WO2020015393A1 - LIN28/let-7信号通路抑制剂在制备调控PD-L1表达的药物中的应用 - Google Patents

LIN28/let-7信号通路抑制剂在制备调控PD-L1表达的药物中的应用 Download PDF

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WO2020015393A1
WO2020015393A1 PCT/CN2019/081835 CN2019081835W WO2020015393A1 WO 2020015393 A1 WO2020015393 A1 WO 2020015393A1 CN 2019081835 W CN2019081835 W CN 2019081835W WO 2020015393 A1 WO2020015393 A1 WO 2020015393A1
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expression
lin28
tumor
application
regulating
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French (fr)
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赵勇
陈艳莲
谢晨
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中山大学
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Priority claimed from CN201810797573.XA external-priority patent/CN109045036B/zh
Priority claimed from CN201811534321.4A external-priority patent/CN109675035A/zh
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/50—Pyridazines; Hydrogenated pyridazines
    • A61K31/5025—Pyridazines; Hydrogenated pyridazines ortho- or peri-condensed with heterocyclic ring systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/04—Antineoplastic agents specific for metastasis

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  • the invention belongs to the field of medicine, and particularly relates to the application of a LIN28 / let-7 signaling pathway inhibitor in the preparation of a medicine for regulating the expression of PD-L1, in particular to a [1,2,4] triazolo [4,3-B Application of] pyridazine derivatives in the preparation of antitumor drugs.
  • RNA-binding protein LIN28 is mostly at a high expression level in tumor cells, and it is related to tumor deterioration and poor prognosis.
  • LIN28 inhibits the expression of let-7 in cells by interfering with the maturation of let-7 precursors, and let-7 also inhibits LIN28 protein expression through post-transcriptional inhibition.
  • the Let-7 family is a small non-coding RNA (miRNA) family. It includes a total of 12 family members. It plays an important role in promoting cell differentiation and inhibiting tumor cell proliferation. It is considered to be an important tumor suppressor molecule.
  • Let-7 is mostly expressed at low levels in tumor cells, and its expression level can often be used to indicate the degree of tumor progression.
  • LIN28 / let-7 directly forms a negative feedback pathway, and is involved in regulating various aspects of physiological functions of cells, and is one of the important targets for tumor treatment research.
  • PD-L1 programmed death ligand-1
  • PD-L1 is one of the most important targets.
  • PD-L1 is mostly over-expressed and binds to programmed death receptor-1 (PD-1) during the cellular immune response, thereby inhibiting the activity and function of T cells, enabling tumor cells to Avoid surveillance and attacks by the immune system, further worsening growth.
  • PD-1 / PD-L1 has achieved good results in clinical trials such as lung cancer and melanoma, only less than 40% of patients can respond to antibody therapy.
  • the exact cause of the low response rate is still unknown, it may be due to individual differences in PD-L1 expression levels in different patients, or it may be due to the complexity of the tumor microenvironment. Therefore, the development of new treatment methods is extremely urgent for tumor treatment. For this reason, we urgently need to thoroughly understand the specific regulated pathway of PD-L1 in tumor cells.
  • An object of the present invention is to provide a technical solution capable of effectively regulating PD-L1 expression and effectively achieving an antitumor effect in response to the technical problems to be solved above.
  • the present invention provides the use of a LIN28 / let-7 signaling pathway inhibitor in the preparation of a medicament for regulating the expression of PD-L1.
  • the LIN28 / let-7 signaling pathway inhibitor means: any compound, complex, composition, complex that can effectively inhibit and / or block the LIN28 / let-7 signaling pathway Wait.
  • the present invention promotes let-7 processing and maturation by inhibiting the expression of LIN28 protein in tumor cells, thereby further inhibiting PD-L1 protein expression.
  • the LIN28 / let-7 signaling pathway inhibitor is preferably, but not limited to, the [1,2,4] triazolo [4,3-B] pyridazine derivative of the following formula (I) or a pharmaceutically acceptable salt thereof:
  • the invention also provides a method for regulating the expression of PD-L1.
  • the method can regulate the expression of PD-L1 by blocking the LIN28 / let-7 signaling pathway, and the method is used for non-therapeutic and / or non-diagnostic purposes.
  • the present invention also provides a method for regulating PD-L1 expression, which is not limited to non-therapeutic purposes (and / or not limited to non-diagnostic purposes), that is, a method for regulating PD-L1 expression, which can be achieved by blocking LIN28 / let -7 signaling pathway to regulate PD-L1 expression.
  • a medicament for regulating PD-L1 expression by inhibiting the LIN28 / let-7 signaling pathway which comprises a LIN28 / let-7 signaling pathway inhibitor or a pharmaceutically acceptable salt thereof
  • the LIN28 / let-7 signaling pathway inhibitor or a pharmaceutically acceptable salt thereof is preferably present in a therapeutically effective amount and as an active ingredient.
  • the invention may also include one or more pharmaceutically acceptable carrier substances and / or diluents.
  • the medicament can be made into a suitable dosage form, such as, but not limited to, tablets, pills, capsules, liquid preparations such as injection solutions, and the like.
  • LIN28 / let-7 signaling pathway inhibitor is preferably but not limited to a [1,2,4] triazolo [4,3-B] pyridazine derivative of the following formula (I) Or its pharmaceutically acceptable salt:
  • the compound of formula (I) above is a small molecule drug, and its full name is N-methyl-N- (3- (3-methyl- [1,2,4] triazolo [4,3-b] pyridazine- 6-yl) phenyl) acetamide, high water solubility, good thermal stability, and low cytotoxicity.
  • the above-mentioned "regulating PD-L1 expression” particularly refers to regulating PD-L1 levels of tumor cells, and in particular, suppressing PD-L1 levels of tumor cells.
