WO2021120898A1 - 一种icd诱导剂-ido抑制剂缀合物及制备方法与应用 - Google Patents

一种icd诱导剂-ido抑制剂缀合物及制备方法与应用 Download PDF

Info

Publication number
WO2021120898A1
WO2021120898A1 PCT/CN2020/125929 CN2020125929W WO2021120898A1 WO 2021120898 A1 WO2021120898 A1 WO 2021120898A1 CN 2020125929 W CN2020125929 W CN 2020125929W WO 2021120898 A1 WO2021120898 A1 WO 2021120898A1
Authority
WO
WIPO (PCT)
Prior art keywords
nlg919
ido inhibitor
inhibitor conjugate
icd inducer
dox
Prior art date
Application number
PCT/CN2020/125929
Other languages
English (en)
French (fr)
Inventor
姜新义
陈晨
张蕊
杜微
张晶
唐春伟
Original Assignee
山东大学
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 山东大学 filed Critical 山东大学
Publication of WO2021120898A1 publication Critical patent/WO2021120898A1/zh

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • the present disclosure relates to an ICD inducer-IDO inhibitor conjugate, and a preparation method and application.
  • the immune escape of tumors contributes to the occurrence and development of tumors.
  • the mechanisms of tumor immune escape include changes in the tumor itself, changes in the tumor-induced microenvironment, and the tumor microenvironment promotes tumor development.
  • immunotherapy targeting the immune escape link has achieved remarkable success in tumor treatment. Immunotherapy involves multiple factors and links, and is related to the changes of tumor cells and the tumor microenvironment with complex mechanisms. At present, it is still facing greater challenges in clinical practice.
  • TAA tumor-associated antigen
  • tumor cells often suppress T cells through a series of changes (such as loss of antigen expression, endocytic antigen or antigen shedding) The activation of the body, thus avoiding the surveillance of the body's immune system and opportunistic growth.
  • tumor cells can undergo "antigenic drift” like viruses, leading to epitope mutations that change the antigenicity of tumors, and then evade T cell-mediated attacks.
  • tumor cells transform from non-immunogenic cells to immunogenic cells while undergoing apoptosis, and thereby stimulate the body's anti-tumor immune killing effect.
  • the phenomenon is called immunogenic death of tumor cells.
  • certain anthracycline compounds and chemotherapeutic drugs such as oxaliplatin not only induce tumor cell apoptosis, but can also cause immunogenic cell death (ICD).
  • calreticulin Exposure to the cell surface stimulates the phagocytosis of dendritic cells (DC); adenosine triphosphate is released to recruit DCs into tumor foci; the high migration rate group protein B1 promotes the formation of stable binding between DCs and dying tumor cells, and induces the body to produce specific T cells for anti-tumor immunity.
  • DC dendritic cells
  • adenosine triphosphate is released to recruit DCs into tumor foci
  • the high migration rate group protein B1 promotes the formation of stable binding between DCs and dying tumor cells, and induces the body to produce specific T cells for anti-tumor immunity.
  • IDO Indoleamine 2,3-dioxygenase
  • Trp tryptophan
  • Kyn kynurenine pathway
  • IDO is closely related to tumor immune escape. It can mediate tumor immune escape through a variety of mechanisms: tryptophan depletion inhibits local T cell proliferation, tryptophan metabolites promote T cell apoptosis, and induce regulatory T cell proliferation.
  • tryptophan depletion inhibits local T cell proliferation
  • tryptophan metabolites promote T cell apoptosis
  • induce regulatory T cell proliferation In view of the important role that IDO plays in the formation and maintenance of tumor immune tolerance, it has become a new target for anti-tumor immunotherapy.
  • the combination medication can achieve the synergy between the ICD induction effect of chemotherapeutics and the immune-related metabolic regulation effect of IDO inhibitors, so as to achieve better tumor treatment effects.
  • the current research on the combination of ICD inducers and IDO inhibitors mostly uses additional carriers for co-delivery of the two drugs.
  • this additional carrier performs co-delivery.
  • the delivery method is more cumbersome.
  • the purpose of the present disclosure is to provide an ICD inducer-IDO inhibitor conjugate and a preparation method and application.
  • the conjugate is used to deliver the ICD inducer and IDO inhibitor at the same time. Convenience.
  • the present disclosure provides an ICD inducer-IDO inhibitor conjugate, the structural formula of which is as follows:
  • the present disclosure uses succinic acid, a reactant in the tricarboxylic acid cycle in the body, that is, the two carboxyl groups of succinic acid as a bridge that can produce a cleavable link with Dox and NLG919, so that the two drugs form a Dox-NLG919 conjugate .
  • succinic acid a reactant in the tricarboxylic acid cycle in the body
  • the two carboxyl groups of succinic acid as a bridge that can produce a cleavable link with Dox and NLG919, so that the two drugs form a Dox-NLG919 conjugate .
  • the present disclosure provides a method for preparing an ICD inducer-IDO inhibitor conjugate.
  • NLG919 and succinic anhydride are esterified to obtain NLG919-SA, and NLG919-SA and Dox are amidated to obtain ICD Inducer-IDO inhibitor conjugate;
  • the present disclosure proves through experiments that using succinic acid can prepare an ICD inducer-IDO inhibitor conjugate. Secondly, the reaction sequence of succinic anhydride with NLG919 and Dox also affects the successful synthesis of the ICD inducer-IDO inhibitor conjugate. The present study found that NLG919 was synthesized with succinic anhydride and then reacted with Dox to successfully prepare ICD inducer-IDO inhibitor conjugate, and if the order is reversed, ICD inducer-IDO inhibitor conjugate cannot be obtained.
  • the present disclosure provides an application of the above-mentioned ICD inducer-IDO inhibitor conjugate in the preparation of tumor treatment drugs.
  • the present disclosure provides an application of the above-mentioned ICD inducer-IDO inhibitor conjugate in a single-drug delivery system.
  • the present disclosure provides an immunogenic cell death (ICD) inducer-tryptophan metabolism blocker conjugate that can achieve similar single-drug delivery during tumor treatment.
  • the conjugate includes ICD, which has a strong effect on T cell immune response, induces chemotherapeutics, and blocks the metabolism of tryptophan to kynurenine indoleamine 2,3 dioxygenase (IDO) inhibitor.
  • ICD immunogenic cell death
