WO2024017310A1 - 一种防风草内酯衍生物及其药物组合物在肾脏纤维化防治中的应用 - Google Patents

一种防风草内酯衍生物及其药物组合物在肾脏纤维化防治中的应用 Download PDF

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WO2024017310A1
WO2024017310A1 PCT/CN2023/108239 CN2023108239W WO2024017310A1 WO 2024017310 A1 WO2024017310 A1 WO 2024017310A1 CN 2023108239 W CN2023108239 W CN 2023108239W WO 2024017310 A1 WO2024017310 A1 WO 2024017310A1
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acid
renal
application according
formula
compound
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French (fr)
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龙海波
鲍世琦
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南方医科大学珠江医院
合肥尚德药缘生物科技有限公司
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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
    • A61K31/365—Lactones
    • 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
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics

Definitions

  • the invention belongs to the field of medicinal chemistry, and specifically relates to the application of an Ovatodiolide derivative and its pharmaceutical composition in the prevention and treatment of renal fibrosis (RF) and/or kidney diseases related thereto.
  • RF renal fibrosis
  • CKD Chronic kidney disease
  • ESRD end-stage renal disease
  • RF is the common pathological path for all CKD (including primary and secondary glomerular diseases, tubular, interstitial and vascular diseases, and chronic rejection of transplanted kidneys) to end-stage renal disease (ESRD).
  • ECM extracellular matrix
  • glomerulosclerosis glomerulosclerosis and renal tubular atrophy are the main manifestations, indicating that the renal injury response has entered the final pathway marked by tissue structure modification and organ function loss.
  • RAAS renin-angiotensin-aldosterone system
  • SGLT sodium-glucose synergy transporter
  • the present invention provides a compound of formula (I) or a pharmaceutically acceptable salt thereof in the preparation of medicaments for treating and/or preventing renal fibrosis and/or renal diseases related thereto.
  • the structure of the compound of formula (I) is as follows:
  • the R 1 , R 2 , and R 3 are each independently selected from H, halogen, C 1-6 alkyl, and C 1-6 alkoxy.
  • Each R 4 is independently selected from halogen, C 1-6 alkyl, C 1-6 alkoxy; n is selected from 0, 1, 2, 3 or 4.
  • R 1 , R 2 , and R 3 are selected from methyl, ethyl, and propyl.
  • the pharmaceutically acceptable salts include acid addition salts formed by the compound of formula (I) and inorganic acids: for example, hydrochloric acid, hydrofluoric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, Pyrosulfate, phosphoric acid or nitric acid, or hydrogen sulfate; or acid addition salts with organic acids such as formic acid, acetic acid, acetoacetic acid, pyruvic acid, trifluoroacetic acid, propionic acid, butyric acid, hexanoic acid, heptanoic acid , undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxybenzoyl)benzoic acid, camphoric acid, cinnamic acid, cyclopentanepropionic acid, digluconic acid, 3-hydroxy- 2-naphthoic acid, nicotinic acid, paracetamol, pectic acid, pers
  • organic acids such as formic
  • the compound of formula (I) has the following structure:
  • the pharmaceutically acceptable salt of the compound of formula (I) has the following structure:
  • the kidney disease is selected from the group consisting of diabetic nephropathy, primary and secondary glomerulonephritis, hereditary kidney disease, acute kidney injury, chronic renal failure, lupus nephritis, renal vasculitis, renal vasculitis, Glomerulosclerosis, hypertensive nephropathy (nephrosclerosis), interstitial nephritis, autosomal dominant polycystic kidney disease, Alport syndrome, analgesic nephropathy, renal allografts associated with ischemia-reperfusion or rejection damage.
  • the kidney disease is selected from various primary and secondary glomerular diseases, tubulointerstitial diseases, inherited kidney diseases, renovascular diseases, acute kidney injury, chronic renal failure, and Renal allograft injury related to ischemia-reperfusion or rejection, etc.
  • the medicament contains 0.1 wt% to 99 wt%, preferably 0.5 wt% to 90 wt% of the compound of formula (I) or a pharmaceutically acceptable salt thereof.
  • the medicament further includes a pharmaceutically acceptable carrier and/or excipient.
  • the pharmaceutical composition further includes one or more additional prophylactic/therapeutic drugs.
  • the drug can be administered via both injection and oral administration.
  • injectable forms include Including intravenous injection and intramuscular injection; the medicine can be prepared into oral preparations, injection preparations, external preparations, etc., such as injections, tablets, pills, capsules, etc.
  • the medicament may be prepared as a single dose dosage form or a divided dose dosage form.
  • renal fibrosis in the present invention is the same as “renal fibrosis”.
  • the present invention provides a compound (I) or a pharmaceutically acceptable salt thereof for the preparation of drugs for preventing/treating renal fibrosis and/or renal diseases related thereto, and it has been experimentally verified that the compound ACT004 of the present invention can regulate RALB expression, inhibiting the activation of the transforming growth factor (TGF)- ⁇ 1/Smad signaling pathway, STAT3 signaling pathway and NF- ⁇ B signaling pathway, inhibiting the EMT process of renal tubular epithelial cells, delaying renal fibrosis, and thereby preventing and treating related kidney diseases. role.
  • TGF transforming growth factor
  • Figure 1 shows the picture of ACT004 improving renal fibrosis in UUO model and IRI model.
