatography (eluent: dichloromethane: ethyl acetate = 10:1) gave 0.16 g of white solid, yield 36.1%, mp 192-194°C. ¹ H-NMR (300MHz, DMSO-d ₆ ), δ (ppm): 11.41 (s, 1H), 9.01 (s, 1H), 8.94 (s, 1H), 7.22 (dd, J=6.0, 2.5Hz, 1H), 7.17(t, J＝8.8Hz, 1H), 7.09(brs, 1H), 6.91–6.81(m, 1H), 3.22–3.07(m, 2H), 1.06(t, J＝7.1Hz, 3H ); ¹³ C-NMR (75MHz, DMSO-d ₆ ), δ (ppm): 155.43, 152.30, 150.36, 141.87, 138.83, 137.71, 125.16, 121.71 (d, J = 7.1Hz), 116.00 (d, J = 22.8Hz), 107.10(d, J＝22.2Hz), 34.47, 15.08. HRMS(ESI): m/z[M+H] ⁺ .Calcd for C ₁₂ H ₁₃ BrFN ₆ O ₃ : 387.0211; Found: 387.0213.

Example 2

N-(3-bromo-4-fluorophenyl)-4-(3-n-propylureido)-N'-hydroxyl-1,2,5-oxadiazole-3-carboxamidine (I-2) preparation

Using VII-1 (0.4g, 1.17mmol), n-propyl isocyanate (VIII-2) (119mg, 1.4mmol), 60% sodium hydride (140mg, 3.51mmol) as raw materials, the operation is the same as I-1, column Chromatographic separation (eluent, dichloromethane: ethyl acetate = 10:1) gave 0.15 g of a white solid, yield 32%, mp 185-187°C. ¹ H-NMR (300MHz, DMSO-d ₆ ), δ (ppm): 11.41 (s, 1H), 9.02 (s, 1H), 8.94 (s, 1H), 7.22 (dd, J=6.1, 2.7Hz, 1H), 7.18(t, J=8.8Hz, 1H), 7.12(brs, 1H), 6.87-6.82(m, 1H), 3.31-3.01(m, 2H), 1.49-1.39(m, 2H), 0.87 (t, J=7.4Hz, 3H). ¹³ C-NMR (75MHz, DMSO-d ₆ ), δ(ppm): 155.45, 152.34 (d, J=9.7Hz), 150.34, 141.81, 138.81, 137.69, 125.17 ,121.66(d,J=13.3Hz),115.98(d,J=23.2Hz),107.12(d,J=21.9Hz),41.30,22.61,11.22.HRMS(ESI):m/z[M+H] ⁺ .Calcd for C ₁₃ H ₁₅ BrFN ₆ O ₃ : 401.0368; Found: 401.0365.

Example 3

N-(3-bromo-4-fluorophenyl)-4-(3-isopropylureido)-Nˊ-hydroxy-1,2,5-oxadiazole-3-carboxamidine (I-3) preparation

Using VII-1 (0.4g, 1.17mmol), isopropyl isocyanate (VIII-3) (119mg, 1.4mmol), 60% sodium hydride (140mg, 3.51mmol) as raw materials, the operation is the same as I-1, column Chromatographic separation (eluent, dichloromethane:ethyl acetate=10:1) gave 0.31 g of white solid, yield 66.1%, mp 177-179°C. ¹ H-NMR (300MHz, DMSO-d ₆ ), δ (ppm): 11.42 (s, 1H), 9.02 (s, 1H), 8.83 (s, 1H), 7.22 (dd, J=6.2, 2.8Hz, 1H), 7.16(t, J=8.8Hz, 1H), 7.00(brs, 1H), 6.83(m, 1H), 3.81-3.73(m, 1H), 1.11(d, J=6.5Hz, 6H). ¹³ C-NMR (75MHz, DMSO-d ₆ ), δ (ppm): 155.69, 152.06 (d, J=67.2Hz), 149.75, 141.19, 138.96, 137.22, 125.40, 121.98 (d, J=6.8Hz), 115.99(d, J=23.2Hz), 107.15(d, J=22.1Hz), 41.66, 22.32.HRMS(ESI):m/z[M+H] ⁺ .Calcd for C ₁₃ H ₁₅ BrFN ₆ O ₃ : 401.0368; Found: 401.0372.