  • the bridging substance of the conjugate provided in the present disclosure is succinic acid.
  • Succinic acid connects the ICD inducer and the tryptophan metabolism blocker together through an ester bond and an amide bond.
  • the ester bond and the amide bond are easily accessible. It is hydrolyzed by esterases and amidases that are abundant in tumors, thereby releasing ICD inducers and tryptophan metabolism blockers.
  • FIG. 1 is a proton nuclear magnetic resonance spectrum ( 1 H NMR) chart of NLG919-SA prepared in Example 1 of the disclosure.
  • Example 2 is an electrospray mass spectrometry (ESI-MS) diagram of NLG919-SA prepared in Example 1 of the disclosure.
  • ESI-MS electrospray mass spectrometry
  • Example 5 is an electrospray mass spectrometry (ESI-MS) diagram of the product prepared in Example 2 of the disclosure.
  • Example 6 is an electrospray mass spectrometry (ESI-MS) diagram of the product prepared in Example 3 of the disclosure.
  • Figure 7 is a comparison of the Dox-NLG919 conjugate inhibiting the growth of breast tumors in vivo in Example 4 of the disclosure, the treatment by the Dox, NLG919, and Dox-NLG919 conjugates, and the comparison with the control treatment with PBS.
  • FIG. 8 is a graph of fluorescence quantitative detection of tumors in mice treated with Dox, NLG919, and Dox-NLG919 conjugates and treated with PBS control in Example 4 of the disclosure.
  • Figure 9 shows the fluorescence quantitative detection of Dox, NLG919, Dox-NLG919 conjugate treatment and the in vivo fluorescence intensity of mice treated with PBS control in Example 4 of the disclosure.
  • Figure 10 shows the quantitative detection of the tumor volume in mice treated with Dox, NLG919, and Dox-NLG919 conjugates and PBS control in Example 4 of the disclosure.
  • Figure 11 shows the tumor weights reduced by treatment with Dox, NLG919, and Dox-NLG919 conjugates compared with control treatment with PBS in Example 4 of the disclosure.
  • Figure 12 shows the average weight of mice during treatment with Dox, NLG919, and Dox-NLG919 conjugates in comparison with the control treatment with PBS in Example 4 of the disclosure.
  • Figure 13 shows the survival rate of 4T1 cells in Example 5 of the present disclosure after treatment with different concentrations of Dox, NLG919, and Dox-NLG919 conjugates (concentrations ranging from 0-18ug/mL).
  • the present disclosure proposes an ICD inducer-IDO inhibitor conjugate, as well as a preparation method and application.
  • a typical embodiment of the present disclosure provides an ICD inducer-IDO inhibitor conjugate, the structural formula of which is as follows:
  • the present disclosure uses succinic acid, a reactant in the tricarboxylic acid cycle in the body, that is, the two carboxyl groups of succinic acid as a bridge that can produce a cleavable link with Dox and NLG919, so that the two drugs form a Dox-NLG919 conjugate .
  • succinic acid a reactant in the tricarboxylic acid cycle in the body
  • the two carboxyl groups of succinic acid as a bridge that can produce a cleavable link with Dox and NLG919, so that the two drugs form a Dox-NLG919 conjugate .
  • Another embodiment of the present disclosure provides a method for preparing an ICD inducer-IDO inhibitor conjugate, wherein NLG919 is esterified with succinic anhydride to obtain NLG919-SA, and NLG919-SA is amidated with Dox The reaction obtains the ICD inducer-IDO inhibitor conjugate;
  • the present disclosure proves through experiments that using succinic acid can prepare an ICD inducer-IDO inhibitor conjugate. Secondly, the reaction sequence of succinic anhydride with NLG919 and Dox also affects the successful synthesis of the ICD inducer-IDO inhibitor conjugate. The present study found that NLG919 was synthesized with succinic anhydride and then reacted with Dox to successfully prepare ICD inducer-IDO inhibitor conjugate, and if the order is reversed, ICD inducer-IDO inhibitor conjugate cannot be obtained.
  • the synthetic route is as follows:
  • the catalyst for the esterification reaction is 4-dimethylaminopyridine (DMAP) and N,N-diisopropylethylamine (DIPEA).
  • the temperature of the esterification reaction is room temperature.
  • the room temperature mentioned in the present disclosure refers to the ambient temperature in the room, which is generally 15-30°C.
  • the solvent for the esterification reaction is dichloromethane.
  • the purification process of NLG919-SA is: adding the mixture after the esterification reaction to a saturated ammonium chloride solution, extracting with dichloromethane, and extracting the organic phase Dissolve in a mixed solution of ethanol and chloroform for recrystallization.
  • the extracted organic phase is dried with sodium sulfate and then recrystallized.
  • the volume ratio of ethanol to chloroform is 1:3.8-4.2.
  • the recrystallization temperature is -22 to -18°C.
  • the catalyst for the amidation reaction is N,N-diisopropylethylamine and HBTU.
  • the amidation reaction time is 20-28 hours.
  • hydrochloric acid is added to terminate the reaction.
  • the material after the amidation reaction is washed with water, and then purified by silica gel column chromatography.
  • the mobile phase of silica gel column chromatography is a mixture of dichloromethane and methanol.
  • the volume ratio of dichloromethane to methanol is 10:0.9 ⁇ 1.1, the purification effect is better.
  • the third embodiment of the present disclosure provides an application of the above-mentioned ICD inducer-IDO inhibitor conjugate in the preparation of tumor treatment drugs.
  • the fourth embodiment of the present disclosure provides an application of the above-mentioned ICD inducer-IDO inhibitor conjugate in a single-drug delivery system.
  • Dox-NLG919 conjugate inhibits the growth of breast tumors in vivo
  • the 4T1 cells were seeded on a 96-well plate (8000cells/wel1) with a volume of 100 ⁇ L per well. After seeding, the 96-well plate was cultured overnight at 37°C in a 5% carbon dioxide incubator, and 100 ⁇ L of Dox, NLG919, Dox-NLG919 conjugate (concentration range of 0-18 ⁇ g/mL). After culturing in a 37°C, 5% carbon dioxide incubator for 48 hours, add 10 ⁇ L of CCK-8 solution to each well, incubate in a 37°C, 5% carbon dioxide incubator for 2 hours, and measure at 450nm with an enzyme-linked immunosorbent assay. The absorbance value is used to calculate the cell survival rate. The results are shown in Figure 13.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Chemical & Material Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicinal Preparation (AREA)

