CN111333653A

CN111333653A - ICD inducer-IDO inhibitor conjugate, preparation method and application

Info

Publication number: CN111333653A
Application number: CN201911306005.6A
Authority: CN
Inventors: 姜新义; 陈晨; 张蕊; 杜微; 张晶; 唐春伟
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2019-12-16
Filing date: 2019-12-16
Publication date: 2020-06-26
Also published as: WO2021120898A1

Abstract

The present disclosure provides an ICD inducer-IDO inhibitor conjugate, its preparation method and application, and its conjugate structural formula is

The preparation method comprises the following steps: NLG919 and succinic anhydride are subjected to esterification reaction to obtain NLG919-SA, and NLG919-SA and Dox are subjected to amidation reaction to obtain an ICD inducer-IDO inhibitor conjugate.

Description

ICD inducer-IDO inhibitor conjugate, preparation method and application

Technical Field

The disclosure relates to an ICD inducer-IDO inhibitor conjugate, a preparation method and application thereof.

Background

The statements herein merely provide background information related to the present disclosure and may not necessarily constitute prior art.

Immune escape from tumors contributes to the development of tumors, wherein the mechanisms of immune escape from tumors include changes in the tumor itself, changes in the tumor-induced microenvironment, and promotion of tumor development by the tumor microenvironment. In recent years, immunotherapy against immune escape links has achieved significant success in tumor therapy. Immunotherapy involves multiple factors and links, and has a complex connection with changes of tumor cells and tumor microenvironment, so that the immunotherapy still faces a great challenge in the clinical practice process.

The production of Tumor Associated Antigens (TAAs) is an important factor in activating T cells, however, tumor cells often inhibit T cell activation through a series of changes (such as loss of antigen expression, endocytic antigen or antigen shedding) to evade the surveillance and opportunistic growth of the body's immune system. In addition, tumor cells can "antigen drift" like viruses, leading to antigenic epitope mutations that alter the antigenicity of the tumor, which in turn evades T cell-mediated attacks.

Recently, it has been found that when a tumor is treated with a specific chemical or radiation, tumor cells undergo apoptosis and are transformed from non-immunogenic cells to immunogenic cells, thereby triggering an anti-tumor immune killing effect in the body, which is called immunogenic death of tumor cells. Research shows that certain anthracyclines and oxaliplatin and other therapeutic drugs not only induce tumor cell apoptosis, but also can cause Immunogenic Cell Death (ICD), and release 3 types of signals through inducing tumor cell autophagy: calreticulin is exposed on the cell surface and stimulates phagocytosis of Dendritic Cells (DCs); adenosine triphosphate is released, and DC are recruited to enter a tumor focus; the high migration rate family protein B1 promotes DC and dying tumor cells to form stable combination, and induces the organism to generate specific T cell anti-tumor immunity.

Indoleamine 2,3-dioxygenase (IDO) is the only rate-limiting enzyme outside the liver that catalyzes the catabolism of tryptophan (Trp) along the kynurenine (Kyn) pathway. IDO is closely related to tumor immune escape, and can mediate tumor immune escape through multiple mechanisms, wherein tryptophan exhaustion inhibits local T cell proliferation, and tryptophan metabolites promote T cell apoptosis, induce regulatory T cell proliferation and the like. Given the important role played by IDO in the development and maintenance of tumor immune tolerance, IDO has become a new target for anti-tumor immunotherapy. However, the results of preclinical studies suggest that the IDO inhibitor has weak tumor killing activity alone and the inhibition rate is 30-50%, and the combination of the IDO inhibitor and chemotherapeutic drugs is an important strategy for improving the clinical treatment effect of the IDO inhibitor.

The synergistic effect of ICD induction effect of the chemotherapeutic drug and the immune-related metabolic regulation effect of the IDO inhibitor can be realized by the combined administration, so that better tumor treatment effect is achieved. To the knowledge of the inventors of the present disclosure, most of the current studies on the combination of ICD inducer and IDO inhibitor are to use an additional vector for co-delivery of both drugs, however, the studies of the inventors of the present disclosure find that the manner of co-delivery using such an additional vector is complicated.

Disclosure of Invention

In order to overcome the defects of the prior art, the present disclosure aims to provide an ICD inducer-IDO inhibitor conjugate, and a preparation method and application thereof, wherein the ICD inducer and IDO inhibitor are delivered simultaneously in a conjugate manner, so that convenience is provided.

