WO2018177114A1 - 一种水杨酸基富马酸盐衍生物及在治疗帕金森症和其他神经退行性疾病应用 - Google Patents

一种水杨酸基富马酸盐衍生物及在治疗帕金森症和其他神经退行性疾病应用 Download PDF

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WO2018177114A1
WO2018177114A1 PCT/CN2018/078618 CN2018078618W WO2018177114A1 WO 2018177114 A1 WO2018177114 A1 WO 2018177114A1 CN 2018078618 W CN2018078618 W CN 2018078618W WO 2018177114 A1 WO2018177114 A1 WO 2018177114A1
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fumarate
salicylic acid
disease
derivative
structural formula
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PCT/CN2018/078618
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English (en)
French (fr)
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杨立川
托马斯鲍比
郭明跃
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重庆纽源生物科技有限公司
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Priority to JP2019551467A priority Critical patent/JP2020515536A/ja
Priority to EP18774681.3A priority patent/EP3611157A4/en
Priority to US16/485,487 priority patent/US20200048182A1/en
Publication of WO2018177114A1 publication Critical patent/WO2018177114A1/zh
Priority to US17/105,948 priority patent/US11866401B2/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C67/00—Preparation of carboxylic acid esters
    • C07C67/28—Preparation of carboxylic acid esters by modifying the hydroxylic moiety of the ester, such modification not being an introduction of an ester group
    • C07C67/29—Preparation of carboxylic acid esters by modifying the hydroxylic moiety of the ester, such modification not being an introduction of an ester group by introduction of oxygen-containing functional groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C69/00—Esters of carboxylic acids; Esters of carbonic or haloformic acids
    • C07C69/52—Esters of acyclic unsaturated carboxylic acids having the esterified carboxyl group bound to an acyclic carbon atom
    • C07C69/593—Dicarboxylic acid esters having only one carbon-to-carbon double bond
    • C07C69/60—Maleic acid esters; Fumaric acid esters

Definitions

  • the present invention provides a new class of compounds, and more particularly to a salicylic acid fumarate derivative and its use in the treatment of Parkinson's disease.
  • Neurodegenerative disease is a disease state in which cellular neurons of the brain and spinal cord are lost.
  • the brain and spinal cord are made up of neurons, which have different functions, such as controlling movement, processing sensory information, and making decisions. Cells in the brain and spinal cord are generally not regenerated, so excessive damage can be devastating and irreversible.
  • Neurodegenerative diseases are caused by the loss of neurons or their myelin, which deteriorate over time to cause dysfunction. Neurodegenerative diseases are divided into two groups according to phenotype: one class affects exercise, such as cerebellar ataxia; and one class affects memory and related dementia.
  • Neurodegenerative diseases are a state of disease in which neuronal cells of the brain and spinal cord are lost. Neuronal cells generally do not regenerate, and excessive damage may be devastating and irreversible. Neurodegenerative diseases can worsen over time, eventually leading to dysfunction. Alzheimer's disease and Parkinson's disease are the two most prevalent diseases, causing great suffering to patients and their families.
  • Jianhua chao provides a technical solution for fumaric acid and its derivatives and is granted a US patent (publication number: US20140194427A1).
  • the fumaric acid derivative provided by Jianhua chao personal name
  • the fumaric acid derivative has a therapeutic effect on a neurodegenerative disease, such as multiple sclerosis, amyotrophic lateral sclerosis, Parkinson's disease , neurogenic chorea or Alzheimer's disease.
  • the US Food and Drug Administration has approved a new two-time oral drug, dimethyl fumarate (DMF, trade name: Tecfidera) for the treatment of relapsing-remitting multiple sclerosis (RRMS).
  • DMF dimethyl fumarate
  • Tecfidera trade name: Tecfidera
  • RRMS relapsing-remitting multiple sclerosis
  • NMSS American Society of Multiple Sclerosis
  • two dosage forms of interferon beta-1a Avonex and Rebif
  • beta-1b Betaseron and Extavia
  • the drug also includes Copaxone acetate, Gilenya and Aubagio.
  • Natalizumab (Tysabri) is also approved for RRMS treatment, but with restrictions.
  • Novantrone was approved for secondary progressive, progressive relapsing, and relapsing-remitting MS treatment.
  • Ampicra is approved for improving the walking ability of MS patients.