Abstract

提供了一种ICD诱导剂-IDO抑制剂缀合物及制备方法与应用，缀合物结构式为（I）其制备方法为：NLG919与丁二酸酐进行酯化反应获得NLG919-SA，NLG919-SA与Dox进行酰胺化反应获得ICD诱导剂-IDO抑制剂缀合物。采用缀合物的方式同时递送ICD诱导剂和IDO抑制剂，更为方便。

Description

一种ICD诱导剂-IDO抑制剂缀合物及制备方法与应用 技术领域
本公开涉及一种ICD诱导剂-IDO抑制剂缀合物及制备方法与应用。
背景技术
这里的陈述仅提供与本公开有关的背景信息，而不必然构成现有技术。
肿瘤的免疫逃逸有助于肿瘤的发生发展，其中，肿瘤免疫逃逸的机制包括肿瘤自身的改变、肿瘤诱导微环境的改变以及肿瘤微环境促进肿瘤的发展。近年来，针对免疫逃逸环节的免疫疗法于肿瘤治疗中取得了显著的成功。免疫疗法涉及多个因素和环节，与肿瘤细胞自身和肿瘤微环境的改变均有机制复杂的联系，目前在临床实践过程中仍面临着较大的挑战。
肿瘤相关抗原(tumor associated antigen,TAA)的产生是激活T细胞的一个重要因素，然而，肿瘤细胞常常通过一系列改变(如抗原表达的丢失、内吞抗原或抗原脱落等方式)来抑制T细胞的活化，从而躲避机体免疫系统的监视并伺机生长。另外，肿瘤细胞可像病毒一样发生“抗原漂移”，导致抗原表位突变而改变肿瘤的抗原性，继而逃避T细胞介导的攻击。
近来研究发现，用特定化学药物或放射线治疗肿瘤时，肿瘤细胞在发生凋亡的同时，从非免疫原性细胞转化为免疫原性细胞，并由此激发机体内抗肿瘤的免疫杀伤效应，此现象被称之为肿瘤细胞的免疫原性死亡。研究发现某些蒽环类化合物和奥沙利铂等化疗药物不仅诱导肿瘤细胞凋亡，而且可以引起免疫原性细胞死亡(ICD)，通过诱导肿瘤细胞自噬，释放3类信号：钙网蛋白暴露在细胞表面，刺激树突状细胞(DC)吞噬；三磷酸腺苷释放，招募DC进入肿瘤灶；高迁徙率族蛋白B1促进DC与垂死肿瘤细胞形成稳定结合，诱导机体产生特异性的T细胞抗肿瘤免疫。
吲哚胺2,3-双加氧酶(indoleamine 2,3-dioxygenase，IDO)是肝脏外唯一催化色氨酸(Trp)沿犬尿氨酸(Kyn)途径分解代谢的限速酶。IDO与肿瘤免疫逃逸密切相关，可通过多种机制介导肿瘤免疫逃逸:色氨酸耗竭抑制局部T细胞增殖，色氨酸代谢产物促进T细胞凋亡，诱导调节性T细胞增殖等。鉴于IDO在肿瘤免疫耐受的形成和维持中所发挥的重要作用，其已经成为抗肿瘤免疫治疗的新靶点。然而，临床前研究结果提示，IDO抑制剂单用的肿瘤杀伤活性较弱，抑制率在30％～50％，为提高IDO抑制剂的临床治疗效果，将其与化疗药物联合使用是一种重要的策略。
联合用药可实现化疗药的ICD诱导作用与IDO抑制剂的免疫相关代谢调节作用的协同，从而达到更佳的肿瘤治疗效果。据本公开发明人所知，目前对于ICD诱导剂和IDO抑制剂的联合用药的研究多为利用附加载体进行两药的共递送，然而，经本公开发明人研究发现，这种附加载体进行共递送的方式较为繁琐。
发明内容
为了解决现有技术的不足，本公开的目的是提供一种ICD诱导剂-IDO抑制剂缀合物及制备方法与应用，采用缀合物的方式同时递送ICD诱导剂和IDO抑制剂，更为方便。
为了实现上述目的，本公开的技术方案为：
第一方面，本公开提供了一种ICD诱导剂-IDO抑制剂缀合物，其结构式如下：
Figure PCTCN2020125929-appb-000001
本公开利用体内三羧酸循环中的反应物琥珀酸，即丁二酸的两个羧基作为能与Dox及NLG919产生易裂解连接部分的桥联物，从而使得两药形成Dox-NLG919缀合物。据本公开发明人基于目前的研究发现，采用丁二酸作为桥联物时，能够获得Dox和NLG919缀合物，而采用其他二酸或多酸，难以获得Dox和NLG919缀合物。
第二方面，本公开提供了一种ICD诱导剂-IDO抑制剂缀合物的制备方法，将NLG919与丁二酸酐进行酯化反应获得NLG919-SA，NLG919-SA与Dox进行酰胺化反应获得ICD诱导剂-IDO抑制剂缀合物；
NLG919-SA的结构式为
Figure PCTCN2020125929-appb-000002
ICD诱导剂-IDO抑制剂缀合物的结构式为
