CN111116614B - Covalent link between immunoregulatory factor and taxane, albumin nano-preparation and preparation method thereof - Google Patents

Abstract

The invention belongs to the field of pharmaceutical chemistry and pharmaceutical preparations, and relates to a covalent linker of an immune regulatory factor and taxane and a preparation method of an albumin nano preparation thereof; according to the invention, fatty acid anhydride is respectively linked with hydroxyl of indoleamine 2, 3-dioxygenase inhibitor NLG919 and hydroxyl of taxane compounds through ester bond condensation reaction to obtain the link, the link is prepared into an albumin nano preparation, and the link is delivered to tumors by utilizing the enhanced permeation and retention effects of the nano preparation on the tumors. The linker delivered to the tumor cells is used as a prodrug of NLG919 and taxane compounds, and is hydrolyzed into NLG919 and taxane compounds by esterase, so that the purpose of delivering the two drugs to the tumor cells together is achieved. NLG919 achieves synergistic tumor treatment by inhibiting IDO and taxane chemotherapy.

Description

Covalent links between immunomodulatory factors and taxanes and their albumin nano-preparations and Preparation

technical field

The invention belongs to the field of medicinal chemistry and pharmaceutical preparations, and relates to a covalent link between an immunoregulatory factor and a taxane, an albumin nano-preparation and a preparation method thereof. Enhanced penetration and retention effects, delivery of the linker to tumors, and synergistic treatment of tumors with taxane chemotherapy.

Background technique

In recent years, the incidence of malignant tumors has risen sharply, and the mortality rate is high. According to the data of the World Health Organization, 36% of new cancer patients are Chinese every year, and malignant tumors have become a major chronic disease affecting the life expectancy of Chinese people. Traditional antineoplastic drugs (such as paclitaxel, doxorubicin, etc.) have strong toxic effects. Chemotherapy inhibits tumor growth, and is also accompanied by drug resistance and recurrence of tumors.

The prior art discloses that albumin exists in large quantities in blood plasma, and its isoelectric point is 4.7-4.9. It is an acidic protein, easily soluble in water, and stable between pH 4-9. Human serum albumin is the most abundant protein in the human body. Due to its natural structure, biodegradability, non-toxicity, and high biocompatibility, it has been approved for clinical use by the U.S. Food and Drug Administration (FDA). It is regarded as an ideal drug delivery carrier.

Taxane compounds, including paclitaxel and docetaxel, have strong anti-tumor activity against a variety of tumors, by strengthening tubulin polymerization and inhibiting microtubule depolymerization, leading to the formation of stable non-functional microtubule bundles, thereby Inhibits cell mitosis and proliferation. However, its poor water solubility greatly limits its application in tumor therapy. Taxane compounds have a high affinity with serum albumin, and paclitaxel-albumin nanosuspension (trade name Abraxane) prepared with human serum albumin as a carrier was approved by the FDA in 2005.

IDO is an important regulatory protein that participates in the formation of tumor immunosuppressive microenvironment and promotes tumor cell growth. IDO is highly expressed in a variety of tumor tissues. It is the rate-limiting enzyme for the catabolism of tryptophan through the kynurenic acid pathway. It catalyzes the degradation of the essential amino acid tryptophan and accumulates its metabolites, resulting in cell cycle arrest and Effector T cells die, increasing the number of regulatory T cells. Immunomodulator NLG919 is an IDO inhibitor that reduces the metabolism of tryptophan, enabling effector T cells to perform their normal functions and kill tumor cells.

Based on the current state of the art, the inventors of the present application intend to provide a covalent link between an immunomodulatory factor and a taxane and an albumin nano-preparation thereof, so as to achieve the effect of synergistic treatment of tumors with taxane-based chemotherapy.

Contents of the invention

The purpose of the present invention is to provide a covalent link between an immunomodulatory factor and a taxane and its albumin nano-preparation and its preparation method. The prepared albumin nano-preparation can achieve the effect of synergistic treatment of tumors with taxane chemotherapy .

The present invention synthesizes a covalent link between NLG919 and taxane; the link is obtained by condensing the immunoregulatory factor IDO inhibitor NLG919 and taxane compounds through ester bond condensation reaction by using fatty acid anhydride; further the link Preparation of albumin nano-preparation, using the enhanced penetration and retention effect of nano-preparation on tumor, delivering the link to the tumor; the link delivered to tumor cells, as the prodrug of NLG919 and taxane compounds, through Esterase is hydrolyzed into NLG919 and taxane compounds, which can achieve the purpose of co-delivery of the above two drugs to tumor cells; the NLG919 reduces its inhibition of effector T cells by inhibiting IDO, and achieves synergy with taxane chemotherapy The effect of treating tumors.

specific,

再与紫杉醇或多烯紫杉醇的羟基通过酯键缩合反应得到共价链接物。The present invention provides a covalent link between NLG919 and paclitaxel, which is characterized in that fatty acid NLG919 is obtained by condensation of fatty acid anhydride and the hydroxyl group of NLG919, namely

Then it reacts with the hydroxyl group of paclitaxel or docetaxel through ester bond condensation reaction to obtain a covalent link.

More specifically, the linker of the present invention is a compound obtained by condensation reaction of the carboxyl group of NLG919 fatty acid and the 2'-hydroxyl group of paclitaxel or docetaxel through an ester bond condensation reaction, and the structure of this type of linker is shown in the general formula II,

in,

R ₁ is phenyl or tert-butoxy;

m and n are 0-10;

_R5 is hydrogen, methyl, ethyl or propyl.