  • Biogen Idec said that in the DEFINE study, the recurrence and disability rates of DMF users were reduced by 49% and 38%, respectively, while the recurrence rate of CONFIRM study patients was reduced by 34%. Both studies have shown that DMF can significantly reduce brain damage compared with placebo. Dr. Robert Fox, MD, director of the Mellen Multiple Sclerosis Center at the Cleveland Clinic, said in a statement from Biogen Idec: "In clinical trials, patients with DMF have reduced disease activity compared to patients in the placebo group, regardless of whether they were early MS The patient is still a more precise patient. The drug provides doctors with another important treatment option for patients with different MS stages.
  • XManuj Ahuja personal name
  • other experiments have verified the DMF and its effect on the active metabolite monomethyl fumarate (MMF) on Nrf2 signal and its 1-methyl-4-phenyl-1,2,3,6-four
  • MMF active metabolite monomethyl fumarate
  • MPTP 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
  • DMF and MMF show protection against MPTP neurotoxicity due to their unique Nrf2-mediated antioxidant, anti-inflammatory, and mitochondrial functions and biological effects, but MMF does not consume glutathione, inhibit mitochondria and sugar Fermentation function.
  • the pathogenesis of PD includes oxidative damage, inflammation and mitochondrial dysfunction.
  • the applicant provided a new class of compounds based on the defects of DMF combined with previous findings, and verified the compounds to include Parkinson's disease (PD), Alzheimer's disease (AD) and Huntington's disease (HD).
  • PD Parkinson's disease
  • AD Alzheimer's disease
  • HD Huntington's disease
  • the neurodegenerative disease has good activity.
  • the new compound provided by the applicant is a salicylic acid fumarate derivative, and its structural formula (A) is:
  • R1 in the structural formula (A) is one of H + , Na + , K + or NH 4 +
  • R 2 is an esterified product of fumaric acid.
  • esterified fumaric acid represented by the R 2 group in the above structural formula is monomethyl fumarate, monoethyl fumarate, monopropyl fumarate, and rich.
  • the salicylic acid fumarate derivative is methyl salicylic acid fumarate, and its structural formula is:
  • the salicylic acid fumarate derivative is ethyl salicylic acid fumarate, and its structural formula is:
  • the R 1 group may be one of H + , Na + , K + or NH 4 + depending on the specific use of the salicylic acid group by the applicant. Whether or not the fumarate derivative needs to react with a salicylic acid fumarate derivative to form a salt. If the applicant does not react the salicylic acid fumarate derivative with a base, then R 1 will be an H + , and if reacted with a NaOH solution, the R 1 group will be a Na + , the other of the R 1 groups The change is also similar to the above.
  • the R 1 group has no direct effect on the activity of the salicylic acid fumarate derivative, but R 1 will change the solubility of the salicylic acid fumarate derivative. Thereby affecting the absorption of the gastrointestinal administration of the salicylic acid fumarate derivative.
  • the method is an esterification reaction, but salicylic acid has two groups of a hydroxyl group and a carboxyl group. Under normal circumstances, the stability of the carboxyl group is even worse.
  • the applicant has first protected the carboxyl group by esterification with tert-butanol in salicylic acid in the experimental design. It is then reacted with a fumaric acid monoester. Finally, t-butanol was removed to obtain the target product.
  • the method provided by the applicant includes the following steps:
  • the beneficial technical effect of the present invention is that the present invention provides a salicylic acid-based fumarate derivative having a structural formula in which salicylic acid and fumaric acid are combined by an esterification reaction, and the experiment is carried out according to the applicant. verification. It has been demonstrated that such compounds have good activity in the treatment of diseases of neurodegenerative diseases.
  • Figure 6 is a high performance liquid chromatogram of MSF
  • Figure 7 is a high performance liquid chromatogram of ESF
  • Figure 8 The drug content of the MSF and ESF in the blood at a dose of 100 mg/kg;
  • Figure 9 The drug content of the MSF and ESF in the liver at a dose of 100 mg/kg;
  • Figure 10 The drug content of MSF and ESF in the brain at a dose of 100 mg/kg;
  • Figure 11 The drug content of MSF and ESF in the striatum of the brain under different doses
  • NMR spectra generated by the instrument workstation, which are colored, and the text and image overlap problems appear in the black and white part.