Figure PCTCN2020125929-appb-000003
本公开通过实验证实，采用丁二酸能够制备ICD诱导剂-IDO抑制剂缀合物。其次，丁二 酸酐与NLG919、Dox的反应顺序也影响ICD诱导剂-IDO抑制剂缀合物的成功合成，本公开研究发现，先将NLG919与丁二酸酐合成，然后再与Dox反应可以成功制备ICD诱导剂-IDO抑制剂缀合物，而若顺序调换，则无法获得ICD诱导剂-IDO抑制剂缀合物。
第三方面，本公开提供了一种上述ICD诱导剂-IDO抑制剂缀合物在制备肿瘤治疗药物中的应用。
第四方面，本公开提供了一种上述ICD诱导剂-IDO抑制剂缀合物在单药递送系统中的应用。
本公开的有益效果为：
1.本公开提供了一种用于瘤治疗过程中可实现类似单药递送的免疫原性细胞死亡(ICD)诱导剂-色氨酸代谢阻断剂缀合物，该缀合物包括能够引起强烈效应T细胞免疫反应的ICD诱导化疗药、阻断色氨酸代谢为犬尿氨酸的吲哚胺2,3双加氧酶(IDO)抑制剂。
2.本公开提供的缀合物的桥连物为丁二酸，丁二酸通过酯键和酰胺键将ICD诱导剂和色氨酸代谢阻断剂连接在一起，同时酯键和酰胺键易被瘤内大量存在的酯酶与酰胺酶水解，从而释放ICD诱导剂和色氨酸代谢阻断剂。
3.本公开首次合成了一种从属不同作用机制并可协同抗癌的Dox-NLG919缀合物，为协同递药带来了极大的便利。
附图说明
构成本公开的一部分的说明书附图用来提供对本公开的进一步理解，本公开的示意性实施例及其说明用于解释本公开，并不构成对本公开的不当限定。
图1为本公开实施例1制备的NLG919-SA的核磁共振氢谱( 1H NMR)图。
图2为本公开实施例1制备的NLG919-SA的电喷雾质谱(ESI-MS)图。
图3为本公开实施例1制备的Dox-NLG919缀合物的核磁共振氢谱( 1H NMR)图。
图4为本公开实施例1制备的Dox-NLG919缀合物的电喷雾质谱(ESI-MS)图。
图5为本公开实施例2制备产物的电喷雾质谱(ESI-MS)图。
图6为本公开实施例3制备产物的电喷雾质谱(ESI-MS)图。
图7为本公开实施例4中Dox-NLG919缀合物抑制体内乳腺肿瘤生长，通过Dox、NLG919、Dox-NLG919缀合物治疗，以及用PBS对照治疗的比较。
图8为本公开实例4中通过Dox、NLG919、Dox-NLG919缀合物治疗，以及用PBS对照治疗的小鼠体内肿瘤的荧光定量检测图。
图9为本公开实例4中通过荧光定量检测Dox、NLG919、Dox-NLG919缀合物治疗，以及用PBS对照治疗的小鼠体内荧光强度。
图10为本公开实例4中通过荧光定量检测Dox、NLG919、Dox-NLG919缀合物治疗，以及用PBS对照治疗的小鼠体内肿瘤体积的大小。
图11为本公开实例4中通过用Dox、NLG919、Dox-NLG919缀合物治疗与用PBS对照治疗比较减少的肿瘤重量。
图12为本公开实例4中与用PBS对照治疗比较，用Dox、NLG919、Dox-NLG919缀合物治疗期间小鼠的平均重量。
图13为本公开实例5中4T1细胞通过用不同浓度的Dox、NLG919、Dox-NLG919缀合物(浓度范围为0-18ug/mL)，治疗后的存活率。
具体实施方式
应该指出，以下详细说明都是示例性的，旨在对本公开提供进一步的说明。除非另有指明，本文使用的所有技术和科学术语具有与本公开所属技术领域的普通技术人员通常理解的相同含义。
需要注意的是，这里所使用的术语仅是为了描述具体实施方式，而非意图限制根据本公开的示例性实施方式。如在这里所使用的，除非上下文另外明确指出，否则单数形式也意图包括复数形式，此外，还应当理解的是，当在本说明书中使用术语“包含”和/或“包括”时，其指明存在特征、步骤、操作、器件、组件和/或它们的组合。
鉴于利用附加载体进行两药共递送的方式较为繁琐的不足，本公开提出了一种ICD诱导剂-IDO抑制剂缀合物及制备方法与应用。
本公开的一种典型实施方式，提供了一种ICD诱导剂-IDO抑制剂缀合物，其结构式如下：
Figure PCTCN2020125929-appb-000004