The synthetic method of linker described in the present invention, specific steps are as follows:

Synthetic route 1

a. Fatty acidation of NLG919:

Select fatty acid anhydride, use pyridine, dichloromethane, THF, DMF or DMSO as a solvent, and react under heating conditions to obtain fatty acidated NLG919; preferably, the reaction conditions are NLG919 and fatty acid anhydride in pyridine solution, heated to 60 ℃ reaction for 48 hours;

b. Esterification of taxane compounds with fatty acidated NLG919:

Commonly used condensing agents are dicyclohexylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 2-(7-benzotriazole oxide)- N,N,N',N'-tetramethyluronium hexafluorophosphate, etc.; preferably, the condensing agent is dicyclohexylcarbodiimide.

The covalent link between NLG919 and paclitaxel according to the present invention is characterized in that fatty acidated NLG919 is obtained by condensation of fatty acid anhydride and hydroxyl of NLG919, and then obtained by condensation reaction with hydroxyl of paclitaxel or docetaxel through ester bond;

The linker is a compound obtained by condensing the carboxyl group of fatty acidated NLG919 and the 7-hydroxyl group of paclitaxel or docetaxel through an ester bond condensation reaction; the structure of this type of linker is shown in the general formula III:

in,

R ₁ is phenyl or tert-butoxy;

m and n are 0-10;

_R5 is hydrogen, methyl, ethyl or propyl.

The synthetic method of described linker, comprises steps:

Synthetic route 2

a. Protection of the 2' hydroxyl group of the taxane compound:

Hydroxyl protective agents include triisopropylsilyl, triethylsilyl, diphenyl tert-butylsilyl, tert-butyldimethylsilyl chloride, etc.; preferably, the protective agent is tert-butyldimethyl Chlorosilane; b. Esterification of taxane compound after protection with fatty acid NLG919:

Commonly used condensing agents are dicyclohexylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 2-(7-benzotriazole oxide)- N,N,N',N'-tetramethyluronium hexafluorophosphate, etc.; preferably, the condensing agent is dicyclohexylcarbodiimide;

c. Selective removal of the protecting group of the 2' hydroxyl group in the taxane compound structure:

Reaction conditions, tetrabutylammonium fluoride and THF, room temperature.

The covalent link between NLG919 and paclitaxel according to the present invention is obtained by condensation of fatty acid anhydride and the hydroxyl group of NLG919 to obtain fatty acidified NLG919, and then reacted with the hydroxyl group of paclitaxel or docetaxel through ester bond condensation reaction;

The linker is obtained by condensing the carboxyl group of fatty acidated NLG919 with the 2' and 7, or 2' and 10', or 2', 7 and 10' hydroxyl groups of paclitaxel or docetaxel through ester bond condensation reaction Compound; The structure of this type of linker is shown in the general formula IV:

in,

R ₁ is phenyl or tert-butoxy;

m and n are 0-10;

_R5 is hydrogen, methyl, ethyl or propyl.

The synthesis method of this type of linker comprises steps:

Synthetic route 3

的酯化反应：a. 2'-protected taxane compound and fatty acidated NLG919, ie

The esterification reaction:

Commonly used condensing agents are dicyclohexylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 2-(7-benzotriazole oxide)- N,N,N',N'-tetramethyluronium hexafluorophosphate, etc.; preferably, the condensing agent is dicyclohexylcarbodiimide.

The covalent link between NLG919 and paclitaxel according to the present invention is characterized in that fatty acidated NLG919 is obtained by condensation of fatty acid anhydride and hydroxyl of NLG919, and then obtained by condensation reaction with hydroxyl of paclitaxel or docetaxel through ester bond;

The linker is a compound obtained by condensing the carboxyl group of fatty acidated NLG919 and the 7 and 10' hydroxyl groups of paclitaxel or docetaxel through an ester bond condensation reaction; the structure of this type of linker is shown in the general formula V:

in,

R ₁ is phenyl or tert-butoxy;

m and n are 0-10;

_R5 is hydrogen, methyl, ethyl or propyl.

The synthetic method of this class linker, concrete steps are as follows:

Synthetic route 4

a. Esterification reaction of taxane compound with fatty acidated NLG919:

Commonly used condensing agents are dicyclohexylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride, 2-(7-benzotriazole oxide)- N,N,N',N'-tetramethyluronium hexafluorophosphate, etc.; preferably, the condensing agent is dicyclohexylcarbodiimide;

b. Selective removal of the protecting group of the 2'-position hydroxyl in the taxane compound structure:

Reaction conditions, tetrabutylammonium fluoride and THF, room temperature.

The present invention adopts the above-mentioned covalent link between NLG919 and taxane compounds to prepare albumin nano-preparation;

Described albumin nano-preparation is made through following technical scheme:

The preparation contains the linker and albumin; the mass ratio of the linker to albumin is 1:6-1:20; preferably, the mass ratio is 1:9-1:15;

The albumin is one or a mixture of human serum albumin, recombinant human serum albumin and bovine serum albumin; preferably, the albumin is human serum albumin.

The preparation method of the above-mentioned albumin nano-preparation containing the linker comprises the steps of:

(1) albumin is dissolved in water;

(2) dissolving the linker in an organic solvent;

(3) mixing the aqueous solution of step (1) with the organic solution of step (2) to obtain a white emulsion;

(4) The white emulsion is homogenized under high pressure, so that the particle size is controlled at 50-1000nm;

(5) Remove the organic solvent from the emulsion (4) under reduced pressure.

