CN105646207A - 阿魏酸与细辛醚类似物拼合的化合物及其制备方法与应用 - Google Patents
阿魏酸与细辛醚类似物拼合的化合物及其制备方法与应用 Download PDFInfo
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- CN105646207A CN105646207A CN201610095203.2A CN201610095203A CN105646207A CN 105646207 A CN105646207 A CN 105646207A CN 201610095203 A CN201610095203 A CN 201610095203A CN 105646207 A CN105646207 A CN 105646207A
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- Prior art keywords
- ferulic acid
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- compound
- asaricin
- chloride
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- KSEBMYQBYZTDHS-HWKANZROSA-M (E)-Ferulic acid Natural products COC1=CC(\C=C\C([O-])=O)=CC=C1O KSEBMYQBYZTDHS-HWKANZROSA-M 0.000 title claims abstract description 27
- KSEBMYQBYZTDHS-UHFFFAOYSA-N ferulic acid Natural products COC1=CC(C=CC(O)=O)=CC=C1O KSEBMYQBYZTDHS-UHFFFAOYSA-N 0.000 title claims abstract description 27
- 229940114124 ferulic acid Drugs 0.000 title claims abstract description 27
- 235000001785 ferulic acid Nutrition 0.000 title claims abstract description 27
- QURCVMIEKCOAJU-UHFFFAOYSA-N trans-isoferulic acid Natural products COC1=CC=C(C=CC(O)=O)C=C1O QURCVMIEKCOAJU-UHFFFAOYSA-N 0.000 title claims abstract description 27
- 150000001875 compounds Chemical class 0.000 title claims abstract description 22
- KSEBMYQBYZTDHS-HWKANZROSA-N ferulic acid Chemical compound COC1=CC(\C=C\C(O)=O)=CC=C1O KSEBMYQBYZTDHS-HWKANZROSA-N 0.000 title claims abstract description 22
- 238000002360 preparation method Methods 0.000 title claims abstract description 13
- RKFAZBXYICVSKP-AATRIKPKSA-N alpha-asarone Chemical class COC1=CC(OC)=C(\C=C\C)C=C1OC RKFAZBXYICVSKP-AATRIKPKSA-N 0.000 title abstract 2
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- RZWZRACFZGVKFM-UHFFFAOYSA-N propanoyl chloride Chemical compound CCC(Cl)=O RZWZRACFZGVKFM-UHFFFAOYSA-N 0.000 claims abstract description 4
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Abstract
