WO2011047595A1 - 羟基苯并吡喃酮类化合物在制备治疗白血病药物中的应用 - Google Patents

羟基苯并吡喃酮类化合物在制备治疗白血病药物中的应用 Download PDF

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WO2011047595A1
WO2011047595A1 PCT/CN2010/077334 CN2010077334W WO2011047595A1 WO 2011047595 A1 WO2011047595 A1 WO 2011047595A1 CN 2010077334 W CN2010077334 W CN 2010077334W WO 2011047595 A1 WO2011047595 A1 WO 2011047595A1
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李靖
孟坤
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北京盛诺基医药科技有限公司
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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
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the invention relates to the application of hydroxybenzopyrone compounds in the preparation of medicaments for treating leukemia
  • This invention relates to the use of hydroxybenzopyrone compounds.
  • the compounds of the invention are useful in the prevention and treatment of chronic myeloid leukemia.
  • the invention also relates to pharmaceutical compositions comprising these compounds, and to the use of said compounds, alone or in combination with other agents, for the treatment of chronic myeloid leukemia. Background technique
  • Estrogen is a steroid hormone produced by the endocrine system in the body and plays an important role in the reproductive system, cardiovascular system, central nervous system, immune system and skeletal system. Recent studies have shown that estrogen exerts biological effects through two signaling pathways, the nuclear pathway and the cell membrane pathway; estrogen and its receptor signal transduction system are widely involved in cell growth, differentiation and apoptosis in multiple system tissues in vivo. . A large number of studies have confirmed that estrogen receptor ER-ct is present in leukemia cells of leukemia patients, and its receptor expression level is correlated with clinical treatment prognosis; ER-ct-mediated signal transduction pathway is likely to be involved in leukemia The formation and development process.
  • estrogen receptors are divided into two subtypes, alpha and beta; wherein ER-ct includes three homologs, ER-a66, ER-a46 and ER-a36.
  • the ER-a36 is transcribed from a promoter located in the first intron of the ER-a66 gene and encoded by ER- (a partial exon of x66.
  • ER-CL36 lacks two transcriptional domains) , retaining the DNA binding domain and the dimerization functional region; more importantly, the hormone ligand binding region of ER-a36 lacks a partial helix region, thus completely changing its specificity and affinity for binding to hormone ligands.
  • ER-CL36 is mainly distributed in the cell membrane and cytoplasm, and is present in a small amount in the nucleus. ER-CL36 can regulate cell growth and apoptosis through estrogen cell membrane signal transduction pathway (1. ZY, Wang; XT, Zhang; P , Shen; BW, Loggie; YC, Chang; TF, Deuel. A variant of estrogen receptor- ⁇ , hER-a36: Transduction of estrogen- and antiestrogen-dependent membrane-initiated mitogenic signaling. PNAS. 2006, 103(24): 9063-9068. 2. LMJ, Lee; J, Cao; H, Deng; P, Chen; Z, Gatalica; ZY, Wang. ER-a36, a novel variant of ER- ⁇ , is expressed in ER-positive and - Negative human breast carcinomas. Anticancer Res. 2008, 28(lB): 479-483). Summary of the invention
  • the compounds of the present invention are useful as a system for regulating estrogen receptor bioinformatics and are useful for the prevention and treatment of chronic myelogenous leukemia.
  • the present invention relates to a compound of formula (I), a pharmaceutically acceptable salt or pharmaceutical composition thereof for improving, preventing and treating white blood in preparation
  • RR 2 and R 3 are independently of each other selected from the group consisting of hydrogen, methyl, ethyl, propyl, and isopropyl.
  • the compound of formula (I) is: 3,5,7-trihydroxy-2-(4-methoxyphenyl)-8-(3-methyl-2- Butene-)-4H-benzopyran-4-one (aka: Icaridin, Icaritin), also a compound of formula (I)
  • the compound of formula (I) is a demethylated product of a compound of formula ( ⁇ ), ie, demethylated icariin of formula ( ⁇ ): 3, 5, 7 - Trihydroxy-2-(4-hydroxyphenyl)-8-3-methyl-2-butene-)-4H-benzopyran-4-one.
  • the leukemia refers to Chronic myeloid leukemia (CML).
  • CML Chronic myeloid leukemia
  • RR 2 and R 3 are independently of each other selected from the group consisting of hydrogen, methyl, ethyl, propyl, and isopropyl.
  • the compound of formula (I) is: 3,5,7-trihydroxy-2-(4-methoxyphenyl)-8-(3-methyl-2- Butene-)-4H-benzopyran-4-one (aka: Epimedium, Icaritin), that is, the compound of formula (I) has the structure of ( ⁇ ):
  • the compound of formula (I) is a demethylated product of a compound of formula ( ⁇ ), ie, demethylated icariin of formula ( ⁇ ): 3, 5, 7 -trihydroxy-2-(4-hydroxyphenyl)-8-(3-methyl-2-butene-)-4H-benzopyran-
  • the ER- ⁇ subtype includes ER-a36, ER-a46 and ER-a66.