As a preference, the above preparation method also includes the step of dehydrating the nano-preparation solution obtained in step (5); preferably, the dehydration treatment is freeze-drying;

Preferably, the concentration of the albumin aqueous solution described in step (1) is 0.5%-5% (w/v), preferably 1%-2% (w/v);

Preferably, the ratio of the linker in step (2) to the total protein in step (2) is 1:9-1:15;

Preferably, the ratio of the organic solvent in step (2) to the water in step (1) is 1:20-1:50;

Preferably, the organic solvent in the step (2) is formed by mixing a hydrophobic organic solvent and a hydrophilic organic solvent; the hydrophobic organic solvent can be chloroform, dichloromethane or a mixed solvent of the two; the The hydrophilic organic solvent can be absolute ethanol, methanol, propylene glycol or a mixed solvent of two or three thereof; the ratio of the organic solvent to the aqueous solution in step (1) is 1:20-1:50;

Preferably, the high-pressure homogenization described in step (4) has a pressure range of 10000psi--20000psi, and the number of cycles is 10-20 times.

Embodiments of the present invention will be described in detail below in conjunction with the accompanying drawings.

Description of drawings

Fig. 1 Survival curve of melanoma-bearing C57 female mice after intravenous administration; tumor cells were inoculated on day 0, and treatment started on day 6. (n=10, p<0.01).

Fig. 2 is a curve graph of body weight versus time after intravenous administration of melanoma-bearing C57 female mice; n=10, tumor cells were inoculated on day 0, and treatment was started on day 6.

Fig. 3 Survival curve of BALB/c female mice bearing breast cancer cells after intravenous administration; tumor cells were inoculated on day 0, and treatment started on day 6. (n=10, p<0.01).

Fig. 4 is a graph of body weight versus time after intravenous administration of BALB/c female mice bearing breast cancer cells; n=10, tumor cells were inoculated on day 0, and treatment was started on day 6.

detailed description

The following examples further illustrate and explain the present invention, but not as a limitation of the present invention.

Embodiment 1: synthetic compound 4

Synthetic route 5

Reagents and conditions:

a. Glutaric anhydride, anhydrous pyridine, react at 60°C for 48 hours

b. paclitaxel, dicyclohexylcarbodiimide, 4-dimethylaminopyridine, methylene chloride, overnight at room temperature; the synthesis of compound 2 (fatty acidated NLG919) in Example 1:

Put NLG919 (0.1g) and glutaric anhydride (0.2g) in a 50mL eggplant-shaped bottle, add 5mL of anhydrous pyridine to dissolve, stir and react at 60°C for 48 hours; TLC detects the progress of the reaction. After the reaction is complete, cool to room temperature. Pyridine was removed under reduced pressure, and the residue was dissolved in dichloromethane. After silica gel column chromatography (dichloromethane:methanol=60:1), Compound 2 was obtained as a white solid, 0.11 g (yield: 80%).

NMR analysis: ¹ H NMR (400MHz, cdcl ₃ ) δ13.01 (m, 1H), 7.86–7.76 (m, 1H), 7.55 (d, J＝6.9Hz, 1H), 7.38 (m, 2H), 7.28 (d,J=2.5Hz,1H),7.21–7.16(m,1H),5.55–5.32(m,1H),3.73(m,1H),2.27–1.93(m,7H),1.90-1.71(m ,6H),1.37(t,J=11.1Hz,1H),1.11(m,5H).

Mass Spectrometry ESI-MS m/z[MH] ⁻ ; Calcd. for C ₂₃ H ₂₈ N ₂ O ₄ : 395.2; Found: 395.4

The synthesis of compound 4 in embodiment 1:

Add compound 2 (0.1g), paclitaxel (compound 3, 0.22g), dicyclohexylcarbodiimide (77mg), 4-dimethylaminopyridine (4mg) in sequence to a 50mL eggplant-shaped bottle, and wash with 12mL anhydrous Dichloromethane dissolved. The reaction was stirred overnight at room temperature; after the reaction was completed, the solvent was spin-dried, the residue was redissolved in dichloromethane, washed with saturated brine, the organic phase was dried with anhydrous sodium sulfate, filtered, the solvent was spin-dried, and silica gel column chromatography (dichloromethane :methanol=80:1) Compound 1 was obtained as a white solid, 0.15g (yield: 49%).

NMR analysis: ¹ H NMR (400MHz, cdcl ₃ ) δ8.15(d, J=6.6Hz, 2H), 7.74(d, J=7.5Hz, 1H), 7.67(d, J=9.5Hz, 2H), 7.59(dd,J=10.0,6.7Hz,1H),7.49(m,5H),7.43–7.29(m,9H),7.20(m,2H),6.31(d,J=18.3Hz,1H),6.23 (t,J=9.0Hz,1H),5.98(d,J=9.9Hz,1H),5.70(d,J=6.4Hz,1H),5.58–5.47(m,1H),5.09–4.90(m, 3H), 4.45(dt, J=13.3, 8.7Hz, 1H), 4.32(d, J=8.3Hz, 1H), 4.23(t, J=7.9Hz, 1H), 3.82(d, J=7.0Hz, 1H),2.61–2.40(m,7H),2.36(d,J＝7.1Hz,1H),2.31(m,2H),2.23(d,J＝8.5Hz,5H),2.15–1.99(m,3H ),1.95(s,3H),1.93–1.84(m,5H),1.76–1.70(m,4H),1.51–1.39(m,1H),1.37(s,1H),1.24(m,5H), 1.17(m,6H)..

Mass spectrometry ESI-MS m/z [ _M +H] ⁺ Calcd. for _C70H77N3O17 : _1232.5 ; found: _1232.2 .

