WO2018184274A1 - 一种增强TRPV4-KCa2.3复合体耦联度的化合物及其在抗高血压中的应用 - Google Patents

一种增强TRPV4-KCa2.3复合体耦联度的化合物及其在抗高血压中的应用 Download PDF

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WO2018184274A1
WO2018184274A1 PCT/CN2017/084079 CN2017084079W WO2018184274A1 WO 2018184274 A1 WO2018184274 A1 WO 2018184274A1 CN 2017084079 W CN2017084079 W CN 2017084079W WO 2018184274 A1 WO2018184274 A1 WO 2018184274A1
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compound
trpv4
domain
protein
ion channel
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马鑫
何东旭
唐春雷
张鹏
陈震
蔡燕飞
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江南大学
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Priority to US15/773,553 priority Critical patent/US10336712B2/en
Priority to RU2019124088A priority patent/RU2718913C9/ru
Priority to EP17904901.0A priority patent/EP3556747B8/en
Publication of WO2018184274A1 publication Critical patent/WO2018184274A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/498—Pyrazines or piperazines ortho- and peri-condensed with carbocyclic ring systems, e.g. quinoxaline, phenazine
    • 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
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/517—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim ortho- or peri-condensed with carbocyclic ring systems, e.g. quinazoline, perimidine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/12—Antihypertensives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/70—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings condensed with carbocyclic rings or ring systems
    • C07D239/72—Quinazolines; Hydrogenated quinazolines
    • C07D239/86—Quinazolines; Hydrogenated quinazolines with hetero atoms directly attached in position 4
    • C07D239/88—Oxygen atoms
    • C07D239/90—Oxygen atoms with acyclic radicals attached in position 2 or 3

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  • the invention relates to the technical field of antihypertensive drugs, in particular to a compound for enhancing the spatial coupling degree of the endothelial cell ion channel complex TRPV4-KCa2.3 in inhibiting hypertension.
  • Hypertension is a lifelong disease, and patients generally need to take medication every day.
  • Diuretics were introduced in the 1960s, beta-blockers were introduced in the 1970s, calcium channel antagonists and angiotensin-converting enzyme inhibitors (ACEI) were introduced in the 1980s.
  • ACEI angiotensin-converting enzyme inhibitors
  • diuretics such as hydrochlorothiazide, bumetanide, indapamide and diuretic compound preparations;
  • calcium channel antagonists such as nifedipine, amlodipine, diltiazem, verapamil, etc.;
  • ⁇ -blockers such as propranolol, atenolol, metoprolol, labetalol, etc.;
  • angiotensin converting enzyme inhibitors such as captopril, enalapril, benazepril, lisinopril, etc.;
  • angiotensin II receptor antagonists such as losartan, valsartan, telmisartan, olmesartan and the like.
  • Hypertensive drugs with different mechanisms have their own advantages and disadvantages because of their differences in target. In the treatment of hypertension, it is necessary to select drugs suitable for patients. Most patients often adopt a combination after a drug fails to achieve therapeutic effects. The way of treatment.
  • the ransient receptor potential vanilloid 4 (TRPV4) is a member of the TRP channel vanilloid receptor subfamily and is a non-selective cation channel.
  • the TRPV4 channel has six transmembrane alpha helix domains, S1-S6, respectively. There is a pore-loop region between S5 and S6 that regulates the passage of ions, and both the N-terminus and the C-terminus are in the cell.
  • the TRPV4 channel must function as a functional homomeric or heteromeric tetramer in the transduction of the signal.
  • the N-terminus of the TRPV4 channel contains at least three ankyrin binding sites, and the ankyrin interacts with the TRPV4 channel, which inhibits the IP3 receptor and regulates the release of intracellular Ca 2+ .
  • the TRPV4 channel has a very high Ca 2+ permeability and is abundantly expressed in vascular endothelial cells, which acts as a Ca 2+ channel and is involved in endothelial cell signaling.
  • Small-conductance calcium-activated potassium channel SKca is divided into three classes of KCa2.1, KCa2.2, and KCa2.3, in which KCa2.3 is mainly expressed in neurons and glial cells as well as vascular smooth muscle cells and endothelial cells, KCa2.3 It plays an important role in the physiological activities of the human body, especially during the relaxation of smooth muscle. Sustained activation of KCa2.3 causes the membrane potential of vascular endothelial cells to continue to be superimposed and then connected to nearby smooth muscle. Blocking or inhibiting KCa2.3 greatly increases vascular resistance, produces peripheral blood vessel resistance, and increases blood pressure.
