Use of compounds in the preparation of drugs for treating tumors

Abstract

This invention relates to a compound, a method for preparing the compound, and its use in the preparation of a medicament for treating tumors. Based on the above technical solution, it can be concluded that: this disclosure identifies a small molecule inhibitor targeting JMJD1C; small molecule inhibitors of JMJD1C, represented by JI-4, can selectively kill leukemia cells.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and more specifically, to the use of a compound in the preparation of a medicament for treating tumors. Background Technology

[0002] Acute myeloid leukemia (AML) is the most common type of leukemia in Chinese adults, with highly variable prognoses among its subtypes. Mixed lineage leukemia-rearranged AML (MLLr AML) accounts for approximately 5% of adult AML and 10% of infant AML, and has a moderate to poor prognosis. Furthermore, there is evidence that MLLr AML patients do not benefit from hematopoietic stem cell transplantation, and currently popular immunotherapies are not effective against MLLr AML. The most promising strategy for MLLr AML is currently targeted therapy.

[0003] Several chromatin-associated proteins are essential for the maintenance of MLLr AML, such as the histone H3K79 methyltransferase DOT1L, the PRC2 complex containing the histone H3K27 methyltransferase component, histone demethylases LSD1 (KDM1A), KDM4C, and histone modification-binding proteins BRD4, RNF20, MENIN, LEDGF, and CBX8. These essential proteins for MLLr AML maintenance are potential drug targets for MLLr AML. Small molecule inhibitors targeting some of these proteins have already been successfully developed. For example, JQ1 and I-BET151 can inhibit the interaction between BRD4 homomyocytes; EPZ004777 can inhibit DOT1L-mediated H3K79 methylation; MI-2 and MI-3 can inhibit menin-MLL interaction; GSK126, EPZ-6438, and EI1 can inhibit EZH2-mediated H3K27 methylation; and ORY-1001 can inhibit LSD1-mediated H3K4 demethylation. Many small molecule inhibitors have entered clinical trials, but leukemia cells quickly develop drug resistance. A multi-target synergistic targeting strategy may overcome drug resistance to some extent. More drug targets and specific inhibitors are needed.

[0004] Histone H3 lysine 9 (H3K9) demethylase JMJD1C is also an essential protein for the maintenance of MLLr AML and a good drug target for a variety of hematopoietic malignancies, including MLLr AML leukemia.

[0005] Sroczunska et al. (Blood. 2014 Mar 20; 123(12):1870-82.) knocked out 319 chromatin-related proteins using an shRNA library in a mouse AML model induced by the MLL-AF9 fusion oncogene and in bone marrow of mice as a control. The results showed that JMJD1C was the most critical for the survival of MLL-AF9 leukemia cells, but had no significant effect on normal bone marrow, demonstrating the strongest potential as a drug target. In contrast, Brd4, a key molecule for leukemia maintenance and a drug target, ranked 29th among the key genes for MLL-AF9 survival. Further in vitro and in vivo experiments showed that MLL-AF9 fusion oncogene-induced mouse leukemia cells and human leukemia cell lines were sensitive to JMJD1C knockout, with a significant decrease in cell proliferation. In contrast, normal bone marrow cells and c-Kit+ / Sca-1+ / Lin- hematopoietic stem cells were not sensitive to JMJD1C knockout. Zhu et al. (J Clin Invest. 2016 Mar 1; 126(3): 997-1011.) conducted another screening for MLL-AF9 AML, targeting 149 MLL-AF9 target genes. JMJD1C ranked third among differentially expressed genes in MLL-AF9 leukemia stem cells and normal stem cells, with HOXA9 and HOXA10 ranking first and second, respectively. JMJD1C knockout in vitro resulted in weakened MLL-AF9 leukemia cell colony formation, increased apoptosis and terminal differentiation, and the production of more neutrophil- and macrophage-like cells, leading to a decrease in hematopoietic stem cell frequency in vivo. JMJD1C is essential for the maintenance of leukemia, but not for the initiation of leukemia. These results suggest that JMJD1C is a key factor in the maintenance of MLL ectopic AML.

[0006] Chen et al. (Genes Dev. 2015 Oct 15; 29(20): 2123-39.) found that JMJD1C is also crucial for AML containing the AML1 / ETO fusion gene. JMJD1C can interact with the AML1 / ETO complex. JMJD1C is essential for the survival of the AML1 / ETO fusion gene cell lines Kasumi-1 and SKNO-1. These results indicate that JMJD1C is also a key factor in the maintenance of AML1 / ETO AML.