Example 4

N-(3-bromo-4-fluorophenyl)-4-(3-n-butylureido)-N'-hydroxy-1,2,5-oxadiazole-3-carboxamidine (I-4) preparation

Using VII-1 (0.4g, 1.17mmol), n-butyl isocyanate (VIII-4) (139mg, 1.4mmol), 60% sodium hydride (140mg, 3.51mmol) as raw materials, the operation is the same as I-1, column Chromatographic separation (eluent, petroleum ether: ethyl acetate = 5:1) gave 0.24 g of a white solid, yield 49.8%, mp 158-160°C. ¹ H-NMR (300MHz, DMSO-d ₆ ), δ (ppm): 11.40 (s, 1H), 8.99 (s, 1H), 8.90 (s, 1H), 7.21 (dd, J=6.1, 2.7Hz, 1H), 7.16(t, J=8.8Hz, 1H), 7.08(brs, 1H), 6.83(m, 1H), 3.13-3.03(m, 2H), 1.44-1.37(m, 2H), 1.33-1.20 (m,2H),0.88(t,J=7.2Hz,3H). ¹³ C-NMR(75MHz,DMSO-d ₆ ),δ(ppm):155.46,152.32(d,J=6.1Hz),150.32, 141.78, 138.82, 137.65, 125.17, 121.70(d, J=6.8Hz), 115.97(d, J=23.2Hz), 107.12(d, J=22.0Hz), 39.16, 31.43, 19.43, 13.59.HRMS(ESI) :m/z[M+H] ⁺ .Calcd for C ₁₄ H ₁₇ BrFN ₆ O ₃ :415.0524; Found: 415.0526.

Example 5

Preparation of N-(3-bromo-4-fluorophenyl)-4-(3-n-hexylureido)-N′-hydroxy-1,2,5-oxadiazole-3-carboxamidine (I-5)

Using VII-1 (0.4g, 1.17mmol), n-hexyl isocyanate (VIII-5) (178mg, 1.4mmol), 60% sodium hydride (140mg, 3.51mmol) as raw materials, the operation is the same as I-1, column layer Analysis and separation (eluent, dichloromethane: ethyl acetate = 12:1) gave 0.28 g of a white solid, yield 54.1%, mp 146-148°C. ¹ H-NMR (300MHz, DMSO-d ₆ ), δ (ppm): 11.42 (s, 1H), 9.02 (s, 1H), 8.92 (s, 1H), 7.22 (dd, J=6.1, 2.6Hz, 1H), 7.17(t, J=8.9Hz, 1H), 7.10(brs, 1H), 6.85(m, 1H), 3.13-3.07(m, 2H), 1.46-1.39(m, 2H), 1.27(brs ,6H),0.87(t,J=6.5Hz,3H). ¹³ C-NMR(75MHz,DMSO-d ₆ ),δ(ppm):155.46,152.34,150.31,141.76,138.82,137.68,125.17,121.70( d,J=6.8Hz),115.96(d,J=23.2Hz),107.10(d,J=22.2Hz),39.53,30.94,29.31,25.96,22.04,13.88.HRMS(ESI):m/z[M +H] ⁺ .Calcd for C ₁₆ H ₂₁ BrFN ₆ O ₃ :443.0837; Found: 443.0848.