  • the specific information has been described in the first embodiment of the specification; in the NMR spectrum, Chemical The Chinese meaning of Formula is: “Chemical formula”, the Chinese meaning of Exact Mass is “precise quality”, the Chinese translation of Molecular Weight is: “molar quality”;
  • the Chinese meaning of protein is “protein”
  • the Chinese meaning of Blood is “blood”
  • the Chinese meaning of Liver is “liver”
  • the Chinese meaning of Cortex is “cerebral cortex”
  • the meaning of striatum It is “the striatum of the anterior side of the brain.”
  • Test compound A and compound B the specific data is as follows:
  • MSF Metal salicyl fumarate, methyl salicyl fumarate
  • ESF Ethyl salicyl fumarate, ethyl salicyl fumarate
  • MSF and ESF (200 mg/kg/d) also showed significant dopamine neuronal protection in a model of chronic MPTP-induced mouse dopamine neuronal injury, and protection of dopamine neurons was close to that of the blank control. Level (specifically as shown in Figure 4).
  • the concentration of MPTP metabolite MPP+ in brain tissue was determined by HPLC.
  • the content of MPP+ in the striatum of the ESF (500 mg/kg)-administered group and the blank group was the same, which ruled out the possibility of MSF and ESF affecting the absorption and metabolism of MPTP in vivo and in the brain. It confirmed its neuroprotective effect (specifically as shown in Figure 5).
  • a reliable high-performance liquid phase MSF and ESF analysis method (specifically shown in Figures 6 and 7) was established to determine the MSF and ESF levels in peripheral blood, liver, and brain tissue, demonstrating that MSF and ESF were injected into mice via the abdominal cavity. It is able to enter the brain tissue through the blood-brain barrier, and maintain the original molecular structure of MSF and ESF in the brain tissue (specifically as shown in Figure 8, Figure 9, Figure 10, Figure 11), inferring MSF and ESF molecules It directly produces neuroprotective pharmacological activity.