本公开利用体内三羧酸循环中的反应物琥珀酸，即丁二酸的两个羧基作为能与Dox及NLG919产生易裂解连接部分的桥联物，从而使得两药形成Dox-NLG919缀合物。据本公开发明人基于目前的研究发现，采用丁二酸作为桥联物时，能够获得Dox和NLG919缀合物，而采用其他二酸或多酸，难以获得Dox和NLG919缀合物。
本公开的另一种实施方式，提供了一种ICD诱导剂-IDO抑制剂缀合物的制备方法，将NLG919与丁二酸酐进行酯化反应获得NLG919-SA，NLG919-SA与Dox进行酰胺化反应获得ICD诱导剂-IDO抑制剂缀合物；
NLG919-SA的结构式为
Figure PCTCN2020125929-appb-000005
ICD诱导剂-IDO抑制剂缀合物的结构式为
Figure PCTCN2020125929-appb-000006
本公开通过实验证实，采用丁二酸能够制备ICD诱导剂-IDO抑制剂缀合物。其次，丁二酸酐与NLG919、Dox的反应顺序也影响ICD诱导剂-IDO抑制剂缀合物的成功合成，本公开研究发现，先将NLG919与丁二酸酐合成，然后再与Dox反应可以成功制备ICD诱导剂-IDO抑制剂缀合物，而若顺序调换，则无法获得ICD诱导剂-IDO抑制剂缀合物。
其合成路线如下所示：
Figure PCTCN2020125929-appb-000007
该实施方式的一种或多种实施例中，酯化反应的催化剂为4-二甲氨基吡啶(DMAP)和N,N-二异丙基乙胺(DIPEA)。
该系列实施例中，酯化反应的温度为室温。本公开所述室温是指室内的环境温度，一般为15～30℃。
该实施方式的一种或多种实施例中，酯化反应的溶剂为二氯甲烷。
该实施方式的一种或多种实施例中，NLG919-SA的纯化过程为：将酯化反应后的混合物液加入至饱和氯化铵溶液中，采用二氯甲烷进行萃取，将萃取的有机相溶于乙醇与氯仿的混合溶液中进行重结晶。
该系列实施例中，萃取后的有机相采用硫酸钠干燥后进行重结晶。
该系列实施例中，乙醇与氯仿的体积比为1:3.8～4.2。
该系列实施例中，重结晶的温度为-22～-18℃。
该实施方式的一种或多种实施例中，酰胺化反应的催化剂为N,N-二异丙基乙胺和HBTU。
该实施方式的一种或多种实施例中，酰胺化反应时间为20～28h。
该实施方式的一种或多种实施例中，酰胺化反应设定时间后，添加盐酸终止反应。
该实施方式的一种或多种实施例中，酰胺化反应后的物料进行水洗，然后采用硅胶柱层析进行纯化。
该系列实施例中，硅胶柱层析的流动相为二氯甲烷与甲醇的混合液。当二氯甲烷与甲醇的体积比为10:0.9～1.1时，纯化效果更好。
本公开的第三种实施方式，提供了一种上述ICD诱导剂-IDO抑制剂缀合物在制备肿瘤治疗药物中的应用。
本公开的第四种实施方式，提供了一种上述ICD诱导剂-IDO抑制剂缀合物在单药递送系统中的应用。
为了使得本领域技术人员能够更加清楚地了解本公开的技术方案，以下将结合具体的实施例详细说明本公开的技术方案。
实施例1
(1)NLG919-SA的合成：
取一个洁净干燥的25mL磨口圆底烧瓶，精密称定一定量的0.048g NLG919、0.00083g DMAP，溶于3mL二氯甲烷中，搅拌条件下加入一定量的0.019g丁二酸酐和33μL DIPEA，然后于室温下密封搅拌过夜。反应混合液倒入至饱和的3mL氯化铵溶液中，混匀后，用二氯甲烷萃取3次，并将合并的有机相层用硫酸钠干燥，粗产物溶于乙醇：氯仿＝1:4的有机试剂中，于-20℃的条件下得结晶物，白色结晶固体即为所制得的0.061g NLG919-SA(产量为95％)，结构表征如图1～2所示。
(2)Dox-NLG919缀合物的合成：
取一个洁净干燥的25mL磨口圆底烧瓶，精密称定一定量的0.0217g NLG919-SA、0.0324g HBTU，精密量取一定量的14.25μL DIPEA，溶于无水9mLDMF中，搅拌条件下反应10min；另精密称定一定量的0.0393g Dox·HCl，加入至上述反应液中，搅拌条件下反应24h。然后，加入适量0.1N HCl以终止反应，去除反应液中的水相层，有机相层被水洗2次后，用无水硫酸钠干燥过夜；将干燥产物置于硅胶柱内，干法上样，用配比为二氯甲烷：甲醇＝10:1的流动相洗脱，点板观察以收集产物，合并洗脱下来的产物并做减压蒸干处理，最终得到所制0.0417g Dox-NLG919(产率为81％)缀合物，结构表征如图3～4所示。