Example 6

Preparation of N-(3-bromo-4-fluorophenyl)-4-(3-cyclohexylureido)-Nˊ-hydroxy-1,2,5-oxadiazole-3-carboxamidine (I-6)

Using VII-1 (0.4g, 1.17mmol), cyclohexyl isocyanate (VIII-6) (175mg, 1.4mmol), 60% sodium hydride as (140mg, 3.51mmol) raw materials, the operation is the same as I-1, column Chromatographic separation (eluent, petroleum ether: ethyl acetate = 6:1) yielded 0.17 g of a white solid, yield 32.9%, mp 189-191°C. ¹ H-NMR (300MHz, DMSO-d ₆ ), δ (ppm): 11.40 (s, 1H), 9.01 (s, 1H), 8.84 (s, 1H), 7.27–7.20 (m, 1H), 7.20- 7.12(m,1H),7.02(m,1H),6.90-6.76(m,1H),3.58-3.39(m,1H),1.86-1.73(m,2H),1.72-1.59(m,2H), 1.39-1.05(m,6H). ¹³ C-NMR(75MHz,DMSO-d ₆ ),δ(ppm):155.49,151.88(d,J=65.8Hz),150.12,141.67,138.63,137.45,125.16,121.66 (d, J=6.5Hz),115.99(d,J=23.2Hz),107.14(d,J=21.8Hz),48.28,32.48,25.05,24.20.HRMS(ESI):m/z[M+H] ⁺ .Calcd for C ₁₆ H ₁₉ BrFN ₆ O ₃ :441.0681; Found: 441.0683.

Example 7

N-(3-bromo-4-fluorophenyl)-4-(3-n-octylureido)-N'-hydroxyl-1,2,5-oxadiazole-3-carboxamidine (I-7) preparation

With VII-1 (0.4g, 1.17mmol), n-octyl isocyanate (VIII-7) (217mg, 1.4mmol), 60% sodium hydride (140mg, 3.51mmol), the same operation as I-1, column chromatography Separation (eluent, petroleum ether: ethyl acetate = 6:1) gave 0.27 g of a light yellow solid, yield 49.0%, mp 130-132°C. ¹ H-NMR (300MHz, DMSO-d ₆ ), δ (ppm): 11.42 (s, 1H), 9.02 (s, 1H), 8.91 (s, 1H), 7.27–7.20 (m, 1H), 7.17 ( t,J＝8.8Hz,1H),7.10(m,1H),6.91-6.79(m,1H),3.13-3.04(m,2H),1.45-1.42(m,2H),1.26(brs,10H) ,0.86(t,J=6.2Hz). ¹³ C-NMR(75MHz,DMSO-d ₆ ),δ(ppm):155.58,152.32(d,J=11.9Hz),149.90,141.29,138.81,137.38,125.26 ,121.81(d,J=6.7Hz),115.88(d,J=23.1Hz),107.09(d,J=22.0Hz),39.52,39.25,31.15,29.18,28.58,26.19,22.00,13.78.HRMS(ESI ):m/z[M+H] ⁺ .Calcd for C ₁₈ H ₂₅ BrFN ₆ O ₃ : 471.1150; Found: 471.1147.

Example 8

N-(3-bromo-4-fluorophenyl)-4-(3-tert-butylureido)-N′-hydroxy-1,2,5-oxadiazole-3-carboxamidine (I-8) preparation

With VII-1 (0.4g, 1.17mmol), tert-butyl isocyanate (VIII-8) (139mg, 1.4mmol), 60% sodium hydride (140mg, 3.51mmol), the same operation as I-1, column chromatography Separation (eluent, dichloromethane:ethyl acetate=20:1) gave 0.18g of white solid, yield 37.0%, mp 174-175°C. ¹ H-NMR (300MHz, DMSO-d ₆ ), δ (ppm): 11.41 (s, 1H), 9.00 (s, 1H), 8.67 (s, 1H), 7.22-7.20 (m, 1H), 7.20– 7.13(m,1H),6.93(s,1H),6.84–6.75(m,1H),1.29(s,9H). ¹³ C-NMR(75MHz,DMSO-d ₆ ),δ(ppm):155.49, 152.31, 150.53(d, J=65.0Hz), 141.50, 138.69, 137.62, 125.25, 121.67(d, J=7.0Hz), 115.98(d, J=28.3Hz), 107.14(d, J=17.4Hz), 49.98, 28.58. HRMS(ESI): m/z[M+H] ⁺ .Calcd for C ₁₄ H ₁₇ BrFN ₆ O ₃ : 415.0524; Found: 415.0525.