Example 2: Preparation and evaluation of albumin nano-formulations containing compound 4

Human serum albumin was dissolved in water and mixed evenly, the protein concentration was 1.5% (w/v), and 30 g of 1.5% albumin aqueous solution (w/v) was weighed in a 50 mL beaker. Weigh 30.0 mg of compound 4 and dissolve it in an organic solvent (chloroform: absolute ethanol = 85:15), the drug dosage is 1:15 to the protein dosage, and the ratio of the organic solvent to the water in the protein aqueous solution is 1:44; Stir to prepare coarse milk to obtain a white emulsion; homogenize the white emulsion under a pressure of 20,000 psi to control the particle size at 50-200 nm; rotary evaporate the obtained compound 4 albumin nanoparticle solution, remove the organic solvent, and use 0.22 μm sterile membrane filtration. Freeze-dry to obtain the freeze-dried powder of the albumin nanoparticles of compound 4; the relevant parameters of the prepared nano-preparation are shown in Table 1,

Table 1

Pharmacodynamic evaluation in vivo: compound 4 albumin nano-preparation was prepared according to the method described in this example;

Tumor model establishment: 6-8 week old C57 female mice were subcutaneously inoculated with B16-OVA cells (5×10 ⁵ cells/mouse). When the tumor volume reached 50 mm, they ^were randomly divided into four groups (compound 4 albumin nano-preparation group, paclitaxel albumin nano-preparation and NLG919 mixed solution group, paclitaxel albumin nano-preparation group, NLG919 aqueous solution group and control group) 10 rats in each group were intravenously injected with compound 4 albumin nano-preparation, the mixture of paclitaxel albumin nano-preparation and NLG919, paclitaxel albumin nano-preparation, NLG919 and phosphate buffer saline, and the dosage was 10 mg paclitaxel/kg, The amount of NLG919 contained is 2.56mg/kg. Dosing once every 3 days, a total of 5 administrations; make the survival curve and the curve of body weight versus time; , paclitaxel albumin nano-preparation group, NLG919 aqueous solution group and control group, the median survival times were: 35 days, 27 days, 25 days, 22 days and 21 days; The paclitaxel albumin nano-preparation and the mixture of paclitaxel albumin nano-preparation and NLG919 have better anti-tumor effect; The toxicity of the preparation is low;

In vivo pharmacodynamic evaluation: Compound 4 albumin nano-preparation was prepared according to the method described in this example. Tumor model establishment: BALB/c female mice at 6-8 weeks were subcutaneously inoculated with 4T1 cells (5×10 ⁵ cells/mouse); when the tumor volume reached 50 mm ³ , they were randomly and evenly divided into four groups (compound 4 white Protein nano preparation group, paclitaxel albumin nano preparation and NLG919 mixed solution group, paclitaxel albumin nano preparation group, NLG919 aqueous solution group and control group), 10 rats in each group, intravenous injection of compound 4 albumin nano preparation, paclitaxel albumin For the mixed solution of nano-preparation and NLG919, paclitaxel albumin nano-preparation, NLG919 and phosphate buffer solution, the dosage is 10 mg paclitaxel/kg, and the amount of NLG919 is 2.56 mg/kg. Administered once every 3 days, a total of 5 administrations. Make the survival curve and the change curve of body weight to time; As shown in Figure 3, the compound 4 albumin nano-preparation group, the mixed solution group of paclitaxel albumin nano-preparation and NLG919, paclitaxel albumin nano-preparation group, NLG919 aqueous solution group and control group The median survival times were: 45 days, 38 days, 35 days, 33 days and 22 days. The results showed that the compound 4 albumin nano-preparation group had better anti-tumor effect than paclitaxel albumin nano-preparation alone, paclitaxel albumin nano-preparation and NLG919 mixture. As shown in Figure 4, after administration for 5 times, the body weight of the mice did not decrease, confirming that the compound 4 albumin nano-preparation has low toxicity.

Embodiment 3: the synthesis of compound 7

Synthetic route 6

Reagents and conditions:

a. Suberic anhydride, anhydrous pyridine, react at 60°C for 48 hours

b. Docetaxel, dicyclohexylcarbodiimide, 4-dimethylaminopyridine, dichloromethane, overnight at room temperature;

Synthesis of compound 5 (fatty acidated NLG919) in embodiment 3:

Put NLG919 (0.1g) and suberic anhydride (0.23g) in a 50mL eggplant-shaped bottle, add 5mL of anhydrous pyridine to dissolve, and stir at 60°C for 48 hours. TLC detection reaction proceeds. After the reaction is complete, cool to room temperature. Pyridine was removed under reduced pressure, and the residue was dissolved in dichloromethane. After silica gel column chromatography (dichloromethane:methanol=60:1), compound 5 was obtained as a white solid, 0.13g (yield: 87%).

The synthesis of compound 7 in embodiment 3:

Add compound 5 (0.1g), docetaxel (compound 6, 0.19g), dicyclohexylcarbodiimide (75mg), 4-dimethylaminopyridine (6mg) sequentially to a 50mL eggplant bottle, and use 15mL Anhydrous dichloromethane was dissolved. The reaction was stirred overnight at room temperature. After the reaction was completed, the solvent was spin-dried, the residue was redissolved in dichloromethane, washed with saturated brine, the organic phase was dried over anhydrous sodium sulfate, filtered, the solvent was spin-dried, and silica gel column chromatography (dichloromethane:methanol=80: 1) Compound 7 was obtained as a white solid, 0.17 g (yield: 59%).

Example 4: Preparation and Evaluation of Albumin Nano-Preparations Containing Compound 7

Bovine serum albumin was dissolved in water and mixed evenly, the protein concentration was 2.0% (w/v), and 30 g of 2.0% albumin aqueous solution (w/v) was weighed in a 50 mL beaker. Weigh 45.0 mg of compound 7 and dissolve it in an organic solvent (dichloromethane: absolute ethanol = 90:10), the amount of drug to the amount of protein = 1:13, the ratio of organic solvent to water in protein aqueous solution is 1:50 . Stir at high speed to prepare coarse milk to obtain a white emulsion; homogenize the white emulsion under a pressure of 18000 psi to control the particle size at 50-500 nm; rotary evaporate the obtained compound 7 albumin nanoparticle solution, remove the organic solvent, and use 0.22 μm sterile membrane filter. Freeze-dry to obtain the lyophilized powder of albumin nanoparticles of compound 7. The relevant parameters of the prepared nano-preparation are shown in Table 2.