  • TRPV4 and KCa2.3 have physical interactions on vascular endothelial cells, and Ca 2+ enters cells through TRP channels to activate these potassium channels, which then cause vasodilation.
  • Ca 2+ enters cells through TRP channels to activate these potassium channels, which then cause vasodilation.
  • its specific interaction site has not been studied clearly. It is of great significance for the development of hypertension drugs by searching for its interaction sites and finding that the compounds acting on its sites.
  • the Applicant has provided an application of a compound which enhances the spatial coupling degree of the endothelial cell ion channel complex TRPV4-KCa2.3 to inhibit hypertension.
  • the present invention prepares a specific compound capable of simultaneously acting on these two sites by searching for the domain of the interaction site of the endothelial cell ion channel complex TRPV4-KCa2.3, and found that the compound can enhance TRPV4-KCa2.
  • the spatial coupling degree of the complex is of great significance for the development of hypertension drugs.
  • the Applicant provides a compound that enhances the spatial coupling of the endothelial cell ion channel complex TRPV4-KCa2.3, the domain of the interaction site of the endothelial cell ion channel complex TRPV4-KCa2.3 is TRPV4 protein
  • the Applicant also provides the use of the compounds described above which act on the domains of the TRPV4 and KCa2.3 interaction sites, thereby enhancing the spatial coupling of the complex TRPV4-KCa2.3.
  • the Applicant also provides the use of another such compound which inhibits hypertension.
  • the Applicant also provides the use of the AR2 domain of the TRPV4 protein of the interaction site of the endothelial cell ion channel complex TRPV4-KCa2.3 and the 17C domain of the KCa2.3 protein for inhibiting hypertension.
  • the present invention firstly finds the interaction site domain of TRPV4 protein and KCa2.3 protein by means of domain site mutation, and then screens compounds which can enhance the spatial coupling degree of TRPV4-KCa2.3 complex, and develops new preparation. The method of the compound is finally applied to a hypertensive mouse model to test its effect on hypertension.
  • the domain AR2 of TRPV4 and the domain 17C of KCa2.3 are the interaction sites of the interaction, and the compounds which act on these two sites simultaneously are prepared and found by the hypertensive mouse model.
  • the compound acts to inhibit hypertension by enhancing the spatial coupling degree of the TRPV4-KCa2.3 complex.
  • the invention has great significance for the development of hypertension drugs.
  • Figure 1 is a schematic diagram showing the three-dimensional structure of TRPV4 protein and KCa2.3 protein and their functional regions;
  • Example 2 is a diagram showing the results of searching for interaction sites between TRPV4 protein and KCa2.3 protein by fluorescence resonance energy transfer and random optical reconstruction microscopy in Example 2 of the present invention
  • Example 3 is a graph showing the results of double-target immunoprecipitation of the compound prepared in Example 1;
  • Example 4 is a graph showing the results of detecting the change in the coupling degree of the TRPV4-KCa2.3 complex after the compound prepared in Example 1 was applied to a hypertensive mouse model by using a fluorescence resonance energy transfer technique in Example 3 of the present invention;
  • Fig. 5 is a graph showing the results of detecting changes in blood pressure of the compound prepared in Example 1 after acting on three hypertensive mouse models.
  • HEK293 cells were purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences.
  • mice 8 week old C57BL/6 J male mice, SPF grade, purchased from Changzhou Cavans Experimental Animal Co., Ltd.
  • Plasmids and primers Plasmid templates containing CFP-TRPV4 whole gene or YFP-KCa2.3 whole gene were donated by Luo Guixiang Biomedical College, The Chinese University of Hong Kong; primers were purchased from Shanghai Shenggong Bioengineering Co., Ltd.
  • AR2 YGRKKRRQRRRTGKTCLPKALLNLSNGRNDTIPVLLDIAERTGNMREFINSPFRDIYY;
  • YGRKKRRQRRRRKLELTKAEKHVHNFMM was purchased from Shanghai Shenggong Bioengineering Co., Ltd.