实施例2
NLG919-顺-乌头酸酐(CA)的合成
取一个洁净干燥的25mL磨口圆底烧瓶，精密称定一定量的0.048g NLG919、0.00083g DMAP，溶于3mL二氯甲烷中，搅拌条件下加入一定量的0.030g CA和33μL DIPEA，然后于室温下密封搅拌过夜。反应混合液倒入至饱和的3mL氯化铵溶液中混匀，用二氯甲烷萃取3次，并将合并的有机相层用硫酸钠干燥，将干燥产物置于硅胶柱内，干法上样，用配比为乙醇：氯仿＝1:4的流动相洗脱，点板观察以收集产物，合并洗脱下来的产物并做减压蒸干处理。所得产物经质谱鉴定，结构表征如图5所示，无目标产物的分子离子峰438.17。
实施例3
取一个洁净干燥的25mL磨口圆底烧瓶，精密称定一定量的116.0mg Dox·HCl和22.0mg SA溶解于10.0mL无水DMF中，再加入33.5μL三乙胺，氮气保护下避光反应24h。然后反应混合液与100.0mL冷乙酸乙酯混合，冷酸性饱和氯化钠(pH 2-3)洗涤。收集有机层并用硫酸钠干燥，将干燥产物置于硅胶柱内，干法上样，用配比为氯仿：甲醇：乙酸＝17：3：1的流动相洗脱，点板观察以收集产物，合并洗脱下来的产物并做减压蒸干处理。之后所得产物与NLG919反应，将28.2mg NLG919溶于3mL无水DCM中，然后将64.4mg Dox-SA、57.3mg EDC·HCl、1.2mg DMAP加至上述溶液中，室温氮气避光条件下反应72h。减压干燥除去溶剂DCM，然后将干燥产物置于硅胶柱内，干法上样，用配比为乙醇：氯仿＝1:4的流动相洗脱，点板观察以收集产物，合并洗脱下来的产物并做减压蒸干处理。所得产物经质谱鉴定，结构表征如图6所示，无目标产物的分子离子峰908.3561。
实施例4
Dox-NLG919缀合物体内抑制乳腺肿瘤的生长
为了评估Dox-NLG919缀合物在体内抑制肿瘤生长的效力，建立一个乳腺癌的动物模型。在BALB/C小鼠的第四乳房垫下注射4T1细胞，4T1细胞在BALB/C小鼠体内自发产生肿瘤，且这种肿瘤的特性与人体中的乳腺癌十分相近。将注射过4T1细胞的BALB/c小鼠每周两次使用Dox、NLG919、Dox-NLG919缀合物治疗，以及用PBS对照治疗。虽然在PBS对照组中小鼠的肿瘤大小在第七天显著增加，但在整个治疗过程中，用Dox和NLG919治疗的小鼠中的肿瘤生长被抑制，用Dox-NLG919缀合物治疗的小鼠中的肿瘤生长被显著消除(图7)。并且对使用Dox、NLG919、Dox-NLG919缀合物以及用PBS对照治疗后的小鼠体内的肿瘤进行荧光定量检测(图8)，检测到使用Dox-NLG919缀合物治疗的小鼠体内荧光定量最弱(图9)、肿瘤体积定量最小(图10，图11)。这些结果证明了Dox-NLG919缀合物在体内治疗乳腺肿瘤的强效力和功效。
与来自PBS治疗的对照组的那些相比，使用Dox、NLG919、Dox-NLG919缀合物治疗的 小鼠的平均重量没有显著降低(小于15％的损失)(图12)，表明Dox-NLG919缀合物在体内具有良好的耐受性。
实验例5
Dox-NLG919缀合物的体外抗肿瘤活性试验
将4T1细胞接种于96孔板上(8000cells/wel1)，每孔体积100μL，接种后，将96孔板于37℃、体积分数为5％的二氧化碳的培养箱中培养过夜，加入100μL的Dox、NLG919、Dox-NLG919缀合物(浓度范围为0-18μg/mL)。于37℃、5％的二氧化碳的培养箱中培养48h后，每孔加入10μL CCK-8溶液，于37℃、5％的二氧化碳的培养箱中培养2h，用酶联免疫检测仪于450nm处测其吸光度值，计算细胞存活率。结果如图13所示，从测定结果得Dox、NLG919、Dox-NLG919缀合物对4T1细胞的增值均有较好的抑制作用，但Dox-NLG919缀合物对4T1细胞抑制作用比游离的Dox和NLG919更强。
以上所述仅为本公开的优选实施例而已，并不用于限制本公开，对于本领域的技术人员来说，本公开可以有各种更改和变化。凡在本公开的精神和原则之内，所作的任何修改、等同替换、改进等，均应包含在本公开的保护范围之内。