Table 2

In vivo pharmacodynamic evaluation: Compound 7 albumin nano-preparation was prepared according to the method described in this example. Tumor model establishment: 6-8 week old C57 female mice were subcutaneously inoculated with B16-OVA cells (5×10 ⁵ cells/mouse). When the tumor volume reached 50 ^mm3 , they were randomly divided into four groups (compound 7 albumin nano-preparation group, docetaxel albumin nano-preparation and NLG919 mixed solution group, docetaxel albumin nano-preparation group, NLG919 aqueous solution group) and control group), 10 rats in each group, respectively intravenous injection of compound 7 albumin nano-preparation, docetaxel albumin nano-preparation and NLG919 mixed solution, docetaxel albumin nano-preparation, NLG919 and phosphate buffer saline, administration The dose was 10 mg paclitaxel/kg containing 5.11 mg NLG919/kg. Administered once every 3 days, a total of 5 administrations. The results showed that the median survival time of compound 7 albumin nano-preparation group, docetaxel albumin nano-preparation and NLG919 mixture group, docetaxel albumin nano-preparation group, NLG919 aqueous solution group and control group were respectively: 35.5 days, 27.5 days, 26 days, 24 days and 20 days. The results showed that the compound 7 albumin nano-preparation group had better anti-tumor effect than the simple docetaxel albumin nano-preparation and the mixture of docetaxel albumin nano-preparation and NLG919. After administration for 5 times, the compound 7 albumin nano-preparation did not cause weight loss in mice, confirming that the compound 7 albumin nano-preparation has low toxicity.

Embodiment 5: the synthesis of compound 10

Synthetic route 7

Reagents and conditions:

a. Tert-butyldimethylsilyl chloride, dry dichloromethane, room temperature for 5 hours.

b. Paclitaxel, dicyclohexylcarbodiimide, 4-dimethylaminopyridine, dichloromethane, overnight at room temperature

c. Tetrabutylammonium fluoride, THF, room temperature for 3 hours.

Synthesis of Compound 8:

Paclitaxel (compound 3, 0.5 g) was placed in a 50 mL eggplant-shaped bottle, dissolved in 15 mL of anhydrous dichloromethane, and then 0.3 mL of tert-butyldimethylsilyl chloride was added dropwise. The reaction was stirred at room temperature for 5 hours. TLC detection reaction proceeds. After the reaction was completed, the solvent and unreacted tert-butyldimethylchlorosilane were removed under reduced pressure, the residue was dissolved in dichloromethane, and compound 8 was obtained by silica gel column chromatography (dichloromethane:methanol=100:1) as White solid, 0.40 g (yield: 70%).

Synthesis of compound 9:

Add compound 2 (0.1g), compound 8 (0.25g), dicyclohexylcarbodiimide (63mg), 4-dimethylaminopyridine (5mg) in sequence to a 50mL eggplant-shaped bottle, and add 10mL of anhydrous dichloro Methane dissolves. The reaction was stirred overnight at room temperature. After the reaction was completed, the solvent was spin-dried, the residue was redissolved in dichloromethane, washed with saturated brine, the organic phase was dried over anhydrous sodium sulfate, filtered, the solvent was spin-dried, and silica gel column chromatography (dichloromethane:methanol=80: 1) Compound 9 was obtained as a white solid, 0.21 g (yield: 62%).

Synthesis of Compound 10:

Dissolve compound 9 (0.1g) in 6 mL of anhydrous THF, add tetrabutylammonium fluoride, stir at room temperature for 3 hours, spin off the solvent, re-dissolve the residue in dichloromethane, wash with saturated brine, and wash the organic phase with Dry over sodium sulfate, filter, spin to dry the solvent, silica gel column chromatography (dichloromethane:methanol=80:1) to obtain compound 10 as a white solid, 63 mg (yield: 72%).

Example 6: Preparation and Evaluation of Albumin Nano-Preparation Containing Compound 10

The recombinant human serum albumin was dissolved in water and mixed evenly, the protein concentration was 1.0% (w/v), and 30 g of 1.0% albumin aqueous solution (w/v) was weighed in a 50 mL beaker. Weigh 30.0 mg of compound 10 and dissolve it in an organic solvent (chloroform:propylene glycol=85:15), the amount of drug to the amount of protein=1:10, and the ratio of organic solvent to water in protein aqueous solution is 1:40. Stir at high speed to prepare colostrum to obtain a white emulsion; homogenize the white emulsion under a pressure of 18000psi to control the particle size at 50-200nm; rotary evaporate the obtained compound 10 albumin nanoparticle solution, remove the organic solvent, and use 0.22 μm sterile membrane filter. Freeze-dry to obtain the lyophilized powder of albumin nanoparticles of Compound 10. The relevant parameters of the prepared nano-preparation are shown in Table 3;

table 3

Example 7: Synthesis of Compounds 12, 13 and 14

Synthetic route 8

Reagents and conditions:

a. Dicyclohexylcarbodiimide, 4-dimethylaminopyridine, dichloromethane, room temperature for 48 hours

Synthesis of Compounds 11, 12 and 13 in Example 7

Add compound 5 (1.72g), compound 7 (0.50g), dicyclohexylcarbodiimide (0.81g), 4-dimethylaminopyridine (50mg) in sequence to a 50mL eggplant-shaped bottle, and use 30mL of anhydrous di Chloromethane dissolves. The reaction was stirred overnight at room temperature. After the reaction was completed, the solvent was spin-dried, the residue was redissolved in dichloromethane, washed with saturated brine, the organic phase was dried over anhydrous sodium sulfate, filtered, the solvent was spin-dried, and silica gel column chromatography (dichloromethane:methanol=100: 1-60: 1) sequentially separated to obtain compound 11 as a white solid, 0.19g (yield: 28%), compound 12 as a white solid, 51mg (yield: 8%) and compound 13 as a white solid, 25mg (yield : 3%).