In order to achieve the purpose, the technical scheme of the disclosure is as follows:

in a first aspect, the present disclosure provides an ICD inducer-IDO inhibitor conjugate having the structural formula:

the present disclosure utilizes the reactant succinic acid, i.e., the two carboxyl groups of succinic acid, in the tricarboxylic acid cycle in vivo as a bridge that can create an easily cleavable linking moiety with Dox and NLG919, allowing the two drugs to form a Dox-NLG919 conjugate. Based on the current research, the inventor of the present disclosure finds that when succinic acid is used as a bridging substance, Dox and NLG919 conjugates can be obtained, and when other diacid or polyacid is used, Dox and NLG919 conjugates are difficult to obtain.

In a second aspect, the present disclosure provides a method for preparing an ICD inducer-IDO inhibitor conjugate, including performing an esterification reaction between NLG919 and succinic anhydride to obtain NLG919-SA, and performing an amidation reaction between NLG919-SA and Dox to obtain the ICD inducer-IDO inhibitor conjugate;

the structural formula of NLG919-SA is

The structural formula of the ICD inducer-IDO inhibitor conjugate is shown in the specification

The present disclosure demonstrates experimentally that ICD inducer-IDO inhibitor conjugates can be prepared using succinic acid. Secondly, the reaction sequence of succinic anhydride and NLG919 and Dox also influences the successful synthesis of the ICD inducer-IDO inhibitor conjugate, and the research of the disclosure finds that the ICD inducer-IDO inhibitor conjugate can be successfully prepared by synthesizing the NLG919 and the succinic anhydride and then reacting with the Dox, and the ICD inducer-IDO inhibitor conjugate cannot be obtained if the sequence is changed.

In a third aspect, the present disclosure provides a use of the ICD inducer-IDO inhibitor conjugate in preparing a medicament for treating tumors.

In a fourth aspect, the present disclosure provides a use of the ICD inducer-IDO inhibitor conjugate described above in a single drug delivery system.

The beneficial effect of this disclosure does:

1. the present disclosure provides an Immunogenic Cell Death (ICD) inducer-tryptophan metabolism blocker conjugate for achieving similar single-drug delivery during treatment of neoplasms, the conjugate comprising an ICD-induced chemotherapeutic capable of eliciting a strong effector T cell immune response, an

indoleamine

2,3 dioxygenase (IDO) inhibitor that blocks the metabolism of tryptophan to kynurenine.

2. The conjugate provided by the disclosure has a bridging substance which is succinic acid, the succinic acid connects the ICD inducer and the tryptophan metabolism blocker together through ester bonds and amido bonds, and meanwhile, the ester bonds and the amido bonds are easily hydrolyzed by esterase and amidase which are abundantly present in tumors, so that the ICD inducer and the tryptophan metabolism blocker are released.

3. The method synthesizes a Dox-NLG919 conjugate which is dependent on different action mechanisms and can synergistically resist cancer for the first time, and brings great convenience for synergistic drug delivery.

Drawings

The accompanying drawings, which are included to provide a further understanding of the disclosure, illustrate embodiments of the disclosure and together with the description serve to explain the disclosure and are not to limit the disclosure.

FIG. 1 is the NMR spectrum of NLG919-SA prepared in example 1 of the disclosure ((R))¹H NMR) graph.

FIG. 2 is an electrospray mass spectrometry (ESI-MS) plot of NLG919-SA prepared in example 1 of the present disclosure.

FIG. 3 is a nuclear magnetic resonance hydrogen spectrum of Dox-NLG919 conjugate prepared according to example 1 of the disclosure (C: (C))¹H NMR) graph.

FIG. 4 is an electrospray mass spectrometry (ESI-MS) plot of the Dox-NLG919 conjugate prepared according to example 1 of the present disclosure.

FIG. 5 is an electrospray mass spectrometry (ESI-MS) plot of a product prepared according to example 2 of the present disclosure.

FIG. 6 is an electrospray mass spectrometry (ESI-MS) plot of a product prepared according to example 3 of the present disclosure.

FIG. 7 is a comparison of the inhibition of breast tumor growth in vivo by Dox-NLG919 conjugate treatment by Dox, NLG919, Dox-NLG919 conjugate, and treatment with PBS control in example 4 of the disclosure.

Figure 8 is a fluorescent quantitative detection map of tumors in mice treated with Dox, NLG919, Dox-NLG919 conjugate, and treated with PBS control in example 4 of the disclosure.

