WO2012103783A1 - 一种青蒿素衍生物及其药用盐的应用 - Google Patents

一种青蒿素衍生物及其药用盐的应用 Download PDF

Info

Publication number
WO2012103783A1
WO2012103783A1 PCT/CN2012/070401 CN2012070401W WO2012103783A1 WO 2012103783 A1 WO2012103783 A1 WO 2012103783A1 CN 2012070401 W CN2012070401 W CN 2012070401W WO 2012103783 A1 WO2012103783 A1 WO 2012103783A1
Authority
WO
WIPO (PCT)
Prior art keywords
cells
leukemia
aml
kasumi
acute
Prior art date
Legal status (The legal status 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 status listed.)
Ceased
Application number
PCT/CN2012/070401
Other languages
English (en)
French (fr)
Inventor
糜坚青
李英
彭宇
张瑜
聂瑞敏
刘静静
王瑾
王月英
蔡循
李阳
陈赛娟
王振义
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Shanghai Institute of Materia Medica of CAS
Ruinjin Hospital Affiliated to Shanghai Jiaotong University School of Medicine Co Ltd
Original Assignee
Shanghai Institute of Materia Medica of CAS
Ruinjin Hospital Affiliated to Shanghai Jiaotong University School of Medicine Co Ltd
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 Shanghai Institute of Materia Medica of CAS, Ruinjin Hospital Affiliated to Shanghai Jiaotong University School of Medicine Co Ltd filed Critical Shanghai Institute of Materia Medica of CAS
Priority to US13/982,684 priority Critical patent/US20130317095A1/en
Publication of WO2012103783A1 publication Critical patent/WO2012103783A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
    • C07D493/12—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains three hetero rings
    • C07D493/18—Bridged systems
    • 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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C57/00—Unsaturated compounds having carboxyl groups bound to acyclic carbon atoms
    • C07C57/02—Unsaturated compounds having carboxyl groups bound to acyclic carbon atoms with only carbon-to-carbon double bonds as unsaturation
    • C07C57/13—Dicarboxylic acids
    • C07C57/145—Maleic acid