  • A Masson staining of renal tissue of UWO model and IRI model;
  • B Immunohistochemical staining of ACT004 on renal tissue of UU0 model and IR1 model, detecting Fibronectin and Collagen. l.
  • a-SMA Western blot method detects the effect of ACT004 on the renal tissue of UUC model and IRI model Fibronectin, Collagen l, protein expression levels and statistical graphs of a-SMA
  • D Western blot method detects the effect of different doses of ACT004 Protein expression levels and statistical diagrams of Fibronectin, Collagen 1, and a-SMA in renal tubular epithelial cells induced by TGF ⁇ 1 (*, p ⁇ 0.05).
  • Figure 2 shows that ACT004 improves renal fibrosis by inhibiting the TGF- ⁇ 1/Smad, STAT3, and NF- ⁇ B signaling pathways.
  • B Western blot method detects the protein expression levels and statistical diagrams of p-Smad2, p-Smad3, p-Stat3, and p-NF- ⁇ B in kidney tissue of IRI model detected by ACT004.
  • C Western blot method to detect the effects of different concentration gradients of ACT004 on renal tubular epithelial cells after induction with or without TGF- ⁇ 1 for 1 hour on p-Smad2, p-Smad3, p-Stat3, and p-NF- ⁇ B Protein expression levels and statistical graphs (*, p ⁇ 0.05; **, p ⁇ 0.01; ***, p ⁇ 0.001).
  • FIG. 3 is a diagram showing the process of RALB promoting renal fibrosis and renal tubular epithelial cell EMT.
  • A Western blot method detects the protein expression level and statistical chart of RALB acting on ACT004 in the kidney tissue of UUO model and IRI model;
  • B RALB stable overexpression cell line Efficiency;
  • C Western blot method detects the protein expression levels and statistical graphs of Fibronectin, Collagen I, and a-SMA in RALB renal tubular epithelial cells stably overexpressing RALB;
  • D Western blot method detects p in RALB renal tubular epithelial cells stably overexpressing -Protein expression levels and statistical graphs of Smad2, p-Ssmd3, p-Stat3, and p-NFKB (*, p ⁇ 0.05; **, p ⁇ 0.01; ***, p ⁇ 0.001).
  • the compound of formula (I) of the present invention or its pharmaceutically acceptable salt can be prepared with reference to CN111303178, wherein the compound with the following structure (code number ACT004) is obtained by referring to the preparation of compound 10:
  • fibrotic matrix In the initial stage of kidney injury, the production of fibrotic matrix is a tissue damage repair mechanism. After mild injury, this part of fibrotic matrix will be gradually absorbed without affecting the structure and function of the kidney.
  • CKD the continued deposition of fibrotic matrix escapes regulatory suppression, leading to the collapse of tissue and organ structures, reduced blood flow supply, reduced organ function, and ultimately reduced renal tissue repair function, causing renal failure. Therefore, the main characteristic of RF is scar formation caused by excessive deposition of ECM.
  • the pathological manifestation is the proliferation of ECM-producing cells such as fibroblasts and myofibroblasts, resulting in the excessive production and accumulation of collagen fibers and adhesive proteins, and the progressive development of kidney disease. glomerulosclerosis and interstitial fibrosis, ultimately leading to loss of renal function.
  • the complex multi-factor-regulated mechanism of RF has not been fully elucidated.
  • Chronic inflammatory response and the renal tubular epithelial cell to mesenchymal transition (TEMT) accompanying the injury repair process are two important mechanisms.
  • Mononuclear macrophages, Notch, and Wnt Hedgehog signaling pathway, TGF- ⁇ 1, basic fibroblast growth factor, angiotensin II and other cells and mediators, growth factors and signaling pathways are directly or indirectly involved in this process.
  • Injury to RTECs is an early event that initiates the fibrotic response, and there are many regulatory factors, including a variety of transcription factors, growth factors, cytokines, hormones and extracellular signals.
  • TGF- ⁇ 1 is the most important growth factor that initiates the occurrence and development of EMT among many fibrotic factors, and regulates the EMT process through the Smad-dependent classical pathway.
  • RTECs that undergo EMT lose their normal material transport function, and the cells undergo G2/M arrest, resulting in a decrease in proliferation and repair ability, and significant secretion phenotype transformation, including the expression levels of multiple pro-fibrotic factors including TGF- ⁇ 1. Significantly increased. Through the profibrotic factors they secrete, RTECs that undergo EMT interact with fibroblasts in the renal interstitium and induce the proliferation and activation of interstitial fibroblasts, thereby promoting the progression of RF.
  • selective blocking of TEMT can also significantly reduce the expression levels of multiple inflammatory factors in the kidney and the infiltration of inflammatory cells in the interstitial area, effectively delaying RF in various CKD models. form.
  • mice 60 8-week-old C57 mice, SPF grade, were provided by the Experimental Animal Center of Zhujiang Hospital, Southern Medical University, Guangzhou. Before the experiment, the animals were allowed to adapt to the indoor environment for 7 days, with a room temperature of 18°C to 22°C and a relative humidity of 65%. All mice had free access to water and were fed conventional feed (protein content 3.14 mg/g).
  • the immortal renal tubular epithelial cell line derived from mice was a gift from the research group of Professor Nie Xiaoli, School of Traditional Chinese Medicine, Southern Medical University.