FIG. 9 is a graph of fluorescence intensity in mice treated with Dox, NLG919, Dox-NLG919 conjugate, and PBS control, quantified by fluorescence in example 4 of the disclosure.

FIG. 10 is a graph of the quantitative detection of tumor volume in mice treated with Dox, NLG919, Dox-NLG919 conjugate, and treated with PBS control by fluorescence in example 4 of the disclosure.

Figure 11 is the tumor weight reduction by treatment with Dox, NLG919, Dox-NLG919 conjugate compared to treatment with PBS control in example 4 of the disclosure.

Figure 12 is the average weight of mice during treatment with Dox, NLG919, Dox-NLG919 conjugate compared to control treatment with PBS in example 4 of the disclosure.

FIG. 13 is the survival rate of 4T1 cells after treatment with different concentrations of Dox, NLG919, Dox-NLG919 conjugate (concentration range 0-18ug/mL) in example 5 of the present disclosure.

Detailed Description

It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments according to the present disclosure. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.

In view of the disadvantage of complicated mode of co-delivery of two drugs by using additional vector, the present disclosure provides an ICD inducer-IDO inhibitor conjugate, and a preparation method and application thereof.

In one exemplary embodiment of the present disclosure, an ICD inducer-IDO inhibitor conjugate is provided having the structural formula:

In another embodiment of the disclosure, a method for preparing an ICD inducer-IDO inhibitor conjugate is provided, in which NLG919 and succinic anhydride are subjected to esterification reaction to obtain NLG919-SA, and NLG919-SA and Dox are subjected to amidation reaction to obtain the ICD inducer-IDO inhibitor conjugate;

the structural formula of NLG919-SA is

The synthetic route is as follows:

in one or more embodiments of this embodiment, the catalyst for the esterification reaction is 4-Dimethylaminopyridine (DMAP) and N, N-Diisopropylethylamine (DIPEA).

In this series of examples, the temperature of the esterification reaction was room temperature. The room temperature refers to indoor ambient temperature, and is generally 15-30 ℃.

In one or more embodiments of this embodiment, the solvent for the esterification reaction is dichloromethane.

In one or more embodiments of this embodiment, the purification process for NLG919-SA is: adding the mixture liquid after the esterification reaction into a saturated ammonium chloride solution, extracting by adopting dichloromethane, dissolving the extracted organic phase into a mixed solution of ethanol and chloroform, and recrystallizing.

In this series of examples, the extracted organic phase was dried over sodium sulfate and then recrystallized.

In the series of embodiments, the volume ratio of ethanol to chloroform is 1: 3.8-4.2.

In this series of examples, the temperature of recrystallization was-22 to-18 ℃.

In one or more embodiments of this embodiment, the catalyst for the amidation reaction is N, N-diisopropylethylamine and HBTU.

In one or more embodiments of this embodiment, the amidation reaction time is 20 to 28 hours.

In one or more embodiments of this embodiment, the amidation reaction is terminated after a set time by adding hydrochloric acid.

In one or more embodiments of this embodiment, the amidation reaction mass is washed with water and then purified using silica gel column chromatography.

In the series of examples, the mobile phase of the silica gel column chromatography is a mixed solution of dichloromethane and methanol. When the volume ratio of the dichloromethane to the methanol is 10: 0.9-1.1, the purification effect is better.

In a third embodiment of the present disclosure, there is provided a use of the ICD inducer-IDO inhibitor conjugate described above in the preparation of a medicament for treating tumors.

In a fourth embodiment of the present disclosure, there is provided a use of the ICD inducer-IDO inhibitor conjugate described above in a single drug delivery system.

In order to make the technical solutions of the present disclosure more clearly understood by those skilled in the art, the technical solutions of the present disclosure will be described in detail below with reference to specific embodiments.

Example 1

(1) Synthesis of NLG 919-SA:

a clean dry 25mL ground round bottom flask was taken, accurately weighed out a certain amount of 0.048g NLG919 and 0.00083g DMAP, dissolved in 3mL dichloromethane, added with a certain amount of 0.019g succinic anhydride and 33 μ LDIPEA under stirring, and then stirred under sealed conditions at room temperature overnight. The reaction mixture was poured into saturated 3mL ammonium chloride solution, mixed well, extracted 3 times with dichloromethane and the combined organic layers were dried over sodium sulfate and the crude product was dissolved in ethanol: in an organic reagent of chloroform-1: 4, crystals are obtained at the temperature of-20 ℃, white crystal solids are obtained, namely 0.061g NLG919-SA (yield is 95%), and the structural characterization is shown in figures 1-2.