  • the DMF of the control drug is different. DMF can be metabolized into monomethyl metabolite MMF (Monomethyl fumarate, monomethyl fumarate) after entering the body to produce pharmacological activity.
  • MSF and ESF like the control DMF, have the traditional fumarate activation target Nrf2, which produces a mechanism of activating intracellular antioxidant stress.
  • Nrf2 the traditional fumarate activation target

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)

Abstract

申请人提供的全新化合物为水杨酸基富马酸盐衍生物，其结构通式(A)为：其中，结构通式(A)中R1为H +、Na +、K +或NH4 +中的一种，R 2为富马酸的酯化物。其具有水杨酸与富马酸通过酯化反应相结合的结构通式，根据申请人通过实验验证。证明了该类化合物在治疗神经退行性疾病类疾病中具有良好的活性。

Description

一种水杨酸基富马酸盐衍生物及在治疗帕金森症和其他神经退行性疾病应用 技术领域
本发明提供了一类新化合物，更具体的说涉及一种一种水杨酸基富马酸盐衍生物及其在治疗帕金森症应用。
技术背景
神经退行性疾病(Neurodegenerative disease)是一大脑和脊髓的细胞神经元丧失的疾病状态。大脑和脊髓由神经元组成，神经元有不同的功能，如控制运动，处理感觉信息，并作出决策。大脑和脊髓的细胞一般是不会再生的，所以过度的损害可能是毁灭性的，不可逆转的。神经退行性疾病是由神经元或其髓鞘的丧失所致，随着时间的推移而恶化，以导致功能障碍。神经退行性疾按表型分为两组：一类影响运动，如小脑性共济失调；一类影响记忆以及相关的痴呆症。
随着老龄化加剧，神经退行性疾病患病率也节节攀升。神经退行性疾病是一类大脑和脊髓的神经元细胞丧失的疾病状态，神经元细胞一般不会再生，过度的损害可能是毁灭性的，不可逆转的。神经退行性疾病会随着时间的推移而恶化，最终导致功能障碍，阿尔茨海默病和帕金森病是患病率最高的两种疾病，为病人和家属带来极大痛苦。
以往的研究表明，在体外和体内模型中，神经元可吸收注射的α-突触核蛋白-形成PD特征性路易体的蛋白质。2014年Recasens A(人名)等发表了作者将提取自PD患者黑质的路易体注射至小鼠和猕猴的黑质或纹状体。研究发现α-突触核蛋白在宿主神经元积累，同时，4至17个月时，多巴胺能神经末端出现神经变性，与PD病理类似，并且发表在2014年的《神经病学年报》(学术期刊信息：Recasens A,Dehay B,Bove J,et al.Lewy body extracts from Parkinson disease brains trigger-synuclein pathology and neurodegeneration in mice and monkeys.Annals of Neurology.2014，75:351-362.)上。
同年，Unterberger U等提出了一种机制，即在PD中α-突触核蛋白如何在神经元间扩散。该研究小组使用了一种可区分正常和异常类型α-突触核蛋白的抗体来确定人神经元转移至相邻细胞的蛋白病理类型。同一研究小组进行的一项相关研究使用抗体来检测脑脊液中的异常突触核蛋白，这种方法将有助于PD的诊断以及其他突触核蛋白病的早期诊断，并且发表在2014年《疾病神经生物学》杂志(学术期刊信息：Unterberger U,Lachmann I,
Figure PCTCN2018078618-appb-000001
T,et al.Detection of disease-associated alpha-synuclein in the cerebrospinal fluid:a feasibility study.Neurobiology of Disease.2014，33:329-334.)上。
Jianhua chao提供了一种富马酸及其衍生物的技术方案并获得美国专利授权(公告号： US20140194427A1)，在上述技术方案中，Jianhua chao(人名)提供的富马酸衍生物，具体如结构通式(Ⅰ)所示，Jianhua chao(人名)声称所述富马酸衍生物对神经退行性疾病具有治疗作用，具体如多发性硬化症，肌萎缩性脊髓侧索硬化症，帕金森氏病、神经性舞蹈病或阿尔茨海默氏病。