[0007] Sroczunska et al. (Blood. 2014 Mar 20; 123(12): 1870-82.) found that not only AML cells containing MLL ectopic genes and AML1 / ETO fusion genes are sensitive to JMJD1C knockout, but other hematopoietic malignancies, including the acute lymphoblastic leukemia (ALL) cell line SEM (B-ALL), the chronic myelogenous leukemia (CML) cell line K562, and the lymphoma cell line U-937, are also sensitive to JMJD1C knockout. Peeken et al. (Blood. 2018 Mar 8. pii: blood-2017-10-810622.) found that myeloproliferative neoplasms are also sensitive to JMJD1C knockout.

[0008] In summary, JMJD1C is a promising drug target for various hematopoietic malignancies. It exhibits 1) tumor-targeting specificity: JMJD1C knockout specifically kills tumor cells rather than normal cells; and 2) broad-spectrum tumor targeting: various hematopoietic malignancies with different cytogenetic alterations are sensitive to JMJD1C knockout. Whether JMJD1C is an essential protein for other malignancies remains unclear. Nevertheless, there are currently no small-molecule inhibitors targeting JMJD1C. Developing small-molecule inhibitors targeting JMJD1C is of great significance for targeted therapy of hematopoietic malignancies, especially leukemia with molecular genetic abnormalities such as MLL ectopic tumors, as well as other malignancies. Summary of the Invention

[0009] This disclosure provides the pharmaceutical use of histone demethylase inhibitor JI-4 and its structural analogs in inhibiting hematopoietic malignancies.

[0010] This disclosure provides the use of a compound in the preparation of a medicament for treating tumors, wherein the compound is as shown in formula (1):

[0011]

[0012] In equation (1), A is Q is

[0013] R1 is H, methyl, formyl,

[0014] R2 is

[0015] Through the above technical solutions, this disclosure identifies a class of small molecule inhibitors targeting JMJD1C; these JMJD1C small molecule inhibitors can selectively kill leukemia cells and have good therapeutic effects on leukemia.

[0016] Other features and advantages of this disclosure will be described in detail in the following detailed description section. Attached Figure Description

[0017] The accompanying drawings are provided to further illustrate the present disclosure and form part of the specification. They are used together with the following detailed description to explain the present disclosure, but do not constitute a limitation thereof. In the drawings:

[0018] Figure 1 The study identified 10 small molecule compounds obtained through virtual screening that selectively inhibited the proliferation of various malignant abnormal cell lines in the hematopoietic system.

[0019] Figure 2 It represents the half-maximal inhibitory rate (IC50) of JI-4 and its structural analogues against various malignant abnormal cell lines of the hematopoietic system and other cell lines.

[0020] Figure 3 The in vitro enzyme activity assay determined the inhibition of JMJD1C demethylase activity by the small molecule inhibitor JI-4.

[0021] Figure 4 The study determined the inhibition of JMJD1C demethylase activity by the small molecule inhibitor JI-4 and its structural analogues on JMJD1C demethylase activity in vivo.

[0022] Figure 5 The colony formation assay measures the effect of JI-4 structural analogues on cell colony formation.

[0023] Figure 6 The mouse experiments confirmed the inhibitory effect of the small molecule inhibitor JI-4 of JMJD1C and its structural analogues on leukemia. Detailed Implementation

[0024] The specific embodiments of this disclosure will be described in detail below with reference to the accompanying drawings. It should be understood that the specific embodiments described herein are for illustration and explanation only and are not intended to limit this disclosure.

[0025] This disclosure provides the use of a compound in the preparation of a medicament for treating tumors, wherein the compound is as shown in formula (1):

[0026]

[0027] In equation (1), A is Q is

[0028] R1 is H, methyl, formyl,

[0029] R2 is

[0030] Preferably, A is

[0031] Preferably, Q is

[0032] Preferably, R1 is

[0033] Preferably, R2 is

[0034] Preferably, the compound is any one of formulas a1-a14:

[0035]

[0036]

[0037]

[0038]

[0039]

[0040]

[0041]

[0042] This disclosure also provides the use of the compounds described above in the preparation of medicaments for treating tumors.

[0043] The tumor can be a malignant tumor of the hematopoietic system, more specifically leukemia, myeloproliferative neoplasm, myelodysplastic syndrome, multiple myeloma, or lymphoma.

[0044] The present invention will be further described in detail below through examples.

[0045] Example 1

[0046] Identification of potential small molecule inhibitors of JMJD1C that selectively inhibit leukemia cells.

[0047] The top 10 small molecule compounds obtained through virtual screening from nearly 200,000 natural components and effective ingredients of traditional Chinese medicine have the following structural formulas t1-t10, all of which were purchased from MolPort.