本发明属医药技术领域,公开了阿魏酸与细辛醚类似物拼合的化合物及其制备方法和用途。化合物的结构如下所示,其制备主要以阿魏酸为起始原料,经乙酰化、氯代成乙酰阿魏酸酰氯;以1,2,4-三甲氧基苯为原料,与丙酰氯或丙酸酐经傅克酰基化反应制得2,4-二羟基-5-甲氧基苯丙酮,然后与乙酰阿魏酸酰氯缩合四步反应得到目标化合物,操作简单、后处理方便、收率较高。目标化合物具有促进神经细胞生长的作用,可以用于抑郁、阿尔兹海默症等神经退行性疾病治疗。
Description
技术领域:
本发明属于医药技术领域,涉及到阿魏酸与细辛醚类似物拼合的化合物及其制备方法和应用。
背景技术:
阿尔茨海默病(Alzheimer’sdisease,AD)是一种慢性中枢神经系统的退行性疾病,是最常见的老年性痴呆类型。临床早期表现主要为患者记忆力的减退和生活自理能力的下降,最终导致认知功能障碍和缺失、神经行为异常及生活自理能力的完全丧失,病程一般为6~12年,常因并发感染而死亡。2010年国际阿尔茨海默病研究中心(Alzheimer’sDiseaseInternational,ADI)报告统计全世界约有3560万人患痴呆,约占全球总人口的0.5%,痴呆人数占60岁以上人数的5%~7%,预计2050年痴呆患者数将达到11540万人。2010年全球用于痴呆的治疗费用高达6040亿美元,预测至2030年痴呆的治疗费用开支将会增加85%。而据世界卫生组织推算,到2020年,AD将成为我国疾病负担排名第4位的疾病。AD不仅严重影响患者的身体健康和生活质量,同时也给患者的家庭和社会带来沉重的负担,AD病因病机的阐明和预防治疗手段的研究已成为亟待解决的医学难题和社会难题。
在哺乳动物大脑中,神经干细胞的增殖和自我更新持续发生于整个生命过程,对神经发生非常关键。在慢性应激的条件下,神经干细胞增殖和神经发生水平下降,是包括抑郁症、AD等神经退行性疾病的发病机理之一。而长期服用抗抑郁药物或者环境刺激可以促进神经干细胞增殖并逆转由应激引起的神经发生缺陷。此外,一些研究表明,在小鼠应激模型中,抗抑郁药的药效至少在一定程度上依赖于其对神经发生水平的增强。因此,促进神经发生被认为是治疗抑郁症、AD等神经退行性疾病的有效手段。神经发生受到多种胞外因子和胞内信号通路调控。在神经干细胞微环境中的生长因子和神经营养因子的刺激下,细胞内信号通路包括ERK和Akt级联反应等很重要。研究表明,能够调控这些因子和信号通路的化合物可以促进神经干细胞增殖。
天然产物具有各种生物活性,可以在生理和病理条件下调控大脑的功能。已报道许多天然产物具有多种细胞靶标(CurrMedChem.2013,20,1673-1685.),因此在治疗包括精神疾病在内的复杂疾病方面具有优势。细辛醚和阿魏酸分别是中药石菖蒲和当归的活性成分。一直以来,石菖蒲和当归被用于调控情绪的传统中药复方。我们之前的研究发现细辛醚促进神经发生(AgingCell.2015,14,784-796.)。此外,研究表明,细辛醚还具有保护神经元和抗炎的作用(EurJPharmacol.2010,635,96-102;BiomolTher(Seoul).2014,22,17-26)。阿魏酸是一种抗氧化剂,具有保护神经元和促进神经发生的作用。有趣的是,两个化合物的拼接可能具有双重或叠加的生物学活性,在疾病治疗中具有优势。
发明内容:
本发明提供了一个有效的用于抑郁、AD等神经退行性疾病治疗的化合物(CG402),所述化合物的结构为:
本发明所述的化合物通过如下方法制备:
(1)阿魏酸酚羟基乙酰化
阿魏酸与乙酸酐,在合适的溶剂和催化剂作用下反应得乙酰化阿魏酸。
(2)(E)-3-(4-乙酰氧基-3-甲氧基苯基)丙烯酰氯合成
乙酰化的阿魏酸在合适的溶剂中,在催化剂作用下,与氯化试剂反应制得酰氯。
(3)细辛醚类似物制备(傅克酰化反应)
1,3,4-三甲氧基苯与丙酰氯、无水三氯化铝,无溶剂或在合适溶剂中反应,反应液倾入到冰水中,分取有机相,有机相再用碱液萃取,合并碱液再调酸性,析出固体,抽滤,即得。
(4)缩合成酯
将步骤(2)制得的乙酰化阿魏酸酰氯与步骤(3)制得的细辛醚类似物在合适溶剂和碱(缚酸剂)作用下反应,反应液倾入冰水中,分取有机相,无水硫酸钠干燥后,蒸去有机溶剂,即得粗品,乙醇重结晶得精品。
步骤(1)中,所述的溶剂为二氯甲烷、氯仿、甲苯、二甲苯、四氢呋喃、2-甲基四氢呋喃、甲基叔丁基醚、乙酸乙酯、乙酸丁酯等常用溶剂,优选二氯甲烷、氯仿;催化剂为酸或碱,所述的酸为硫酸、盐酸、冰醋酸等,所述的碱为三乙胺、吡啶等。