Example 9

Preparation of N-(4-fluorophenyl)-4-(3-ethylureido)-N′-hydroxy-1,2,5-oxadiazole-3-carboxamidine (I-9)

Preparation of 4-amino-N-(4-fluorophenyl)-Nˊ-hydroxy-1,2,5-oxadiazole-3-carboxamidine (VI-2)

Compound IV (10g, 61.53mmol)) was dissolved in ethanol (300mL), and 4-fluoroaniline (V-2) (7.52g, 67.68mmol)) was added, followed by an aqueous solution of NaHCO ₃ (12.92g, 153.83mmol) (150mL water), heated up to 60°C, TLC detected the reaction, after the IV reaction was complete, evaporated the solvent under reduced pressure, dissolved ethyl acetate (100mL), washed three times with water (30mL×3), washed twice with saturated NaCl (30mL×3 2), dried over anhydrous Na ₂ SO ₄ overnight, evaporated the solvent under reduced pressure, recrystallized from ethyl acetate/n-hexane to obtain 6.91 g of pink solid, yield 43%, mp214-215°C. ¹ H-NMR(300MHz,DMSO-d ₆ ),δ(ppm):11.25(s,1H),8.71(s,1H),7.05-6.99(m,2H),6.83-6.81(m,2H), 6.24(s,2H).

4-(4-fluorophenyl)-3-[(4-amino)-1,2,5-oxadiazol-3-yl]-1,2,4-oxadiazol-5(4H)-one (VII-2)

Dissolve compound VI-2 (10g, 42.16mmol) in ethyl acetate (120mL), add N,N'-carbonyldiimidazole (10.25g, 63.24mmol), stir the reaction at 25°C, follow the reaction by TLC until the starting material reacts After completion, wash with 1mol/L HCl (150mL×2), dry over anhydrous sodium sulfate, evaporate the solvent under reduced pressure, recrystallize from methanol to obtain 10.9g white solid, yield 99%, mp203-204°C. ¹ H-NMR (300MHz, DMSO-d ₆ ), δ (ppm): 7.65-7.64 (m, 2H), 7.42-7.37 (m, 2H), 6.60 (s, 2H).

Preparation of N-(4-fluorophenyl)-4-(3-ethylureido)-N′-hydroxy-1,2,5-oxadiazole-3-carboxamidine (I-9)

Using VII-2 (0.3g, 1.15mmol), ethyl isocyanate (VIII-1) (98mg, 1.38mmol), 60% sodium hydride (138mg, 3.45mmol) as raw materials, the operation is the same as I-1, column layer Analysis and separation (eluent, dichloromethane: ethyl acetate = 10:1) gave 0.13 g of a white solid, yield 36.7%, mp 177-179°C. ¹ H-NMR(300MHz,DMSO-d ₆ ),δ(ppm):11.24(s,1H),8.90(s,1H),8.83(s,1H),7.08(brs,1H),7.00(m, 2H), 6.88(m, 2H), 3.22–3.05(m, 2H), 1.07(t, J=7.1Hz, 3H). ¹³ C-NMR(75MHz, DMSO-d ₆ ), δ(ppm): 159.68 ,156.51,151.07(d,J=167.7Hz),141.39,139.46,136.07,123.31(d,J=8.0Hz),114.90(d,J=22.5Hz),34.3414.90.HRMS(ESI):m/ z[M+H] ⁺ .Calcd for C ₁₂ H ₁₄ FN ₆ O ₃ :309.1106; Found: 309.1103.

Example 10

Preparation of N-(4-fluorophenyl)-4-(3-n-propylureido)-N′-hydroxy-1,2,5-oxadiazole-3-carboxamidine (I-10)