  • the compounds of the formula (1), ( ⁇ ), (III), and pharmaceutically acceptable salts or pharmaceutical compositions thereof are modulators of the estrogen receptor ER-a and ER- ⁇ subtypes.
  • compound of formula (I) includes formula (I) as defined herein, and all embodiments, preferred embodiments, more preferred embodiments, and particularly preferred embodiments of the compounds, including those disclosed in specific names or examples. Compounds, each of these compounds are particularly preferred embodiments of the compounds defined by the formula.
  • compound of the present invention means any compound of the formula (I) as defined above.
  • the invention also relates to salts, polymorphs, solvates and hydrates of the compounds of the invention.
  • pharmaceutically acceptable salt as used herein, unless otherwise stated, includes salts which may be present in the compounds of the invention.
  • treatment refers to a process of reversing, alleviating, inhibiting a disease or a condition, or preventing such a disease or condition.
  • treatment refers to the act of treatment, wherein the treatment is as defined above.
  • the invention further relates to a pharmaceutical composition
  • a pharmaceutical composition comprising a compound of formula (I) and a pharmaceutically acceptable carrier.
  • the pharmaceutical composition may be administered orally in the form of a tablet, a capsule, a pill, a powder, a sustained release, a solution, or a suspension; the pharmaceutical composition may also be administered parenterally in the form of a sterile solution, suspension or emulsion.
  • the pharmaceutical composition may also be administered topically in the form of an ointment or cream; the pharmaceutical composition may also be administered rectally in the form of a suppository.
  • the pharmaceutical composition can be formulated as a unit dosage form suitable for Single dose of precise dose.
  • the pharmaceutical composition includes a conventional pharmaceutical carrier or excipient and a compound of the present invention as an active ingredient.
  • the pharmaceutical compositions may include other medical or pharmaceutical formulations, carriers, adjuvants, and the like.
  • Suitable pharmaceutical carriers include inert diluents or fillers, water, and various organic solvents.
  • the pharmaceutical composition may contain other ingredients such as flavoring agents, binders, excipients and the like, if necessary.
  • the tablets may contain various excipients, for example citric acid and various disintegrating agents such as starch, alginic acid and certain complex silicates and binders such as sucrose, gelatin and arabic may be used. gum.
  • lubricants such as magnesium stearate, sodium lauryl sulfate and talc are often used in tablets.
  • a similar type of solid composition can also be used with soft and hard filled capsules.
  • Preferred materials include lactose or milk sugar and high molecular weight polyethylene glycol.
  • the aqueous suspension or elixirs may be combined with various sweetening or flavoring agents, pigments or dyes, if desired, emulsifiers or suspending agents, and diluents such as water, ethanol , propylene glycol, glycerol or a combination thereof is administered in combination.
  • Exemplary parenteral administration forms include solutions or suspensions of the active compounds in sterile aqueous solutions, for example, propylene glycol or aqueous dextrose. Such dosage forms can be suitably buffered if desired.
  • the aqueous composition of the present invention may include other pharmaceutically acceptable solutes, including additives and other drugs, as the case may be. Suitable additives are those well known in the art including, but not limited to, antioxidants, antibacterials, surfactants, chelating agents, sugars, and preservatives.
  • the aqueous composition of the present invention can be administered by injection, wherein the injection can be intramuscular, intravenous or subcutaneous.
  • the compounds of the invention may be administered alone in the intended route of administration and standard pharmaceutical practice, but are often administered with suitable pharmaceutical excipients, diluents or carriers known in the art. If appropriate, adjuvants may be added, including adjuvants, antioxidants, flavoring agents, or coloring agents.
  • suitable pharmaceutical compositions to provide immediate, delayed, modified, sustained, pulsatile or controlled release, depending on the particular route of administration and the specificity of the release profile, and Need to respond.
  • oral refers to an oral administration method in which the oral use amount of the animal is expressly specified, for example, in a food or beverage, directly into the oral cavity, or freely selected.
  • animal refers to warm-blooded animals such as dogs and humans that have adaptive functions in the animal kingdom.
  • Typical oral solid forms can include tablets, powders, multiparticulate formulations (granules), capsules, chewables, lozenges, films, patches, and the like.
  • Typical oral liquid (including semi-solid and gelatinous) forms can include solutions, elixirs, gels, sprays, liquid-filled chews, and the like.
  • Other oral administration forms may also be employed in which the active agent is suspended in a liquid or semi-solid carrier phase, such as a suspension.
  • parenteral administration means that the route of administration does not pass through the mouth.
  • parenteral administration may include topical and transdermal, rectal, vaginal, nasal, inhalation, and parenteral administration (requiring administration of the skin barrier through needle and needle-free methods, including implants and Reservoir).
  • compositions of the invention include tablets.
  • the tablets are prepared by a process selected from the group consisting of direct compression or wet, dry or melt granulation, melt coagulation processes and extrusion.