Example 8: Preparation and Evaluation of Albumin Nano-Preparation Containing Compound 11

Human serum albumin was dissolved in water and mixed evenly, the protein concentration was 1.5% (w/v), and 30 g of 1.5% albumin aqueous solution (w/v) was weighed in a 50 mL beaker. Weigh 30.0 mg of compound 11 and dissolve it in an organic solvent (chloroform: absolute ethanol = 85:15), the drug dosage to protein dosage = 1:15, and the ratio of organic solvent to water in protein aqueous solution is 1:45. Stir at high speed to prepare colostrum to obtain a white emulsion; homogenize the white emulsion under a pressure of 18000psi to control the particle size at 50-200nm; rotary evaporate the obtained compound 11 albumin nanoparticle solution, remove the organic solvent, and use 0.22 μm sterile membrane filter. Freeze-dry to obtain the lyophilized powder of albumin nanoparticles of Compound 11. The relevant parameters of the prepared nano-preparation are shown in Table 4.

Table 4

In vivo pharmacodynamic evaluation: Compound 11 albumin nano-preparation was prepared according to the method described in this example. Tumor model establishment: 6-8 week old C57 female mice were subcutaneously inoculated with B16-OVA cells (5×10 ⁵ cells/mouse). When the tumor volume reached 50 ^mm3 , they were randomly divided into four groups (compound 11 albumin nano-preparation group, docetaxel albumin nano-preparation and NLG919 mixed solution group, docetaxel albumin nano-preparation group, NLG919 aqueous solution group) and control group), 10 rats in each group, respectively intravenous injection of compound 8 albumin nano-preparation, the mixture of docetaxel albumin nano-preparation and NLG919, docetaxel albumin nano-preparation, NLG919 and phosphate buffer saline, administration The dose was 10 mg docetaxel/kg containing 5.40 mg NLG919/kg. Administered once every 3 days, a total of 5 administrations. The results showed that the median survival time of compound 11 albumin nano-preparation group, docetaxel albumin nano-preparation and NLG919 mixed solution group, docetaxel albumin nano-preparation group, NLG919 aqueous solution and control group were respectively: 36 days , 27.5 days, 25 days, 23 days and 21 days. The results showed that the compound 11 albumin nano-preparation group had better anti-tumor effect than the simple docetaxel albumin nano-preparation and the mixture of docetaxel albumin nano-preparation and NLG919. After administration for 5 times, the compound 11 albumin nano-preparation did not cause weight loss in mice, which confirmed that the compound 11 albumin nano-preparation had low toxicity.

Example 9: Preparation and Evaluation of Albumin Nano-Preparation Containing Compound 12

Human serum albumin was dissolved in water and mixed evenly, the protein concentration was 1.5% (w/v), and 30 g of 1.5% albumin aqueous solution (w/v) was weighed in a 50 mL beaker. Weighed 45.0 mg of compound 12 and dissolved it in an organic solvent (chloroform: absolute ethanol = 86:14), the drug dosage to protein dosage = 1:10, and the ratio of organic solvent to water in protein aqueous solution was 1:44. Stir at high speed to prepare colostrum to obtain a white emulsion; homogenize the white emulsion under a pressure of 18000psi to control the particle size at 50-300nm; rotary evaporate the obtained compound 12 albumin nanoparticle solution, remove the organic solvent, and use 0.22 μm sterile membrane filter. Freeze-dry to obtain the freeze-dried rice powder of albumin nanoparticles of compound 12. The relevant parameters of the prepared nano-preparation are shown in Table 5,

table 5

In vivo pharmacodynamic evaluation: Compound 12 albumin nano-preparation was prepared according to the method described in this example. Tumor model establishment: 6-8 week old C57 female mice were subcutaneously inoculated with B16-OVA cells (5×10 ⁵ cells/mouse). When the tumor volume reached 50 mm, they ^were randomly divided into four groups (compound 12 albumin nano-preparation group, docetaxel albumin nano-preparation and NLG919 mixed solution group, docetaxel albumin nano-preparation group, NLG919 aqueous solution and Control group), 10 rats in each group, were intravenously injected with compound 12 albumin nano-preparation, the mixture of docetaxel albumin nano-preparation and NLG919, polyenol albumin nano-preparation, NLG919 and phosphate buffer saline, the dosage To contain 10 mg docetaxel/kg, contain 2.70 mg NLG919/kg. Administered once every 3 days, a total of 5 administrations. The results showed that the median survival time of compound 12 albumin nano-preparation group, docetaxel albumin nano-preparation and NLG919 mixture group, docetaxel albumin nano-preparation group, NLG919 solution group and control group were respectively: 33 days, 26.5 days, 25 days, 22 days and 20 days. The results showed that the compound 12 albumin nano-preparation group had a better anti-tumor effect than the simple docetaxel albumin nano-preparation and the mixture of docetaxel albumin nano-preparation and NLG919. After administration for 5 times, the compound 12 albumin nano-preparation did not cause weight loss in mice, confirming that the compound 12 albumin nano-preparation has low toxicity.