Definitions

  • the invention belongs to the field of medicine, in particular to the application of an artemisinin derivative and a pharmaceutically acceptable salt thereof.
  • Leukemia is a malignant tumor of the blood system. It is a malignant clonal disease of hematopoietic stem cells, which seriously endangers human health.
  • acute leukemia is a rapidly developing disease that causes a large accumulation of immature blood cells in bone marrow and blood, including acute non-lymphocytic leukemia (ANLL) and acute lymphocytic leukemia (ALL).
  • ANLL acute non-lymphocytic leukemia
  • ALL acute lymphocytic leukemia
  • Acute myelocytic leukemia or acute non-lymphocytic leukemia (ANLL) includes all non-lymphocytic-derived acute leukemia. It is formed by mutations in pluripotent stem cells or karyotypes of slightly differentiated precursor cells. One type of disease is a clonal malignant disease of the hematopoietic system.
  • the WHO classification criteria for acute myeloid leukemia are as follows: 1.
  • AML chromosomal translocation, characterized by the production of AML1-ETO fusion gene; (2) AML with abnormal bone marrow eosinophils, inv (16) (pl3; q22) or t (16; 16) (pl3; Q22); (3) AML is accompanied by t ( 15; 17) (q22: ql2) , (PML-RAR a ) and variant; (4) AML is accompanied by l lq23
  • AML multi-lineage hematopoietic AML
  • Treatment-related AML and MDS 4. AML without further classification, the definition of most subtypes is almost the same as the corresponding disease in the FAB classification, diagnostic criteria
  • (l) AML micro-differentiation type, SPFAB classification of AML-M0 primordial cells are similar to L2 type cells under light microscope, nucleoli are obvious, cytoplasm is transparent, basophilic, no hobby Azure particles and Auer bodies.
  • Myeloperoxidase (MPO) and Sudan black B were positive ⁇ 3%. Under electron microscopy, the MPO is (+), and the myeloid markers such as CD33 or CD13 can be (+).
  • lymphoid antigens are (-), but sometimes CD7+, TdT+ ;
  • AML is not mature, classified AML-M1: undifferentiated granulocytes (type I + 11) account for 90% of bone marrow non-erythroid cells Above, at least 3% of cells are peroxidase stained (+);
  • AML has mature type, classified AML-M2: protoblasts (type I + 11) account for 30% of bone marrow non-erythroid cells ⁇ 89%, monocytes ⁇ 20%, other granulocytes >10%;
  • AML-M4 classified by SPFAB The primordial cells in the bone marrow account for more than 30% of non-erythroid cells. The granulocytes account for 30% ⁇ 80% at each stage, and mononuclear cells at each stage>20%, CD14 Positive
  • Acute monocytic leukemia classified AML-M5: primary mononuclear, young mononuclear ⁇ 30%, CD14 positive in bone marrow non-erythroid cells; (6) acute erythroleukemia, SPFAB classified AML-M6: in bone marrow Young red blood cells ⁇ 50%, non-erythroid cells primordial cells (type I+11) ⁇ 30%; (7) acute megakaryoblastic leukemia, SPFAB classified AML-M7: primitive megakaryocytes in bone marrow ⁇ 30%, CD41, CD6 nCD42 is positive.
  • the object of the present invention is to provide an artemisinin derivative, artemisinylamine and a pharmaceutically acceptable salt thereof, and provide a novel therapeutic drug for leukemia patients.
  • the artemisinin derivative of the present invention is an artemisinyl ether amine having the following structure:
  • the inventive artemisinin derivative arsenic ether and its pharmaceutically acceptable salt can be used for preparing a medicament for treating leukemia, in particular for preparing a medicament for treating acute leukemia, more particularly for preparing acute myeloid leukemia for treatment.
  • Drug a pharmaceutically acceptable salt for preparing a medicament for treating leukemia, in particular for preparing a medicament for treating acute leukemia, more particularly for preparing acute myeloid leukemia for treatment.
  • the acute myeloid leukemia comprises reproducible genetics AML in abnormal AML is associated with t (8; 21 ) (q22; q22), (AML1/ETO) subtype and AML with t ( 15; 17) (q22: ql2) , (PML-RAR a ) subtype And AML-M2 subtypes and AML-M5 subtypes in AML not otherwise classified.
  • Figure 1 is a graph showing the growth inhibition curves of acute myeloid leukemia cells treated with SM1044, wherein (a) is Kasumi-1 cells; (b) is NB4-R1 cells; (c) is HL60 cells; (d) U937 cells.
  • Figure 2 is a graph showing the apoptosis rate of acute myeloid leukemia cells treated with SM1044 by flow cytometry, wherein (a) is Kasumi-1 cells; (b) is NB4-R1 cells; (c) is HL60 Cells; (d) U937 cells.
  • Figure 3 is a flow cytometry analysis of mitochondrial transmembrane potential of acute myeloid leukemia cells treated with SM1044, wherein (a) is Kasumi-1 cells; (b) is NB4-R1 cells; (c) HL60 cells; (d) U937 cells.
  • Figure 4 is a diagram showing the results of apoptosis-related proteins in acute myeloid leukemia cells treated with SM1044 by Western blot, wherein (a) is NB4-R1 cells; (b) is HL60 cells; (c) is U937 cells.
  • Fig. 5A is an electrophoresis pattern of SM1044-induced Kasumi-1 cell fusion protein AML1-ETO degradation assay
  • Figure 5B is an electropherogram showing SM1044-induced changes in Kasumi-1 cell fusion protein AML1-ETO at mRNA levels.
  • Figure 6 is a diagram showing the results of western blot analysis of the expression of highly expressed oncogenes in c-myc ⁇ 3 ⁇ 4 HL60 cells treated with SM1044 in HL60 cells.
  • Figure 7 is a graph showing the cell cycle results of SM1044 block acute myeloid leukemia, wherein (a) is Kasumi-1 cells; (b) is HL60 cells.
  • Figure 8 is a graph showing the growth curve of SM1044 inhibiting acute myeloid leukemia cell xenografts in mice, wherein (a) is Kasumi-1 cells; (b) is HL60 cells; (c) is U937 cells. detailed description The present invention will be further described below in conjunction with specific embodiments. It is to be understood that the following examples are merely illustrative of the invention and are not intended to limit the scope of the invention.
  • the cell lines used in the following examples were derived from the Shanghai Blood Institute and included the following cell lines:
  • Kasumi-1 cells are AML with t (8; 21 ) (q22; q22), (AML1/ETO) subtype cell line, NB4-R1 cells are AML with t ( 15; 17) (q22: ql2), (PML-RAR a ) subtype retinoic acid resistant cell line, HL60 cell is AML-M2 subtype cell line, and U937 cell is AML-M5 subtype cell line.
  • Example 1 Preparation of artemisinin derivative arsenic ether amine maleate
  • This example is prepared from the known compound hydroxyethyl ether (Reference: Li Ying et al., Pharmacological Journal, 1981, 16: 429-439), which is first prepared with p-toluenesulfonate and then in solvent dimethylformamide. The reaction with ammonia water gives the artemisinyl ether amine.
  • the reaction route is:
  • the resulting artemisinide amine is then reconstituted into the maleate salt.
  • SM1044 was first dissolved in tri-distilled water at a concentration of 1 mg/ml, and then the typical acute myeloid leukemia cell lines Kasumi-1, NB4-K HL60 and U937 cells were selected for experiments.
  • a blank group, a non-medicated control group, and different concentration dosing groups were set, and three parallel holes were set for each concentration.
  • MTT (5 mg/ml) was added and culture was continued for 4 h.
  • IC 5Q was 0.17 ⁇ (Kasumi-1 cells, Figure 1 (a)), 0.021 ⁇ (NB4-R1 cells, Figure 1 (b) )), 0.04 ⁇ (HL60 cells, Figure 1 (c)), 0.02 ⁇ (U937 cells, Figure 1 (d)), indicating that SM1044 can inhibit leukemic cell proliferation.
  • Kasumi-1, NB4-K HL60 and U937 cells were seeded in cell culture dishes at a concentration of 3 x 10 5 to 5 x 10 5 /ml.