Figure PCTCN2018078618-appb-000002
美国食品药品管理局(FDA)已批准一种新的每日2次口服药物富马酸二甲酯(DMF，商品名：Tecfidera)用于治疗复发缓解型多发性硬化症(RRMS)。DMF成为众多已获批的MS治疗药物之一。根据美国多发性硬化症学会(NMSS)资料，两种剂型的干扰素β-1a(Avonex和Rebif)以及两种剂型的β-1b(Betaseron和Extavia)已获准用于RRMS治疗，此外获批的药物还包括醋酸格拉替雷(Copaxone)、芬戈莫德(Gilenya)和特立氟胺(Aubagio)。那他珠单抗(Tysabri)也获准用于RRMS治疗，但附带了限制条件。米托蒽醌(Novantrone)获准用于继发进展型、进展复发型和复发缓解型MS治疗。达伐吡啶(Ampyra)被批准用于改善MS患者的行走能力。
Biogen Idec公司称，在DEFINE研究中，DMF服用者复发率和残疾率分别减少49％和38％，而CONFIRM研究服药者复发率减少34％。两项研究均表明，与安慰剂相比，DMF可显著减少大脑损伤。克利夫兰医学中心Mellen多发性硬化症中心医学主任Robert Fox博士在Biogen Idec公司的声明中称：“在临床试验中，与安慰剂组患者相比，DMF治疗患者疾病活动性下降，无论他们是MS早期患者还是更为确切的患者。该药物为医生提供了针对不同MS阶段患者的又一种重要治疗选择。
但是，美国FDA称，DMF可降低淋巴细胞计数，但没有证据表明服药患者感染增加。建议在开始治疗前以及此后每年监测患者的淋巴细胞计数。面部潮红和胃肠道不适是最常见不良反应。
XManuj Ahuja(人名)等通过实验验证了DMF及其影响活性代谢产物富马酸单甲酯(MMF)对Nrf2信号及其1-甲基-4-苯基-1,2,3,6-四氢吡啶(1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine，MPTP)诱导的帕金森病(PD)的影响。研究证明DMF和MMF表现出对MPTP的神经毒性的保护作用是由于他们独特的Nrf2介导的抗氧化、抗炎、和线粒体功能和生物效应，但MMF不消耗谷胱甘肽，抑制线粒体和糖酵解功能。PD的发病机制包括氧化损伤、炎症和线粒体功能障碍等，XManuj Ahuja(人名)等认为MMF才是治疗PD真正的活性成分，并且发表在2016年的《神经科学》杂志(期刊信息：Manuj Ahuja,Navneet Ammal  Kaidery,Lichuan Yang,Noel Calingasan,Natalya Smirnova,Arsen Gaisin,Irina N.Gaisina,Irina Gazaryan,Dmitry M.Hushpulian,Ismail Kaddour-Djebbar,Wendy B.Bollag,John C.Morgan,XRajiv R.Ratan,XAnatoly A.Starkov,M.Flint Beal,and Bobby Thomas1.Distinct Nrf2Signaling Mechanisms of Fumaric Acid Esters and Their Role in Neuroprotection against 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine-Induced Experimental Parkinson’s-Like Disease.The Journal of Neuroscience.2016,36(23):6332–6351.)上。
发明内容
申请人基于DMF存在的缺陷结合前期的发现，提供了一类全新的化合物，并且验证了该类化合物对包括帕金森病(PD)、阿尔茨海默病(AD)和亨廷顿病(HD)等的神经退行性疾病具有良好的活性。
申请人提供的全新化合物为水杨酸基富马酸盐衍生物，其结构通式(A)为：
Figure PCTCN2018078618-appb-000003
其中，结构通式(A)中R1为H +、Na +、K +或NH4 +中的一种，R 2为富马酸的酯化物。
在实际的研究中，申请人进一步的确认上述结构式中R 2基团所指的富马酸的酯化物为富马酸单甲酯、富马酸单乙酯、富马酸单丙酯、富马酸单异丙酯、富马酸单丁酯、富马酸单叔酯中一种。
在确定R 2基团的前提下，申请人进一步的确认所述水杨酸基富马酸盐衍生物为甲基水杨酸基富马酸盐，其结构式为：
Figure PCTCN2018078618-appb-000004
在确定R 2基团的前提下，申请人进一步的确认所述水杨酸基富马酸盐衍生物为乙基水杨酸基富马酸盐，其结构式为：
Figure PCTCN2018078618-appb-000005