  • the tumor includes various tumors, including but not limited to osteosarcoma, breast cancer, cervical cancer, thyroid cancer, esophageal cancer, liver cancer, gastric cancer, kidney cancer, pancreatic cancer, head and neck cancer, lymphoma, glioma, uterus Endometrial cancer, bile duct cancer, bone cancer, gallbladder cancer, rectal cancer, lung cancer, colon cancer, prostate cancer, skin cancer, ovarian cancer, neuroblastoma, melanoma, keratinocyte cancer, skin cancer, nasopharyngeal cancer , Eye cancer, etc.
  • the present invention also provides an application of the [1,2,4] triazolo [4,3-B] pyridazine derivative of the formula (I) in the preparation of an antitumor drug.
  • the medicament can be applied to the treatment and / or prevention of different types of tumors, including but not limited to any one of breast cancer, cervical cancer, osteosarcoma or lung adenocarcinoma.
  • the medicament can significantly inhibit the growth of tumor cells and the proliferation of solid tumors.
  • the medicament can inhibit the PD-L1 level of tumor cells, especially the surface PD-L1 level.
  • the medicament includes the compound of the above formula (I) as an active ingredient and a pharmaceutically acceptable carrier.
  • the medicament may be made into a suitable dosage form, such as, but not limited to, tablets, pills, capsules, liquid preparations such as injection solutions and the like.
  • the present invention also provides the use of the compound of formula (I) in the preparation of a medicament for inhibiting the expression level of PD-L1 in tumor cells.
  • the tumor is any one of breast cancer, cervical cancer, osteosarcoma or lung adenocarcinoma.
  • the tumor is any one of breast cancer, cervical cancer, osteosarcoma or lung adenocarcinoma.
  • the medicament can improve the immune activity of the body, including but not limited to increasing the proportion of CD8 + lymphocytes and CD69 + lymphocytes in tumor-infiltrating T lymphocytes, and inhibiting PD-1 + CD8 + lymphocytes. proportion.
  • the invention also provides a method for inhibiting the expression level of PD-L1 in tumor cells, which uses a LIN28 / let-7 signaling pathway inhibitor, in particular a compound of formula (I), as an active ingredient of a medicament.
  • the invention also provides a method for inhibiting the growth of tumor cells, which uses a LIN28 / let-7 signaling pathway inhibitor, in particular a compound of formula (I), as a pharmaceutical active ingredient.
  • Figure 1 shows the results of the luciferase activity after homogenization.
  • FIG. 2 shows the results of flow cytometry to detect the inhibitory effect of let-7 on PD-L1 protein expression.
  • FIG. 3 shows the results of detecting the effect of LIN28 on the expression level of PD-L1 by flow cytometry.
  • FIG. 4 shows the results of detecting PD-L1 expression level of mouse tumor cells by flow cytometry.
  • FIG. 5 shows the results of the cell proliferation assay detected by CCK-8.
  • Figure 6 shows the effects of compounds of formula (I) on the level of PD-L1 protein on the cell surface by flow cytometry.
  • Figure 7 is the curve of weight gain and solid tumor growth in tumor-bearing mice.
  • FIG. 8 shows the results of flow cytometry to detect the proportion of tumor infiltrating T lymphocytes in tumor-bearing mice.
  • reagents, materials, and instruments used in the present invention are all commercially available, and the parameters of conventional operation steps are known to those skilled in the art and can be implemented.
  • the compound of the formula (I) referred to in the present invention is a [1,2,4] triazolo [4,3-B] pyridazine derivative, and its full name is N-methyl-N- (3- (3-methyl -[1,2,4] triazolo [4,3-b] pyridazin-6-yl) phenyl) acetamide, the molecular formula is C 15 H 15 N 5 O; the relative molecular weight is 281.13, and the chemical structural formula is as follows :
  • the starting material 1 (purchased from J & K Chemical) (0.65 g, 3.40 mmol) was dissolved in toluene (40 mL), and 3-methyl-4H-1,2,4-triazole-4-amine (purchased from J & K Chemical) (0.33 g, 3.36 mmol), and the reaction was refluxed through a Dean-Stark trap overnight (22 hours). After cooling to room temperature, an appropriate amount of silica gel was added to the mixture, and the solvent was spin-dried. Flash column chromatography using MeOH / CH 2 Cl 2 (0-10%) provided compound 3 (0.85 g, 92%) as a pale yellow solid.
  • Example 2 is only described by taking osteosarcoma, breast cancer, and cervical cancer cells as examples, but considering the structural commonness of the cells, those skilled in the art can understand that the PD- L1 can also be applied to other types of tumor cells.
  • the human kidney epithelial cell line 293T, the osteosarcoma cell line U2OS, the human breast cancer cell line MCF-7, the cervical cancer cell line Hela, and the mouse breast cancer cell line TUBO were purchased from the China Typical Species Collection (CTCC).
  • CCC China Typical Species Collection
  • Female 4-6 week old BALB / C mice were purchased from Animal Experiment Center of Sun Yat-sen University.
  • the Let-7 family is a class of small non-coding RNAs that inhibits the expression of multiple proteins through post-transcriptional inhibition. After sequence alignment analysis, we found that there are multiple potential let-7 target sites on PD-L1. To verify that let-7 regulates its protein expression by targeting the PD-L1 sequence, we performed experiments in 293T cells using a dual fluorescence reporter system. We cloned the wild-type sequence of PD-L1 containing the let-7 target site (or a mutant sequence mutating the let-7 target site) into the plasmid of the dual fluorescence reporter system to construct a reporter vector.