反应温度为-10-80℃,反应0.5-10小时;
优选室温(20-35℃),反应时间优选1-2小时。
步骤(2)中,所述的溶剂为二氯甲烷、氯仿、甲苯、二甲苯、四氢呋喃、2-甲基四氢呋喃、甲基叔丁基醚、乙酸乙酯、乙酸丁酯等常用溶剂,优选甲苯;所述的催化剂为碱,如吡啶、三乙胺等,氯化试剂为二氯亚砜、三氯化磷、五氯化磷、三氯氧磷、草酰氯等,优选二氯亚砜;
反应温度为15-110℃,优选100-110℃,反应时间为1-10小时,优选3-5小时。
步骤(3)中,所述的溶剂为:二氯甲烷、氯仿、甲基叔丁基醚,优选无溶剂或二氯甲烷;萃取碱液为氢氧化钠(钾)或碳酸钠(钾)水溶液;
反应温度0-80℃,优选20-40℃,反应时间为1-12h,优选1-2小时。
步骤(4)中,所述的溶剂为:二氯甲烷、氯仿、甲苯、二甲苯、四氢呋喃、2-甲基四氢呋喃、甲基叔丁基醚、乙酸乙酯、乙酸丁酯等常用溶剂,优选二氯甲烷;碱(缚酸剂)为有机碱如三乙胺、吡啶或无机碱如碳酸氢钠(钾)、碳酸钠(钾)等,优选三乙胺;
反应温度-10-50℃优选0-25℃,反应时间0.5-5小时,优选1-2小时。
本发明根据细辛醚的结构推测其进入体内很可能会发生甲氧基的去甲基化和丙烯基的氧化等代谢反应,因此,设计了细辛醚的类似物,然后与阿魏酸结合,同时,考虑到增加化合物的血脑屏障通过性,将阿魏酸结构上的酚羟基做成酯,得到了一个目标化合物。
结果表明,本发明制备的化合物能够促进神经干细胞增殖,并在慢性温和应激小鼠模型中产生抗抑郁作用。能够激活ERK并增强EGF的表达。可作为具有潜力的新型药物来治疗抑郁症、AD等神经退行性疾病。
附图说明:
图1为CG402促进神经干细胞增殖
(A)CG402的化学结构
(B-C)在含有1ng/mlEGF和1ng/mlbFGF的培液中,用不同浓度的CG402处理单层贴壁培养的成体海马神经干细胞(B)或胚胎神经干细胞(C)。24小时后,加入EdU,并于2小时后固定细胞。检测EdU+细胞,并统计其在所有细胞中的比例。N=5次独立实验。
(D)在分化的条件下,用不同浓度的CG402处理胚胎神经干细胞5天,检测Tuj1+(左图)或GFAP+(右图)细胞,并计算其在所有细胞中的比例(E)小鼠给药CG402或溶剂对照14天,在第15天注射BrdU。在海马齿状回(DentateGyrus,DG)对BrdU和DAPI进行染色,并统计BrdU+细胞数。每组N=5。
(F-J)小鼠给药CG402、溶剂对照或阳性对照氟西汀14天,在第11-14天每日注射BrdU,第16和18天分别进行悬尾实验(G)和强迫游泳实验(F),第21天小鼠灌流。对海马齿状回中BrdU、Dcx和DAPI进行染色,并统计BrdU+Dcx+细胞数(H)及其在所有BrdU+细胞中的比例(I)。(J)强迫游泳实验中小鼠的不动时间和其DG中BrdU+Dcx+细胞数之间的Pearson相关性分析。(F-G)每组N=8-9。(H-I)每组N=5。
定量结果表示为平均值±标准误;*P<0.05,**P<0.01,***P<0.001,双尾t检验或one-wayANOVA分析。
图2为在慢性应激模型中,CG402缓解小鼠抑郁样行为表现
(A)慢性应激模型中行为学实验和免疫组化分析实验设计和流程
(B)CG402和氟西汀改善由应激导致的小鼠毛发状态受损。每组N=10-12
(C)强迫游泳实验。通过测量不动时间来测定抑郁相关表现。每组N=10-12
(D-E)在DG中对BrdU、Dcx和DAPI染色,统计BrdU+Dcx+的数目(D)和其在所有BrdU+细胞中的比例(E)。每组N=5-6。
定量结果表示为平均值±标准误;*P<0.05,**P<0.01,***P<0.001,one-wayANOVA分析。
图3为CG402激活ERK级联反应并增加EGF的表达来促进神经干细胞增殖
(A)胚胎神经干细胞在含有1ng/mlEGF和1ng/mlbFGF的培液中培养24小时,并用50μMCG402处理一定时间。用免疫印迹法检测蛋白样品中的磷酸化ERK1/2、Akt和GSK3。总ERK、Akt和GSK3的量作为上样量的对照。4次独立实验得到类似的结果。
(B)向胚胎神经干细胞中加入MEK抑制剂U0126(U;0.1uM),ERK抑制剂FR180204(F;1uM),PI3K抑制剂LY294002(L;1uM)或Akt抑制剂MK-2206(M;3uM)预处理30分钟,再加入CG402孵育24小时。最后2小时加入EdU,染色并定量统计。所有实验组分别与该图中的只加有CG402的实验组比较。N=5次独立实验。