针对上述所有的水杨酸基富马酸盐衍生物中，R 1基团可以是H +、Na +、K +或NH4 +中的一种，这取决于申请人在具体使用水杨酸基富马酸盐衍生物是否需要用碱与水杨酸基富马酸盐衍生物反应生成盐。如果申请人不将水杨酸基富马酸盐衍生物于碱反应，那么R 1将是一个H +，如果与NaOH溶液反应，则R 1基团为一个Na +，R 1基团的其他变化也是上述类似的情况。根据申请人目前的研究，可以发现R 1基团对水杨酸基富马酸盐衍生物的活性并没有直接的影响，但是，R 1将改变水杨酸基富马酸盐衍生物的溶解度，从而影响水杨酸基富马酸盐衍生物胃肠给药的吸收。
关于水杨酸基富马酸盐衍生物的制备方法，本方法是进行酯化反应，但是由于水杨酸有羟基和羧基两个基团。在一般的情况下，羧基的稳定性更差，为了实现定向的反应，申请人在实验的设计上，先用叔丁醇于水杨酸发生酯化反应，将羧基进行了保护。然后与富马酸单酯反应。最后脱去叔丁醇，获得目标产物。申请人提供的方法包括以下步骤：
1)以叔丁醇与水杨酸进行酯化反应，保护水杨酸的羧基，得到中间产物Ⅰ；
2)将中间产物Ⅰ与富马酸衍生物进行酯化反应，得到中间产物Ⅱ；
3)将中间产物Ⅱ在酸性条件下脱去叔丁醇，得到目标产物Ⅲ；
其中，化学反应式如下：
Figure PCTCN2018078618-appb-000006
申请人通过药理实验验证了水杨酸基富马酸盐衍生物在制备治疗帕金森病药物中的应用。
本发明的有益技术效果是：本发明提供了一种水杨酸基富马酸盐衍生物，其具有水杨酸与富马酸通过酯化反应相结合的结构通式，根据申请人通过实验验证。证明了该类化合物在治疗神经退行性疾病类疾病中具有良好的活性。
附图说明
图1 化合物A(甲基水杨酸基富马酸盐)的MR图谱；
图2 化合物B(乙基水杨酸基富马酸盐)的MR图谱；
图3 MSF和ESF对MPTP诱导的小鼠多巴胺神经元损伤模型中的安全性考察；
图4 MSF和ESF对MPTP诱导的小鼠多巴胺神经元损伤模型中对多巴胺神经元保护作用；
图5 MSF和ESF对MPTP代谢物MPP+的浓度的影响；
图6 MSF的高效液相图谱；
图7 ESF的高效液相图谱；
图8 MSF和ESF在给药量100mg/kg条件下，在血液中的药物含量；
图9 MSF和ESF在给药量100mg/kg条件下，在肝脏中的药物含量；
图10 MSF和ESF在给药量100mg/kg条件下，在脑中的药物含量；
图11 MSF和ESF在不同给药量条件下，在脑的纹状体种的药物含量；
图12 在敲除了Nrf2基因的转基因老鼠应用MPTP诱导的小鼠多巴胺神经元损伤模型中，对多巴胺神经元的保护作用；
图13 体外实验中MSF和ESF对细胞内谷胱甘肽的浓度的影响。
其中，附图1、2为NMR图谱为仪器工作站生成，为彩色，调整为黑白色后部分出现文字及图像重叠问题，其具体信息已经被记载在说明书实施例1中了；在NMR图谱中Chemical Formula的中文意思是：“化学分子式”，Exact Mass的中文意思译是“精确质量”，Molecular Weight的中文翻译是：“摩尔质量”；
附图3、4、5、12、13中protein的中文意思是“蛋白质”，Doparmine的中文意思是“多巴胺”；
附图6、7中HPLC chromatogram的中文意思是“高效液相色谱”，signal response(volts)的中文意思是“液相信号相应(volts)”,Retention time(min)的中文意思是“保留时间”；
附图8、9、10中，protein的中文意思是“蛋白质”，Blood的中文意思是“血液”，Liver的中文意思是“肝脏”，Cortex的中文意思是“大脑皮质”，striatum的中文意思是“大脑前侧纹状体”。
具体实施方式
实施例1 水杨酸基富马酸盐衍生物的制备
1.保护水杨酸上的羧基团
将水杨酸(45g，795.9nmol，1个当量)溶于二甲基甲酰胺(DMF)(450ml)中，在0℃(冰水浴)条件下，加入N'N-羰基二咪唑(63.5g),在室温条件下搅拌1h，并缓慢滴加DBU(58.5ml)和叔丁醇(63ml)，然后在室温条件下搅拌2h,反应完成用LC-MS检测(产物图谱如附图1所示)。将反应物导入水中(500ml),并用乙酸乙酯提取三次(3*800ml)。合并有机层，用分别用水洗涤3次(3*800ml)、再用盐水(1000ml)洗涤一次，用无水Na 2SO 4干燥，过滤，滤液浓缩至无有机溶剂，将浓缩物经过硅胶柱层析纯化(石油醚：乙酸乙酯＝100:1作为流动相)，得到 产品(52g产品，收率82.5％)，产品为淡黄色油状(给名为：化合物A3)。其化学反应式如下：
Figure PCTCN2018078618-appb-000007
2.富马酸单甲酯与化合物A3酯化反应