  • Reagents primary anti-TRPV4 (sc-47527), purchased from Santa Cruz; primary anti-rabbit anti-KCa2.3 (APC-025), purchased from Alomone; secondary anti-Alexa Fluor 647 and Alexa Fluor 488 was purchased from Invitrogen.
  • the point mutation kit QuickChangeTM was purchased from Stratagene; the DNA product purification kit and plasmid extraction kit were purchased from Beijing Tiangen Biochemical Technology Co., Ltd.; DH5 ⁇ competent cells were purchased from Beijing Tiangen Biochemical Technology Co., Ltd.;
  • biotinylated JNc-440 synthesis steps are as follows:
  • TP6032-S8 8% high-salt feed (TP6032-S8) was purchased from Nantong Trophy Feed Technology Co., Ltd.; L-NAME (Nitro-L-arginine) (N5501-5G) was purchased from Sigma; angiotensin 2 (ANG-2) (A107852) purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.;
  • the PCR reaction system used was: 0.5 ⁇ l of template (CFP-TRPV4 whole gene or YFP-KCa2.3 whole gene), 25 ⁇ l of Prim Star HS, 0.5 ⁇ l of each of the upstream and downstream primers, and 50 ⁇ l of H 2 O.
  • the PCR reaction procedure was: pre-denaturation at 95 ° C for 2 min, denaturation at 95 ° C for 30 s, annealing at 55 ° C for 30 s, extension at 68 ° C for 5 min, cycle 30 times, and fully extended at 68 ° C for 10 min.
  • the PCR product purification kit was used for purification. The purified product was digested with DpnI enzyme.
  • the digestion reaction system was: 0.4 ⁇ l of DpnI enzyme, 5 ⁇ l of PCR purified product, 2 ⁇ buffer 2 ⁇ l, and ddH 2 O 12.6 ⁇ l.
  • the digestion conditions were 37 ° C for 1 h, and the inactivation conditions were 80 ° C for 20 min.
  • the digested product is transformed into DH5a competent cells, and the selected monoclonal cells are expanded, and the plasmid is extracted by a plasmid extraction kit, and the sample is subjected to gene sequencing.
  • the mutant plasmid was transfected into HEK293 cells, and the transfected cells were plated in a small dish. After 24 hours, the coupling of the two proteins before and after the mutation was detected by FRET AB mode of laser confocal microscopy.
  • the TRPV4- ⁇ AR2H and KCa2.3 ⁇ 17C plasmids were constructed according to the above gene mutation method and transfected into In HEK293 cells, cells were fixed with PBS containing 3% paraformaldehyde and 0.1% glutaraldehyde after 24 hours, and then washed with 0.1% sodium borohydride diluted with PBS. The cells were blocked, permeabilized in blocking solution (3% BSA and 0.2% Triton X-100 in PBS), incubated with primary antibody at 4 degrees overnight, washed three times, and then incubated with secondary antibody for 45 minutes at room temperature. .
  • Goat anti-TRPV4 and rabbit anti-KCa2.3 were used as primary antibodies, and the secondary antibodies were Alexa Fluor 647 and Alexa Fluor 488.
  • the two proteins were co-localized by random optical reconstruction microscopy (STORM) to further verify the accuracy of the FRET results.
  • TRANSM random optical reconstruction microscopy
  • Figure 2a shows that the FRET phenomenon is significantly reduced when the AR2 domain of TRPV4 or the 17C domain of KCa2.3 is mutated compared to other mutation sites.
  • the ultra-high-definition imaging experiment of Figure 2b was further verified. It is shown that the domain AR2 of TRPV4 and the domain 17C of KCa2.3 are binding sites of two proteins.
  • red indicates TRPV4 protein
  • green indicates KCa2.3 protein
  • yellow indicates green and red overlap.
  • Example 3 The compound prepared in Example 1 targets the domain AR2 of TRPV4 and the domain 17C of KCa2.3.
  • Example 4 The compound prepared in Example 1 can enhance the spatial coupling degree of endothelial cell ion channel complex TRPV4-KCa2.3
  • mice Eight-week-old male C57BL/6J mice were used. After one week of free diet, they were divided into four groups, namely 8% high salt group, L-NAME group, ANG-2 group and control group. Base blood pressure measurements were performed on 4 groups of mice to determine the baseline blood pressure of the mice. The control group did not change the condition, blood pressure was measured every day; the high-salt group was fed with 8% high-salt diet, and the blood pressure was measured every day; the L-NAME group was given 5% L-NAME solution, and the blood pressure was measured every day; the ANG-2 group was first 4 % chloral hydrate was anesthetized, and the mice were operated by a buried pump on the back.