(C)胚胎神经干细胞用15μMCG402处理一定时间,并用定量实时PCR法检测Egf、Fgf2和Bdnf的mRNA水平。N=6次独立实验。
(D)ELISA法检测慢性应急模型中CG402给药小鼠海马中的EGF水平
(E)CG402通过激活ERK级联反应和EGF表达来促进神经干细胞增殖的示意图。
具体实施方式:
实施例1:
第一步:阿魏酸酚羟基乙酰化
于250mL的三颈瓶中加入阿魏酸7.8g(0.04mol)、三乙胺5.8mL,用100mLCH2Cl2溶解,搅拌均匀。在室温12℃滴入4.2mL醋酸酐,滴完后室温反应1h。TLC检测反应完全。反应液用水洗20mL×3次,无水Na2SO4干燥,过滤除去干燥剂,溶液蒸干得8.6g淡黄色固体。
第二步:(E)-3-(4-乙酰氧基-3-甲氧基苯基)丙烯酰氯合成
于250mL的三颈瓶中加入(E)-3-(4-乙酰氧基-3-甲氧基苯基)丙烯酸8.62g(0.018mol)用50mL甲苯溶解、加入氯化亚砜2.6ml,在110℃反应4h检测反应完全,蒸干溶剂得固体产品。
第三步:细辛醚类似物制备(傅克酰化反应)
于500mL的三颈瓶中加入1,3,4三甲氧基苯33.6g(0.2mol)加入丙酰氯24.4mL(0.28mol),冰浴加入三氯化铝37g(0.28mol),后移入40℃油浴反应2h,石油醚:乙酸乙酯(5:1)为展开剂,TLC检测反应完全,反应液倒入冰水中,水层用乙酸乙酯萃取3次,合并乙酸乙酯萃取液后用5%NaOH洗3次,合并氢氧化钠萃取液用10%盐酸调pH3,析出固体,抽滤干燥得产品8.5g1-(2,4-二羟基-5-甲氧基苯基)丙基-1-酮。1HNMR(CDCl3,400MHz)δ12.69(s,1H),7.09(s,1H),6.53(s,1H),6.30(s,1H),3.89(s,3H),2.97(q,2H,J=7.3Hz),1.25(t,3H,J=7.3Hz).
第四步:缩合成酯
于250mL的三颈瓶中加入1.5g(0.008mol)1-(2,4-二羟基-5-甲氧基苯基)丙基-1-酮,三乙胺1.2ml,再加50mL二氯甲烷,搅拌溶解,冰浴下滴加2.3g(0.01mol)(E)-3-(4-乙酰氧基-3-甲氧基苯基)丙烯酰氯的25mL二氯甲烷溶液。滴完后室温反应1h。反应液倒入水中,有机相用5%NaOH洗3次后用饱和食盐水洗,无水硫酸钠干燥,过滤,蒸干溶剂得2.2g,用乙醇重结晶得精品CG402,收率88.6%。1HNMR(CDCl3,400MHz)δ12.29(s,1H),7.85(d,1H,J=16.0Hz),7.24(d,1H,J=5.4Hz),7.19(dd,2HJ=1.8,8.2Hz),7.10(d,1H,J=8.0Hz),6.83(s,1H),6.60(d,1H,J=16.0Hz),3.90(s,3H),3.85(s,3H),3.04(q,2H,J=7.2Hz),2.35(s,3H),1.28(t,3H,J=7.2Hz).ESI-MSm/z:415.2[M+H]+,437.3[M+Na]+,453.3[M+K]+,413.0[M-H]-。
实施例2:活性实验部分:
实验方法:
(1)小鼠给药和BrdU注射
8周大小鼠灌胃给药CG402、氟西汀或水。给药浓度为CG40220、60或200mg/kg小鼠体重,氟西汀10mg/kg小鼠体重,另参照组给相同体积的水,每日一次,持续给药14天或在慢性应激条件下给药21天。在检测神经干细胞体内增殖的实验中,最后一次给药后分3次腹腔注射150mg/kg小鼠体重的BrdU,每次之间间隔3h,在最后一次BrdU注射后2h,将小鼠麻醉后用4%多聚甲醛灌流(PFA),取全脑进行下一步的实验。在检测神经干细胞体内分化的实验中,在给药的最后5天同时腹腔注射50mg/kg小鼠体重的BrdU,每日一次,7天后将小鼠麻醉后多聚甲醛灌流,取全脑进行下一步的实验。
(2)细胞培养
成年小鼠海马神经干细胞的分离和培养:首先给8周大小小鼠腹腔注射10%(w/v)的水合氯醛,剂量为5ml/kg小鼠体重,麻醉后70%乙醇全身喷洒消毒,剪开颅骨,取出全脑,置于预冷的PBS中,去除小脑,再按中线将剩余部分切开,剥出左右两侧海马。将海马置于500μl预热的PDD(2.5U/ml,papain;1U/ml,dispase;250U/ml,DNase)中,用眼科剪剪碎至小于1mm3的碎块,每只小鼠再加入2ml消化液,吹匀后加入60mm细胞培养皿中铺开,放入37℃细胞培养箱摇床上消化20分钟,连未消化碎块一起吸出至15ml离心管,加入2.5mlNeuroCultNSCbasal培液,用剪过头部的1ml枪头吹打10次,自然沉降30秒后,上清加入40μm滤网过滤,如此重复三次,之后再用5ml培液冲洗滤网,合并所有过滤的细胞。