  • mice undergoing UUO surgery were randomly divided into UUO group, UUO+low-dose ACT004 (12.5mg/kg.d) group, UUO+medium-dose ACT004 (25mg/kg.d) group, and UUO+high-dose ACT004 (50mg/kg.d) group.
  • UUO+low-dose ACT004 (12.5mg/kg.d) group
  • UUO+medium-dose ACT004 25mg/kg.d
  • UUO+high-dose ACT004 50mg/kg.d
  • Administration was administered via intragastric administration starting on the 4th day after the operation.
  • the sham group and the UUO group were given an equal volume of normal saline as a control treatment.
  • the left kidney tissue was collected.
  • mice intraperitoneally with tribromoethanol, make a midline incision or dorsal incision in the abdomen, free the left renal vein, clamp it with an arterial clip, place it in a 37.5°C thermostatic pad for 30 minutes, cover the opening with PBS moistened gauze, and release it after 30 minutes.
  • the artery is clamped, and the abdomen is closed after observing the recovery of renal congestion.
  • mice undergoing IRI surgery were randomly divided into IRI group, IRI+low-dose ACT004 (12.5mg/kg.d) group, IRI+medium-dose ACT004 group (25 mg/kg.d) group, IRI + high-dose ACT004 (50 mg/kg.d) group, plus sham group (sham group), a total of 5 groups, with 6 mice in each group.
  • Administration was given via intragastric administration starting on the 4th day after the operation.
  • the sham group and the IRI group were given an equal volume of normal saline as a control treatment.
  • the right nephrectomy was performed on the 10th day after the operation, and the left kidney tissue was collected on the 11th day after the operation.
  • the medium dose of ACT004 in mice is 25mg/kg ⁇ d, so low-dose (12.5mg/kg.d), medium-dose (25mg/kg.d) and high-dose groups (50mg/kg.d) were established. ).
  • ACT004 is made into a solution with physiological saline and administered once a day for 1 week starting from the 4th day after surgery.
  • the mice in the sham group, UUO group, and IRI group were fed with the same amount of normal saline every day for 1 week.
  • Kidney tissue was collected after 1 week of intragastric administration. Anesthetize with intraperitoneal injection of tribromoethanol at a concentration of 200 mg/kg/time, remove the eyeballs to collect blood, separate the serum, and store at -20°C or -70°C. Make an abdominal incision, use sterile gauze to open the abdominal organs, perfuse cold PBS through the heart until the organs turn white, take out the kidney tissue, weigh the entire left kidney, cut it open along the mid-sagittal plane, rinse it with PBS, and remove the bag. The membrane was fixed in 10% formaldehyde neutral buffer, pending pathological examination.
  • Renal pathology Masson staining was used to observe the renal pathological structure and collagen fiber deposition.
  • TGF- ⁇ 1 cytokine 10ng/ ⁇ L
  • NRK renal tubular epithelial cells
  • fibrous changes and increased fibrosis phenotype expression Fibronectin, Collagen I , ⁇ -SMA,
  • ACT004 intervention 2.5 ⁇ M, 5 ⁇ M, 10 ⁇ M
  • Ras GTPase superfamily member RALB Ras GTPase superfamily member RALB
  • EMT-related indicator ⁇ -SMA EMT-related indicator ⁇ -SMA
  • fibrosis phenotype-related indicators Fibronectin and Collagen I.
  • the plasmid pc-RALB (using the empty vector as the negative control pcDNA3.1) targeting the RALB sequence was constructed in Suzhou Jima Gene Co., Ltd. and transfected into NRK.
  • RALB EMT-related indicator ⁇ -SMA
  • fibrosis phenotype-related indicators Fibronectin, Collagen I.
  • ACT004 improves renal fibrosis by effectively regulating TGF- ⁇ 1/Smad, STAT3, and NF- ⁇ B signaling pathways
  • RALB can promote the EMT process of renal tubular epithelial cells in vitro and may promote renal fibrosis by activating the TGF- ⁇ 1/Smad signaling pathway, STAT3 signaling pathway and NF- ⁇ B signaling pathway.
  • the renal cortex of this animal model was thin and hydronephrosis was obvious, which is a typical obstructive renal fibrosis model.
  • IRI ischemia-reperfusion
  • ACT004 can significantly improve renal fibrosis changes in UUO mice and IRI mice, inhibit the synthesis of renal interstitial matrix proteins Fibronectin, Collagen I, and ⁇ -SMA, and inhibit the occurrence of EMT in renal tubular epithelial cells.
  • the renal protective function of ACT004 is mainly achieved by inhibiting the activation of the TGF- ⁇ 1/Smad signaling pathway, STAT3 signaling pathway and NF- ⁇ B signaling pathway.
  • ACT004 down-regulates the expression of RALB in the kidney tissue of UUO and IRI models in vivo, and overexpression of RALB aggravates renal fibrosis and induces the activation of the TGF- ⁇ 1/Smad signaling pathway, STAT3 signaling pathway and NF- ⁇ B signaling pathway.
  • ACT004 regulates the expression of RALB, inhibits the activation of TGF- ⁇ 1/Smad signaling pathway, STAT3 signaling pathway and NF- ⁇ B signaling pathway, inhibits the EMT process of renal interstitial tubular epithelial cells, and plays a role in delaying renal fibrosis, and then treat related kidney diseases.