Claims (10)

  1. 一种ICD诱导剂-IDO抑制剂缀合物，其特征是，结构式如下：
    Figure PCTCN2020125929-appb-100001
  2. 一种ICD诱导剂-IDO抑制剂缀合物的制备方法，其特征是，将NLG919与丁二酸酐进行酯化反应获得NLG919-SA，NLG919-SA与Dox进行酰胺化反应获得ICD诱导剂-IDO抑制剂缀合物；
    NLG919-SA的结构式为
    Figure PCTCN2020125929-appb-100002
    ICD诱导剂-IDO抑制剂缀合物的结构式为
    Figure PCTCN2020125929-appb-100003
  3. 如权利要求2所述的ICD诱导剂-IDO抑制剂缀合物的制备方法，其特征是，酯化反应的催化剂为4-二甲氨基吡啶和N,N-二异丙基乙胺；
    优选的，酯化反应的温度为室温。
  4. 如权利要求2所述的ICD诱导剂-IDO抑制剂缀合物的制备方法，其特征是，酯化反应的溶剂为二氯甲烷。
  5. 如权利要求2所述的ICD诱导剂-IDO抑制剂缀合物的制备方法，其特征是，NLG919-SA的纯化过程为：将酯化反应后的混合物液加入至饱和氯化铵溶液中，采用二氯甲烷进行萃取，将萃取的有机相溶于乙醇与氯仿的混合溶液中进行重结晶；
    优选的，萃取后的有机相采用硫酸钠干燥后进行重结晶；
    优选的，乙醇与氯仿的体积比为1:3.8～4.2；
    优选的，重结晶的温度为-22～-18℃。
  6. 如权利要求2所述的ICD诱导剂-IDO抑制剂缀合物的制备方法，其特征是，酰胺化反应的催化剂为N,N-二异丙基乙胺和HBTU。
  7. 如权利要求2所述的ICD诱导剂-IDO抑制剂缀合物的制备方法，其特征是，酰胺化反应设定时间后，添加盐酸终止反应。
  8. 如权利要求2所述的ICD诱导剂-IDO抑制剂缀合物的制备方法，其特征是，酰胺化反应后的物料进行水洗，然后采用硅胶柱层析进行纯化；
    优选的，硅胶柱层析的流动相为二氯甲烷与甲醇的混合液；进一步优选的，二氯甲烷与甲醇的体积比为10:0.9～1.1。
  9. 一种权利要求1所述的ICD诱导剂-IDO抑制剂缀合物在制备肿瘤治疗药物中的应用。
  10. 一种权利要求1所述的ICD诱导剂-IDO抑制剂缀合物在单药递送系统中的应用。
PCT/CN2020/125929 2019-12-16 2020-11-02 一种icd诱导剂-ido抑制剂缀合物及制备方法与应用 WO2021120898A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201911306005.6A CN111333653A (zh) 2019-12-16 2019-12-16 一种icd诱导剂-ido抑制剂缀合物及制备方法与应用
CN201911306005.6 2019-12-16

Publications (1)

Publication Number Publication Date
WO2021120898A1 true WO2021120898A1 (zh) 2021-06-24

Family

ID=71177725

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2020/125929 WO2021120898A1 (zh) 2019-12-16 2020-11-02 一种icd诱导剂-ido抑制剂缀合物及制备方法与应用

Country Status (2)

Country Link
CN (1) CN111333653A (zh)
WO (1) WO2021120898A1 (zh)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111333653A (zh) * 2019-12-16 2020-06-26 山东大学 一种icd诱导剂-ido抑制剂缀合物及制备方法与应用
CN111437399B (zh) * 2020-05-27 2021-07-09 山东大学 一种基因与化学小分子共递送系统及在肿瘤治疗中的应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103547579A (zh) * 2011-04-15 2014-01-29 新联基因公司 用作ido抑制剂的稠合咪唑衍生物
CN105418733A (zh) * 2015-06-23 2016-03-23 天津药物研究院有限公司 一种穿膜多肽与抗肿瘤药偶联物的制备及应用
WO2017023667A1 (en) * 2015-07-31 2017-02-09 University Of Pittsburgh - Of The Commonwealth System Of Higher Education Immunostimulatory nanocarrier
CN111333653A (zh) * 2019-12-16 2020-06-26 山东大学 一种icd诱导剂-ido抑制剂缀合物及制备方法与应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106074379B (zh) * 2016-07-11 2019-01-25 中国科学院上海药物研究所 聚乙二醇化奥沙利铂前药及其制备方法和用途
GB2552774A (en) * 2016-07-12 2018-02-14 Evox Therapeutics Ltd EV-Mediated delivery of binding protein-small molecule conjugates
EP3624810A4 (en) * 2017-05-18 2021-02-17 The Regents of The University of California NANO-ACTIVATED IMMUNOTHERAPY FOR CANCER DISEASES