  • 293T cells were seeded in a 96-well plate at a cell density of 1 ⁇ 10 4 / well, and 4 wells were seeded in each group. After the cells are adhered, the modified reporter vector and let-7 overexpression vector (represented by let-7a, -7c, and -7e) or a randomized control EV are simultaneously subjected to transient overexpression, and the culture is replaced after 6 hours of transfection. base. After 48 hours, the fluorescence values of firefly luciferase and Renilla luciferase were measured using a multifunctional microplate reader.
  • FIG. 1 The change in luciferase activity after homogenization is shown in FIG. 1.
  • let-7 significantly inhibited the luciferase activity of the wild-type PD-L1 sequence; when the let-7 target site on the PD-L1 sequence was mutated, let-7 inhibited luciferase The effect is cancelled.
  • the experimental results show that let-7 targets PD-L1 sequence, thereby inhibiting post-transcriptional translation of PD-L1.
  • PD-L1 When PD-L1 is located on the surface of the cell membrane, it binds PD-1 to inhibit the functional activity of T cells, helping tumor cells to escape immunely. Therefore, we use flow cytometry to detect PD-L1 expression (for example, on the surface of cell membranes).
  • U2OS, MCF-7, and Hela cells in 6-well plates at a seeding density of 2 ⁇ 10 5 / well. After the cells are adhered, each well is added with 2 ⁇ g of the corresponding plasmid (the control vector EV, the experimental group let-7a, -7b, -7c, -7d, -7e, -7f, or -7i overexpression vector). The complex was stained and the medium was changed after 6 hours.
  • IFN- ⁇ was added to each well to a final concentration of 10 ng / mL to enhance the background expression level of PD-L1 for easy detection.
  • the culture was stopped and the cells were collected, and the cell samples were placed on ice and incubated with APC (Allophycocyanin) -labeled PD-L1 antibody.
  • APC Allophycocyanin
  • let-7 family members As shown in Figure 2, all members of the let-7 family that overexpress can significantly inhibit PD-L1 expression in cells, indicating that let-7 has a negative regulatory effect on the protein level of PD-L1. And let-7 family members play different roles in different kinds of cells.
  • LIN28 inhibits let-7 expression by inhibiting precursor processing of let-7.
  • U2OS, MCF-7, and Hela cells were seeded in 6-well plates at a seeding density of 2 ⁇ 10 5 / well. After the cells were adhered, LIN28 protein was knocked down by siRNA, and the medium was changed after 6 hours. Twenty-four hours before sample collection, IFN- ⁇ was added to each well to a final concentration of 10 ng / mL to enhance the background expression level of PD-L1 for easy detection. After 24 hours, the culture was stopped and cells were collected.
  • knockdown of LIN28A or LIN28B by siRNA can significantly inhibit the PD-L1 expression level of cells, indicating that LIN28 has a positive regulatory effect on PD-L1 protein expression.
  • [1,2,4] triazolo [4,3-B] pyridazine derivatives of formula (I) increase the expression level of let-7 by blocking the interaction between LIN28 and let-7.
  • mice Derivatives of PBS (experimental group) or an equal volume of PBS (control group) containing 0.1% DMSO (dimethyl sulfoxide) were introduced into mice. Tail vein injections were performed every two days for a total of six injections. Twenty-four hours after the last injection, the mice were sacrificed by cervical dislocation, and the tumors were stripped and digested into single cells with DNase and collagenase. The expression levels of PD-L1 in tumor cells were analyzed by flow cytometry. The experimental data were processed and analyzed with FlowJo software.
  • DMSO dimethyl sulfoxide
  • Human breast cancer cell line MCF-7, cervical cancer cell line Hela, osteosarcoma cell line U2OS, lung adenocarcinoma cell line A549, and mouse breast cancer cell line TUBO were purchased from China Typical Species Collection (CTCC).
  • mice Female 4-6 week old BALB / C mice were purchased from Animal Experiment Center of Sun Yat-sen University.
  • MCF-7, Hela, U2OS and A549 cells were seeded in 96-well plates, respectively, at a seeding density of 1 ⁇ 10 4 / well, and each cell was seeded in 4 wells. After waiting for 6 hours for the cells to adhere, the medium was carefully removed, and 100 ⁇ L of a medium containing a compound of formula (I) at a final concentration of 68 mg / L or an equal volume of a medium containing 0.1% DMSO was added. The medium was changed every 48 hours. After 72 hours, the culture was terminated, 10 ⁇ L of CCK8 solution was added to each well, and the cells were returned to the 37 ° C cell incubator for 1 hour, and then the absorbance at 450 nm was measured with a microplate reader.
  • the experimental results show that the compound of formula (I) can significantly inhibit the proliferation of many types of tumor cells, including breast cancer, cervical cancer, osteosarcoma and lung adenocarcinoma cells.
  • PD-L1 is a cell membrane surface protein that inhibits the differentiation, proliferation, and function of T cells by binding to PD-1. Therefore, we use flow cytometry to detect the protein of PD-L1 (for example, on the surface of cell membranes). Level.
  • MCF-7, Hela, U2OS and A549 cells were seeded into 6-well plates at a seeding density of 1 ⁇ 10 5 / well. After waiting 6 hours for the cells to adhere, the medium was carefully removed, and 2 mL of a medium containing a compound of formula (I) at a final concentration of 68 mg / L or an equal volume of a medium containing 0.1% DMSO was added. The culture medium was replaced once after 48 hours, and IFN- ⁇ was added at a final concentration of 10 ng / mL to enhance the background expression level of PD-L1, which is convenient for detection.
  • the culture was terminated, and the cells were collected and incubated with APC-labeled PD-L1 antibody on ice for 30 minutes, and then the protein level of PD-L1 was detected by flow cytometry.
  • the experimental data were processed and analyzed with FlowJo software.
  • mice 5 ⁇ 10 5 TUBO cells were inoculated into the third pair of breast subcutaneously under the right forelimb armpit of female 4-6 week old BALB / C mice.