  • Each pump was injected with 2.88 mg/ml ANG-2 solution 200 ul. After embedding, the suture was to be cured. After about 3 days, the blood pressure measurement was continued for about 4 weeks. The blood pressure of the high-salt group was stable at 120 mmHg, and the blood pressure of the L-NAME and ANG-2 groups was stable at around 130 mmHg for about one week. The blood pressure no longer rises and the modeling is considered successful.
  • the mesenteric endothelial cells of the primary hypertensive model mice were cultured in a constant temperature incubator, JNc-440 (10 ⁇ M) was added, and the cells were co-cultured for 96 hours.
  • the staining was carried out according to the immunostaining method described in Example 2, and then the coupling of the two proteins before and after the mutation was detected by the FRET AB mode of the laser confocal microscope.
  • Example 5 Effect of the compound prepared in Example 1 on three hypertensive model mice
  • mice Three hypertensive mouse models were constructed according to the method described in Example 4. After successful modeling, JNc-440 (1 mg/kg) was injected through the tail vein, and the blood pressure of the mice was measured.

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Abstract

本申请提供了一种增强内皮细胞离子通道复合体TRPV4-KCa2.3空间耦联度的化合物及其在抗高血压中的应用。

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一种增强TRPV4-KCa2.3复合体耦联度的化合物及其在抗高血压中的应用 技术领域
本发明涉及抗高血压药物技术领域，尤其是涉及一种增强内皮细胞离子通道复合体TRPV4-KCa2.3空间耦联度的化合物在抑制高血压中的应用。
背景技术
中国产业信息网发布的《2014-2019年中国抗高血压药物市场运营监测与发展前景研究报告》指出：高血压是一个终身疾病，一般病人需要终身每天服药。高血压药物发展经历了几个时代，60年代推出利尿剂，70年代推出β-受体阻滞剂，80年代推出钙通道拮抗剂和血管紧张素转化酶抑制剂(ACEI)等，90年代又开发出更具有特异性的血管紧张素II受体拮抗剂(沙坦类)，之后相继有多个单方和复方制剂获美国FDA批准上市，成为高血压治疗的一线药物。
抗高血压单药发展相对成熟，根据机理大致可分为五类：
(1)利尿剂：如氢氯噻嗪、布美他尼、吲达帕胺及利尿剂类复方制剂等；
(2)钙离子通道拮抗剂：如硝苯地平、氨氯地平、地尔硫卓、维拉帕米等；
(3)β受体阻滞剂：如普萘洛尔、阿替洛尔、美托洛尔、拉贝洛尔等；