Using VII-2 (0.4g, 1.53mmol), n-propyl isocyanate (VIII-2) (157mg, 1.84mmol), 60% sodium hydride (183mg, 4.59mmol) as raw materials, the operation is the same as I-1, column Chromatographic separation (eluent, dichloromethane:ethyl acetate=10:1) gave a white solid 0.17g, yield 34.7%, mp 175-176°C. ¹ H-NMR(300MHz,DMSO-d ₆ ),δ(ppm):11.25(s,1H),8.91(s,1H),8.85(s,1H),7.11(brs,1H),7.00(m, 2H), 6.88(m, 4.9Hz, 2H), 3.10-3.04(m, 2H), 1.50-1.40(m, 2H), 0.87(t, J=7.4Hz, 3H). ¹³ C-NMR(75MHz, DMSO-d ₆ ), δ(ppm): 159.56, 156.40, 151.28(d, J=153.7Hz), 141.68, 139.39, 136.36, 123.13(d, J=7.9Hz), 114.88(d, J=22.4Hz) ,41.25,22.60,11.22.HRMS(ESI):m/z[M+H] ⁺ .Calcd for C ₁₃ H ₁₆ FN ₆ O ₃ :323.1262; Found: 323.1269.

Example 11

Preparation of N-(4-fluorophenyl)-4-(3-isopropylureido)-Nˊ-hydroxy-1,2,5-oxadiazole-3-carboxamidine (I-11)

Using VII-2 (0.4g, 1.53mmol), isopropyl isocyanate (VIII-3) (157mg, 1.84mmol), 60% sodium hydride (183mg, 4.59mmol) as raw materials, the operation is the same as I-1, column Chromatographic separation (eluent, dichloromethane:ethyl acetate=20:1) gave 0.20 g of white solid, yield 40.8%, mp 183-185°C. ¹ H-NMR (300MHz, DMSO-d ₆ ), δ (ppm): 11.22 (s, 1H), 8.86 (s, 1H), 8.81 (s, 1H), 7.00 (brs, 3H), 6.87 (brs, 2H),3.99-3.57(m,1H),1.11(brs,6H). ¹³ C-NMR(75MHz,DMSO-d ₆ ),δ(ppm):159.76,156.58,150.63(d,J＝136.0Hz) ,141.07,139.57,135.91,123.43(d,J=6.0Hz),114.92(d,J=21.8Hz),41.63,22.35.HRMS(ESI):m/z[M+H] ⁺ .Calcd for C ₁₃ H ₁₆ FN ₆ O ₃ : 323.1262; Found: 323.1269.

Example 12

Preparation of N-(4-fluorophenyl)-4-(3-n-butylureido)-N′-hydroxy-1,2,5-oxadiazole-3-carboxamidine (I-12)

Using VII-2 (0.4g, 1.53mmol), n-butyl isocyanate (VIII-4) (182mg, 1.84mmol), 60% sodium hydride (183mg, 4.59mmol) as raw materials, the operation is the same as I-1, column Chromatographic separation (eluent, dichloromethane:ethyl acetate=20:1) gave 0.15 g of white solid, yield 28.8%, mp 149-151°C. ¹ H-NMR (300MHz, DMSO-d ₆ ), δ (ppm): 11.26 (s, 1H), 8.89 (s, 1H), 8.86 (s, 1H), 7.08 (brs, 1H), 7.05-6.93 ( ¹³ C -NMR (75MHz, DMSO-d ₆ ), δ(ppm): 159.75, 156.58), 151.08(d, J=189.7Hz), 141.17, 139.54, 135.96, 123.41(d, J=8.0Hz), 114.92(d , J＝22.5Hz), 39.06, 31.18, 19.29, 13.46. HRMS (ESI): m/z[M+H] ⁺ .Calcd for C ₁₄ H ₁₈ FN ₆ O ₃ : 337.1419; Found: 337.1420.

Example 13

Preparation of N-(4-fluorophenyl)-4-(3-n-hexylureido)-N′-hydroxy-1,2,5-oxadiazole-3-carboxamidine (I-13)