Example 10: Preparation and Evaluation of Albumin Nano-Preparation Containing Compound 13

Human serum albumin was dissolved in water and mixed evenly, the protein concentration was 1.5% (w/v), and 30 g of 1.5% albumin aqueous solution (w/v) was weighed in a 50 mL beaker. Weighed 30.0 mg of compound 13 and dissolved it in an organic solvent (chloroform: absolute ethanol = 87:13), the drug dosage to protein dosage = 1:15, and the ratio of organic solvent to water in protein aqueous solution was 1:44. Stir at high speed, prepare colostrum, and obtain white emulsion; homogenize the white emulsion under a pressure of 20,000 psi, so that the particle size is controlled at 50-200 nm; rotary evaporate the obtained compound 13 albumin nanoparticle solution, remove the organic solvent, and use 0.22 μm sterile membrane filter. Freeze-dry to obtain the lyophilized powder of albumin nanoparticles of Compound 13. The relevant parameters of the prepared nano-preparation are shown in Table 6,

Table 6

In vivo pharmacodynamic evaluation: Compound 13 albumin nano-preparation was prepared according to the method described in this example. Tumor model establishment: 6-8 week old C57 female mice were subcutaneously inoculated with B16-OVA cells (5×10 ⁵ cells/mouse). When the tumor volume reached 50 ^mm3 , they were randomly divided into four groups (compound 13 albumin nano-preparation group, docetaxel albumin nano-preparation and NLG919 mixed solution group, docetaxel albumin nano-preparation group, NLG919 aqueous solution group) and control group), 10 rats in each group, were intravenously injected with compound 13 albumin nano-preparation, the mixture of docetaxel albumin nano-preparation and NLG919, docetaxel albumin nano-preparation, NLG919 and phosphate buffer saline, administration The dose was 10 mg docetaxel/kg containing 8.10 mg NLG919/kg. Give 1 medicine every 3 days, a total of 5 administrations. The results showed that the median survival time of compound 13 albumin nano-preparation group, docetaxel albumin nano-preparation and NLG919 mixture group, docetaxel albumin nano-preparation group, NLG919 aqueous solution group and control group were respectively: 39 days, 27.5 days, 25 days, 23 days and 21 days. The results showed that the compound 13 albumin nano-preparation group had a better anti-tumor effect than the simple docetaxel albumin nano-preparation and the mixture of docetaxel albumin nano-preparation and NLG919. After administration for 5 times, the compound 13 albumin nano-preparation did not cause weight loss in mice, confirming that the compound 13 albumin nano-preparation has low toxicity.

Embodiment 11: the synthesis of compound 15

Synthetic route 9

Reagents and conditions:

a. Paclitaxel, dicyclohexylcarbodiimide, 4-dimethylaminopyridine, dichloromethane, overnight at room temperature;

b. Tetrabutylammonium fluoride, THF, room temperature for 3 hours.

Synthesis of compound 14 in embodiment 11

Add compound 2 (0.1g), compound 9 (0.41g), dicyclohexylcarbodiimide (80mg), 4-dimethylaminopyridine (5mg) in sequence to a 50mL eggplant-shaped bottle, and add 10mL of anhydrous dichloro Methane dissolves. The reaction was stirred overnight at room temperature. After the reaction was completed, the solvent was spin-dried, the residue was redissolved in dichloromethane, washed with saturated brine, the organic phase was dried over anhydrous sodium sulfate, filtered, the solvent was spin-dried, and silica gel column chromatography (dichloromethane:methanol=80: 1) Compound 11 was sequentially isolated as a white solid, 0.25 g (yield: 58%).

Synthesis of compound 15 in embodiment 11

Compound 14 (0.2 g) was dissolved in 8 mL of anhydrous THF, tetrabutylammonium fluoride was added, and stirred at room temperature for 3 hours. The solvent was spin-off, the residue was re-dissolved in dichloromethane, washed with saturated brine, the organic phase was dried over anhydrous sodium sulfate, filtered, the solvent was spin-dried, and silica gel column chromatography (dichloromethane:methanol=50:1) gave the compound 15 was a white solid, 0.13 g (yield: 72%).

Example 12: Preparation and Evaluation of Albumin Nano-Preparation Containing Compound 15

Human serum albumin was dissolved in water and mixed evenly, the protein concentration was 1.5% (w/v), and 30 g of 1.5% albumin aqueous solution (w/v) was weighed in a 50 mL beaker. Weighed 30.0 mg of compound 15 and dissolved it in an organic solvent (chloroform: absolute ethanol = 87:13), the drug dosage to protein dosage = 1:15, and the ratio of organic solvent to water in protein aqueous solution was 1:44. Stir at high speed, prepare colostrum, and obtain white emulsion; homogenize the white emulsion under a pressure of 20,000 psi, so that the particle size is controlled at 50-200 nm; rotate the obtained compound 15 albumin nanoparticle solution, remove the organic solvent, and use 0.22 μm sterile membrane filter. Freeze-dry to obtain the lyophilized powder of albumin nanoparticles of Compound 15. The relevant parameters of the prepared nano-preparation are shown in Table 7,

Table 7

In vivo pharmacodynamic evaluation: Compound 15 albumin nano-preparation was prepared according to the method described in this example. Tumor model establishment: 6-8 week old C57 female mice were subcutaneously inoculated with B16-OVA cells (5×10 ⁵ cells/mouse). When the tumor volume reached 50 mm, they ^were randomly divided into 5 groups (compound 15 albumin nano-preparation group, paclitaxel albumin nano-preparation and NLG919 mixed solution group, paclitaxel albumin nano-preparation group, NLG919 aqueous solution group and control group) 10 rats in each group were intravenously injected with compound 15 albumin nano-preparation, the mixture of paclitaxel albumin nano-preparation and NLG919, paclitaxel albumin nano-preparation, NLG919 and phosphate buffer saline, and the dosage was 10 mg paclitaxel/kg, Contains 8.10mg NLG919/kg. Give 1 medicine every 3 days, a total of 5 administrations. The results showed that the median survival time of compound 15 albumin nano preparation group, paclitaxel albumin nano preparation and NLG919 mixture group, paclitaxel albumin nano preparation group, NLG919 aqueous solution group and control group were 43 days and 26 days respectively , 24 days, 20 days and 19 days. The results showed that the compound 15 albumin nano-preparation group had a better anti-tumor effect than the simple docetaxel albumin nano-preparation and the mixture of docetaxel albumin nano-preparation and NLG919. After administration for 5 times, the compound 15 albumin nano-preparation did not cause weight loss in mice, which confirmed that the compound 15 albumin nano-preparation had low toxicity.