  • 100 ⁇ L PBS or an equivalent volume containing a compound of formula (I) ([1,2,4] triazolo [4,3-B] pyridazine derivative) with a final concentration of 68 mg / L 0.1% DMSO in PBS was injected into mice via tail vein injection (treatment agent accounted for 40 mg / kg of body weight of the mice).
  • the dosing cycle was 12 days, with a tail vein injection every two days.
  • the mice were weighed and the tumor size was measured, and the weight change curve and tumor growth curve of the mice were recorded.
  • mice received injection of a compound of formula (I) ([1,2,4] triazolo [4,3-B] pyridazine derivative). There was no significant difference in body weight compared with the control group, but the tumor volume growth was significantly slower compared with the control group, and the final tumor volume was also significantly smaller than the control group, indicating that the compound of formula (I) significantly inhibited the growth of solid tumors.
  • a compound of formula (I) [1,2,4] triazolo [4,3-B] pyridazine derivative
  • T lymphocytes Infiltration of lymphocytes into tumors is considered to be a specific immune response of the body to tumors.
  • the detection of the proportion of T lymphocytes infiltrating tumors can reflect the effect of drugs activating the body's immune system.
  • the majority of cells in tumor infiltrating lymphocytes are CD3 positive, and CD8 + T cells are the main ones.
  • tumor-infiltrating CD8 + T cells mostly expressed PD-1, manifested as failed T lymphocytes, weakened proliferation ability, and impaired immune cell function.
  • mice 24 hours after the last administration of the mice, the mice were sacrificed by cervical dislocation and tumors were isolated.
  • the tumor was digested into single cells with collagenase and DNase, and lymphocytes were then enriched with lymphocyte separation solution.
  • Cell surface markers were simultaneously detected with CD3-FITC, CD8-PerCP, PD-1-PE, and CD69-APC. Label, and finally use flow cytometry to detect different labels.
  • the experimental data were processed and analyzed with FlowJo software.
  • mice The results of detecting tumor infiltrating T lymphocytes in mice are shown in FIG. 8.
  • a compound of formula (I) [1,2,4] triazolo [4,3-B] pyridazine derivative
  • the proportion of CD8 + T cells was significantly increased, and the proportion of CD8 + PD-1 + T cells with a failing phenotype was significantly decreased.
  • CD69 + T cells an early activation marker of immune cells