[0048]

[0049]

[0050] (that is, formula a3)

[0051]

[0052]

[0053] (that is, formula a2)

[0054]

[0055]

[0056] First, small molecule compounds that selectively inhibit acute leukemia were screened using cell proliferation experiments. Cell lines containing the ectopic MLL (mixed-lineage leukemia) gene, including MOLM-13, THP-1 (MLL-AF9 fusion gene), MV4-11, SEM (MLL-AF4 fusion gene), HL-60, and KG-1, were used.

[0057] Cell proliferation assays: The top 10 potential JMJD1C small molecule inhibitors identified through virtual screening were selected, and their inhibitory effects on various hematopoietic cell lines were examined. These hematopoietic cell lines (HL-60, Jurkat, K562, Kasumi-1, KG-1, MOLM-13, MV4-11, SEM, THP-1, etc.) were obtained from the Leibniz Institute for Cell Biology (DSMZ - German Collection of Microorganisms and Cell Cultures). Cells were cultured at 37°C in 5% CO2 according to the culture methods provided on the DSMZ website (https: / / www.dsmz.de / catalogues / catalogue-human-and-animal-cell-lines.html). The culture medium (RPMI 1640, α-MEM) and fetal bovine serum were obtained from Thermal Fisher.

[0058] Cells were seeded at a concentration of 30,000 cells / ml at 100 μL in 96-well V-bottom plates to ensure exponential growth during 6 days of culture. Twenty-four hours after seeding, various small molecule compounds (dissolved in DMSO) were added at 10 μM and incubated for 6 days; the DMSO concentration did not exceed 0.1%. Chemiluminescence was then measured using an ATP assay kit (LT07-121, Lonza) to assess ATP levels and reflect cell proliferation. The ATP content of the DMSO-treated group was set as 100%, and the drug-treated groups were expressed as a percentage (% of control) relative to the DMSO-treated group. Each value represents the mean ± standard deviation of the experimental data, with at least three independent replicates. An asterisk on the bar chart indicates a statistically significant difference (i.e., P < 0.05).

[0059] like Figure 1 As shown, after treating various cell lines with 10 μM of 10 small molecule compounds for 6 days, two small molecule compounds, #4 (formula t4) and #8 (formula t8), were found to inhibit cell lines other than KG-1. In the study by Chen et al., gene knockout of JMJD1C inhibited all tested cell lines except KG-1, including Kasumi-1, MOLM-13, NB-4, HNT-34, CMV, MV4-11, HEL, NOMO-1, HL-60, and THP-1. The study by Sroczunska et al. showed that gene knockout of JMJD1C inhibited cell proliferation and colony formation in MOLM-13, MV4-11, THP-1, K562, U937, and SEM. Based on these results, small molecule compounds #4 (formula t4, also known as formula a3) and #8 (formula t8, also known as formula a2) could not inhibit the KG-1 cell line at a concentration of 10 μmol, indicating that small molecule compounds #4 and #8 specifically inhibited JMJD1C-dependent leukemia cells.

[0060] Considering that #4 and #8 differ by only one fluorine atom in their structure, further structural analogues #11-#22 were obtained through similarity search based on the common structure of #4 and #8, namely formulas a1, a4-a14.

[0061] Structural analogs #11-#22, namely formulas a1, a4-a14, all exhibit leukemia cell inhibitory activity, as shown in the following results. Figure 2 As shown. Formula #16 (i.e., formula a1) exhibited the best leukemia cell inhibitory activity. Cell lines were expanded, and the IC50 values ​​of #4, #8, and #16 against leukemia cell lines HL-60, Jurkat, K562, Kasumi-1, KG-1, MOLM-13, MV4-11, SEM, and THP-1 were measured. Results are as follows. Figure 3 As shown, Figure 3 The IC50 values ​​of #4 (a3), #8 (a2), and #16 (a1) for various leukemia cell lines are shown.

[0062] Example 2

[0063] In vitro enzyme activity assays: In vitro enzyme activity assays confirmed the inhibitory effect of the obtained small molecule compound on the demethylase activity of the JMJD1C recombinant protein. Then, Western blot was used to detect changes in histone methylation modification. Figure 4 describe.