  • the tablet core portion of the compositions of the present invention may be single or multi-layered, coated with a suitable coating material well known in the art.
  • the oral liquid form of the compound of the present invention is preferably a solution or suspension in which the active compound is sufficiently dissolved or partially dissolved.
  • the solution comprises the active compound and a pharmaceutically acceptable solvent suitable for oral administration.
  • the solvent is a solvent which exhibits good solubility for the compounds of the present invention. These solvents typically comprise a major portion of the formulation, i.e., greater than 50% by weight, preferably greater than 80%, such as 95%.
  • the solution further comprises an adjuvant or an additive.
  • the additive or adjuvant is a taste masking agent, a palatant, a flavoring agent, an antioxidant, a stabilizer, a texture modifier, a viscosity modifier, and a solubilizer.
  • Another embodiment is a process for the preparation of a preferred oral liquid form of a compound of the invention wherein, in a suitable temperature range, the preferred components are optionally mixed by mechanical or ultrasonic agitation in a manner which facilitates dissolution rate.
  • the preparation of the compound of the present invention is isolated and extracted from the raw material of the Chinese medicinal material.
  • Preparation of the compound of the present invention With reference to the method described in another invention patent "Preparation method of icariin" (Meng Kun, CN 101302548A), the chemical ingredient icariin is isolated and extracted from the Chinese medicinal material Epimedium. That is, the compounds of the present examples are distinct.
  • Demethylated icariin was purchased from Shanghai Yousi Biotechnology Co., Ltd.
  • the inhibitory effect of compounds on CML cells and the induction of apoptosis The proliferation inhibition rate of K562 cells and primary cells derived from bone marrow of CML patients was observed by MTT assay. The bone marrow cells of CML patients were divided into blast phase and chronic phase. Normal human bone marrow cells were collected as control. The percentage of apoptotic cells after primary cells in the bone marrow of CML patients was detected by flow cytometry.
  • the concentration of K562 cells was adjusted to lx10 7 /ml with sterile saline.
  • Each NOD-SCID mouse was injected with Kx cells 2x10 6 /0.2 mL via the tail vein.
  • the tumor-bearing mice were randomly divided into four groups; the blank control group was given the same amount of solvent; the positive control group was given Imatinib (Glivec, Gleevec) 50 ⁇ ⁇ / ⁇ ; the experimental group was given two concentrations of the compound.
  • Tumor-bearing mice were intraperitoneally injected with different drugs once a day for 45 consecutive days. The number of peripheral blood leukocytes and the number of CD45 + leukemia cells in mice were measured weekly. The pathological changes of mouse spleens were observed by HE staining after death, and the expression of ERct-36 receptor in mouse spleen leukemia cells was detected by immunohistochemistry. .
  • Flow cytometry and indirect immunofluorescence microscopy were used to detect the ratio of ERa-36 receptor-expressing cells in ⁇ 562 cells without intervention and 8 ⁇ compound.
  • Western blot was used to detect ERa-36 receptor in K562 cells after different concentrations of compounds. The amount of protein expression.
  • the experimental results show that the compound having the structure of formula (I), especially the formula ( ⁇ ), can inhibit the proliferation of CML cells in a concentration-dependent manner, and induce apoptosis of bone marrow cells of CML patients in a concentration-dependent manner; Peripheral white blood cell count and CD45+ leukemia cell ratio; can also significantly reduce the spleen weight of leukemia mice; can also inhibit the expression of ERa-36 receptor protein in K562 cells in a concentration-dependent manner, thus indicating the structure of formula (I)
  • the compound can treat chronic myeloid leukemia.
  • Fig. 1 Effect of compound ( ⁇ ) on proliferation inhibition of K562 cells MTT assay.
  • Figure 2 Compound (I I) inhibits proliferation of bone marrow mononuclear cells from bone marrow primary leukemia cells and 11 non-hematologic tumor patients in 19 patients with MML.
  • IC 8 is a compound ( ⁇ ) 8 ⁇ ⁇ / ⁇ group
  • IC 4 is a compound ( ⁇ ) 4 ⁇ ⁇ / ⁇ group
  • Con is a blank control group.
  • Ima 50 is the Imatinib 50 g/g group.
  • IC 8 is compound (II) 8 g/g group
  • IC 4 is compound (II) 4 g/g group
  • Con is blank control group
  • Control is a blank control group
  • IC162 8 is compound (II) 8 g/g group
  • IC162 4 is compound (II) 4 g/g group
  • Ima 50 is Imatinib5 ( ⁇ g/g group).
  • Con is a blank control group
  • IC 8 is compound (II) 8 g/g group
  • IC 4 is compound (II) 4 g/g group
  • Ima 50 is Imatinib 5 ( ⁇ g/g group (compared with blank control group) , * P ⁇ 0.05; compared with the positive control group, # P ⁇ 0.05; compared with the compound (II) 4 g / g group, ⁇ P > 0.05).