Claims (11)

1. A covalent linkage of an immunomodulatory factor and a taxane, characterized in that the immunomodulatory factor is indoleamine 2,3-dioxygenase inhibitor NLG919; the taxane is The taxane compound is selected from paclitaxel or docetaxel; the covalent link is a compound obtained by linking the fatty acid anhydride with the hydroxyl group of NLG919 and the hydroxyl group of the taxane compound through an ester bond condensation reaction; Its general structural formula includes taxane and fatty acidated NLG919 with different carbon chain lengths, and its structure is shown in general formula I:

in, R ₁ is phenyl or tert-butoxy; R ₂ , R ₃ , R ₄ are H or

At least one of R ₂ , R ₃ , R ₄ is

group, m and n are 0-10; _R5 is hydrogen, methyl, ethyl or propyl. 2. A method for preparing a covalent link between an immunomodulatory factor and a taxane, characterized in that: Synthetic route 1

Wherein, R ₁ and R ₅ are the same as those described in claim 1, and m and n are 0-10; Reagents and conditions in the synthetic route 1 include: a. Fatty acidation of NLG919: Select fatty acid anhydride, use pyridine, dichloromethane, tetrahydrofuran (THF), N,N-dimethylformamide (DMF) or dimethyl sulfoxide (DMSO) as solvent, react under heating conditions to obtain fatty acid NLG919, which is

b. Esterification of taxane compounds with fatty acidated NLG919: The condensing agent used is dicyclohexylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride or 2-(7-benzotriazole oxide)- N,N,N',N'-Tetramethyluronium hexafluorophosphate. 3. A method for preparing a covalent link between an immunomodulatory factor and a taxane, characterized in that: Synthetic route 2

Wherein, R ₁ and R ₅ are the same as those described in claim 1, and m and n are 0-10; Reagents and conditions in the synthetic route 2 include: a. Protection of the 2' hydroxyl group of the taxane compound: Select tert-butyldimethylsilyl chloride as the hydroxyl protecting agent; b. Esterification reaction of protected taxane compound with fatty acidated NLG919: Using dicyclohexylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride or 2-(7-benzotriazole oxide)-N,N, N', N'-tetramethyluronium hexafluorophosphate is the condensation agent; c. Selective removal of the protecting group of the 2' hydroxyl group in the taxane compound structure: Reaction conditions: tetrabutylammonium fluoride and THF, room temperature. 4. A method for preparing a covalent link between an immunomodulatory factor and a taxane, characterized in that: Synthetic route 3

Wherein, R ₁ and R ₅ are the same as those described in claim 1, and m and n are 0-10; Reagents and conditions in the synthetic route 3 include: a. Esterification reaction of taxane compound with fatty acidated NLG919: Using dicyclohexylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride or 2-(7-benzotriazole oxide)-N,N, N', N'-Tetramethyluronium hexafluorophosphate is the condensation agent, fatty acid NLG919 is

5. A method for preparing a covalent link between an immunomodulatory factor and a taxane, characterized in that: Synthetic route 4

Wherein, R ₁ and R ₅ are the same as those described in claim 1, and m and n are 0-10; Reagents and conditions in the synthetic route 4 include: a. Esterification reaction of taxane compound with fatty acidated NLG919: Using dicyclohexylcarbodiimide, 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride or 2-(7-benzotriazole oxide)-N,N, N', N'-Tetramethyluronium hexafluorophosphate is the condensation agent, fatty acid NLG919 is

b. Selective removal of the protecting group of the 2'-position hydroxyl in the taxane compound structure: The reaction conditions are: tetrabutylammonium fluoride and THF, room temperature. 6. the albumin nano-preparation prepared by the covalent link of a kind of immunoregulatory factor and taxane described in claim 1, it is characterized in that, by the covalent linkage of the immunomodulatory factor described in claim 1 and taxane Valence linkers and albumin composition. 7. albumin nano preparation according to claim 6, is characterized in that, the mass ratio of described linker and albumin is 1:6-1:20. 8. The albumin nano-preparation according to claim 6, characterized in that the particle size of the albumin nano-preparation is 50-1000nm. 9. The albumin nano-preparation according to claim 6, wherein the albumin is selected from one or more mixtures of human serum albumin, recombinant human serum albumin and bovine serum albumin. 10. by the preparation method of the albumin nano-preparation described in claim 6, it is characterized in that, comprises the step: (1) albumin is dissolved in water; (2) dissolving the covalent link between the immunoregulatory factor and taxane described in claim 1 in an organic solvent; (3) the aqueous solution of step (1) is mixed with the organic solution of step (2); (4) the mixed solution obtained by step (3) is homogenized under high pressure; (5) removing the organic solvent from the product obtained in step (4) under reduced pressure. 11. The preparation method of albumin nano-preparation according to claim 10, characterized in that the concentration of the albumin aqueous solution is 0.5%-5% w/v.

Images