(4)血管紧张素转化酶抑制剂：如卡托普利、依那普利、贝那普利、赖诺普利等；
(5)血管紧张素II受体拮抗剂：如氯沙坦、缬沙坦、替米沙坦、奥美沙坦等。
不同机制的高血压药品因为其作用靶点的差异，均有各自的优劣势，在高血压治疗中需要选择适合病人的药品，大部分患者在一种药物达不到治疗效果后，往往采取联合治疗的方式。
瞬时感受器电位离子通道香草素受体家族4(ransient receptor potential vanilloid4，TRPV4)是TRP通道香草素受体亚家族的成员，是一种非选择性阳离子通道。TRPV4通道具有六个跨膜α螺旋结构域，分别为S1-S6，在S5和S6之间有一个调控离子通过的孔环区域，其N末端和C末端均在细胞内。TRPV4通道在信号的转导作用必须形成功能性的同聚或异聚四聚体方可发挥作用。TRPV4通道的N末端包含至少三个锚蛋白结合部位，锚蛋白可以与TRPV4通道相互作用，可以抑制 IP3受体从而调节细胞内Ca2+的释放。TRPV4通道具有非常高的Ca2+通透性，大量表达于血管内皮细胞中，其作为Ca2+通道，参与内皮细胞的信号传导。
小电导钙激活钾离子通道SKca分为三类KCa2.1、KCa2.2、KCa2.3，其中KCa2.3主要表达分布在神经元和胶质细胞以及血管平滑肌细胞和内皮细胞其中，KCa2.3在人体的生理活动中起重要的作用，尤其是在平滑肌的松弛过程中。KCa2.3的持续激活导致了血管内皮细胞的膜电位持续超级化，然后连接到附近的平滑肌。阻断或抑制KCa2.3会大大增加血管阻力，产生血管外周阻力，增加血压。
研究表明TRPV4和KCa2.3在血管内皮细胞上存在物理相互作用，Ca2+通过TRP通道进入细胞激活这些钾离子通道，然后引起血管的舒张。但是，其具体的相互作用位点仍未研究清楚，通过寻找其相互作用位点并发现作用于其位点的化合物对于高血压药物的研发具有重大的意义。
发明内容
针对现有技术存在的上述问题，本申请人提供了一种增强内皮细胞离子通道复合体TRPV4-KCa2.3空间耦联度的化合物抑制高血压的应用。本发明通过寻找内皮细胞离子通道复合体TRPV4-KCa2.3的相互作用位点的结构域，制备了能同时作用于这两个作用位点的特异性化合物，发现该化合物能增强TRPV4-KCa2.3复合体的空间耦联度，并对高血压药物的研发具有重大的意义。
本发明的技术方案如下：
本申请人提供了一种增强内皮细胞离子通道复合体TRPV4-KCa2.3空间耦联度的化合物，所述内皮细胞离子通道复合体TRPV4-KCa2.3的相互作用位点的结构域为TRPV4蛋白的AR2结构域及KCa2.3蛋白的17C结构域，所述化合物为通式(1)所示化合物，该化合物编号为JNc-440：
Figure PCTCN2017084079-appb-000001
所述的增强内皮细胞离子通道复合体TRPV4-KCa2.3空间耦联度的化合物的制备方法具体步骤如下：
(1)丙二胺首先经叔丁氧羰基保护单个氨基：
Figure PCTCN2017084079-appb-000002
(2)将另一裸露的氨基与二苯基乙酰氯成酰胺：
Figure PCTCN2017084079-appb-000003
(3)酰胺产物在酸性条件下脱除叔丁氧羰基保护：
Figure PCTCN2017084079-appb-000004
(4)脱除氨基保护的化合物再与4-喹唑啉酮-2-甲酸乙酯发生胺酯交换得到目的化合物：
Figure PCTCN2017084079-appb-000005
本申请人还提供了所述的化合物的应用，所述化合物能作用于TRPV4和KCa2.3相互作用位点的结构域，从而增强复合体TRPV4-KCa2.3的空间耦联度。
本申请人还提供了另一种所述的化合物的应用，所述化合物能抑制高血压。
本申请人还提供了所述的内皮细胞离子通道复合体TRPV4-KCa2.3的相互作用位点的TRPV4蛋白的AR2结构域及KCa2.3蛋白的17C结构域在抑制高血压中的应用。
本发明有益的技术效果在于：
本发明首先通过结构域位点突变的方法寻找TRPV4蛋白和KCa2.3蛋白的相互作用位点结构域，然后筛选能增强TRPV4-KCa2.3复合体空间耦联度的化合物，并研发新的制备该化合物的方法，最后将该化合物作用于高血压小鼠模型，检测其对高血压的疗效。