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Abstract

本发明涉及一种式(I)化合物或其药学上可接受的盐在制备治疗和/或预防肾脏纤维化和/或与其相关的肾脏疾病的药物中的应用。所述式(I)化合物结构如下：

Description

一种防风草内酯衍生物及其药物组合物在肾脏纤维化防治中的应用
本申请要求2022年7月20日向中国国家知识产权局提交的，专利申请号为202210861718.4，发明名称为“一种防风草内酯衍生物及其药物组合物在肾脏纤维化防治中的应用”的在先申请的优先权。所述申请的全文通过引用的方式结合于本申请中。
技术领域
本发明属于药物化学领域，具体涉及一种防风草内酯(Ovatodiolide)衍生物及其药物组合物在肾脏纤维化(RF)和/或与其相关的肾脏疾病防治中的应用。
背景技术
慢性肾脏病(CKD)是一个严重危害人类健康的全球性重大公共卫生问题，影响着多达10-15％的人口，我国现有成年CKD患者约1.3亿。而且，随着糖尿病、高血压、肥胖和老龄化人口的增加，全球范围内CKD的发病率逐年增加。1990年至2017年间，全球所有年龄段人群的CKD患病率上升了29.3％，CKD已成为日益增长的健康和社会负担。
不论肾脏的原发疾病是什么，CKD一旦发生，将持续进展，最终导致终末期肾脏病(ESRD)。ESRD在病理学上表现为RF。RF是所有CKD(包括原发性、继发性肾小球疾病，肾小管、间质和血管疾病以及移植肾脏慢性排斥性病变等)发展至终末期肾脏病(ESRD)的共同病理归途，以细胞外基质(ECM)大量沉积、肾小球硬化及肾小管萎缩为主要表现，预示肾损伤反应进入了以组织结构改建和器官功能丧失为标志的最终通路。
迄今为止，除病因、饮食治疗外，几乎不存在一种经批准的专门针对RF的治疗方法。
(1)临床上现有的抗RF治疗策略，主要是基于血流动力学机制、减低肾小球滤过压，包括肾素-血管紧张素-醛固酮系统(RAAS)阻断剂、钠葡萄糖协同转运体(SGLT)-2抑制剂。然而，RAAS阻断虽能延缓却无法完全阻断CKD的进展，而且容易诱发高钾血症、急性肾损伤等，达不到令人满意的效果；SGLT-2抑制剂虽然具有一定的肾脏保护作用，但在CKD的中后期(3b-5期)应用受限。
(2)过去的十余年间，虽然基于分子机制的抗RF治疗方法很多，包括针对血管内皮细胞的增殖及功能障碍(内皮素受体拮抗剂等)、炎症细胞的浸润及炎症微环境(CCL2选择性抑制剂等)、肾小管上皮细胞(RTECs)的上皮-间充质转分化(EMT)及相关分泌表型(NF-κB抑制剂等)，以及成纤维细胞的活化(转化生长因子-β1拮抗剂等)等，但是，这些临床试验基本以失败告终。
正因为如此，CKD患者一旦出现RF，必将不可避免地朝着ESRD进展，最终需依赖透析治疗或肾脏移植生存，给家庭和社会带来极大的经济负担。因此，如何有效地预防、治疗RF已成为世界医学界共同关注的问题，积极研发疗效显著、毒副作用小、质量可控和使用方便的治疗RF的新药具有十分重要的意义。
目前未见关于本发明提供的式(I)防风草内酯衍生物或其药物组合物应用于预防、治疗RF和/或与其相关的肾脏疾病的报道。
发明内容
为了改善现有技术存在的技术问题，本发明提供一种式(I)化合物或其药学上可接受的盐在制备治疗和/或预防肾脏纤维化和/或与其相关的肾脏疾病的药物中的应用，所述式(I)化合物结构如下：
所述式(I)中：
所述R1、R2、R3各自独立地选自H、卤素、C1-6烷基，C1-6烷氧基。
每个R4各自独立地选自卤素、C1-6烷基、C1-6烷氧基；n选自0，1，2，3或4。
根据本发明的实施方案，优选地，所述R1、R2、R3选自甲基、乙基、丙基。
根据本发明的实施方案，所述药学上可接受的盐包括所述式(I)化合物与无机酸形成的酸加成盐：例如盐酸、氢氟酸、氢溴酸、氢碘酸、硫酸、焦硫酸、磷酸或硝酸，或硫酸氢盐；或者与如下有机酸形成的酸加成盐：例如甲酸、乙酸、乙酰乙酸、丙酮酸、三氟乙酸、丙酸、丁酸、己酸、庚酸、十一烷酸、月桂酸、苯甲酸、水杨酸、2-(4-羟基苯甲酰基)苯甲酸、樟脑酸、肉桂酸、环戊烷丙酸、二葡糖酸、3-羟基-2-萘甲酸、烟酸、扑酸、果胶酯酸、过硫酸、3-苯基丙酸、苦味酸、特戊酸、2-羟基乙磺酸、衣康酸、氨基磺酸、三氟甲磺酸、十二烷基硫酸、乙磺酸、苯磺酸、对甲苯磺酸、甲磺酸、2-萘磺酸、萘二磺酸、樟脑磺酸、柠檬酸、酒石酸、硬脂酸、乳酸、草酸、丙二酸、琥珀酸、苹果酸、己二酸、藻酸、马来酸、富马酸、D-葡糖酸、扁桃酸、抗坏血酸、葡庚酸、甘油磷酸、天冬氨酸、磺基水杨酸、半硫酸或硫氰酸。
优选地，所述式(I)化合物为如下结构：
更优选地，所述式(I)化合物药学上可接受的盐为如下结构：
根据本发明的实施方案，所述肾脏疾病选自糖尿病性肾病，原发性以及继发性肾小球肾炎、遗传性肾脏疾病、急性肾损伤、慢性肾衰竭、狼疮肾炎、肾血管炎、肾小球硬化、高血压性肾病(肾硬化)、间质性肾炎、常染色体显性多囊肾、Alport综合症、镇痛剂肾病、与缺血再灌注或排斥反应相关的肾脏同种异体移植损伤。