  • Figure 8 Effect of compound (II) on ERct-36 receptor expression in spleen of tumor-bearing mice.
  • A, B, C and D were blank control group, Imatinib5 ( ⁇ g/g group, compound (II) 4 g/g group and compound ( ⁇ ) 8 ⁇ ⁇ / ⁇ group, respectively.
  • Figure 9 Effect of compound (II) on the expression of ERct-36 receptor in ⁇ 562 cell membrane.
  • the present invention relates to a compound of formula (I), or a pharmaceutically acceptable salt or pharmaceutical composition thereof, for use in the treatment of chronic myeloid leukemia.
  • the compound ( ⁇ ) of the examples will now be described in detail as an example.
  • the concentration of the compound ( ⁇ ) used in the other experiments was 0 ⁇ , 4 ⁇ , 8 ⁇ , 16 ⁇ , 32 ⁇ , 64 ⁇ .
  • the experimental results show that the example compound ( ⁇ ) can inhibit the proliferation of CML cells in a concentration-dependent manner and induce apoptosis of bone marrow cells of CML patients in a concentration-dependent manner.
  • the specific results are shown in Figure 1-3.
  • the experimental results show that the example compound ( ⁇ ) can significantly reduce the peripheral blood leukocyte count and the proportion of CD45+ white blood cells; the example compound ( ⁇ ) can significantly reduce the spleen volume of leukemia mice and reduce the spleen weight of leukemia mice.
  • the results also showed that the expression of ERct-36 receptor in the spleen of the blank control group was higher, and the expression of ERct-36 receptor was not observed in the spleen of the treated compound ( ⁇ ).
  • Example Compound (II) was applied to ⁇ 562 cells at a concentration of 8 ⁇ M, and the ratio of cells expressing the ERa-36 receptor was detected by immunofluorescence and flow cytometry.
  • the compound of the example ( ⁇ ) was treated with 0 ⁇ , 4 ⁇ , 8 ⁇ , 16 ⁇ , 32 ⁇ and 64 ⁇ , and the expression of ERa-36 was detected by Western blot.

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Description

说明书 羟基苯并吡喃酮类化合物在制备治疗白血病药物中的应用 技术领域
本发明涉及羟基苯并吡喃酮类化合物的用途。本发明化合物适用于预防和治 疗慢性粒细胞性白血病。本发明还涉及包含这些化合物的药物组合物， 并且涉及 使用所述化合物， 单独使用或与其他药剂联合使用， 治疗慢性粒细胞性白血病。 背景技术