  • the compound of formula (I) [1,2,4] triazolo [4,3-B] pyridazine was derived Material) can significantly improve the body's immune activity and improve antitumor effects.
  • the compound of formula (I) disclosed in the present invention can effectively inhibit tumor cell growth and PD-L1 expression, inhibit tumor growth and activate the immune system, and can be used as an antitumor drug.

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Abstract

本发明公开了LIN28/let-7信号通路抑制剂在制备调控PD-L1表达的药物中的应用。

Description

LIN28/let-7信号通路抑制剂在制备调控PD-L1表达的药物中的应用 技术领域
本发明属于医药领域，具体涉及LIN28/let-7信号通路抑制剂在制备调控PD-L1表达的药物中的应用，特别是一种[1,2,4]***并[4,3-B]哒嗪衍生物在制备抗肿瘤药物中的应用。
背景技术
随着癌症发生率的升高，以及癌症发生群体的逐渐年轻化，癌症已成为严重威胁国民生命健康的疾病。现代生命科学研究中，如何治疗并治愈癌症已成为亟待突破的研究重点。
在细胞生长过程中，一些重要分子的异常表达和积累往往容易导致癌症的发生发展。其中，RNA结合蛋白LIN28在肿瘤细胞中多处于高表达水平，并与肿瘤的恶化以及较差的预后效果有相关关系。LIN28通过干扰let-7前体的加工成熟从而抑制细胞中let-7的表达水平，而let-7也通过转录后抑制的方式抑制LIN28的蛋白表达。Let-7家族是一类小非编码RNA(miRNA)家族，一共包括12个家族成员，具有促进细胞分化、抑制肿瘤细胞增殖等重要作用，被认为是重要的肿瘤抑制分子。Let-7在肿瘤细胞中多为低表达水平，其表达水平常能用于指示肿瘤的恶化程度。LIN28/let-7直接形成负反馈通路，并参与调控细胞的多方面生理功能，是肿瘤治疗研究的重要靶标之一。
肿瘤治疗方法有多种，传统方法主要包括外科手术、化学治疗和放射线治疗，但由于诊断及治疗手段的局限性，绝大部分肿瘤都无法简单通过这三种方法得到根治。除了传统手段外，治疗方法还包括近年来由于疗效显著而极受关注的肿瘤免疫治疗。越来越多的研究表明，在放、化疗过程中，辐射或药物的使用会造成大量DNA损伤，从而导致肿瘤细胞表面的程序性死亡配体-1(PD-L1)异常上调，进而抑制病人的免疫***，最终导致病人出现对辐射或药物的耐受性。因此，如何在放、化疗过程中结合免疫治疗以控制PD-L1的水平，已成为肿瘤治疗中的一大关键。
在肿瘤免疫治疗中，程序死亡配体-1(PD-L1)是极其重要的靶标之一。在肿瘤细胞中，PD-L1多呈过表达状态，并通过在细胞免疫应答过程中与程序死亡受体-1(PD-1)结合，从而抑制T细胞的活性与功能发挥，使肿瘤细胞得以躲避免疫***的监控与攻击，进一步恶化增长。目前，针对PD-1/PD-L1的单抗治疗虽然在临床试验如肺癌和黑色素瘤等的治疗上取得良好疗效，但其中只有不足40％的患者能对抗体治疗产生应答。低应答率的确切原因仍不得而知，有可能是因为不同患者中PD-L1的表达水平存在个体差异，也有可能是由于肿瘤微环境的复杂性。因此，开发新型治疗方法对于肿瘤治疗显得极为迫切。也正因此，我们急需透 彻了解PD-L1在肿瘤细胞中的具体受调控通路。
发明内容
本发明的目的是针对以上要解决的技术问题，提供一种能够有效调控PD-L1表达、有效实现抗肿瘤效应的技术方案。
为此，本发明提供了LIN28/let-7信号通路抑制剂在制备调控PD-L1表达的药物中的应用。
根据本发明所述的应用，所述LIN28/let-7信号通路抑制剂意指：可有效抑制和/或阻断LIN28/let-7信号通路的任何化合物、复合物、组合物、络合物等。
特别地，本发明通过抑制肿瘤细胞中LIN28蛋白的表达，来促进let-7加工成熟，从而进一步抑制PD-L1蛋白表达。
LIN28/let-7信号通路抑制剂优选但不限于以下式(I)的[1,2,4]***并[4,3-B]哒嗪衍生物或其药学上可接受的盐：
Figure PCTCN2019081835-appb-000001
本发明还提供了一种调控PD-L1表达的方法，该方法可通过阻断LIN28/let-7信号通路来调控PD-L1的表达，该方法用于非治疗目的和/或非诊断目的。
本发明还提供了一种不限于非治疗目的(和/或不限于非诊断目的)调控PD-L1表达的方法，即一种调控PD-L1表达的方法，该方法可通过阻断LIN28/let-7信号通路来调控PD-L1的表达。
在本发明的另一方面，还提供了式(I)的[1,2,4]***并[4,3-B]哒嗪衍生物或其药学上可接受的盐在制备通过抑制LIN28/let-7信号通路调控PD-L1表达的药物中的应用：
Figure PCTCN2019081835-appb-000002
在本发明的另一方面，还提供了一种通过抑制LIN28/let-7信号通路调控PD-L1表达的药物，其包括LIN28/let-7信号通路抑制剂或其药学上可接受的盐，该LIN28/let-7信号通路抑制剂或其药学上可接受的盐优选以治疗有效量存在，并且作为活性成分。在进一步优选 的实施例中，本发明还可以包括一种或多种药用载体物质和/或稀释剂。
应当理解的是，所述药物可制成适当的剂型，例如但不限于片剂、丸剂、胶囊剂、液体制剂如注射液等。
根据本发明所述的药物，其中所述LIN28/let-7信号通路抑制剂优选但不限于以下式(I)的[1,2,4]***并[4,3-B]哒嗪衍生物或其药学上可接受的盐：
Figure PCTCN2019081835-appb-000003