Using VII-2 (0.4g, 1.53mmol), n-hexyl isocyanate (VIII-5) (234mg, 1.84mmol)), 60% sodium hydride (183mg, 4.59mmol) as raw materials, the operation is the same as I-1, column Chromatographic separation (eluent, petroleum ether: ethyl acetate = 6:1) gave 0.20 g of a white solid, yield 35.7%, mp 155-157°C. ¹ H-NMR(300MHz,DMSO-d ₆ ),δ(ppm):11.26(s,1H),8.90(s,1H),8.87(s,1H),7.10(brs,1H),7.00(m, 2H),6.87(m,2H),3.13-3.07(m,2H), ^1.53-1.37 (m,2H),1.35-1.20(m,6H),0.87(t,J=6.3Hz,3H). C-NMR (75MHz, DMSO-d ₆ ), δ(ppm): 159.69, 156.52, 151.07(d, J=171.9Hz), 141.29, 139.43, 136.06, 123.30(d, J=7.9Hz), 114.86(d , J＝22.5Hz), 39.31, 30.85, 29.14, 25.85, 21.97, 13.79. HRMS(ESI): m/z[M+H] ⁺ .Calcd for C ₁₆ H ₂₂ FN ₆ O ₃ : 365.1732; Found: 365.1736 .

Example 14

Preparation of N-(4-fluorophenyl)-4-(3-cyclohexylureido)-N'-hydroxyl-1,2,5-oxadiazole-3-carboxamidine I-14)

Using VII-2 (0.4g, 1.53mmol), cyclohexyl isocyanate (VIII-6) (230mg, 1.84mmol), 60% sodium hydride (183mg, 4.59mmol) as raw materials, the operation is the same as I-1, column Chromatographic separation (eluent, petroleum ether: ethyl acetate = 8:1) gave 0.16 g of a white solid, yield 29.1%, mp 201-203°C. ¹ H-NMR (300MHz, DMSO-d ₆ ), δ (ppm): 11.27 (s, 1H), 8.88 (s, 1H), 8.83 (s, 1H), 7.19-6.93 (m, 3H), 6.86 ( dd, J＝8.9, 4.8Hz, 2H), 3.58-3.39(m, 1H), 1.93-1.73(m, 2H), 1.73-1.59(m, 2H), 1.41-1.03(m, 6H). ¹³ C -NMR (75MHz, DMSO-d ₆ ), δ (ppm): 159.56, 156.39, 150.79 (d, J=88.2Hz), 141.63, 139.32, 136.33, 123.09 (d, J=8.1Hz), 114.90 (d, J＝22.5Hz), 48.19, 32.51, 25.09, 24.19. HRMS (ESI): m/z[M+H] ⁺ .Calcdfor C ₁₆ H ₂₀ FN ₆ O ₃ : 363.1575; Found: 363.1577.

Example 15

Preparation of N-(4-fluorophenyl)-4-(3-n-octylureido)-N′-hydroxy-1,2,5-oxadiazole-3-carboxamidine (I-15)

Using VII-2 (0.3g, 1.15mmol), n-octyl isocyanate (VIII-7) (214mg, 1.38mmol), 60% sodium hydride (138mg, 3.45mmol) as raw materials, the operation is the same as I-1, column Chromatographic separation (eluent, dichloromethane:ethyl acetate=15:1) gave 0.15 g of light yellow solid, yield 33.3%, mp 130-132°C. ¹ H-NMR(300MHz,DMSO-d ₆ ),δ(ppm):11.27(s,1H),8.90(s,1H),8.87(s,1H),7.10(brs,1H),7.00(m, 2H), 6.87(m, 2H), 3.13-3.06(m, 2H), 1.51-1.37(m, 2H), 1.27(brs, 10H), 0.86(t, J＝6.4Hz, 3H). ¹³ C- NMR (75MHz, DMSO-d ₆ ), δ(ppm): 159.65, 156.48, 151.07(d, J=162.8Hz), 141.35, 139.37, 136.14, 123.23(d, J=7.8Hz), 114.83(d, J =22.5Hz), 39.44, 38.89, 31.18, 29.24, 28.62, 26.23, 22.02, 13.83.HRMS(ESI):m/z[M+H] ⁺ .Calcd for C ₁₈ H ₂₆ FN ₆ O ₃ :393.2045; Found :393.2056.