  • the control group (0 ⁇ ) and the different concentrations of the drug-added groups were separately cultured for 24 h and 48 h, and the cells to be detected were collected.
  • the cells were washed twice with pre-cooled phosphate buffer (PBS) and then mixed with 200 ⁇ l of binding buffer (binding buffer). Resuspend, add 5 ⁇ ⁇ V-FITC and 5 ⁇ propidium iodide ( ⁇ ), mix gently, incubate at room temperature for 15 min in the dark, and measure by flow cytometry in lh.
  • PBS pre-cooled phosphate buffer
  • binding buffer binding buffer
  • SM 1044 acted on Kasumi-1 (Fig. 2 (a)), NB4- 1 (Fig. 2 (b)), HL60 (Fig. 2 (c)) and U937 cells (Fig. 2 (d)).
  • Apoptosis can be induced within ⁇ 48 hours. It indicated that SM1044 could induce apoptosis of leukemia cells, and the proportion of apoptotic cells increased with the increase of drug concentration and prolongation of treatment time.
  • Example 4 SM1044 leukemia cells induced loss of mitochondrial transmembrane potential Assay Kasumi-1, NB4- K HL60 and U937 cells 3x l0 5 ⁇ 5x l0 5 cells / ml were seeded in cell culture dishes.
  • the control group (0 ⁇ ) and the different concentration dosing groups were set up, cultured for 24 h and 48 h, respectively, and 20 nM DiOC 6 (3) was added at 37 ° C for 15 min in the dark, and the cells were washed twice with PBS. The cells were resuspended in 100 ⁇ PBS and detected by flow cytometry.
  • DiOC 6 (3)-negative cells are cells in which the mitochondrial transmembrane potential is lost.
  • SM1044 makes Kasumi-1 (Fig. 3 (a) and Table 1), NB4- 1 (Fig. 3 (b) and Table 2), HL60 (Fig. 3 (c) and Table 3) and U937 (Fig. 3 (d) ) and Table 4) Loss of mitochondrial transmembrane potential in cells, while potential loss is time- and concentration-dependent. Apoptosis includes both intrinsic pathways and extrinsic pathways. The loss of mitochondrial transmembrane potential is an important manifestation of the intrinsic pathway of apoptosis. The results indicate that SM 1044 can induce the apoptosis of Kasumi-1, NB4-K HL60 and U937 cells through the intrinsic pathway. Die.
  • Example 5 SM1044 induces apoptosis-related protein formation in leukemia cells
  • NB4-R1, HL60 and U937 cells were seeded in cell culture dishes at a concentration of 3 x 10 5 to 5 x 10 5 /ml.
  • the control group (0 ⁇ ) and the different concentrations of the drug-added group were set up, and the total protein was extracted after 24 hours of culture.
  • the anti-apoptosis-related proteins (PARP, caspase-3, caspase-8, caspase-9) were obtained by western blot.
  • the antibody detects changes in the amount of apoptosis-related proteins.
  • the Caspase family plays a very important role in mediating apoptosis.
  • apoptosis There are two main pathways for apoptosis: one is to activate the cell's apoptotic enzyme caspase-8 by extracellular signal; the other is to activate caspase-9 by releasing mitochondrial apoptotic enzyme activator. These activated caspases in turn activate the apoptosis-executing protein caspase-3, which is a substrate for caspase-3.
  • caspase-3 and caspase were activated by apoptosis-related proteins in NB4-R1 (Fig. 4 (a)), HL60 (Fig. 4 (b)) and U937 (Fig. 4 (c)) cells after treatment with SM1044.
  • SM1044 induced Kasumi-1 cell fusion protein AML1-ETO degradation assay 1 ⁇ 10 7 Kasumi-1 cells were suspended in 20 ml of medium and seeded in a cell culture dish. The control group (Con) and the different concentration dosing groups were set separately. After 24 h of culture, the total rnRNA was extracted, and the change of the fusion protein at the mRNA level was detected by RT-PCR. As shown in Fig. 5A, the results showed that SM1044 did not affect the fusion gene. Transcription of AML1-ETO.
  • Kasumi-1 cells were then seeded in cell culture dishes at a concentration of 5 x 105/ml.
  • the control group (Con) and the different concentration dosing groups were set separately.
  • the total protein was extracted, and the change of the fusion protein AML1-ETO was detected by Western blot, as shown in Fig. 5B. It indicates that SM1044 degrades the fusion protein AML1-ETO, and when the concentration of SM1044 is 1 ⁇ , the degradation of the fusion protein AML1-ETO can be induced.
  • the fusion protein AML1-ETO which is a characteristic fusion gene of AML-M2b, plays an important role in the development of leukemia and can be used as a target for drug action.
  • the results of this study showed that SM1044 can significantly degrade the fusion protein AML1-ETO, indicating that the fusion protein AML1-ETO can be used as an intracellular target for SM1044.
  • Example 7 SMI 044 inhibits oncogene c-myc expression in HL60 cells HL60 cells were seeded in cell culture dishes at a concentration of 3 x 105/ml. The control group (0 ⁇ ) and the different concentration dosing groups were set up. After 24 hours of culture, the total protein was extracted.
  • oncogene c-myc was detected by anti-c-myc antibody by western blot.
  • One of the hallmarks of HL60 cells is the high expression of the oncogene c-myc.
  • Figure 6 when the concentration of SM1044 is greater than 1 ⁇ , it can completely inhibit the expression of the oncogene c-myc in HL60 cells, indicating that SM1044 is on the oncogene c.
  • the expression of -myc has an inhibitory effect.
  • Example 8 SM1044 block leukemia cell cycle test
  • Kasumi-1 and HL60 cells were seeded in cell culture dishes at a concentration of 5 x 10 5 /ml.
  • the control group (Con) and the different concentration dosing groups were set separately.
  • the cells were collected, washed twice with PBS, and fixed with 70% cold ethanol at 4 ° C overnight.
  • the cells were washed with PBS, resuspended in PBS containing 10 mg/ml RNase, incubated at 37 °C for 30 min, and then added with 50 ⁇ M ⁇ propidium iodide ( ⁇ ) for DNA staining. Flow cytometry within 1 h. Analyze the distribution of cellular DNA content.
  • Example 9 SM1044 inhibits mouse leukemia cell xenograft growth test
  • mice model of acute myeloid leukemia cell line Kasumi-1, HL60 and U937 cells was established, subcutaneously injected with lx lO 7 leukemia cells, the mice grew to a length of about 5 mm, and the mice were divided into two groups, respectively Control group (Con), different concentrations of the drug group. Each day, the intraperitoneal injection was 0.125 ml (Kasumi-1 and HL60 cells) or 0.1 ml (U937 cells), and the control group was injected with the same amount of normal saline for 18 to 35 days. The mouse mass was measured daily. . The results showed that in the mouse xenograft model of Kasumi-1 (Fig. 8 (a)), HL60 (Fig.
  • Artemisinin is a sesquiterpene lactone with a peroxy group extracted from the plant Artemisia annua L., artesunate, artemether and dihydroartemisinin. ) are all derivatives of artemisinin. At present, the above artemisinin drugs have become the first line of drugs for the treatment of malaria worldwide.
  • SM1044 can inhibit leukemia cell proliferation and induce apoptosis, with time and dose. Dependence. SM1044 can be used for the preparation of drugs for the treatment of leukemia, especially for the treatment of acute leukemia, more particularly for the preparation of drugs for the treatment of acute myeloid leukemia.
  • the artemisinin derivative of the invention has a strong ability to induce apoptosis of leukemia, and can inhibit tumor cells in a short time and a small dose, and has less toxic and side effects, and has important significance for clinical treatment of leukemia.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Medicinal Chemistry (AREA)
  • Public Health (AREA)
  • Epidemiology (AREA)
  • Hematology (AREA)
  • Oncology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)