将富马酸单甲酯(10.4g)加入二甲基甲酰胺(DCM)(100毫升)加入HATU(45.6克)和DIEA(31克)的混合溶液。反应混合物搅拌30分钟，然后在室温条件下将化合物A3(10克)加入上述反应混合物中于室温下搅拌16h，LC-MS显示反应完毕。反应混合物淬火水(200毫升)的提取与DCM(2*200毫升)。合并有机层用盐水(300毫升)，无水Na2SO4干燥，过滤，滤液浓缩除去有机溶剂。产物经硅胶柱层析纯化(石油醚:乙酸乙酯＝30:1)得到产品(12.6克，收率79.9％)，产品为淡黄色油(给名为，化合物A4)。其反应式如下：
Figure PCTCN2018078618-appb-000008
3.甲基水杨酸基富马酸盐的制备
在0℃(冰水浴)条件下，将化合物A4(5.6克)加入DCM(100毫升)、2当量盐酸(2mol)和乙醚(25毫升)的混合液溶液；反应混合物在室温下搅拌过夜。TCL显示反应完成。除去反应溶剂，加水(100毫升)，用DCM提取3次(3*150毫升)；合并有机层，用盐水洗涤，用无水Na2SO4干燥脱水。产物经硅胶柱层析纯化(石油醚:乙酸乙酯＝2:1)，得到粗产品，将粗产品用正己烷混合(结晶)过夜，过滤得到纯产品A(4.5克，收率为98.4％)；经过MR和MS检测，确认纯产品A为甲基水杨酸基富马酸盐。其反应式如下：
Figure PCTCN2018078618-appb-000009
4.富马酸单乙酯与化合物A3酯化反应(化合物B2)
将富马酸单乙酯(10.4g)加入二甲基甲酰胺(DCM)(100毫升)加入HATU(45.6克)和DIEA(31克)的混合溶液。反应混合物搅拌30分钟，然后在室温条件下将化合物A3(10 克)加入上述反应混合物中于室温下搅拌16h，LC-MS显示反应完毕。反应混合物淬火水(200毫升)的提取与DCM(2*200毫升)。合并有机层用盐水(300毫升)，无水Na2SO4干燥，过滤，滤液浓缩除去有机溶剂。产物经硅胶柱层析纯化(石油醚:乙酸乙酯＝30:1)得到产品(12.8g,收率79.9％)，产品为淡黄色油(给名为，化合物B2)。其反应式如下：
Figure PCTCN2018078618-appb-000010
5.乙基水杨酸基富马酸盐的制备(化合物B)
在0℃(冰水浴)条件下，将化合物B2(5.6克)加入DCM(100毫升)、2当量盐酸(2mol)和乙醚(25毫升)的混合液溶液；反应混合物在室温下搅拌过夜。TCL显示反应完成。除去反应溶剂，加水(100毫升)，用DCM提取3次(3*150毫升)；合并有机层，用盐水洗涤，用无水Na2SO4干燥脱水。产物经硅胶柱层析纯化(石油醚:乙酸乙酯＝2:1)，得到粗产品，将粗产品用正己烷混合(结晶)过夜，过滤得到纯产品A(4.0g,收率86.5％)；经过MR和MS检测，确认纯产品B为甲基水杨酸基富马酸盐。其反应式如下：
Figure PCTCN2018078618-appb-000011
6.对化合物A和化合物B进行检测，具体数据如下：
MR检测
化合物A: 1H NMR(400MHz,DMSO):δ13.18(s,1H),7.98～7.96(m,1H),7.71～7.67(m,1H),7.46～7.42(m,1H),7.32～7.30(m,1H),6.99～6.98(m,2H),3.80(s,3H)；(NMR图谱具体如图1所示)。通过MR检测可以确认化合物A为甲基水杨酸基富马酸盐。
化合物B: 1H NMR(400MHz,DMSO):δ13.16(s,1H),7.98～7.96(m,1H),7.70～7.67(m,1H),7.45～7.41(m,1H),7.31～7.29(m,1H),6.97～6.96(m,2H),4.27～4.22(m,2H),1.29～1.26(m,3H)。(NMR图谱具体如图2所示)。通过MR检测可以确认化合物B为乙基水杨酸基富马酸盐。
7.申请人采用上述合成工艺路线以水杨酸单酯和富马酸进行酯化反应，分别合成得到了丙基水杨酸基富马酸酯(化合结构式如结构式B)、异丙基水杨酸基富马酸酯(化合结构式 如结构式C)和丁基水杨酸基富马酸酯(化合结构式如结构式D)。经过申请预实验，认为丙基水杨酸基富马酸酯、异丙基水杨酸基富马酸酯和丁基水杨酸基富马酸酯的药理活性次于甲基水杨酸基富马酸酯以及乙基水杨酸基富马酸酯。所以对于这三个化合物的药理研究不再累述。
但是，从甲基水杨酸基富马酸酯以、乙基水杨酸基富马酸酯、丙基水杨酸基富马酸酯、异丙基水杨酸基富马酸酯和丁基水杨酸基富马酸酯等5个化合物的合成路线可以知道，是要对富马酸上的羧基进行有效的保护，就可以合成水杨酸基富马酸酯盐的衍生物。验证了申请人提供的合成工艺路线针对水杨酸基富马酸盐衍生物的合成是有效通用的。
Figure PCTCN2018078618-appb-000012
实施例2 MSF和ESF对多巴胺神经元的保护作用