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103547579A (zh) * 2011-04-15 2014-01-29 新联基因公司 用作ido抑制剂的稠合咪唑衍生物
CN105418733A (zh) * 2015-06-23 2016-03-23 天津药物研究院有限公司 一种穿膜多肽与抗肿瘤药偶联物的制备及应用
WO2017023667A1 (en) * 2015-07-31 2017-02-09 University Of Pittsburgh - Of The Commonwealth System Of Higher Education Immunostimulatory nanocarrier
CN111333653A (zh) * 2019-12-16 2020-06-26 山东大学 一种icd诱导剂-ido抑制剂缀合物及制备方法与应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
CHEN YICHAO, SUN JINGJING, HUANG YIXIAN, LU BINFENG, LI SONG: "Improved Cancer Immunochemotherapy via Optimal Co-delivery of Chemotherapeutic and Immunomodulatory Agents", MOLECULAR PHARMACEUTICS, AMERICAN CHEMICAL SOCIETY, US, vol. 15, no. 11, 5 November 2018 (2018-11-05), US, pages 5162 - 5173, XP055821425, ISSN: 1543-8384, DOI: 10.1021/acs.molpharmaceut.8b00717 *
MALLICK ABHIK, MORE PIYUSH, GHOSH SOUGATA, CHIPPALKATTI ROHAN, CHOPADE BALU A., LAHIRI MAYURIKA, BASU SUDIPTA: "Dual Drug Conjugated Nanoparticle for Simultaneous Targeting of Mitochondria and Nucleus in Cancer Cells", APPLIED MATERIALS & INTERFACES, AMERICAN CHEMICAL SOCIETY, US, vol. 7, no. 14, 15 April 2015 (2015-04-15), US, pages 7584 - 7598, XP055821448, ISSN: 1944-8244, DOI: 10.1021/am5090226 *
ZHU YONG ET AL.: "Amplification of tumor antigen presentation by NLGplatin to improve chemoimmunotherapy", INTERNATIONAL JOURNAL OF PHARMACEUTICS, vol. 573, 19 November 2019 (2019-11-19), XP086033733, DOI: 10.1016/j.ijpharm.2019.118736 *

Also Published As

Publication number Publication date
CN111333653A (zh) 2020-06-26

Similar Documents

Publication Publication Date Title
WO2021120898A1 (zh) 一种icd诱导剂-ido抑制剂缀合物及制备方法与应用
EP3341375B1 (en) Process for preparing parp inhibitor, crystalline forms, and uses thereof
JP6989505B2 (ja) Ｍａｌｔ１阻害剤およびその使用
WO2021129160A1 (zh) 聚乙二醇化的icd诱导剂-ido抑制剂纳米缀合物及制备方法与应用
RU2546663C2 (ru) Комплексное соединение металл-сален и способ его получения
CN110256313B (zh) 一种光敏剂前药化合物及其制备方法和应用
CN103547564A (zh) 自磁性金属salen络合物化合物
CN107522772A (zh) 短肽、其作为疫苗佐剂的应用及以所述短肽作为疫苗佐剂的疫苗
RU2583900C2 (ru) Соединения 2-арилбензофуран-7-формамида, способ их получения и применение
CA3012846A1 (en) Max binders as myc modulators and uses thereof
CN103781760B (zh) 金属salen络合物化合物、局部麻醉药剂及抗恶性肿瘤药剂
Zhu et al. Synthesis and evaluation of new 5-aminolevulinic acid derivatives as prodrugs of protoporphyrin for photodynamic therapy
JP2021502986A (ja) 新規イミダゾピリミジン化合物およびそれらの使用
CN110577526A (zh) 溴域结构蛋白抑制剂的盐及其制备方法和应用
KR101138438B1 (ko) 스피루리나로부터 클로로필 ａ 및 광민감제 제조방법
Kang et al. Discovery of a novel water-soluble, rapid-release triptolide prodrug with improved drug-like properties and high efficacy in human acute myeloid leukemia
Liu et al. Novel octapeptide-DTX prodrugs targeting MMP-7 as effective agents for the treatment of colorectal cancer with lower systemic toxicity
WO2022022354A1 (zh) 聚乙二醇偶联药物增效剂、其制备方法及用途
WO2018227886A1 (zh) 新型吲哚胺2,3-双加氧化酶抑制剂
CN103288916B (zh) 丹参酮类化合物的衍生物及合成方法及用途
CN105153055A (zh) 苯丙烯酰化1，5-二芳基-1，2，4-三氮唑衍生物、其制备方法及医药用途
KR101106756B1 (ko) 스피루리나로부터 클로로필 ａ 및 광민감제 제조방법
CN110124057A (zh) 一种包含谷氨酰胺修饰的环糊精的抗肿瘤药物或药物载体
WO2019056375A1 (zh) 酸敏感的吉非替尼轴向取代酞菁硅配合物及其制备方法和在医药上的应用
CN109438525B (zh) 具有化疗和光疗抗肿瘤作用的化合物及其制备方法和应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 20903196

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 20903196

Country of ref document: EP

Kind code of ref document: A1