Abstract

本发明公开了一种青蒿素衍生物及其药用盐的应用。该青蒿素衍生物二蒿乙醚基胺及其药用盐能够抑制白血病细胞的增殖，阻滞白血病细胞周期，诱导白血病细胞凋亡。本发明的青蒿素衍生物二蒿乙醚基胺及其药用盐能用于制备治疗白血病的药物，特别是用于制备治疗急性白血病的药物，更特别是用于制备治疗急性髓细胞性白血病的药物。

Description

一种青蒿素衍生物及其药用盐的应用
技术领域
本发明属于医学领域，具体地说，涉及一种青蒿素衍生物及其药用盐的应用。 背景技术
白血病是血液系统的恶性肿瘤， 是一类造血干细胞的恶性克隆性疾病， 严重 危害人类健康。 其中， 急性白血病是一类急速发展的疾病， 其导致骨髓和血 液中未成熟的血细胞大量积累， 包括急性非淋巴细胞白血病 （ANLL) 和急性 淋巴细胞性白血病 （ALL)。
急性髓细胞性白血病 （acute myelocytic leukemia, AMD或急性非淋巴细胞 白血病 （ANLL) 包括所有非淋巴细胞来源的急性白血病。 它是多能干细胞或已 轻度分化的前体细胞核型发生突变所形成的一类疾病，是造血系统的克隆性恶性 疾病。 急性髓细胞性白血病的 WHO分型标准如下： 1、 伴有重现性遗传学异常 AML， 包括 （1 ) AML伴有 t (8； 21 ) (q22; q22) ， (AML1/ETO) 。 以具有 t
(8； 21 )染色体易位， 产生 AML1-ETO融合基因为特点； （2) AML伴有骨髓异 常嗜酸粒细胞， inv ( 16) (pl3 ; q22) 或 t ( 16； 16) (pl3 ; q22) ； (3 ) AML 伴有 t ( 15; 17) (q22: ql2) , (PML-RAR a )及变异型； （4) AML伴有 l lq23
(MLL) 异常； 2、 伴多系病态造血 AML; 3、 治疗相关 AML和 MDS; 4、 不另 做分类的 AML，其多数亚型的定义与 FAB分类中的相应病种几乎相同，诊断标准 根据受累的主要细胞系列和成熟程度： （l ) AML微分化型， SPFAB分类的 AML-M0: 原始细胞在光镜下类似 L2型细胞， 核仁明显， 胞浆透明， 嗜碱性， 无 嗜天青颗粒及 Auer小体。髓过氧化物酶（MPO)及苏丹黑 B阳性 <3%。在电镜下， MPO为 （+ ) ， CD33或 CD13等髓系标志可呈 （+ ) 。 通常淋巴系抗原为 （-) ， 但有时 CD7+、 TdT+; (2) AML无成熟型， 分类的 AML-M1： 未分化原 粒细胞 （I型 +11型） 占骨髓非红系细胞的 90%以上， 至少 3%细胞为过氧化物酶染 色 （+ ) ； (3 ) AML有成熟型， 分类的 AML-M2: 原粒细胞 （I型 +11型） 占骨髓非红系细胞的 30%〜89%， 单核细胞<20%， 其他粒细胞 > 10%; (4) 急 性粒单核细胞白血病， SPFAB分类的 AML-M4: 骨髓中原始细胞占非红系细胞的 30%以上， 各阶段粒细胞占 30%〜<80%， 各阶段单核细胞>20%， CD14阳性；
(5 ) 急性单核细胞白血病， 分类的 AML-M5: 骨髓非红系细胞中原单核、 幼单核≥30%， CD14阳性； （6) 急性红白血病， SPFAB分类的 AML-M6: 骨髓中 幼红细胞≥50%， 非红系细胞中原始细胞 （I型 +11型） ≥30%； (7) 急性巨核细胞 白血病， SPFAB分类的 AML-M7:骨髓中原始巨核细胞≥30%， CD41、CD6 nCD42 阳性。
目前治疗白血病标准方法包括常规化疗、 骨髓移植和放射治疗。 但是， 大多 数病人的预后依旧很差。 治疗后的严重副反应 （如感染， 出血， 移植后的排斥反 应等）或疾病的复发严重地影响了患者的生存率。 因此寻找新型的高效的抗肿瘤 药物以提高患者的完全缓解率和生存期， 显得尤为重要。 并且， 由于白血病亚型 不同， 诊断标准也有不同， 治疗方案及预后亦不尽相同。 因此应根据白血病细胞 的形态学、 免疫学和细胞遗传学特点进一步作出分型诊断和亚型诊断。 发明内容
本发明的目的在于提供一种青蒿素衍生物二蒿乙醚基胺及其药用盐的应用， 为白血病患者提供一种新型的治疗药物。
本发明的青蒿素衍生物为二蒿乙醚基胺， 具有如下结构：
Figure imgf000003_0001
经发明人广泛而深入的研究，发现水溶性的青蒿素衍生物二蒿乙醚基胺马来酸盐 (标记为 SM1044) 能够抑制白血病细胞增殖， 阻滞细胞周期， 诱导细胞凋亡。
因此，发明的青蒿素衍生物二蒿乙醚基胺及其药用盐可用于制备治疗白血病 的药物， 特别是用于制备治疗急性白血病的药物， 更特别是用于制备治疗急性髓 细胞性白血病的药物。
根据本发明的优选实施例，所述急性髓细胞性白血病包括伴有重现性遗传学 异常 AML中的 AML伴有 t (8; 21 ) (q22; q22)， （AML1/ETO)亚型和 AML 伴有 t ( 15; 17) (q22: ql2) , (PML-RAR a )亚型以及不另做分类的 AML中 的 AML-M2亚型和 AML-M5亚型。 附图说明