雌激素是由体内内分泌系统产生的一种类固醇激素，在生殖系统、心血管系 统、 中枢神经系统、免疫系统和骨骼系统中都发挥着重要的作用。近年来的研究 表明， 雌激素通过细胞核途径和细胞膜途径两种信号转导通路发挥生物效应； 雌 激素及其受体信号转导系统广泛参与体内多个系统组织中细胞生长、分化和凋亡 过程。 已有大量研究证实雌激素受体 ER-ct存在于白血病患者的白血病细胞中， 且其受体表达水平与临床治疗预后存在相关性； ER-ct介导的信号转导途径很可 能参与了白血病的形成和发展过程。
目前研究证实雌激素受体分为 α和 β两种亚型； 其中 ER-ct包括三个同源异 构体 ER-a66、 ER-a46和 ER-a36。 异构体 ER-a36从位于 ER-a66基因的第一个 内含子中的启动子开始转录，利用 ER-(x66的部分外显子进行编码。因此， ER-CL36 缺失两个转录功能区， 保留了 DNA结合功能区和二聚化功能区； 更为重要的是 ER-a36 的激素配体结合区缺失了部分螺旋区， 从而完全改变了其与激素配体结 合的专一性和亲和力。 ER-CL36 主要分布于细胞膜和细胞浆中， 少量存在于细胞 核中。 ER-CL36可以通过雌激素的细胞膜信号转导途径调节细胞生长和凋亡 (1. ZY, Wang; XT, Zhang; P, Shen; BW, Loggie; YC, Chang; TF, Deuel. A variant of estrogen receptor-α, hER-a36： Transduction of estrogen- and antiestrogen- dependent membrane-initiated mitogenic signaling. PNAS. 2006, 103(24):9063-9068. 2. LMJ, Lee; J, Cao; H, Deng; P, Chen; Z, Gatalica; ZY, Wang. ER-a36, a novel variant of ER-α, is expressed in ER-positive and -negative human breast carcinomas. Anticancer Res. 2008, 28(lB):479-483)。 发明内容
本发明的化合物用作调节雌激素受体生物信息系统，可以用于预防和治疗慢 性粒细胞性白血病。
本发明涉及式 (I)的化合物， 其药学上可接受的盐或药物组合物在制备改善、 预防和治疗白血
Figure imgf000003_0001
(I)
其中，
R R2和 R3彼此独立地选自氢、 甲基、 乙基、 丙基、 异丙基。
在本发明的一个特别优选实施方案中， 式 (I)的化合物是： 3，5，7-三羟基 -2-(4- 甲氧基苯基) -8-(3-甲基 -2-丁烯 -)-4H-苯并吡喃 -4-酮 （又名： 淫羊藿素， Icaritin), 也即式 (I)的化合物
Figure imgf000003_0002
(Π)
在本发明的一个特别优选实施方案中， 式 (I)的化合物是式 （Π) 化合物的去 甲基产物， 即式 (ΠΙ)所示的去甲基淫羊藿素： 3，5，7-三羟基 -2-(4-羟基苯基) -8- 3- 甲基 -2-丁烯 -)-4H-苯并吡喃 -4-酮。
Figure imgf000004_0001
(ΠΙ)
所述白血病是指慢性粒细胞性白血病 (Chronic myeloid leukemia, CML)。 所述的式 (I)化合物，及其药学上可接受的盐或药物组合物， 在制备受雌激素 受体 ER-ct或 ER-β
Figure imgf000004_0002
(I)
其中，
R R2和 R3彼此独立地选自氢、 甲基、 乙基、 丙基、 异丙基。
在本发明的一个特别优选实施方案中， 式 (I)的化合物是： 3，5，7-三羟基 -2-(4- 甲氧基苯基) -8-(3-甲基 -2-丁烯 -)-4H-苯并吡喃 -4-酮 （又名： 淫羊藿素， Icaritin), 也即式 (I)的化合物具 (Π)的结构：
Figure imgf000004_0003
在本发明的一个特别优选实施方案中， 式 (I)的化合物是式 （Π) 化合物的去 甲基产物， 即式 (ΠΙ)所示的去甲基淫羊藿素： 3，5，7-三羟基 -2-(4-羟基苯基) -8-(3- 甲基 -2-丁烯 -)-4H-苯并吡喃 -
Figure imgf000005_0001
(ΠΙ)
所述的式 ω、 （π)、 cm) 化合物， 及其药学上可接受的盐或药物组合物， 在制备受雌激素受体 ER-ct或 ER-β亚型调控的相关疾病的药物中的应用， 所述 ER-α亚型包括 ER-a36， ER-a46和 ER-a66。
所述式 (1)、 （Π)、 (III) 化合物， 及其药学上可接受的盐或药物组合物是作 为雌激素受体 ER-a和 ER-β亚型的调节剂。
术语"式 （I) 化合物"包括如这里定义的式 （I) 以及这化合物的所有实施方 案， 优选实施方案， 更优选实施方案以及特别优选实施方案， 包括有具体名称的 或实施例中公开的化合物，这些化合物的每个都是通式定义的化合物的特别优选 实施方案。 关于"本发明的化合物"是指上述定义的式 （I) 中的任何化合物。
本发明还涉及到本发明的化合物的盐， 多晶型物， 溶剂化物和水合物。 这里 使用的术语"药学上可接受的盐"， 除非另有所述， 包括可以在本发明的化合物中 存在的盐。