(2) Synthesis of Dox-NLG919 conjugate:

precisely weighing a clean and dry 25mL ground round bottom flask, precisely weighing a certain amount of 0.0217g NLG919-SA and 0.0324 g HBTU, precisely weighing a certain amount of 14.25 mu L DIPEA, dissolving in anhydrous 9mLDMF, and reacting for 10min under the stirring condition; a predetermined amount of 0.0393g of Dox & HCl was precisely weighed and added to the above reaction solution, followed by reaction for 24 hours under stirring. Then, adding a proper amount of 0.1N HCl to terminate the reaction, removing an aqueous phase layer in the reaction solution, washing an organic phase layer for 2 times, and drying the organic phase layer overnight by using anhydrous sodium sulfate; putting the dried product into a silica gel column, loading the dried product by a dry method, and adding dichloromethane in the ratio: eluting with methanol 10:1 mobile phase, collecting the product by dot-plate observation, combining the eluted products, and evaporating to dryness under reduced pressure to obtain 0.0417g of Dox-NLG919 (yield 81%) conjugate, and structural characterization is shown in FIGS. 3-4.

Example 2

Synthesis of NLG 919-cis-aconitic anhydride (CA)

A clean, dry 25mL ground round bottom flask was taken, accurately weighed out amounts of 0.048g NLG919, 0.00083g DMAP, dissolved in 3mL dichloromethane, and with stirring added amounts of 0.030g CA and 33. mu.L DIPEA, and then stirred at room temperature overnight under sealed conditions. Pouring the reaction mixture into a saturated 3mL ammonium chloride solution, mixing uniformly, extracting with dichloromethane for 3 times, drying the combined organic phase layer with sodium sulfate, placing the dried product into a silica gel column, loading by a dry method, and mixing with ethanol: eluting with chloroform-1: 4 mobile phase, observing on spot plate to collect product, combining eluted products and evaporating under reduced pressure. The resulting product was identified by mass spectrometry and structurally characterized as shown in figure 5, without the molecular ion peak 438.17 of the target product.

Example 3

A clean and dry 25mL round-bottom flask with a ground opening is taken, a certain amount of 116.0mg of Dox & HCl and 22.0 mg of SA are precisely weighed and dissolved in 10.0mL of anhydrous DMF, 33.5 mu L of triethylamine is added, and the mixture is reacted for 24 hours in a dark place under the protection of nitrogen. The reaction mixture was then mixed with 100.0mL of cold ethyl acetate and washed with cold acidic saturated sodium chloride (pH 2-3). The organic layer was collected and dried with sodium sulfate, the dried product was placed on a silica gel column and loaded by dry method with chloroform: methanol: acetic acid 17: 3: 1, and observing a spot plate to collect the product, and combining the eluted products and performing reduced pressure evaporation treatment. After that, the obtained product was reacted with NLG919, 28.2mg of NLG919 was dissolved in 3mL of anhydrous DCM, and then 64.4mg of Dox-SA, 57.3mg of EDC. HCl and 1.2mg of DMAP were added to the above solution, and reacted for 72 hours under a condition of keeping nitrogen away from light at room temperature. Drying under reduced pressure to remove solvent DCM, placing the dried product in a silica gel column, loading by a dry method, and performing dry-method loading on the dried product by using ethanol: eluting with chloroform-1: 4 mobile phase, observing on spot plate to collect product, combining eluted products and evaporating under reduced pressure. The obtained product is identified by mass spectrometry, the structure is characterized as shown in figure 6, and the molecular ion peak 908.3561 of the target product is absent.

Example 4

Dox-NLG919 conjugate inhibits growth of breast tumors in vivo

To assess the efficacy of the Dox-NLG919 conjugate in inhibiting tumor growth in vivo, an animal model of breast cancer was established. 4T1 cells were injected under the fourth breast pad of BALB/C mice, 4T1 cells spontaneously developed tumors in BALB/C mice, and the characteristics of these tumors were very similar to breast cancer in humans. BALB/c mice injected with 4T1 cells were treated twice weekly with Dox, NLG919, Dox-NLG919 conjugate, and PBS control. Although the tumor size of the mice in the PBS control group increased significantly on day seven, tumor growth was inhibited in the mice treated with Dox and NLG919 and significantly eliminated in the mice treated with Dox-NLG919 conjugate throughout the treatment (fig. 7). And tumors in mice treated with Dox, NLG919, Dox-NLG919 conjugate and with PBS control were quantitatively detected by fluorescence (fig. 8), with the least fluorescence (fig. 9) and tumor volume (fig. 10, fig. 11) in mice treated with Dox-NLG919 conjugate. These results demonstrate the strong efficacy and efficacy of Dox-NLG919 conjugates in treating breast tumors in vivo.