用MSF(Methyl salicyl fumarate，甲基水杨酸基富马酸盐)和ESF(Ethyl salicyl fumarate，乙基水杨酸基富马酸盐)在帕金森病小鼠模型上做治疗试验，采用公认的MPTP诱导的小鼠多巴胺神经元损伤的模型。对照药物采用DMF(Dimethyl fumarate，二甲基富马酸盐)，DMF采用6个剂量组，实验组中MSF和ESF，每个药物10个剂量组，每组10只小鼠。实验组和对照组均采用通过腹腔给药，对照组采用DMF治疗，实验组分别使用了不同剂量的ESF和MSF治疗(具体如附图3所示)。发现了具有统计学意义的显著的保护多巴胺神经元的作用，并呈量效关系。反应了出现保护作用的最低剂量(10mg/kg/d)、最佳剂量(100-200mg/kg/d)、 和开始出现毒性作用的剂量(>500mg/kg/d)。与DMF的效果相比较，MSF和ESF具有更加显著的保护作用，而且，DMF出现保护作用的剂量(50mg/kg/d)比MSF和ESF高，而出现毒性作用的剂量(>200mg/kg/d)又比ESF低，因此，ESF具有更加有效和安全的治疗剂量区间(具体如附图3所示)。
在用于慢性MPTP诱导的小鼠多巴胺神经元损伤的模型时，MSF和ESF(200mg/kg/d)同样显示出了显著的多巴胺神经元保护作用，对多巴胺神经元的保护接近了空白对照的水平(具体如附图4所示)。
用HPLC测定脑组织中MPTP代谢物MPP+的浓度。对于注射了MPTP的模型小鼠，ESF(500mg/kg)给药组和空白组的脑纹状体MPP+含量相同，排除了MSF和ESF对MPTP在体内和脑内吸收及代谢产生影响的可能性，证实了其神经保护作用(具体如附图5所示)。
建立了可靠的高效液相MSF和ESF分析方法(具体如附图6、7所示)对外周血、肝脏、脑组织的MSF和ESF含量进行测定，证明MSF和ESF经腹腔给小鼠注射后能够通过血脑屏障进入脑组织，而且在脑组织中保持MSF和ESF原分子结构不变(具体如附图8、附图9、附图10、附图11所示)，推断MSF和ESF分子本身直接产生神经保护药理活性作用。而对照药DMF却不同，DMF是靠进入体内后迅速代谢成单甲基代谢物MMF(Monomethyl fumarate，单甲基富马酸盐)后才能产生药理活性作用。
MSF和ESF与对照药DMF一样都具有普通富马酸盐(fumarate)传统的激活靶点Nrf2，从而产生激活细胞内抗氧化应激作用机制。通过用敲除了Nrf2基因的转基因老鼠做MPTP实验发现，DMF因失去了作用靶点而失去了多巴胺神经元的保护作用，MSF和ESF却仍然具有显著的保护作用(具体如附图12所示)，从而证实MSF和ESF除了作用于Nrf2外，还有另一个神经保护靶点，从其分子结构中含有水杨基(salicyl)推断，这一新靶点可能与抗炎性反应有关。
通过体外细胞实验发现，对照药DMF有耗竭细胞内谷胱甘肽(GSH，内源性抗氧化多肽)的作用，这可能是导致其毒副作用大的原因。而MSF和ESF对细胞内谷胱甘肽的浓度不产生影响(具体如附图13所示)。

Claims (6)

  1. 一种水杨酸基富马酸盐衍生物，其特征在于：其结构通式(A)为：
    Figure PCTCN2018078618-appb-100001
    其中，结构通式(A)中R1为H +、Na +、K +或NH4 +中的一种，R 2为富马酸的酯化物。
  2. 根据权利要求1所述的水杨酸基富马酸盐衍生物，其特征在于：所述富马酸的酯化物为富马酸单甲酯、富马酸单乙酯、富马酸单丙酯、富马酸单异丙酯、富马酸单丁酯、富马酸单叔酯中一种。
  3. 根据权利要求2所述的水杨酸基富马酸盐衍生物，其特征在于：所述水杨酸基富马酸盐衍生物为甲基水杨酸基富马酸盐，其结构式为：
    Figure PCTCN2018078618-appb-100002
  4. 根据权利要求2所述的水杨酸基富马酸盐衍生物，其特征在于：所述水杨酸基富马酸盐衍生物为乙基水杨酸基富马酸盐，其结构式为：
    Figure PCTCN2018078618-appb-100003
  5. 根据权利要求1所述的水杨酸基富马酸盐衍生物的制备方法，其特征在于：包括以下步骤：
    1)以叔丁醇与水杨酸进行酯化反应，保护水杨酸的羧基，得到中间产物Ⅰ；
    2)将中间产物Ⅰ与富马酸衍生物进行酯化反应，得到中间产物Ⅱ；
    3)将中间产物Ⅱ在酸性条件下脱去叔丁醇，得到目标产物Ⅲ；
    其中，化学反应式如下：
    Figure PCTCN2018078618-appb-100004
  6. 权利要求1-4中任一项所述的水杨酸基富马酸盐衍生物在制备治疗帕金森病药物中的应用。
PCT/CN2018/078618 2017-03-28 2018-03-09 一种水杨酸基富马酸盐衍生物及在治疗帕金森症和其他神经退行性疾病应用 WO2018177114A1 (zh)

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