用于本发明的动词"治疗"， 除非另有所述， 是指逆转、 减轻、 抑制疾病或病 症的过程， 或预防这样的疾病或病症。 这里使用的名词"治疗"是指治疗的行为， 其中的治疗如上面所定义。
本发明还涉及一种药物组合物， 所述的药物组合物包括式（I)的化合物和一 种药学上可接受的载体。 例如， 药物组合物可以以片剂、 胶囊、 丸剂、 粉末、 缓 释剂、 溶液、 悬浮液的形式口服给药； 药物组合物也可以以无菌液、 悬浮液或乳 液的形式肠胃外注射给药； 药物组合物也可以以软膏或霜膏的形式局部给药； 药 物组合物也可以以栓剂的形式直肠给药。药物组合物可以做成单位剂型， 其适于 精确剂量的单一给药。药物组合物包括常规的药物载体或赋形剂和作为活性成分 的本发明的化合物。 另外， 药物组合物可以包括其它医学或药学制剂、 载体、 助 剂等等。
合适的药物载体包括惰性稀释剂或填料、 水和各种有机溶剂。 如果需要， 本 药物组合物可以含有其它的成分， 例如调味剂、 粘合剂、 赋形剂等等。 因此对于 口服给药， 片剂可以包含各种赋形剂， 例如可以使用柠檬酸和各种崩解剂例如淀 粉、 褐藻酸和某些复杂的硅酸盐和粘合剂例如蔗糖、 明胶和阿拉伯胶。 另外， 经 常在片剂中使用润滑剂例如硬脂酸镁、十二烷基硫酸钠和滑石粉。 固体组合物的 一个相似类型也可以使用软的和硬的填充胶囊。优选的材料包括乳糖或奶糖和高 分子量的聚乙二醇。当希望通过可口服给予本发明活性化合物时， 水悬浮液或酏 剂可以与各种增甜剂或调味剂、 色素或染料以及， 如果需要， 乳化剂或悬浮剂， 以及稀释剂例如水、 乙醇、 丙二醇、 甘油或其组合结合给药。
示范性的肠胃外给药形式包括活性化合物在无菌水溶液中的溶液或悬浮液， 例如， 丙二醇或右旋糖水溶液。 如果需要， 这样的剂型可以适当地缓冲。 视情况 而定，本发明的含水组合物可以包括其它药学上可接受的溶质， 包括添加剂及其 它药物。 合适的添加剂是本领域熟知的那些， 包括但不限于， 抗氧化剂、 抗菌药 物、 表面活性剂、 螯合剂、 糖、 以及防腐剂。 本发明的含水组合物可以通过注射 给药， 其中注射可以是肌肉注射、 静脉注射或皮下注射。
在给药的预定途径和标准药物操作方面， 本发明的化合物可以单独给药， 但 常常与本领域公知的合适的药物赋形剂、稀释剂或载体一起给药。如果合适的话， 可以加入辅助剂， 所述的辅助剂包括防腐剂、 抗氧化剂、 调味剂、 或着色剂。 本 发明的化合物可以配制成合适的药物组合物， 以提供直接的、 延迟的、 改性的、 持久的、脉冲的或控制的释放， 取决于具体给药途径以及释放分布的特异性， 与 治疗需要相对应。
本发明的化合物可以按以下路线给药， 例如， 但不限于， 本领域公知的口服 (包括口腔、 舌下等等） 形式。 在这种情况下， "口服"是指口服给药方式， 其中 动物的口服使用量是明文规定的， 例如在食物或饮料中， 直接放入口腔中， 或者 是自由选择使用量。在本发明中， 术语"动物"是指动物界拥有适应性机能的温血 动物， 例如犬和人类。 典型的口服的固体形式可以包括片剂、 粉剂、 多微粒制剂 （颗粒）、 胶囊、 咀嚼剂、 锭剂、 膜剂、 贴片等等。 典型的口服液体（包括半固体和胶质）形式可 以包括溶液、 酏剂、 凝胶剂、 喷雾剂、 液体填充的咀嚼剂等等。 也可以使用其它 口服给药形式， 其中活性剂悬浮在液体或半固体载体相中， 例如悬浮液。
本发明的化合物还可以通过肠胃外给药。术语肠胃外给药是指给药路线不通 过口腔。 优选对于本发明的化合物， 肠胃外给药可以包括局部和经皮、 直肠、 阴 道、 鼻腔、 吸入和注射给药 （需要通过针和无针方法渗透皮肤屏障的给药方式， 包括植入物和储层）。
本发明的组合物包括片剂。 在一种优选实施方案中， 片剂通过选自直接压片 或湿、 干或熔融造粒、 熔融凝结方法和挤出的方法制备。 在另一个实施方案中， 本发明的组合物的片剂核部分可以是单层或多层的，用本领域公知的合适包衣材 料进行包衣。
本发明化合物的口服液形式优选是溶液或悬浮液， 其中活性化合物是充分溶 解或部分溶解的。在一种实施方案中， 溶液包含活性化合物和药学上有先例的适 于口服的溶剂。在一种优选实施方案中， 溶剂是一种对本发明的化合物显现出良 好溶解度的溶剂。这些溶剂通常占配方的主要部分， 即大于 50% (按重量计算）， 优选大于 80%， 例如 95%。 在一种优选实施方案中， 溶液进一步包含辅助剂或 添加剂。 在其一种优选的实施方案中， 添加剂或辅助剂是一种掩味剂、 可口剂、 调味剂、 抗氧化剂、 稳定剂、 质地改性剂、 粘度调节剂和增溶剂。
另一个实施方案是制备本发明化合物的优选口服液形式的方法， 其中在适宜 温度范围内，各个优选组分以一种有利于溶解速度的方式任选通过机械搅拌或超 声波搅拌混合。
本发明化合物的制备从中药材原料中提取分离得到。 本发明的化合物的 制备参照另一发明专利 "淫羊藿素的制备方法"（孟坤， CN 101302548A) 中所 述的方法， 自中药材淫羊藿中分离提取出化学成份淫羊藿素， 也即本分明实施例 化合物。
去甲基淫羊藿素从上海友思生物技术有限公司购买。
生物实验及测试