The mean weight of mice treated with the Dox, NLG919, Dox-NLG919 conjugates was not significantly reduced (less than 15% loss) compared to those from the PBS-treated control group (fig. 12), indicating that the Dox-NLG919 conjugate is well tolerated in vivo.

Experimental example 5

In vitro antitumor Activity assay of Dox-NLG919 conjugates

4T1 cells were seeded in 96-well plates (8000cells/wel1) at a volume of 100. mu.L per well, and after seeding, the 96-well plates were incubated overnight at 37 ℃ in an incubator with 5% carbon dioxide by volume, and 100. mu.L of Dox, NLG919, Dox-NLG919 conjugate (concentration range 0-18. mu.g/mL) was added. Culturing in an incubator containing 5% carbon dioxide at 37 deg.C for 48h, adding 10 μ L of CCK-8 solution into each well, culturing in an incubator containing 5% carbon dioxide at 37 deg.C for 2h, measuring absorbance at 450nm with enzyme linked immunosorbent assay, and calculating cell survival rate. As shown in FIG. 13, the assay results showed that the Dox, NLG919 and Dox-NLG919 conjugates all inhibited proliferation of 4T1 cells well, but the Dox-NLG919 conjugate inhibited 4T1 cells more strongly than free Dox and NLG 919.

The above description is only a preferred embodiment of the present disclosure and is not intended to limit the present disclosure, and various modifications and changes may be made to the present disclosure by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present disclosure should be included in the protection scope of the present disclosure.

Claims

1. An ICD inducer-IDO inhibitor conjugate having the structural formula:

2. a preparation method of an ICD inducer-IDO inhibitor conjugate is characterized in that NLG919 and succinic anhydride are subjected to esterification reaction to obtain NLG919-SA, and NLG919-SA and Dox are subjected to amidation reaction to obtain the ICD inducer-IDO inhibitor conjugate;

the structural formula of NLG919-SA is

3. The process for preparing an ICD inducer-IDO inhibitor conjugate of claim 2 wherein the catalyst for the esterification reaction is 4-dimethylaminopyridine and N, N-diisopropylethylamine;

preferably, the temperature of the esterification reaction is room temperature.

4. The process for preparing an ICD inducer-IDO inhibitor conjugate of claim 2 wherein the solvent of the esterification reaction is dichloromethane.

5. The process for the preparation of ICD inducer-IDO inhibitor conjugate of claim 2 wherein the purification process of NLG919-SA is: adding the mixture liquid after the esterification reaction into a saturated ammonium chloride solution, extracting by adopting dichloromethane, dissolving the extracted organic phase into a mixed solution of ethanol and chloroform, and recrystallizing;

preferably, the extracted organic phase is dried by sodium sulfate and then recrystallized;

preferably, the volume ratio of the ethanol to the chloroform is 1: 3.8-4.2;

preferably, the temperature of recrystallization is from-22 to-18 ℃.

6. The process for preparing an ICD inducer-IDO inhibitor conjugate of claim 2 wherein the catalyst for the amidation reaction is N, N-diisopropylethylamine and HBTU.

7. The process for preparing an ICD inducer-IDO inhibitor conjugate according to claim 2, wherein the reaction is terminated by adding hydrochloric acid after a set time of amidation reaction.

8. The process for preparing an ICD inducer-IDO inhibitor conjugate according to claim 2, wherein the material after the amidation reaction is washed with water and then purified by silica gel column chromatography;

preferably, the mobile phase of the silica gel column chromatography is a mixed solution of dichloromethane and methanol; more preferably, the volume ratio of the dichloromethane to the methanol is 10: 0.9-1.1.

9. Use of the ICD inducer-IDO inhibitor conjugate of claim 1 for the preparation of a medicament for the treatment of tumors.

10. Use of the ICD inducer-IDO inhibitor conjugate of claim 1 in a single drug delivery system.