图 1为急性髓细胞性白血病细胞经 SM1044处理后的生长抑制曲线图，其中， (a) 为 Kasumi-1细胞； （b) 为 NB4-R1细胞； （c) 为 HL60细胞； （d) 为 U937 细胞。
图 2为流式细胞仪检测急性髓细胞性白血病细胞经 SM1044处理后的凋亡率 结果图， 其中， （a)为 Kasumi-1细胞； （b)为 NB4-R1细胞； （c)为 HL60细胞； (d) 为 U937细胞。
图 3为流式细胞仪检测急性髓细胞性白血病细胞经 SM1044处理后的线粒体 跨膜电位结果图， 其中， （a) 为 Kasumi-1 细胞； （b) 为 NB4-R1 细胞； （c) 为 HL60细胞； （d) 为 U937细胞。
图 4为 western blot检测急性髓细胞性白血病细胞经 SM1044处理后的凋亡 相关蛋白结果图， 其中， （a) 为 NB4-R1细胞； （b) 为 HL60细胞； （c) 为 U937 细胞。
图 5A为 SM1044诱导 Kasumi-1细胞融合蛋白 AML1-ETO降解试验的电泳 图； 图 5B为 SM1044诱导 Kasumi-1细胞融合蛋白 AML1-ETO在 mRNA水平的 变化的电泳图。
图 6为 western blot检测 HL60细胞经 SM1044处理后的 c-myc Γ¾ HL60细 胞中高表达的癌基因 表达的蛋白结果图。
图 7 为 SM1044 阻滞急性髓细胞性白血病细胞周期结果图， 其中， （a) 为 Kasumi-1细胞； (b) 为 HL60细胞。
图 8为 SM1044抑制小鼠的急性髓细胞性白血病细胞移植瘤生长曲线图，其 中， （a) 为 Kasumi-1细胞； （b) 为 HL60细胞； （c) 为 U937细胞。 具体实施方式 以下结合具体实施例， 对本发明做进一步说明。 应理解， 以下实施例仅用于 说明本发明而非用于限制本发明的范围。
以下实施例中使用的细胞株来源于上海血液研究所， 包括如下细胞株：
Kasumi-1细胞为 AML伴有 t (8; 21 ) (q22; q22) ， (AML1/ETO) 亚型细胞 株、 NB4-R1细胞为 AML伴有 t ( 15; 17) (q22: ql2) ， （PML-RAR a )亚型 维甲酸抗性细胞株、 HL60细胞为 AML-M2亚型细胞株、 U937细胞为 AML-M5 亚型细胞株。 实施例 1 青蒿素衍生物二蒿乙醚基胺马来酸盐的制备
本实施例从已知化合物羟基蒿乙醚 （参考文献： 李英 等， 药学学报， 1981， 16: 429-439) 出发， 先制成它的对甲苯磺酸酯， 然后在溶剂二甲基甲酰胺中与 氨水反应即得到二蒿乙醚基胺， 反应路线为：
Figure imgf000005_0001
随后将制得的二蒿乙醚基胺再制成马来酸盐。
具体操作如下：
将羟基蒿乙醚的对甲苯磺酸酯（1.54 g)溶于二甲基甲酰胺（10 mL) 中， 再 加氨水 （0.5 mL) 搅拌加热至 40〜50°C， 反应 20 h左右。 TLC检测原料点基本 消失后， 将反应液倒入冰水， 用乙酸乙酯反复提取， 合并有机相， 再用饱和盐水 洗涤， 无水硫酸钠干燥。 减压蒸去溶剂， 残余物通过柱层析 （硅胶， 洗脱剂为乙 酸乙酯 /石油醚 /三乙胺的混合溶剂， 梯度洗脱的浓度为 1/19/1→ 1/10/1， v/v/v) o 得到浅黄色油状产物 0.4 g， 收率 40%。 油状产物用少许乙酸乙酯溶解后， 慢慢 滴入马来酸 /乙酸乙酯溶液至弱酸性， 即有固体析出， 将此马来酸盐过滤， 乙醇 / 石油醚重结晶， 得白色晶体。 熔点： 140〜142°C。 iHNMR (游离碱， 300 MHz， CDC13) δ: 5.40 (s， 2H), 4.82 (d, J = 3.3 Hz, 2H), 3.97 (m, 2H), 3.56 (m, 2H), 2.84 (m, 4H)， 1.43 (s, 6H)， 0.95 (d, J = 6.0 Hz, 6H)， 0.90 (d, J = 7.2Hz, 6H)。 质谱分析 （游离 碱 C34H55NO1Q): m/z 638 (M+l)+ 0 元素分析 （马来酸盐， C38H59NO14): 计算值 为 C 60.53、 H 7.89、 N 1.86； 实测值为 C 60.72、 H 8.00、 N 1.73。
以上分析数据确证二蒿乙醚基胺马来酸盐 （SM 1044) 的结构如下：
Figure imgf000006_0001
实施例 2 SM1044对白血病细胞的抑制试验