1. 化合物对 CML细胞的抑制增值和诱导凋亡效应： 用 MTT实验观察不同浓度的化合物对 K562细胞和来自 CML患者骨髓的原 代细胞的增殖抑制率， CML患者的骨髓细胞分为急变期和慢性期两种， 另采集 正常人骨髓细胞作为对照； 用流式细胞仪检测不同浓度化合物作用于 CML患者 骨髓的原代细胞后凋亡细胞的百分率。
2. 化合物对移植 K562细胞株的 NOD-SCID小鼠的治疗作用：
用无菌生理盐水调整 K562细胞浓度为 l x l07/ml，每只 NOD-SCID小鼠经尾 静脉注射 K562细胞 2x l06/0.2mL。 荷瘤小鼠随机分为四组； 空白对照组给予等 量溶剂； 阳性对照组给予 Imatinib ( Glivec, 格列卫） 50μ§/§ ; 实验组分别给予 两个浓度的化合物。 每日一次给予荷瘤小鼠腹腔注射不同药物， 连续 45 日。 每 周检测小鼠外周血白细胞数和 CD45+白血病细胞数，小鼠死亡后通过 HE染色观 察小鼠脾的病理学变化， 并通过免疫组化检测小鼠脾脏白血病细胞 ERct-36受体 的表达。
3. 化合物对 K562细胞中 ERa-36受体表达的影响：
用流式细胞仪和间接免疫荧光显微镜检测无干预和 8μΜ化合物作用后 Κ562 细胞中表达 ERa-36受体的细胞的比例， 用 Western blot方法检测不同浓度化合 物干预后 K562细胞中 ERa-36受体蛋白表达量情况。
实验结果表明， 具有式 （I ) 结构的化合物特别是式 （Π)， 能以浓度依赖性 的方式抑制 CML细胞增殖， 并以浓度依赖性的方式诱导 CML患者骨髓细胞发 生凋亡； 能明显降低外周血白细胞计数和 CD45+白血病细胞比例； 同时还能明显 减轻白血病小鼠的脾脏重量；还能以浓度依赖性的方式抑制 K562细胞中 ERa-36 受体蛋白表达量， 因此表明式 （I ) 结构的化合物能治疗慢性粒细胞性白血病。 附图说明
图 1 化合物（Π)对 K562细胞的增殖抑制效应 MTT试验。
图 2 化合物（I I)对 19例 CML患者骨髓原代白血病细胞和 11例非血液肿瘤患者 骨髓单个核细胞的增殖抑制效应 MTT试验。
图 3 化合物（I I)对 CML患者骨髓原代白血病细胞凋亡率的影响 (与对照组相比 * P<0.05， ** P<0.01
图 4 化合物（I I)对小鼠外周血白细胞计数随生存时间变化的影响。
图中 IC 8为化合物 (Π)8μ§/§组， IC 4为化合物 (Π)4μ§/§组， Con为空白对照组， Ima 50为 Imatinib50 g/g组。
图 5 化合物（II)对小鼠外周血人 CD45抗原表达随生存时间变化的影响。
图中 IC 8为化合物 (II)8 g/g组， IC 4为化合物 (II)4 g/g组， Con为空白对照组， Ima
50为 Imatinib5(^g/g组。
图 6 化合物（II)对荷瘤小鼠脾脏大小的影响。
图中 Control为空白对照组， IC162 8为化合物 (II)8 g/g组， IC162 4为化合物 (II)4 g/g组， Ima 50为 Imatinib5(^g/g组。
图 7 化合物（II)对荷瘤小鼠脾脏平均重量的影响。
图中 Con为空白对照组， IC 8为化合物 (II)8 g/g组， IC 4为化合物 (II)4 g/g组， Ima 50为 Imatinib5(^g/g组 （与空白对照组比较， * P<0.05; 与阳性对照组比较， # P 〉0.05; 与化合物 (II)4 g/g组比较， △ P〉0.05)。
图 8 化合物（II)对荷瘤小鼠脾脏 ERct-36受体表达的影响。
其中 A、 B、 C和 D分别为空白对照组、 Imatinib5(^g/g组、 化合物 (II)4 g/g组 和化合物 (Π)8μ§/§组。
图 9 化合物（II)对 Κ562细胞膜 ERct-36受体表达的影响。
图 10 化合物（II)对 K562细胞 ERct-36受体蛋白表达的影响。
具体实施方式
下面的实施例说明制备本发明的化合物和方法。 应该理解， 本发明并不受下 面提供的具体内容的限制。
下面的实施例说 (Π)化合物的制备。
Figure imgf000009_0001
3，5，7-三羟基 -2-(4-甲氧基苯基) -8-(3-甲基 -2-丁烯基 -)-4H-苯并吡喃 -4-酮 （又名： 淫羊藿素， Icaritin; 在生物测试实验中又名 IC162和 IC)
该实施例化合物的制备参照另一发明专利 "淫羊藿素的制备方法"（孟坤， CN 101302548A)中所述的方法， 自中药材淫羊藿中分离提取出化学单体淫羊藿 素， 也即本发明实施例化合物。
实施例化合物的生物实验
前面已描述了本发明涉及式（I )的化合物或其药学上可接受的盐或药物组合 物适用于治疗慢性粒细胞性白血病。 现以实施例的化合物 (Π)为例， 对其进行详 细说明。
( 1)实施例化合物（Π)对 CML细胞的抑制增值和诱导凋亡效应：
本实验具体实验方法如前生物实验及测试部分中实验 1 (化合物对 CML细 胞的抑制增值和诱导凋亡效应）所述。 除对 K562细胞周期的影响采用单一浓度 夕卜，其余实验中实施例化合物（Π)所用浓度均为 0μΜ、4μΜ、8μΜ、 16μΜ、32μΜ、 64μΜ。