以上式(I)化合物为小分子药物，其全称为N-甲基-N-(3-(3-甲基-[1,2,4]***并[4,3-b]哒嗪-6-基)苯基)乙酰胺，水溶性高，热稳定性良好，细胞毒性低。
式(I)化合物的合成路线如下：
Figure PCTCN2019081835-appb-000004
上述“调控PD-L1表达”尤其是指调控肿瘤细胞的PD-L1水平，特别是抑制肿瘤细胞的PD-L1水平。该肿瘤包括各种肿瘤，包括但不限于骨肉瘤、乳腺癌、***、甲状腺癌、食道癌、肝癌、胃癌、肾癌、胰腺癌、头颈部癌、淋巴瘤、神经胶质瘤、子宫内膜癌、胆管癌、骨癌、胆囊癌、直肠癌、肺癌、结肠癌、***癌、皮肤癌、卵巢癌、成神经细胞瘤、黑素瘤、角质形成细胞癌、皮肤癌、鼻咽癌、眼癌等中的任意一种。
在另一方面，本发明还提供了式(I)的[1,2,4]***并[4,3-B]哒嗪衍生物在制备抗肿瘤药物中的应用。
根据本发明所述的应用，所述药物能应用于不同类型的肿瘤的治疗和/或预防，包括但不限于乳腺癌、***、骨肉瘤或肺腺癌中的任意一种。
根据本发明所述的应用，所述药物能显著抑制肿瘤细胞的生长，以及实体瘤的增殖。
根据本发明所述的应用，所述药物能抑制肿瘤细胞的PD-L1水平，特别是表面的PD-L1水平。
根据本发明所述的应用，所述药物包括上述式(I)的化合物作为活性成分以及药学上可接受的载体。
应当理解的是，所述药物可制成适当的剂型，例如但不限于片剂、丸剂、胶囊剂、液体 制剂如注射液等。
特别地，本发明还提供了上述式(I)的化合物在制备用于抑制肿瘤细胞的PD-L1表达水平的药物中的应用。优选地，所述肿瘤为乳腺癌、***、骨肉瘤或肺腺癌中的任意一种。
在本发明的另一方面，还提供了上述式(I)的化合物在制备用于抑制肿瘤细胞增殖并重新激活免疫***的药物中的应用。优选地，所述肿瘤为乳腺癌、***、骨肉瘤或肺腺癌中的任意一种。
根据本发明所述的应用，所述药物能提高机体的免疫活性，包括但不限于提高肿瘤浸润T淋巴细胞中CD8+淋巴细胞的比例以及CD69+淋巴细胞的比例，抑制PD-1+CD8+淋巴细胞的比例。
本发明还提供了一种用于抑制肿瘤细胞PD-L1表达水平的方法，其采用LIN28/let-7信号通路抑制剂，特别是式(I)的化合物，作为药物活性成分。
本发明还提供了一种用于抑制肿瘤细胞生长的方法，其采用LIN28/let-7信号通路抑制剂，特别是式(I)的化合物，作为药物活性成分。
附图说明
图1为均一化后的荧光素酶活性变化结果。
图2为流式细胞术检测let-7对PD-L1蛋白表达的抑制作用的结果。
图3为流式细胞术检测LIN28对PD-L1表达水平的影响的结果。
图4为流式细胞术检测小鼠肿瘤细胞的PD-L1表达水平的结果。
图5为CCK-8检测细胞增殖实验结果。
图6为流式细胞术检测式(I)化合物对细胞表面PD-L1蛋白水平的影响。
图7为荷瘤小鼠体重增长及实体瘤增长曲线。
图8为流式细胞术检测荷瘤小鼠中肿瘤浸润T淋巴细胞比例的结果。
具体实施方式
下面将结合一些具体实施例进一步清楚、完整地阐述本发明。在此说明，这里所描述的实施例仅是本发明中的部分实施例，而且以下所描述的实施例仅用于说明本发明，并不用于限制本发明的应用范围。
如未特别指出，本发明所使用的试剂、材料、仪器均可通过商购获得，常规操作步骤参数等均为本领域技术人员所知晓并可实施。
实施例1
式(I)化合物的结构与制备
本发明所称的式(I)化合物为[1,2,4]***并[4,3-B]哒嗪衍生物，其全称为N-甲基-N-(3-(3-甲基-[1,2,4]***并[4,3-b]哒嗪-6-基)苯基)乙酰胺，分子式为C 15H 15N 5O；相对分子量为281.13，化学结构式如下：
Figure PCTCN2019081835-appb-000005
式(I)化合物的合成路线如下：
Figure PCTCN2019081835-appb-000006
式(I)化合物的具体合成步骤如下：
将起始原料1(购自J&K Chemical公司)(0.65g，3.40mmol)溶解在甲苯(40mL)中，加入3-甲基-4H-1,2,4-***-4-胺(购自J&K Chemical公司)(0.33g，3.36mmol)，反应通过迪安-斯达克分水器将混合物回流过夜(22小时)。冷却至室温后，向混合物中加入适量硅胶，旋干溶剂。使用MeOH/CH 2Cl 2(0-10％)进行快速柱层析，得到浅黄色固体化合物3(0.85g，92％)。
将化合物3(0.77g，2.84mmol)溶解在THF(四氢呋喃)(5mL)中，滴加叔丁氧基双(二甲基氨基)甲烷(购自J&K Chemical公司)(1.19mL，5.68mmol)。室温搅拌4.5小时后，向反应混合物中加入适量硅胶并旋干溶剂。使用MeOH/CH 2Cl 2(0-15％)进行快速柱层析，得到淡黄色固体化合物5(0.95g，定量)。
将化合物5(0.2g，0.61mmol)溶解在AcOH(3mL)回流(浴温140℃)过夜(20h)。冷却至室温后，向反应混合物中加入适量硅胶并真空旋干溶剂。使用MeOH/CH 2Cl 2(0-15％)进行快速柱层析，得到为浅黄色固体的CL285032(0.122g，71％)，即本发明所涉及的式(I)化合物。
经质谱和核磁共振分析化学结构。
实施例2
以下实施例2仅以骨肉瘤、乳腺癌、***细胞举例进行说明，但考虑到细胞的结构通性，本领域技术人员可理解，本发明的通过抑制LIN28/let-7信号通路调控PD-L1亦可适用于其他类型的肿瘤细胞。
1.实验材料：
人肾上皮细胞株293T、骨肉瘤细胞株U2OS、人类乳腺癌细胞株MCF-7、***细胞株Hela以及小鼠乳腺癌细胞株TUBO均从中国典型物种保藏中心(CTCC)购买。雌性4-6周龄BALB/C小鼠购自中山大学动物实验中心。
实验中所用质粒均从Addgene购买。
2.双荧光报告***实验验证let-7在PD-L1序列上的靶位点：