Example 16

Preparation of N-(4-fluorophenyl)-4-(3-tert-butylureido)-N′-hydroxy-1,2,5-oxadiazole-3-carboxamidine (I-16)

Using VII-2 (0.4g, 1.53mmol), tert-butyl isocyanate (VIII-8) (182mg, 1.84mmol), 60% sodium hydride (183mg, 4.59mmol) as raw materials, the operation is the same as I-1, column Chromatographic separation (eluent, dichloromethane:ethyl acetate=25:1) gave 0.12 g of white solid, yield 23.1%, mp 176-178°C. ¹ H-NMR (300MHz, DMSO-d ₆ ), δ (ppm): 11.29 (s, 1H), 8.89 (s, 1H), 8.69 (s, 1H), 7.08-6.94 (m, 3H), 6.84 ( dd, J＝8.9, 4.8Hz, 2H), 1.29(s, 9H). ¹³ C-NMR (75MHz, DMSO-d ₆ ), δ(ppm): 159.60, 156.43, 150.48 (d, J＝61.8Hz) ,141.35,139.25,136.30,123.17(d,J=8.0Hz),114.92(d,J=22.5Hz),49.96,28.59.HRMS(ESI):m/z[M+H] ⁺ .Calcd for C ₁₄ H ₁₈ FN ₆ O ₃ : 337.1419; Found: 337.1423.

Example 17

N-(3-chloro-4-fluorophenyl)-4-(3-n-propylureido)-N'-hydroxyl-1,2,5-oxadiazole-3-carboxamidine (VI-3) preparation

Compound IV (8g, 49.22mmol) was dissolved in ethanol (200mL), and 3-chloro-4-fluoroaniline (V-3) (7.88g, 54.14mmol) was added, followed by NaHCO ₃ (0.34g, 123.05mmol) Aqueous solution (100mL water), be warmed up to 60 ℃, TLC detects reaction, after IV reaction is complete, evaporate solvent under reduced pressure, ethyl acetate (80mL) dissolves, washes with water three times (30mL * 3), saturated NaCl washes twice ( 30mL×2), dried and stored overnight in anhydrous Na ₂ SO ₄ , evaporated the solvent under reduced pressure, and recrystallized from ethyl acetate/n-hexane to obtain 6.02g of a yellowish solid, yield 65.03%, mp 160-162°C. ¹ H-NMR (300MHz, DMSO-d ₆ ), δ(ppm): 11.46(s, 1H), 8.89(s, 1H), 7.22(t, J=9.1Hz, 1H), 6.97(dd, J= 6.5,2.6Hz,1H),6.75-6.70(m,1H),6.29(s,2H),3.37(brs,2H).

4-(3-chloro-4-fluorophenyl)-3-[(4-amino)-1,2,5-oxadiazol-3-yl]-1,2,4-oxadiazol-5( Preparation of 4H)-ketone (VII-3)

Dissolve compound VI-3 (6g, 22.09mmol) in ethyl acetate (78mL), add N,N'-carbonyldiimidazole (5.37g, 33.14mmol), react at room temperature (25°C), and monitor the reaction by TLC. Terminate the reaction until the raw materials have reacted. Wash with 1mol/L HCl (80mL×2), dry over anhydrous sodium sulfate, evaporate the solvent under reduced pressure, recrystallize from methanol to obtain 6.37g white solid, yield 97%. mp180-181°C. ¹ H-NMR (300MHz, DMSO-d ₆ ), δ (ppm): 7.99 (dd, J = 6.6, 2.1 Hz, 1H), 7.74-7.76 (m, 1H), 7.63 (t, J = 8.8 Hz, 1H),6.60(s,2H).

N-(3-chloro-4-fluorophenyl)-4-(3-n-propylureido)-N′-hydroxyl-1,2,5-oxadiazole-3-carboxamidine (I-17) preparation