首先将 SM1044溶解于三蒸水中， 浓度为 l mg/ml， 然后选取典型的急性髓细 胞性白血病细胞株 Kasumi-1、 NB4- K HL60和 U937细胞进行实验。 我们将细胞 以 2.5X 105个 /ml的浓度接种于细胞培养皿中。 分别设置空白组、 不加药的对照组、 不同浓度加药组，每种浓度设置 3个平行孔。培养不同时间后加入 MTT (5 mg/ml) 继续培养 4 h。 离心， 吸掉 180 μΐ上清， 每孔加入 180 ^ DMSO, 置于摇床振荡 15 min。 酶标仪检测 570 nm吸光值 (A)， 计算半数抑制浓度 IC5Q。 结果显示四种急性 髓细胞性白血病细胞对 SM1044都敏感， 48 h时， IC5Q分别为 0.17 μΜ (Kasumi-1 细胞， 图 1 (a) )、 0.021 μΜ (NB4-R1细胞， 图 1 (b) )、 0.04 μΜ (HL60细胞， 图 1 (c) )、 0.02 μΜ (U937细胞， 图 1 (d) )， 表明 SM1044能抑制白血病细胞的增殖。 实施例 3 SM1044诱导白血病细胞细胞凋亡试验
将 Kasumi-1、 NB4- K HL60和 U937细胞以 3xl05〜5xl05个 /ml的浓度接 种于细胞培养皿中。 分别设置对照组 （0μΜ)、 不同浓度加药组， 分别培养 24 h 和 48 h， 收集待检测细胞， 预冷的磷酸盐缓冲液 （PBS) 洗 2遍后用 200 μΐ结合 缓冲液（binding buffer)重悬，加入 5 μΙ Αηηεχίη V-FITC和 5 μΐ 碘化丙啶（ΡΙ)， 轻轻混匀， 于室温避光孵育 15 min， l h内流式细胞仪检测。 结果显示不同浓度 的 SM 1044作用于 Kasumi-1 (图 2 (a) )、 NB4- 1 (图 2 (b) )、 HL60 (图 2 (c) ) 和 U937细胞 （图 2 (d) ) 24〜48小时内能诱导细胞凋亡。 表明 SM1044可诱导 白血病细胞凋亡， 凋亡细胞比例随药物浓度的增大和处理时间的延长而增加。 实施例 4 SM1044诱导白血病细胞线粒体跨膜电位丢失试验 将 Kasumi-1、 NB4- K HL60和 U937细胞以 3x l05〜5x l05个 /ml接种于细 胞培养皿中。 分别设置对照组 （0 μΜ)、 不同浓度加药组， 培养分别 24 h和 48 h 后， 加入 20 nM DiOC6(3)于 37°C避光孵育 15 min, PBS洗细胞 2次， 再用 100 μΐ PBS重悬细胞， 流式细胞仪检测。 DiOC6(3)阴性的细胞为线粒体跨膜电位丢失的 细胞。 结果显示 SM1044使 Kasumi-1 (图 3 (a)和表 1 )、 NB4- 1 (图 3 (b )和 表 2)、 HL60 (图 3 (c) 和表 3 ) 和 U937 (图 3 (d) 和表 4) 细胞的线粒体跨膜 电位丢失， 同时电位丢失有时间和浓度的依赖性。 细胞凋亡包括内在途经和外在 途径， 其中线粒体跨膜电位的丢失是细胞凋亡内在途经的重要表现， 结果表明 SM 1044可通过内在途径诱导 Kasumi-1、 NB4- K HL60和 U937细胞的凋亡。
表 1、 SM1044诱导 DiOC6(3)阴性的 Kasumi-1细胞的百分率
Figure imgf000007_0001
48h 2.5% 5.9% 5.6% 16.2% 实施例 5 SM1044诱导白血病细胞凋亡相关蛋白形成试验
将 NB4-R1、 HL60和 U937细胞以 3xl05〜5xl05个 /ml的浓度接种于细胞培 养皿中。 分别设置对照组 （0 μΜ)、 不同浓度加药组， 培养 24 h后抽取细胞总蛋 白， 通过 western blot的方法， 用抗凋亡相关蛋白 （PARP， caspase-3, caspase-8, caspase-9) 的抗体检测凋亡相关蛋白量的变化。 Caspase 家族在介导细胞凋亡的 过程中起着非常重要的作用。 而细胞凋亡的途径主要有两条： 一条是通过胞外信 号激活细胞内的凋亡酶 caspase-8等； 另一条是通过线粒体释放凋亡酶激活因子 激活 caspase-9等。 这些活化的 caspase进而激活凋亡执行蛋白 caspase-3引起细 胞凋亡，其中 PARP是 caspase-3的底物。结果显示经 SM1044处理后，在 NB4-R1 (图 4 (a) ) 、 HL60 (图 4 (b) ) 和 U937 (图 4 (c) ) 细胞中， 凋亡相关蛋 白活化的 caspase-3、 caspase-8禾 Π/或 caspase-9量增加， 表明 SM1044可通过内在 和外在两种途径诱导白血病细胞凋亡。 实施例 6 SM1044诱导 Kasumi-1细胞融合蛋白 AML1-ETO降解试验 将 lxlO7个 Kasumi-1细胞悬于 20 ml培养基中， 接种于细胞培养皿中。分别 设置对照组 （Con)、 不同浓度加药组， 培养 24 h后抽提总 rnRNA, 用 RT-PCR 方法检测融合蛋白在 mRNA水平的变化， 如图 5A所示， 结果表明 SM1044不影 响融合基因 AML1-ETO的转录。 然后将 Kasumi-1细胞以 5xl05个 /ml的浓度接 种于细胞培养皿。 