[0064] In vitro enzyme activity experiments were performed using the following components: 4 μg histone (10223565001, Sigma, Shanghai), 1.5 μg histone demethylase JMJD1C (Wuhan Huamei Biotechnology), and 2 μM small molecule inhibitor (dissolved in DMSO, concentration not exceeding 0.1% of the system). Figure 4 Compounds #1, #2, #3, and #4 in the formula are shown in formulas t1, t2, t3, and t4 (a3), respectively. The solution was prepared with 50 mM Tris-HCl (pH 8.0), 1 mM α-ketoglutarate (α-KG), 100 μM FeSO4, 2 mM ascorbic acid, and a protease inhibitor cocktail. The mixture was incubated at 37°C for 4 hours, then boiled in Laemili loading buffer for 3 minutes, and finally analyzed using Western spectroscopy. blot analysis of changes in histone methylation modification (histone H3 antibody α-H3: catalog number 06-755, batch number 2802577, Millipore; histone H3K9 monomethylation antibody α-H3K9-me1: catalog number 07-450, batch number DAM1791354, Millipore; histone H3K9 dimethylation antibody α-H3K9-me2: catalog number 07-441, batch number 2517832, Millipore; histone H3K9 trimethylation antibody α-H3K9-me3: catalog number 07-442, Millipore; anti-rabbit secondary antibody α-Rabbit: catalog number NA934V, batch number 9568295, GE Healthcare).

[0065] In vitro enzyme activity experiments showed that #4 (formula t4 or a3) could reverse the demethylase activity of JMJD1C.

[0066] Example 3

[0067] In vivo enzyme activity assay: The effects of #4 (formula t4 or a3) and #8 (formula t8 or a2) on the methylation modification of endogenous histone H3K9 were measured by in vivo enzyme activity assay.

[0068] MV4-11 cells were seeded at a density of 200,000-500,000 / ml for 24 hours. Then, 0.05 μM, 0.5 μM, and 5 μM of compounds #4 (formula t4 or a3) and #8 (formula t8 or a2) were added, respectively. After incubation for 48 hours, cells were collected, counted, and lysed with 10,000 / μL RIPA lysis buffer (Cell Signaling). Western blot analysis was performed to detect changes in histone methylation modifications. The antibodies used were the same as those used in in vitro enzyme activity experiments. Results are as follows: Figure 5 As shown, the results indicate that small molecule compounds #4 (formula t4 or a3) and #8 (formula t8 or a2) can upregulate the level of histone H3K9 monomethylation at the whole-cell level.

[0069] Example 4

[0070] Colony formation assay: The effects of #4 and #8 on cell colony formation were measured using a colony formation assay. Results are as follows: Figure 6 describe.

[0071] MV4-11 and MOLM-13 cells were seeded at 200 g / ml in methylcellulose medium (H4434, Stem Cell Technologies), with 10 μM of #4 (formula t4 or a3) or #8 (formula t8 or a2) added simultaneously. After 14–16 days, the number of colonies formed was counted. Each value represents the mean ± standard error or standard deviation of the experimental data, and at least three independent replicates were required. An asterisk on the bar chart indicates a statistically significant difference (i.e., P < 0.05). The results show that small molecule compounds #4 (formula t4 or a3) and #8 (formula t8 or a2) can inhibit colony formation in MV4-11 and MOLM-13 cells.

[0072] Example 5

[0073] Animal experiments: Non-obese diabetic / severe combined immunodeficiency (NOD-SCID) mice (Beijing Vital River Laboratory Animal Technology Co., Ltd.) were used. Results are as follows: Figure 7 describe.

[0074] Mouse experiments identified the JMJD1C small molecule inhibitor #16 (formula a1). Six- to eight-week-old mice (control and treatment, n = 8 each) were irradiated whole-body with a sublethal dose (200 cGy) of radiation, followed by a tail vein injection of 10 million MV4-11 cells. On day 21 post-MV4-11 injection, the powder of #16 (formula a1), the most active of our #4 structural analogues, was dissolved in physiological saline containing 5% dimethyl sulfoxide (DMSO, v / v) and 10% hydroxypropyl β-cyclodextrin (Kleptose HPB, mass / v). The solution was administered intraperitoneally to mice at a dose of 30 mg / kg (5 ml / kg) using a 1 ml syringe. Mice were then observed twice daily. Figure 7 The results showed that #16 (or a1) has leukemia-inhibiting activity.

[0075] The preferred embodiments of the present disclosure have been described in detail above with reference to the accompanying drawings. However, the present disclosure is not limited to the specific details of the above embodiments. Within the scope of the technical concept of the present disclosure, various simple modifications can be made to the technical solutions of the present disclosure, and these simple modifications all fall within the protection scope of the present disclosure.

[0076] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, this disclosure will not describe the various possible combinations separately.

[0077] Furthermore, various different embodiments of this disclosure can be combined in any way, as long as they do not violate the spirit of this disclosure, they should also be regarded as the content disclosed in this disclosure.

Claims

1. The use of the compound in the preparation of a medicament for treating leukemia, characterized in that, The compound is of formula a1:

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