Using VII-3 (0.4g, 1.17mmol), n-propyl isocyanate (VIII-2) (137mg, 1.61mmol), 60% sodium hydride (161mg, 4.02mmol) as raw materials, the operation is the same as I-1, column Chromatographic separation (eluent, dichloromethane: ethyl acetate = 15:1) gave 0.16 g of a light green solid, yield 33.3%, mp 169-171°C. ¹ H-NMR (300MHz, DMSO-d ₆ ), δ (ppm): 11.42 (s, 1H), 9.02 (s, 1H), 8.93 (s, 1H), 7.21 (t, J = 9.1Hz, 1H) ,7.16-7.11(m,1H),7.09(brs,1H),6.87-6.74(m,1H),3.11-3.04(m,2H),1.52-1.40(m,2H),0.87(t,J= 7.4Hz, 3H). ¹³ C-NMR (75MHz, DMSO-d ₆ ), δ (ppm): 154.38, 151.81 (d, J = 92.9Hz), 150.35, 141.84, 138.80, 137.47, 122.36, 121.02 (d, J＝6.8Hz), 118.69(d, J＝18.6Hz), 116.23(d, J＝21.7Hz), 41.30, 22.60, 11.21.HRMS(ESI):m/z[M+H] ⁺ .Calcd for C ₁₃ H ₁₅ ClFN ₆ O ₃ : 357.0873; Found: 357.0875

Example 18

Preparation of N-(3-chloro-4-fluorophenyl)-4-(3-isopropylureido)-N'-hydroxyl-1,2,5-oxadiazole-3-carboxamidine I-18)

Using VII-3 (0.4g, 1.34mmol), isopropyl isocyanate (VIII-3) (137mg, 1.61mmol), 60% sodium hydride (161mg, 4.02mmol) as raw materials, the operation is the same as I-1, column Chromatographic separation (eluent, petroleum ether: ethyl acetate = 3:1) gave 0.40 g of a white solid, yield 83.3%, mp 163-165°C. ¹ H-NMR (300MHz, DMSO-d ₆ ), δ (ppm): 11.42 (s, 1H), 9.03 (s, 1H), 8.83 (s, 1H), 7.21 (t, J = 9.1Hz, 1H) ,7.15-7.06(m,1H),7.00(brs,1H),6.85-6.76(m,1H),3.88-3.61(m,1H),1.11(d,J=6.4Hz,6H). ¹³ C- NMR (75MHz, DMSO-d ₆ ), δ(ppm): 154.39, 151.37(d, J=67.2Hz), 150.26, 141.73, 138.78, 137.43, 122.37, 121.00(d, J=6.8Hz), 118.70(d ,J=23.2Hz), 116.23(d,J=22.1Hz), 41.62,22.59.HRMS(ESI):m/z[M+H] ⁺ .Calcd for C ₁₃ H ₁₅ ClFN ₆ O ₃ :357.0873; Found :357.0875.

Claims (8)

1. the compound of logical formula (I) or its pharmaceutically acceptable salt:

Wherein R¹、R²It is independently represented each other: H, F, Cl, Br, CF₃、CH₃、OCH₃, CN or NO₂；

R³Represent C₁~C₈Straight chained alkyl.

2. the compound of claim 1 or its pharmaceutically acceptable salt, wherein R¹、R²It is independently represented each other F, Cl or Br.

3. the compound of claim 1 or its pharmaceutically acceptable salt, wherein R³Represent C₂~C₅Straight chained alkyl.

4. the preparation method of the compound of claim 1, comprising:

Wherein R¹、R²、R³Definition with claim 1.

5. the compound of claim 1 or its pharmaceutically acceptable salt, wherein pharmaceutically acceptable salt is claim 1 The acid-addition salts that compound and following acid are formed: hydrogen chloride, hydrogen bromide, sulfuric acid, carbonic acid, oxalic acid, citric acid, succinic acid, winestone Acid, phosphoric acid, lactic acid, pyruvic acid, acetic acid, maleic acid, methanesulfonic acid, benzene sulfonic acid, p-methyl benzenesulfonic acid or ferulic acid.

6. a kind of pharmaceutical composition, wherein the compound containing claim 1 or its pharmaceutically acceptable salt and pharmaceutically may be used The carrier of receiving.

7. the compound or its pharmaceutically acceptable salt of claim 1,2 or 3 are in preparation IDO1 and IDO2 double inhibitor medicine Purposes in object.

8. the purposes of claim 7, wherein IDO1 and IDO2 double inhibitor drug is anti-tumor drug.