经本发明人研究发现，TRPV4的结构域AR2和KCa2.3的结构域17C是其相互作用的作用位点，制备同时作用于这两个作用位点的化合物，通过作用高血压小鼠模型发现该化合物通过增强TRPV4-KCa2.3复合体空间耦联度从而起到抑制高血压的效果。本发明对于高血压药物的研发具有重大的意义。
附图说明
图1为TRPV4蛋白和KCa2.3蛋白的三维结构及其功能区域简图；
图2为本发明实施例2中运用荧光共振能量转移和随机光学重构显微技术寻找TRPV4蛋白和KCa2.3蛋白相互作用位点的结果图；
图3为实施例1制备的化合物的双靶点免疫共沉淀结果图；
图4为本发明实施例3中运用荧光共振能量转移技术检测实施例1制备的化合物作用于高血压小鼠模型后TRPV4-KCa2.3复合体耦联度变化的结果图；
图5为实施例1制备的化合物作用于三种高血压小鼠模型后，检测其血压变化的结果图。
具体实施方式
下面结合附图和实施例，对本发明进行具体描述。
以下实施例所使用实验材料如下：
细胞系：HEK293细胞购自中科院上海细胞库。
实验动物：8周龄C57BL/6 J雄性小鼠，SPF级，购自常州卡文斯实验动物有限公司。
质粒和引物：含CFP-TRPV4全基因或YFP-KCa2.3全基因的质粒模板由香港中文大学罗桂祥生物医学院赠送；引物均购自上海生工生物工程有限公司
短肽：
AR2：YGRKKRRQRRRTGKTCLPKALLNLSNGRNDTIPVLLDIAERTGNMREFINSPFRDIYY；
17C：YGRKKRRQRRRRKLELTKAEKHVHNFMM均购自上海生工生物工程有限公司。
试剂：一抗goat anti-TRPV4(sc-47527),购自Santa Cruz公司；一抗rabbit anti-KCa2.3(APC-025),购自Alomone公司；二抗Alexa Fluor 647和Alexa Fluor  488购自Invitrogen公司。
点突变试剂盒QuickChangeTM购自Stratagene公司；DNA产物纯化试剂盒和质粒提取试剂盒均购自北京天根生化科技有限公司；DH5α感受态细胞购自北京天根生化科技有限公司；
生物素化JNc-440合成步骤如下：
(1)化合物JNc-440在三氯氧磷的作用下得到羰基的氯化产物：
Figure PCTCN2017084079-appb-000006
(2)氯代产物与5-氨基-1-戊醇发生亲核取代反应：
Figure PCTCN2017084079-appb-000007
(3)生物素与上一步的产物发生酯化反应得到生物素化的JNc-440：
Figure PCTCN2017084079-appb-000008
8％高盐饲料(TP6032-S8)购自南通特洛菲饲料科技有限公司；L-NAME(Nitro-L-arginine)(N5501-5G)购自Sigma公司；血管紧张素2(ANG-2)(A107852)购自上海阿拉丁生化科技股份有限公司；
其它所有化学试剂均购自国药集团化学试剂有限公司。
实验仪器：大小鼠血压测量仪(BP98A)，购自SINSI公司；OSMOTIC PUMPS (植入式胶囊渗透压泵)购自ALZET公司；凝胶成像系统购自GENE公司；激光共聚焦显微镜购自Leica公司；
实施例1：化合物的制备方法
(1)将化合物1-丙烯二胺(30g,405mmol,1eq)溶解于150mL二氯甲烷中，冰浴搅拌；将2-叔丁基碳酸氢钠(16.1g,73mmol,0.18eq)溶解并稀释于50mL二氯甲烷中；将混合物缓慢倒入烧瓶中，室温搅拌3小时；反应完全后用薄层色谱法(TLC)检测，用50mL二氯甲烷稀释并用水洗涤数次，再用饱和NaCl溶液洗涤，之后用无水Na2SO4进行干燥，浓缩后获得化合物2(19g,27％)；
(2)化合物2(10g,57mmol,1eq)和三乙胺(TEA)(8.7g，86mmol,1.5eq)溶解于100mL二氯甲烷并于冰浴搅拌；二苯基乙酰氯(13.1g,57mmol,1eq)溶解于30mL二氯甲烷中，并缓慢倒入烧瓶内，室温搅拌2.5小时；反应完全后使用TLC检测，进行粗制品浓缩；柱层析法分离(20:1二氯甲烷/甲醇)获得化合物3(13.2g,63％)；