在一些实施方案中，所述肾脏疾病选自各种原发性以及继发性肾小球疾病、肾小管间质疾病、遗传性肾脏疾病、肾血管疾病、急性肾损伤、慢性肾衰竭，以及与缺血再灌注或排斥反应相关的肾脏同种异体移植损伤等。
在一些实施方案中，所述药物中含有0.1wt％～99wt％，优选为0.5wt％～90wt％的式(I)化合物或其药学上可接受的盐。
在一些实施方案中，所述药物还进一步包括药物学上可接受载体和/或赋形剂。
在一些实施方案中，所述药物组合物还包含一种或多种另外的预防/治疗药物。
在一些实施方案中，所述药物可经注射和口服两种形式给药，注射形式包 括静脉注射、肌肉注射；所述药物可以制备成口服制剂、注射制剂、外用制剂等，例如注射液、片剂、丸剂、胶囊等。
根据本发明的实施方案，所述药物可以制备为单剂量剂型或分剂量剂型。
本发明术语“肾脏纤维化”同“肾纤维化”。
本发明的有益效果
本发明提供了一种(I)化合物或其药学上可接受的盐用于制备预防/治疗肾脏纤维化和/或与其相关的肾脏疾病的药物，并经试验验证本发明化合物ACT004可以通过调控RALB的表达，抑制转化生长因子(TGF)-β1/Smad信号通路、STAT3信号通路和NF-κB信号通路的激活，抑制肾小管上皮细胞EMT过程，发挥延缓肾脏纤维化，进而防治与其相关的肾脏疾病的作用。
附图说明
图1为ACT004改善UUO模型和IRI模型肾脏纤维化图，A:UWO模型和IRI模型肾脏组织的Masson染色；B:ACT004作用于UU0模型和IR1模型肾脏组织的免疫组织化学染色，检测Fibronectin、Collagen l、a-SMA的表达；C:Western blot法检测ACT004作用于UUC模型和IRI模型肾脏组织Fibronectin、Collagen l、a-SMA的蛋白表达水平及统计图；D:Western blot法检测不同剂量ACT004作用于TGFβ1诱导的肾小管上皮细胞Fibronectin、Collagen 1、a-SMA的蛋白表达水平及统计图(*，p<0.05)。
图2为ACT004通过抑制TGF-β1/Smad、STAT3、NF-κB信号通路改善肾脏纤维化图，A:Western blot法检测ACT004作用于UUO模型肾脏组织p-Smad2、p-Smad3、p-Stat3、p-NF-κB的蛋白表达水平及统计图；B:Western blot法检测ACT004作用于IRI模型肾脏组织p-Smad2、p-Smad3、p-Stat3、p-NF-κB的蛋白表达水平及统计图；C:Western blot法检测不同浓度梯度ACT004作用于肾小管上皮细胞给予或不给予TGF-β1诱导1h后p-Smad2、p-Smad3、p-Stat3、p-NF-κB 的蛋白表达水平及统计图(*，p<0.05；**，p<0.01；***，p<0.001)。
图3为RALB促进肾脏纤维化及肾小管上皮细胞EMT过程图，A:Western blot法检测ACT004作用于UUO模型和IRI模型肾脏组织RALB的蛋白表达水平及统计图；B:RALB稳定过表达细胞株的效率；C:Western blot法检测稳定过表达RALB肾小管上皮细胞的Fibronectin、Collagen I、a-SMA的蛋白表达水平及统计图；D:Western blot法检测稳定过表达RALB肾小管上皮细胞的p-Smad2、p-Ssmd3、p-Stat3、p-NFKB的蛋白表达水平及统计图(*，p<0.05；**，p<0.01；***，p<0.001)。
具体实施方式
下文将结合具体实施例对本发明的技术方案做更进一步的详细说明。应当理解，下列实施例仅为示例性地说明和解释本发明，而不应被解释为对本发明保护范围的限制。凡基于本发明上述内容所实现的技术均涵盖在本发明旨在保护的范围内。
除非另有说明，以下实施例中使用的原料和试剂均为市售商品，或者可以通过已知方法制备。
实施例1：化合物的制备
本发明式(I)化合物或其药学上可接受的盐可参照CN111303178制备，其中参照化合物10的制备得到以下结构的化合物(代号ACT004)：
其化学名称为(1aS,4Z,7S,9E,10aS,13S,13aS,15aS)-9,15a-二甲基-13-((4-甲基 哌嗪-1-基)甲基)-1a,2,3,8,10a,13,13a,14,15,15a-10氢-5H-4,7-(亚甲基)呋喃[2,3-f]氧杂壬酸[2,3-j][1]氧杂环戊烯-5,12(7H)-二酮富马酸盐。
实施例2：ACT004的抗RF活性测试
在肾脏损伤的初始阶段，纤维化基质产生是组织损伤修复机制，当轻度损伤之后，这部分纤维化基质会被逐渐吸收而不影响肾脏的结构和功能。然而当CKD发生时，纤维化基质持续沉积逃脱监管压制，导致组织器官架构崩解、血流供应减少、脏器功能降低，最终降低肾脏组织修复功能引起肾功能衰竭。所以，RF的主要特点是ECM过度沉积导致瘢痕形成，病理学表现为成纤维细胞和肌成纤维细胞等ECM生成来源细胞增生，以致胶原纤维和黏连蛋白过度产生和堆积，进行性发展为肾小球硬化和肾间质纤维化，最终导致肾脏功能丧失。