Let-7家族是一类小非编码RNA，通过转录后抑制的方式抑制多种蛋白表达。经过序列比对分析，我们发现PD-L1上存在多个潜在的let-7靶位点。为了验证let-7通过靶向PD-L1序列进而调控其蛋白表达，我们利用双荧光报告***在293T细胞中进行实验。我们把PD-L1中含let-7靶位点的野生型序列(或突变let-7靶位点的突变体序列)克隆进双荧光报告***的质粒中，构建报告载体。按1×10 4/孔的细胞密度接种293T细胞于96孔板中，每组实验接种4孔。待细胞贴壁后，把改造后的报告载体和let-7过表达载体(以let-7a、-7c及-7e为代表)或随机对照EV同时进行瞬时过表达，转染6小时后更换培养基。48小时后，使用多功能酶标仪对萤火虫荧光素酶和海肾荧光素酶的荧光值分别进行测定。
均一化后的荧光素酶活性变化如图1所示。与对照组EV相比，let-7显著抑制含野生型PD-L1序列的荧光素酶活性；当PD-L1序列上的let-7靶位点突变后，let-7对荧光素酶的抑制效果解除。实验结果表明，let-7靶向PD-L1序列，从而抑制PD-L1的转录后翻译。
3.流式细胞术检测let-7对PD-L1蛋白表达的抑制作用：
PD-L1位于细胞膜表面时，通过与PD-1结合从而抑制T细胞的功能活性，帮助肿瘤细胞进行免疫逃逸。因此，我们采用流式细胞术的方法检测(例如位于细胞膜表面的)PD-L1的表达水平。我们将U2OS、MCF-7和Hela细胞分别接种于6孔板中，接种密度为2×10 5/孔。待细胞贴壁后，每孔加入含2μg相应质粒(分别为对照载体EV、实验组let-7a、-7b、-7c、-7d、-7e、-7f或-7i的过表达载体)的转染复合物，6小时后更换培养基。在样品收集前24小时，每孔加入IFN-γ使其终浓度为10ng/mL，以增强PD-L1的本底表达水平，便于检测。24小时后终止培养并收集细胞，把细胞样品置于冰上，并与APC(别藻蓝蛋白)标记的PD-L1抗体进行孵育。30分钟后，洗去未标记的抗体，再用流式细胞仪检测PD-L1的表达水平。用FlowJo软件对实验数据进行后续处理分析。
如图2所示，过表达不同的let-7家族成员均能显著抑制细胞的PD-L1表达水平，表明let-7对PD-L1的蛋白水平有负调控作用。且在不同种类的细胞中，起主要抑制作用的let-7家族成员不同。
4.流式细胞术检测LIN28对PD-L1表达水平的影响：
LIN28通过抑制let-7的前体加工从而抑制let-7的表达。在验证let-7对PD-L1的抑制作用后，我们进一步检测LIN28对PD-L1蛋白表达的影响。我们将U2OS、MCF-7和Hela细胞分别接种于6孔板中，接种密度为2×10 5/孔。待细胞贴壁后，利用siRNA对LIN28蛋白进行敲降，6小时后更换培养基。在样品收集前24小时，每孔加入IFN-γ使其终浓度为10ng/mL，以增强PD-L1的本底表达水平，便于检测。24小时后终止培养并收集细胞，把细胞样品置于冰上，并与APC标记的PD-L1抗体进行孵育。30分钟后，洗去未标记的抗体，再用流式细胞仪检测PD-L1的表达水平。用FlowJo软件对实验数据进行后续处理分析。
如图3所示，利用siRNA对LIN28A或LIN28B进行敲降均能显著抑制细胞的PD-L1表达水平，表明LIN28对PD-L1蛋白表达有正调控作用。
5.小鼠肿瘤细胞PD-L1检测实验：
式(I)的[1,2,4]***并[4,3-B]哒嗪衍生物通过阻断LIN28与let-7相互作用从而提高let-7的表达水平。
为进一步验证LIN28/let-7通路在活体中对PD-L1的调控效果，使用式(I)的[1,2,4]***并[4,3-B]哒嗪衍生物处理雌性4-6周龄BALB/C小鼠，进行实验。实验前，我们将5×10 5个TUBO细胞接种于其右前肢第三对乳腺皮下。待小鼠成瘤后，按处理剂占小鼠体重比例为40mg/kg的剂量通过尾静脉注射100μL含式(I)[1,2,4]***并[4,3-B]哒嗪衍生物的PBS(实验组)或等体积的含0.1％DMSO(二甲基亚砜)的PBS(对照组)进入小鼠体内。每两天进行一次尾静脉注射，一共注射六次。最后一次注射后24小时，颈椎脱臼处死小鼠，随即剥离肿瘤并用DNA酶和胶原酶把肿瘤消化成单细胞，用流式细胞术分析小鼠肿瘤细胞的PD-L1表达水平。实验数据用FlowJo软件进行处理分析。
如图4所示，结果表明，式(I)的[1,2,4]***并[4,3-B]哒嗪衍生物(实验组)通过阻断LIN28与let-7相互作用，从而提高let-7的表达水平，能有效降低小鼠肿瘤细胞PD-L1的相对表达量。
实施例3
式(I)化合物的抗肿瘤效果验证
1.实验材料
人类乳腺癌细胞株MCF-7、***细胞株Hela、骨肉瘤细胞株U2OS、肺腺癌细胞株A549，以及小鼠乳腺癌细胞株TUBO均购自中国典型物种保藏中心(CTCC)。
雌性4-6周龄BALB/C小鼠购自中山大学动物实验中心。
2.实验方法
2.1CCK8检测细胞增殖实验：
将MCF-7、Hela、U2OS和A549细胞分别接种于96孔板中，接种密度为1×10 4/孔，每种细胞接种4孔。等6小时待细胞贴壁后，小心撤去培养基，加入100μL含终浓度为68mg/L的式(I)化合物的培养基或等体积的含0.1％DMSO的培养基。培养基每48小时更换一次。72小时后终止培养，每孔加入10μL CCK8溶液，放回37℃细胞培养箱孵育1小时，随后用酶标仪测定在450nm处的吸光度。
细胞培养72小时后，4种细胞的增殖结果如图5所示。
实验结果表明，式(I)的化合物可以显著抑制多种类型肿瘤细胞(包括乳腺癌、***、骨肉瘤和肺腺癌细胞)的增殖。
2.2流式细胞术检测PD-L1实验：
PD-L1是一种细胞膜表面蛋白，它通过与PD-1结合从而抑制T细胞的分化、增殖以及功能行使，因此，我们采用流式细胞术检测(例如位于细胞膜表面的)PD-L1的蛋白水平。
将MCF-7、Hela、U2OS和A549细胞分别接种于6孔板中，接种密度为1×10 5/孔。等6小时待细胞贴壁后，小心撤去培养基，加入2mL含终浓度为68mg/L的式(I)化合物的培养基或等体积的含0.1％DMSO的培养基。培养基48小时后更换一次，同时加入终浓度为10ng/mL的IFN-γ，以增强PD-L1的本底表达水平，便于检测。再经过24小时后终止培养，收集细胞并与APC标记的PD-L1抗体于冰上孵育30分钟，随后用流式细胞仪检测PD-L1的蛋白水平。实验数据用FlowJo软件进行处理分析。