分别设置对照组 （Con)、 不同浓度加药组， 培养 24 h后抽取 细胞总蛋白，通过 western blot的方法，用抗 ETO的抗体检测融合蛋白 AML1-ETO 的变化，如图 5B所示，结果表明 SM1044可降解融合蛋白 AML1-ETO,当 SM1044 浓度为 1 μΜ时即可诱导融合蛋白 AML1-ETO的降解。结果表明 SM1044不影响 融合基因 AML1-ETO的转录， 只诱导融合蛋白的降解。 融合蛋白 AML1-ETO作 为 AML-M2b的特征融合基因， 在白血病的发生中起重要作用， 可将其作为药物 作用的靶点。本研究结果显示， SM1044能明显引起融合蛋白 AML1-ETO的降解， 说明融合蛋白 AML1-ETO可作为 SM1044作用的胞内靶点。 实施例 7 SMI 044抑制 HL60细胞中癌基因 c-myc表达试验 将 HL60细胞以 3xl05个 /ml浓度接种于细胞培养皿中。 分别设置对照组 （0 μΜ)、 不同浓度加药组， 培养 24 h后抽取细胞总蛋白， 通过 western blot的方法， 用抗 c-myc的抗体检测癌基因 c-myc的表达变化。 HL60细胞的一个特点是高表达 癌基因 c-myc,如图 6所示，当 SM1044的浓度大于 1 μΜ时，能够完全抑制 HL60 细胞中的癌基因 c-myc的表达， 表明 SM1044对癌基因 c-myc的表达具有抑制作 用。 实施例 8 SM1044阻滞白血病细胞周期试验
将 Kasumi-1和 HL60细胞以 5xl05个 /ml的浓度接种于细胞培养皿中。 分别 设置对照组（Con)、不同浓度加药组， 培养 24 h (Kasumi-1细胞）或 12h (HL60 细胞） 后收集细胞， PBS洗涤 2次， 加入 70%的冷乙醇 4°C固定过夜。 用 PBS 洗涤细胞， 重悬于含有 10mg/mlRNA酶的 PBS中， 37°C共同孵育 30 min， 再加 入 50 μ^ιηΐ碘化丙啶（ΡΙ)进行细胞 DNA染色， 1 h内流式细胞仪分析细胞 DNA 含量分布。 结果显示， Kasumi-1 (图 7 (a) 和表 5) 和 HL60细胞 （图 7 (b) 和 表 6)经 SM1044处理后被阻滞在 Go/Gi期，显示 Go/Gi期的细胞增多。表明 SM1044 可以阻滞细胞周期， 使细胞停止生长。
表 5、 SM1044对 Kasumi-1细胞周期的影响
Figure imgf000009_0001
s 56.09% 54.61% 47.88% 37.52% 实施例 9 SM1044抑制小鼠的白血病细胞移植瘤生长试验
建立急性髓细胞性白血病细胞株 Kasumi-1、 HL60和 U937细胞小鼠移植瘤模 型， 皮下注射 l x lO7个白血病细胞， 小鼠肿块长到 5 mm左右， 将小鼠分为 2组， 分 别为对照组（Con)、不同浓度加药组。每天予以腹腔注射，每次注射量为 0.125 ml (Kasumi-1和 HL60细胞） 或 0.1 ml (U937细胞）， 对照组注射等量生理盐水， 疗 程为 18〜35天，每日测量小鼠肿块大小。结果显示，在 Kasumi-1 (图 8 (a) )、 HL60 (图 8 (b) )和 U937 (图 8 (c) ) 细胞小鼠移植瘤模型中， 与对照组相比， 加药组 小鼠肿块明显比对照组的小， 说明 SM1044能够在小鼠体内抑制肿瘤细胞的增殖。 青蒿素 (artemisinin)是从植物黄花蒿中提取出的具有过氧基团的倍半萜内酯， 青蒿琥酉^ artesunate)、 蒿甲醚 (artemether)禾卩二氢青蒿素( dihydroartemisinin) 等均 为青蒿素的衍生物。 目前上述青蒿素类药物已成为在世界范围内治疗疟疾的第一 线药物。 虽然近 20年来， 国内外学者开展了青蒿素类化合物的抗肿瘤作用的研 究， 证明青蒿素类化合物体外抗瘤谱广， 但是具体在体内实施中往往疗效不佳， 而最终没能进一步深入研究。我们试图在原有的基础上研究一种新型的青蒿素衍 生物， 以期提高抗肿瘤作用的效果。本实验采用新型的水溶性青蒿素衍生物二蒿 乙醚基胺马来酸盐， 对抑制白血病细胞增殖等试验进行考察， 表明 SM1044能抑 制白血病细胞增殖， 诱导细胞凋亡， 并具有时间和剂量的依赖性。 SM1044能够 用于制备治疗白血病的药物， 特别是用于治疗急性白血病的药物， 更特别是用于 制备治疗急性髓细胞性白血病的药物。
应当理解， 以上实施例仅用以说明本发明的技术方案， 而非对本发明保护范 围的限制， 尽管参照较佳实施例对本发明作了详细地说明， 本领域的普通技术人 员应当理解， 可以对本发明的技术方案进行修改或者等同替换， 如采用二蒿乙醚 基胺的其它水溶性盐， 也能达到本发明的技术效果。本发明的青蒿素衍生物二蒿 乙醚基胺对白血病的诱导凋亡能力较强， 可在短时间、 小剂量内抑制肿瘤细胞， 且毒副作用较小， 对白血病的临床治疗有重要意义。