复杂的多重因素调控的RF发生机制未被完全阐明，慢性炎症反应以及伴随损伤修复过程的肾小管上皮细胞间充质转分化(TEMT)是两个重要机制，单核巨噬细胞、Notch、Wnt和Hedgehog信号通路、TGF-β1、碱性成纤维细胞生长因子、血管紧张素II等多种细胞及介质、生长因子和信号通路直接或间接参与了这一过程。RTECs损伤是启动纤维化反应的早期事件，调控因子众多，包括多种转录因子、生长因子、细胞因子、激素以及细胞外信号。TGF-β1在众多致纤维化因子中是最主要的启动EMT发生发展的生长因子，通过Smad依赖性的经典通路调控EMT过程。发生EMT的RTECs丧失了正常的物质转运功能，细胞发生G2/M阻滞导致增殖修复能力下降，并出现显著的分泌表型转化，包括TGF-β1在内的多种促纤维化因子的表达水平显著上调。通过其分泌的促纤维化因子，发生EMT的RTECs与肾间质中成纤维细胞发生交互作用，并诱导间质成纤维细胞增殖活化，进而促进RF进展。选择性阻断TEMT除了能够保护RTECs功能、抑制肌成纤维细胞形成以外，还可以显著降低肾脏中多种炎性因子的表达水平及间质区炎症细胞的浸润，有效延缓多种CKD模型的RF形成。
2.1实验材料
2.1.1受试药物
ACT004。
2.1.2实验动物
8周龄C57小鼠60只，SPF级，广州南方医科大学珠江医院实验动物中心提供。实验前置动物于室内适应环境7天，室温18℃～22℃，相对湿度65％。所有小鼠均自由进水，常规饲料(蛋白质含量3.14mg/g)饲养。
2.1.3实验细胞株
小鼠来源的肾小管上皮细胞永生株来源于南方医科大学中医药学院聂晓莉教授课题组馈赠。
2.2实验方法
2.2.1 ACT004对单侧输尿管梗阻(UUO)和缺血再灌注(IRI)RF动物模型的治疗作用
2.2.1.1动物模型制作、分组
构建UUO模型方法：在三溴乙醇腹腔注射麻醉下，腹部正中切口手术范围剪毛及消毒，经腹部正中切口后找到左侧输尿管，游离上段输尿管，用4-0丝线进行完整的输尿管梗阻双结扎(UUO组)，假手术组(sham组)小鼠同法暴露和游离输尿管，但不结扎。分层缝合腹部切口，碘伏消毒。行UUO手术小鼠随机分为UUO组、UUO+低剂量ACT004(12.5mg/kg.d)组，UUO+中剂量ACT004(25mg/kg.d)组，UUO+高剂量ACT004(50mg/kg.d)组，加上假手术组(sham组)，共5组，每组6只小鼠。术后第4天开始通过灌胃给药，sham组、UUO组给予等体积的生理盐水作对照处理，术后第11天收集左侧肾脏组织。
构建IRI模型方法：三溴乙醇腹腔麻醉小鼠，腹部正中切口或背部切口，游离左侧肾静脉，动脉夹夹闭，放在37.5℃恒温垫中30min，PBS湿润纱布覆盖开口，30min后松开动脉夹，观察肾脏淤血恢复后关腹。行IRI手术小鼠随机分为IRI组、IRI+低剂量ACT004(12.5mg/kg.d)组，IRI+中剂量ACT004 (25mg/kg.d)组，IRI+高剂量ACT004(50mg/kg.d)组，加上假手术组(sham组)，共5组，每组6只小鼠。术后第4天开始通过灌胃给药，sham组、IRI组给予等体积的生理盐水作对照处理，术后第10天行右肾切除，术后第11天收集左侧肾脏组织。
2.2.1.2药物剂量设定、给药
根据预实验结果，小鼠ACT004的中等剂量为25mg/kg·d，故设立低剂量(12.5mg/kg.d)、中等剂量(25mg/kg.d)和高剂量组(50mg/kg.d)。ACT004用生理盐水配成溶液，术后第4天开始每日灌服1次，1周。sham组、UUO组、IRI组小鼠每日均以等量生理盐水灌服、持续1周。
2.2.1.3标本收集、处理
灌胃处理1周后收集肾脏组织。用三溴乙醇按200mg/kg浓度/1次腹腔注射麻醉，摘取眼球采血，分离血清，﹣20℃或﹣70℃保存。腹部切口，用无菌纱布拨开腹腔脏器，冷PBS经心脏灌注至脏器变白，取肾脏组织，全部左肾称重后，沿正中矢状面剖开，用PBS冲洗干净，除去包膜，投入10％甲醛中性缓冲液中固定，待做病理检测。
2.2.1.4观察指标、测定方法
2.2.1.4.1肾脏病理学：Masson染色观察肾脏病理结构及胶原纤维沉积情况。
2.2.1.4.2蛋白水平的检测：Ras GTP酶超家族成员RALB，EMT相关指标α-SMA、纤维化表型相关指标Fibronectin、Collagen I。
2.2.2 ACT004对TGF-β1诱导肾小管上皮细胞EMT的作用
2.2.2.1细胞模型的制备和分组
构建RF细胞模型的方法：采取人重组TGF-β1细胞因子(10ng/μL)干预肾小管上皮细胞(NRK)48小时，引起NRK呈现纤维样改变、纤维化表型表达升高(Fibronectin、Collagen I、α-SMA,)。加入TGF-β1的同时给予不同浓度的ACT004干预(2.5μM、5μM、10μM),共分为6组：空白对照组、空白对照+ACT004(5μM)组、TGF-β1模型组、TGF-β1+不同浓度ACT004干预组 (2.5μM、5μM、10μM)。