实验结果显示实施例化合物 (Π)能以浓度依赖性的方式抑制 CML细胞增殖， 并以浓度依赖性的方式诱导 CML患者骨髓细胞发生凋亡。 具体结果见图 1-3。
(2) 实施例化合物（I I)对移植 Κ562细胞株的 NOD-SCID小鼠的治疗作用：
本实验具体实验方法如前生物实验及测试部分中实验 2 (化合物对移植 Κ562 细胞株的 NOD-SCID小鼠的治疗作用） 所述。 实施例化合物（I I)采用 4 g/g和 8μ§/§两个剂量。
实验结果显示实施例化合物 (Π)能明显降低外周血白细胞计数和 CD45+白血 病细胞比例； 实施例化合物 (Π)能明显减少白血病小鼠的脾脏体积同时减轻白血 病小鼠的脾脏重量。 实验结果还显示空白对照组小鼠脾脏中 ERct-36受体表达较 多， 实施例化合物 (Π)处理后的小鼠脾脏则未见 ERct-36受体表达。具体结果如图 4-8， 图中 IC 8和 IC162 8表示实施例化合物 (n)8 g/g组， IC 4和 IC162 4表示实 施例化合物 (Ii ^g/g 组， Con 和 Control 表示空白对照组， Ima 50 表示 Imatinib50 g/g组。
(3) 实施例化合物（I I)对 K562细胞中 ERa-36受体表达的影响：
本实验具体实验方法如前生物实验及测试部分中实验 3所述 (化合物对 K562 细胞中 ERa-36受体表达的影响）。实施例化合物（I I)采用 8μΜ浓度作用于 Κ562 细胞， 免疫荧光和流式细胞仪检测表达 ERa-36受体的细胞的比例。 实施例化合 物（Π)采用 0μΜ、4μΜ、8μΜ、 16μΜ、 32μΜ和 64μΜ浓度处理细胞， Western blot 方法检测细胞中 ERa-36表达情况。 实验结果显示 K562细胞膜存在 ERct-36受体强表达情况，实施例化合物（II) 能够显著降低 K562细胞膜 ERct-36受体的表达。 实施例化合物（Π)还能以浓度 依赖性的方式抑制 K562细胞中 ERct-36受体蛋白表达量。 具体结果见图 9-10。

Claims

权利要求书
1. 式 α) 的化合物及其药学上可接受的盐或药物组合物在制备改善、 预防
Figure imgf000012_0001
R R2和 R3彼此独立地选自氢、 甲基、 乙基、 丙基、 异丙基。
2. 根据权利要求 1的所述的应用， 其中的 R1为甲基、 R2和 R3均为氢， 式 (I) 化合物具有如下
Figure imgf000012_0002
(Π)
3. 根据权利要求 1的所述的应用， 其中的 R R2和 R3均为氢， 式 （I) 化 合物具有如下式 （ΠΙ) 。
Figure imgf000012_0003
4. 根据权利要求 1-3任一所述的应用， 所述白血病是慢性粒细胞性白血病 ( Chronic myeloid leukemia, CML )。
5. 式 （I) 的化合物及其药学上可接受的盐或药物组合物在制备受雌激素受 体 ER-ct或 ER-β亚型调控的相关疾病的药物中的应用，
Figure imgf000013_0001
(Π)
7. 根据权利要求 5所述的应用，所述式 (I)化合物中的 R^R2和 R3均为氢， 即 具有式 （III) 结构的化合物：
Figure imgf000013_0002
(ΠΙ)
8. 根据权利要求 5-7任一所述的应用，其特征在于， ER-α亚型包括 ER-a36， ER-a46 禾口 ER-a66。
9. 根据权利要求 8任一所述的应用， 所述式 (1)、 (II) 或 （III) 化合物及其 药学上可接受的盐或药物组合物是作为雌激素受体 ER-a和 ER-β亚型的调节剂。
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CN101528038A (zh) * 2006-10-25 2009-09-09 盛诺基医药科技有限公司 用于治疗与雌激素受体相关的疾病的化合物和方法

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CN102267971A (zh) * 2011-08-03 2011-12-07 华中科技大学 脂环并[c]苯并吡喃酮衍生物及其应用
EP2803665A4 (en) * 2012-01-13 2015-07-08 Beijing Shenogen Pharma Group Ltd SYNTHESIS OF BENZOPYRANE POLYHYDROXY KETONE COMPOUND AND CORRESPONDING ANTI-TUMOR EFFECT
US9221781B2 (en) 2012-01-13 2015-12-29 Beijing Shenogen Pharam Group Ltd. Synthesis of polyhydroxy chromenone compounds and their anti-tumor effects

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