Claims

1、 一种青蒿素衍生物及其药用盐的应用， 其特征在于， 用于制备治疗白血 病的药物， 其中， 所述青蒿素衍生物为二蒿乙醚基胺， 结构为：
Figure imgf000011_0001
2、 如权利要求 1所述的应用， 其特征在于， 所述白血病为急性白血病。
3、如权利要求 2所述的应用， 其特征在于， 所述急性白血病为 AML型急性 髓细胞性白血病。
4、如权利要求 3所述的应用， 其特征在于， 所述 AML型急性髓细胞性白血 病包括： 伴有重现性遗传学异常 AML中的 AML伴有 t (8; 21 ) (q22; q22) ，
(AML1/ETO) 亚型和 AML伴有 t ( 15； 17) (q22: ql2) ， (PML-RAR ) 亚型以及不另做分类的 AML中的 AML-M2亚型和 AML-M5亚型。
PCT/CN2012/070401 2011-01-31 2012-01-16 一种青蒿素衍生物及其药用盐的应用 Ceased WO2012103783A1 (zh)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US13/982,684 US20130317095A1 (en) 2011-01-31 2012-01-16 Use of Artemisine Derivatives and Pharmaceutical Salts Thereof

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN201110034201.X 2011-01-31
CN201110034201XA CN102614168A (zh) 2011-01-31 2011-01-31 一种青蒿素衍生物及其药用盐的应用

Publications (1)

Publication Number Publication Date
WO2012103783A1 true WO2012103783A1 (zh) 2012-08-09

Family

ID=46554622

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2012/070401 Ceased WO2012103783A1 (zh) 2011-01-31 2012-01-16 一种青蒿素衍生物及其药用盐的应用

Country Status (3)

Country Link
US (1) US20130317095A1 (zh)
CN (1) CN102614168A (zh)
WO (1) WO2012103783A1 (zh)

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103202836B (zh) * 2012-01-16 2015-05-13 上海交通大学医学院附属瑞金医院 青蒿素衍生物及其药用盐用于制备治疗急性髓细胞性白血病的药物
CN103202835B (zh) * 2012-01-16 2015-05-13 上海交通大学医学院附属瑞金医院 青蒿素衍生物及其药用盐用于制备治疗急性白血病的药物
CN103202837B (zh) * 2012-01-16 2015-05-13 上海交通大学医学院附属瑞金医院 青蒿素衍生物及其药用盐用于治疗制备白血病的药物
GB201800736D0 (en) * 2018-01-17 2018-02-28 St Georges Hospital Medical School Combination therapy for treatment of leukemia
CN120078895B (zh) * 2025-04-25 2025-09-23 中国中医科学院医学实验中心 青蒿素类化合物在制备抗结节性硬化症相关肾血管平滑肌脂肪瘤药物中的应用

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101421276A (zh) * 2006-04-11 2009-04-29 赛诺菲-安万特 青蒿素衍生物的二聚物，它们的制备与它们的治疗应用
CN102153564A (zh) * 2011-01-31 2011-08-17 中国科学院上海药物研究所 含氮原子的青蒿素二聚体、其制备方法及用途

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5677468A (en) * 1995-06-29 1997-10-14 Hauser, Inc. Artemisinin dimer compounds having anticancer activity

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101421276A (zh) * 2006-04-11 2009-04-29 赛诺菲-安万特 青蒿素衍生物的二聚物，它们的制备与它们的治疗应用
CN102153564A (zh) * 2011-01-31 2011-08-17 中国科学院上海药物研究所 含氮原子的青蒿素二聚体、其制备方法及用途

Also Published As

Publication number Publication date
US20130317095A1 (en) 2013-11-28
CN102614168A (zh) 2012-08-01

Similar Documents

Publication Publication Date Title
Hu et al. Lycorine is a novel inhibitor of the growth and metastasis of hormone-refractory prostate cancer
WO2012103783A1 (zh) 一种青蒿素衍生物及其药用盐的应用
CN107686482A (zh) 新型七环化合物,其合成,活性评价及应用
US11944625B2 (en) Calmodulin inhibitors, Chk2 inhibitors and RSK inhibitors for the treatment of ribosomal disorders and ribosomapathies
CN102274220A (zh) 索拉菲尼在制备逆转肿瘤多药耐药性的药物中的应用
CN102764259A (zh) 血根碱类化合物在制备防治癌症的药物中的用途和药物组合物
Abbasi et al. All-trans retinoic acid-mediated miR-30a up-regulation suppresses autophagy and sensitizes gastric cancer cells to cisplatin
CN111732610A (zh) 一种具有抗肿瘤和抗菌活性的环金属铱配合物及其制备方法和应用
CN102526022A (zh) 表没食子儿茶素没食子酸酯在制备抗肿瘤药物中的应用
CN102552908B (zh) 含青蒿素及青蒿素类衍生物和Bcl-2抑制剂的药物组合物及其应用
CN103251585B (zh) 青蒿素及其衍生物在抑制血小板衍生生长因子受体a中的作用及其应用
CN102106864A (zh) 淫羊藿苷在制备拮抗髓系衍生抑制性细胞药物中的应用
CN114805117B (zh) 抗肿瘤干细胞的紫草素及阿卡宁肟衍生物
CN108030777B (zh) 氯胍在制备抗肿瘤药物中的应用
CN111499649A (zh) 一种具有抗肿瘤活性的苯并二呋喃酮类化合物、制备方法及其用途
CN107286123B (zh) 一种二苯呋喃类化合物的制备方法与应用
Khojastehkiakola et al. Apoptotic effect of antioxidants with silver and titanium dioxide nanoparticles on glioblastoma cancer cells in BALB/C mice
CN103202836B (zh) 青蒿素衍生物及其药用盐用于制备治疗急性髓细胞性白血病的药物
CN103202837B (zh) 青蒿素衍生物及其药用盐用于治疗制备白血病的药物
KR20200090170A (ko) 디시클로플라틴을 함유하는 복합 산물, 이의 제조 및 용도
TWI542348B (zh) 含４－乙醯基－安卓奎諾－ｂ之組合物用於製備抑制癌細胞生長之藥物的用途
CN109481448A (zh) 雷公藤红素在诱导癌细胞自噬上的应用及其与替莫唑胺的联合用药
CN103202835A (zh) 青蒿素衍生物及其药用盐用于制备治疗急性白血病的药物
CN114908094A (zh) 一类反义寡核苷酸分子及其在制备治疗恶性肿瘤药物中的应用
CN112121051A (zh) 莫扎伐坦在制备抗消化道肿瘤药物中的应用

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: 12742069

Country of ref document: EP

Kind code of ref document: A1

WWE Wipo information: entry into national phase

Ref document number: 13982684

Country of ref document: US

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 12742069

Country of ref document: EP

Kind code of ref document: A1