2.2.2.2观察指标、测定方法
蛋白水平的检测：Ras GTP酶超家族成员RALB，EMT相关指标α-SMA、纤维化表型相关指标Fibronectin、Collagen I。
2.2.3探索ACT004抗RF的作用与RALB蛋白的关系
2.2.3.1稳定过表达RALB细胞株的构建
在苏州吉玛基因股份有限公司构建靶向针对RALB序列的质粒pc-RALB(以空载体为阴性对照pcDNA3.1)，转染到NRK中。
2.2.3.2观察指标、测定方法
蛋白水平的检测：RALB、EMT相关指标α-SMA、纤维化表型相关指标Fibronectin、Collagen I。
2.3实验结果
2.3.1小鼠肾组织病理的变化
从图1的A，Masson染色显示UUO模型和IRI模型小鼠肾脏间质增宽、细胞外基质沉积、胶原纤维增加、炎症细胞浸润的情况在ACT004治疗组中明显减少，而纤维化指标Fibronectin、Collagen I、α-SMA的免疫组织化学染色结果非常客观地展示出ACT004治疗对Fibronectin、Collagen I、α-SMA表达的抑制。图C和D的Wb条带反映了ACT004在体内体外水平上均能发挥抑制RF进展的作用，差异具有统计学意义。
2.3.2 ACT004通过有效调节TGF-β1/Smad、STAT3、NF-κB信号通路从而改善肾脏纤维化
从图2的A和B来看，UUO模型和IRI模型肾脏组织p-Smad2、p-Smad3、p-Stat3、p-NFκB表达水平明显上调，ACT004治疗组中表达水平明显降低。C图的条带反映了TGF-β1诱导的肾小管上皮细胞中p-Smad2、p-Smad3、p-Stat3、p-NFκB表达水平升高，ACT004干预后表达降低，差异具有统计学意义。以上结果充分证实了ACT004在体内体外环境下均可抑制肾脏纤维化模型中TGF- β1/Smad信号通路、STAT3信号通路和NF-κB信号通路的激活。
2.3.3ACT004通过调控RALB表达实现抗肾脏纤维化作用
从图3的A来看，UUO模型和IRI模型肾脏组织RALB表达水平明显上调，ACT004治疗组中表达水平明显降低。B图的条带反映了pcRALB组细胞中RALB水平明显上调，表明该稳定过表达细胞株构建成功。在此条件下检测纤维化指标(C)以及p-Smad2、p-Smad3、p-Stat3、p-NFκB指标(D)，纤维化指标Fibronectin、Collagen I、α-SMA以及p-Smad2、p-Smad3、p-Stat3、p-NFκB的蛋白水平均显著上调，差异具有统计学意义。以上结果从正面证实体外环境下，RALB可促进肾小管上皮细胞EMT过程，并且可能是通过激活TGF-β1/Smad信号通路、STAT3信号通路和NF-κB信号通路来促进肾脏纤维化。
2.4讨论
2.4.1动物模型
单侧输尿管梗阻(UUO)实验结束时，该动物模型肾脏皮质菲薄，肾积水明显，属于典型的梗阻性肾脏纤维化模型。缺血再灌注(IRI)实验过程中，微型动脉夹阻断肾蒂后，可见肾脏由鲜红色先变白再变成紫黑色，表示夹闭成功；去除动脉夹，恢复血流灌注后，可见肾脏迅速由紫黑色变成鲜红色，是肾脏缺血再灌注的典型现象。
2.4.2治疗作用
ACT004能够明显改善UUO小鼠和IRI小鼠肾脏纤维化改变，抑制肾脏间质基质蛋白Fibronectin、Collagen I、α-SMA合成，抑制肾小管上皮细胞发生EMT。ACT004的肾脏保护功能主要通过抑制TGF-β1/Smad信号通路、STAT3信号通路和NF-κB信号通路的激活来实现。ACT004在体内水平下调UUO模型和IRI模型肾脏组织RALB的表达，并且RALB过表达后加重肾脏纤维化和诱导TGF-β1/Smad信号通路、STAT3信号通路和NF-κB信号通路的激活。综上，ACT004通过调控RALB的表达，抑制TGF-β1/Smad信号通路、STAT3信号通路和NF-κB信号通路的激活，抑制肾间质肾小管上皮细胞EMT过程，发挥延缓肾 脏纤维化，进而治疗与其相关的肾脏疾病的作用。
以上，对本发明的实施方式进行了说明。但是，本发明不限定于上述实施方式。凡在本发明的精神和原则之内，所做的任何修改、等同替换、改进等，均应包含在本发明的保护范围之内。

Claims (10)

  1. 一种式(I)化合物或其药学上可接受的盐在制备治疗和/或预防肾脏纤维化和/或与其相关的肾脏疾病的药物中的应用，所述式(I)化合物结构如下：
    所述式(I)中，
    所述R1、R2、R3各自独立地选自H、卤素、C1-6烷基，C1-6烷氧基；
    每个R4各自独立地选自卤素、C1-6烷基、C1-6烷氧基；n选自0，1，2，3或4。
  2. 根据权利要求1所述的应用，其特征在于，所述R1、R2、R3选自甲基、乙基、丙基。