如图6所示，经过72小时的式(I)化合物([1,2,4]***并[4,3-B]哒嗪衍生物)处理后，4种细胞的表面PD-L1表达水平都得到了显著抑制。
2.3小鼠体重增长以及实体瘤增长实验
将5×10 5个TUBO细胞接种于雌性4-6周龄BALB/C小鼠右前肢腋窝下第三对乳腺皮下。待小鼠成瘤后，把100μL含终浓度为68mg/L的式(I)化合物([1,2,4]***并[4,3-B]哒嗪衍生物)的PBS或等体积的含0.1％DMSO的PBS通过尾静脉注射的方式注入小鼠体内(处理剂占小鼠体重比例为40mg/kg)。给药周期为12天，每两天进行一次尾静脉注射。在每次注射前，称量小鼠体重并测量肿瘤大小，记录小鼠体重变化曲线以及肿瘤增长曲线。其中，肿瘤体积按照公式“体积＝长×宽 2/2”计算。
小鼠体重增长及实体瘤增长曲线如图7所示，接受式(I)化合物([1,2,4]***并[4,3-B]哒嗪衍生物)注射的荷瘤小鼠体重与对照组相比并无显著差异，但肿瘤体积增长与对照组相比显著缓慢，最终的肿瘤体积也显著小于对照组，说明式(I)的化合物显著抑制实体瘤增长。
2.4小鼠肿瘤浸润T淋巴细胞检测实验：
淋巴细胞浸润到肿瘤中被认为是机体对于肿瘤的一种特异性免疫反应，通过检测肿瘤浸润T淋巴细胞的组成比例能反映药物激活机体免疫***的效果。一般来说，肿瘤浸润淋巴细胞中，绝大多数细胞呈CD3阳性，其中又以CD8+T细胞为主。但是肿瘤浸润CD8+T细胞多高表达PD-1，表现为衰竭型T淋巴细胞，增殖能力减弱，免疫细胞功能发生障碍。
最后一次小鼠给药24小时后，用颈椎脱臼法处死小鼠并分离肿瘤。用胶原酶和DNA酶把肿瘤消化成单细胞，随后用淋巴细胞分离液富集淋巴细胞，并用CD3-FITC、CD8-PerCP、PD-1-PE、以及CD69-APC同时对细胞表面标记物进行标记，最后用流式细胞仪对不同的标记物进行检测。实验数据用FlowJo软件进行处理分析。
小鼠肿瘤浸润T淋巴细胞检测结果如图8所示，持续接受式(I)化合物([1,2,4]***并[4,3-B]哒嗪衍生物)尾静脉注射后，肿瘤浸润淋巴细胞中，CD8+T细胞的比例得到显著增加，且衰竭表型CD8+PD-1+T细胞的比例显著下降。此外，免疫细胞早期活化标志物CD69+T细胞的比例显著上调，说明与对照组相比，式(I)化合物([1,2,4]***并[4,3-B]哒嗪衍生物)能够显著提高机体的免疫活性，提高抗肿瘤效应。
综合以上结果可见，本发明所公开的式(I)化合物能有效抑制肿瘤细胞生长和PD-L1表达，抑制肿瘤生长并激活免疫***，可作为抗肿瘤药物。

Claims (16)

  1. LIN28/let-7信号通路抑制剂在制备调控PD-L1表达的药物中的应用。
  2. 根据权利要求1所述的应用，其特征在于，所述调控PD-L1表达为调控肿瘤细胞的PD-L1水平。
  3. 根据权利要求1所述的应用，其特征在于，所述调控PD-L1表达为抑制肿瘤细胞的PD-L1水平。
  4. 根据权利要求1所述的应用，其特征在于，所述LIN28/let-7信号通路抑制剂为以下式(I)的[1,2,4]***并[4,3-B]哒嗪衍生物或其药学上可接受的盐：
    Figure PCTCN2019081835-appb-100001
  5. 一种调控PD-L1表达的方法，其特征在于，所述方法通过阻断LIN28/let-7信号通路来调控PD-L1的表达。
  6. 根据权利要求5所述的方法，其特征在于，所述调控PD-L1表达为调控肿瘤细胞的PD-L1水平。
  7. 根据权利要求5所述的方法，其特征在于，所述调控PD-L1表达为抑制肿瘤细胞的PD-L1水平。
  8. 式(I)的[1,2,4]***并[4,3-B]哒嗪衍生物或其药学上可接受的盐在制备通过抑制LIN28/let-7信号通路调控PD-L1表达的药物中的应用：
    Figure PCTCN2019081835-appb-100002
  9. 一种通过抑制LIN28/let-7信号通路调控PD-L1表达的药物，其特征在于包括LIN28/let-7信号通路抑制剂或其药学上可接受的盐。
  10. 根据权利要求9所述的药物，其特征在于，所述LIN28/let-7信号通路抑制剂为以下式(I)的[1,2,4]***并[4,3-B]哒嗪衍生物或其药学上可接受的盐：
    Figure PCTCN2019081835-appb-100003
  11. 式(I)的[1,2,4]***并[4,3-B]哒嗪衍生物在制备抗肿瘤药物中的应用：
    Figure PCTCN2019081835-appb-100004
  12. 根据权利要求11所述的应用，其特征在于，所述肿瘤为乳腺癌、***、骨肉瘤或肺腺癌中的任意一种。
  13. 式(I)的[1,2,4]***并[4,3-B]哒嗪衍生物在制备抑制肿瘤细胞的PD-L1表达水平的药物中的应用：
    Figure PCTCN2019081835-appb-100005
  14. 根据权利要求13所述的应用，其特征在于，所述肿瘤为乳腺癌、***、骨肉瘤或肺腺癌中的任意一种。
  15. 式(I)的[1,2,4]***并[4,3-B]哒嗪衍生物在制备抑制肿瘤细胞增殖并重新激活免疫***的药物中的应用：
    Figure PCTCN2019081835-appb-100006
  16. 根据权利要求15所述的应用，其特征在于，所述肿瘤为乳腺癌、***、骨肉瘤或肺腺癌中的任意一种。
PCT/CN2019/081835 2018-07-19 2019-04-09 LIN28/let-7信号通路抑制剂在制备调控PD-L1表达的药物中的应用 WO2020015393A1 (zh)

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