(3)化合物3(8g,22mmol,1eq)溶解于混合溶剂中(40mL：二氯甲烷：三氟乙酸＝4:1)，混合物油浴加热至35℃，1小时；完全反应后使用TLC检测，用氨水调节体系pH至8-9，直至产生固体沉淀；过滤干燥后获得化合物4(5.3g,90％)；
(4)将化合物4(2g,7mmol,2eq)和4-喹唑啉酮-2-甲酸乙酯(0.8g,3.5mmol,1eq)溶解于6mL乙醇中，置于微波反应器中100℃反应45分钟；完全反应后TLC检测，自然冷却溶液获得白色固体沉淀，过滤干燥后获得化合物5。该化合物5即本发明所述的作用于高血压的化合物。
实施例2：寻找TRPV4蛋白和KCa2.3蛋白相互作用位点的结构域
实验方法：根据TRPV4蛋白和KCa2.3蛋白的三维结构及其功能特点选择可能的结合位点(如图1所示)，所选择的结构域均主要用于调节蛋白质-蛋白质之间的关系，是蛋白质交互的平台。突变所选择的结合位点，使结合位点缺失，所用引物如表1所示。
表1
Figure PCTCN2017084079-appb-000009
Figure PCTCN2017084079-appb-000010
所用PCR反应体系为：模板(CFP-TRPV4全基因或YFP-KCa2.3全基因)0.5μl，Prim Star HS 25μl，上下游引物各0.5μl，加H2O补足至50μl。PCR反应程序为：预变性95℃2min，变性95℃30s，退火55℃30s，延伸68℃5min，循环30次，充分延伸68℃10min。PCR结束后用PCR产物纯化试剂盒进行纯化，纯化后产物用DpnI酶进行酶切，酶切反应体系为：DpnI酶0.4μl,PCR纯化产物5μl,10×buffer2μl,ddH2O 12.6μl。酶切条件为37℃1h，灭活条件为80℃，20min。将酶切产物转化DH5a感受态细胞，将筛选出的单克隆细胞扩培后用质粒提取试剂盒提取质粒，送样进行基因测序。
将突变质粒转染至HEK293细胞中，将转染后的细胞铺共聚焦小皿，24小时后通过激光共聚焦显微镜的FRET AB模式检测突变前后两种蛋白的耦联情况。
按照上述基因突变的方法构建TRPV4-△AR2H和KCa2.3△17C质粒，转染至 HEK293细胞中，24小时后细胞用含有3％多聚甲醛和0.1％戊二醛的PBS溶液固定，然后用PBS稀释的0.1％硼氢化钠清洗。细胞在封闭液(在PBS中加入3％BSA和0.2％Triton X-100)中经过封闭、渗透化处理，用一抗4度孵育过夜后，清洗三次，然后在室温下用二抗孵育45分钟。使用goat anti-TRPV4和rabbit anti-KCa2.3作为一抗，二抗是Alexa Fluor 647和Alexa Fluor 488。同时通过随机光学重构显微技术(STORM)将两种蛋白共定位，从而进一步验证FRET结果的准确性。同上方法制备样品，在干净的共聚焦小皿中心处加入大约4μL的成像缓冲液，缓冲液中包含了5％(w/v)葡萄糖，100mM半胱胺酸，0.8mg/mL葡糖氧化酶和40μg/mL过氧化氢酶，溶解在pH为7.5或8的Tris-HCL。在该STORM成像缓冲液中呈现光谱分辨单分子图像。
实验结果：图2a显示，与其它突变位点相比，突变TRPV4的AR2结构域或KCa2.3的17C结构域时，FRET现象均明显下降。图2b的超高清成像实验进一步验证。表明TRPV4的结构域AR2和KCa2.3的结构域17C为两个蛋白的结合位点。
图2a中由蓝色-绿色-黄色-红色，FRET效率依次升高。
图2b中红色表示TRPV4蛋白，绿色表示KCa2.3蛋白，黄色表示绿色和红色重叠。
实施例3：实施例1所制备的化合物以TRPV4的结构域AR2和KCa2.3的结构域17C为靶点。
实验方法：
原代分离C57BL/6 J老鼠肠系膜内皮细胞，置于细胞恒温培养箱培养，加入生物素化JNc-440(10μM/L)或者短肽和生物素化JNc-440(10μM/L)，与细胞共培养96小时。用RIPA裂解得到细胞蛋白，蛋白上清加入10μL链霉亲和素化磁珠，4℃孵育过夜。用磁力架吸附孵育后的悬液，得到亲和素化磁珠-生物素化JNc-440-蛋白复合物。用50μL 1xLoading buffer重悬复合物，沸水浴煮沸5分钟。样品通过SDS-PAGE进行分离，转移蛋白至PVDF膜上，5％BSA室温封闭4h，一抗4度孵育过夜，二抗室温孵育2h，加入ECL显色剂，用蛋白成像系统进行检测分析。