  3. 根据权利要求1所述的应用，其特征在于，所述药学上可接受的盐选自所述式I化合物与无机酸形成的酸加成盐：例如盐酸、氢氟酸、氢溴酸、氢碘酸、硫酸、焦硫酸、磷酸或硝酸，或硫酸氢盐；或者与如下有机酸形成的酸加成盐：例如甲酸、乙酸、乙酰乙酸、丙酮酸、三氟乙酸、丙酸、丁酸、己酸、庚酸、十一烷酸、月桂酸、苯甲酸、水杨酸、2-(4-羟基苯甲酰基)苯甲酸、樟脑酸、肉桂酸、环戊烷丙酸、二葡糖酸、3-羟基-2-萘甲酸、烟酸、扑酸、果胶酯酸、过硫酸、3-苯基丙酸、苦味酸、特戊酸、2-羟基乙磺酸、衣康酸、氨基磺酸、三氟甲磺酸、十二烷基硫酸、乙磺酸、苯磺酸、对甲苯磺酸、甲磺酸、2- 萘磺酸、萘二磺酸、樟脑磺酸、柠檬酸、酒石酸、硬脂酸、乳酸、草酸、丙二酸、琥珀酸、苹果酸、己二酸、藻酸、马来酸、富马酸、D-葡糖酸、扁桃酸、抗坏血酸、葡庚酸、甘油磷酸、天冬氨酸、磺基水杨酸、半硫酸或硫氰酸。
  4. 根据权利要求1所述的应用，其特征在于，所述式(I)化合物为如下结构：
  5. 根据权利要求1所述的应用，其特征在于，所述式(I)化合物药学上可接受的盐为如下结构：
  6. 根据权利要求1-5任一项所述的应用，其特征在于，所述肾脏疾病选自各种原发性以及继发性肾小球疾病、肾小管间质疾病、遗传性肾脏疾病、肾血管疾病、急性肾损伤、慢性肾衰竭，以及与缺血再灌注或排斥反应相关的肾脏同种异体移植损伤等。
  7. 根据权利要求1-5任一项所述的应用，其特征在于，所述药物中含有0.1wt％～99wt％，优选为0.5wt％～90wt％的式I化合物或其药学上可接受的盐。
  8. 根据权利要求1-5任一项所述的应用，其特征在于，所述药物还包括药物学上可接受载体和/或赋形剂。
  9. 根据权利要求1-5任一项所述的应用，其特征在于，所述药物可经注射和 口服两种形式给药，注射形式包括静脉注射、肌肉注射。
  10. 根据权利要求1-5任一项所述的应用，其特征在于，所述药物可以制备成口服制剂、注射制剂、外用制剂等，例如注射液、片剂、丸剂、胶囊。
PCT/CN2023/108239 2022-07-20 2023-07-19 一种防风草内酯衍生物及其药物组合物在肾脏纤维化防治中的应用 WO2024017310A1 (zh)

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Publication number Priority date Publication date Assignee Title
CN109796429A (zh) * 2018-03-02 2019-05-24 郑州大学 穿心莲内酯十氢萘结构修饰衍生物系列iii及其制备方法和用途
CN111303178A (zh) * 2018-12-11 2020-06-19 天津尚德药缘科技股份有限公司 Ovatodiolide衍生物及其盐，制备方法及其在制备抗癌药物中的用途
WO2021236808A1 (en) * 2020-05-19 2021-11-25 Arjil Biotech Holding Company Limited Use of terpenoids in the treatment or prevention of fibrotic diseases

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109796429A (zh) * 2018-03-02 2019-05-24 郑州大学 穿心莲内酯十氢萘结构修饰衍生物系列iii及其制备方法和用途
CN111303178A (zh) * 2018-12-11 2020-06-19 天津尚德药缘科技股份有限公司 Ovatodiolide衍生物及其盐，制备方法及其在制备抗癌药物中的用途
WO2021236808A1 (en) * 2020-05-19 2021-11-25 Arjil Biotech Holding Company Limited Use of terpenoids in the treatment or prevention of fibrotic diseases
WO2021236811A1 (en) * 2020-05-19 2021-11-25 Arjil Biotech Holding Company Limited Use of terpenoids in the treatment or prevention of fibrotic diseases

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ZHANG YAN, YUE ZHANG: "The advances in research on the intervention of triptolide in kidney disease", CHINESE BULLETIN OF LIFE SCIENCES, vol. 24, no. 1, 15 January 2012 (2012-01-15), pages 69 - 73, XP093130234, DOI: 10.13376/j.cbls/2012.01.007 *

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