实验结果：
实验结果如图3显示，生物素化JNc-440能结合TrpV4及KCa2.3蛋白；当加入短肽AR2后，生物素化JNc-440结合TRPV4蛋白能力显著下降。另一方面，相比对照 组，当加入短肽17C后，生物素化JNc-440结合KCa2.3蛋白能力显著下降。结果提示生物素化JNc-440能结合TRPV4和KCa2.3，并且结合区域为AR2及17C。
实施例4：实施例1所制备的化合物能增强内皮细胞离子通道复合体TRPV4-KCa2.3的空间耦联度
实验方法：
选用8周龄雄性C57BL/6J小鼠，自由饮食预适应一周后，分四组，分别是8％高盐组、L-NAME组、ANG-2组和对照组。先对4组小鼠进行基础血压测量，确定小鼠血压基线。对照组条件不改变，每天测量血压；高盐组饲喂8％高盐饲料，后每天测量血压；L-NAME组饮用5％的L-NAME溶液，后每天测量血压；ANG-2组先4％水合氯醛进行麻醉，通过背部埋泵对小鼠进行手术，每个泵注射2.88mg/ml ANG-2溶液200ul，埋入后缝合待痊愈，约3天后开始测量血压持续测量约4周。高盐组血压稳定在120mmHg，L-NAME和ANG-2组血压稳定在130mmHg左右，持续一周左右，血压不再上升即可认为建模成功。
原代分离高血压模型老鼠的肠系膜内皮细胞，置于细胞恒温培养箱培养，加入JNc-440(10μM)，与细胞共培养96小时。根据实施例2所述的免疫染色方法进行染色，然后通过激光共聚焦显微镜的FRET AB模式检测突变前后两种蛋白的耦联情况。
实验结果：如图4所示，化合物作用于三种不同的的高血压小鼠模型的内皮细胞后，TRPV4和KCa2.3蛋白的空间耦联度发生了明显的提高。表明此化合物能增强离子通道复合体TRPV4-KCa2.3的空间耦联度。
实施例5：实施例1所制备的化合物对三种高血压模型小鼠的影响
实验方法：根据实施例4所述的方法构建3种高血压老鼠模型。建模成功后，通过尾静脉注射JNc-440(1mg/kg)，后对小鼠血压进行测量观察。
实验结果：如图5所示，化合物作用于三种不同的的高血压小鼠模型后，其血压发生了明显下降，说明该化合物对该高血压模型有明显的降压作用。

Claims (5)

  1. 一种增强内皮细胞离子通道复合体TRPV4- KCa2.3空间耦联度的化合物，其特征在于：所述内皮细胞离子通道复合体TRPV4- KCa2.3的相互作用位点的结构域为TRPV4蛋白的AR2结构域及KCa2.3蛋白的17C结构域，所述化合物为通式(1)所示化合物：
    Figure PCTCN2017084079-appb-100001
  2. 一种权利要求1所述的增强内皮细胞离子通道复合体TRPV4- KCa2.3空间耦联度的化合物的制备方法，其特征在于：具体步骤如下：
    (1)丙二胺首先经叔丁氧羰基保护单个氨基：
    Figure PCTCN2017084079-appb-100002
    (2)将另一裸露的氨基与二苯基乙酰氯成酰胺：
    Figure PCTCN2017084079-appb-100003
    (3)酰胺产物在酸性条件下脱除叔丁氧羰基保护：
    Figure PCTCN2017084079-appb-100004
    (4)脱除氨基保护的化合物再与4-喹唑啉酮-2-甲酸乙酯发生胺酯交换得到目的化合物：
    Figure PCTCN2017084079-appb-100005
  3. 权利要求1所述的化合物的应用，其特征在于所述化合物能作用于TRPV4和KCa2.3相互作用位点的结构域，从而增强复合体TRPV4- KCa2.3的空间耦联度。
  4. 权利要求1所述的化合物的应用，其特征在于所述化合物能抑制高血压。
  5. 权利要求1所述的内皮细胞离子通道复合体TRPV4- KCa2.3的相互作用位点的TRPV4蛋白的AR2结构域及KCa2.3蛋白的17C结构域在抑制高血压中的应用。
PCT/CN2017/084079 2017-04-07 2017-05-12 一种增强TRPV4-KCa2.3复合体耦联度的化合物及其在抗高血压中的应用 WO2018184274A1 (zh)

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