取部分细胞,与台盼蓝染料1:1混合,加入血细胞计数板计数(只计台盼蓝染色阴性的活细胞)。再以1X105/ml的细胞铺板密度种入24孔板,并加入生长因子(20ng/mlbFGF,20ng/mlEGF,2μg/mlheparin)。每2天加入1/10体积的新鲜培液,6天后大部分神经球可生长至50μm以上,此时可对细胞进行传代,Accutase37℃消化5分钟,以1X105/ml的细胞铺板密度进行传代,由于刚分离出的细胞中有神经细胞等污染,故传代3代及以上的细胞才能进行实验。多余的细胞进行冻存,10%DMSO(v/v),30%SerumReplacement(v/v)。
胚胎神经干细胞的分离和培养:从怀孕12.5的C57BL/6小鼠中取出胚胎后,剥开颅骨,取小鼠胚胎的皮层,消化培养,具体步骤同成体海马神经干细胞,所使用培液为添加B27的DMEM/F12,其中补充10ng/mlbFGF和20ng/mlEGF。
(3)体外EdU掺入实验
神经球在用Accutase消化后,重悬于含1ng/mlbFGF和1ng/mlEGF和B27的DMEM/F12培液(胚胎神经干细胞)中或者含1ng/mlbFGF和1ng/mlEGF的NeuroCultNSC增殖培液中,以4X104/cm2的细胞密度接种到预先包被好的96孔板中(PDL包被过夜,用水洗一遍后,再用laminin包被过夜),培养24小时后,加入CG402,在有抑制剂的实验中,加入CG402之前30分钟用ERK和Akt信号通路抑制剂(U0126,0.1μM;LY294002,1μM;MK-2206,3μM;FR180204,1μM)预处理,加药后共孵育24小时,在第22小时加入10μM的EdU,孵育2小时后收细胞染色。
(4)EdU染色和分析
细胞用4%PFA固定15分钟,PBS洗一遍后,再用含0.1%TritonX-100的PBS打孔处理15分钟,PBS洗一遍,加入配好的EdU染色液染色30分钟,配方下表1所示。
表1
Reaction components | volume |
Click-iT EdU reaction buffer | 5.1ml |
CuSO4 | 240ul |
Alexa Fluor azide | 15ul |
Click-iT EdU buffer additive | 600ul |
total volume | 6ml |
EdU染色完毕后,PBS洗一遍,DAPI(1:5000,PBS)染色5分钟,PBS洗两遍,在operetta上进行检测和分析。
(5)神经干细胞体外分化
神经球在用Accutase消化后,重悬于含10ng/mlbFGF和20ng/mlEGF的NeuroCultNSC增殖培液中,以2.5X104/cm2的细胞密度接种到预先包被好的96孔板中(PDL包被过夜,用水洗一遍后,再用laminin包被过夜),培养24小时后,将培液换成NeuroCultNSC分化培液,同时加入CG402,之后每隔一天更换培液,在培液换成NeuroCultNSC分化培液5天后,对各个谱系的分化标记分子进行染色分析。
(6)免疫印迹实验
神经球在用Accutase消化后,重悬于含1ng/mlbFGF和1ng/mlEGF和B27的DMEM/F12培液,再以3X106/ml的细胞密度接种到预先包被好的12孔板中,培养24小时后,加入CG402,加药2、5、10、30或60分钟后将细胞放至冰上,用预冷的PBS洗两次,加入1XLB,95℃处理5分钟,跑SDS/PAGE胶,转至醋酸纤维膜上,与phospho-ERK,ERK,phospho-Akt,Akt,phospho-GSK3,GSK3一抗和IRDye800CW标记二抗孵育,用Odyssey远红外图像系统检测并在其自带软件上分析。
(7)免疫荧光实验
PFA灌流过的小鼠大脑继续在4%PFA中固定24小时,再在30%蔗糖固定液中静置72小时。处理完毕后,去除小脑部分,将大脑嗅球朝上直立置于滤纸上,-80℃冻存至少24小时。以30μm厚度垂直纵向冰冻切片,按小鼠大脑图谱取DG部分,共切8X9X=72张,共2.16mm,按切片顺序排列,每8片取1张,合并后编为一组脑片,进行染色。切好的脑片置于组织保护液中(30%蔗糖,30%乙二醇,0.1MPB),可在-20℃保存半年以上。染色时取一组脑片,在封闭液(10%驴血清,0.3%TritonX-100,PBS)中室温封闭45分钟,之后加入一抗4℃孵育过夜(一抗稀释比例为:大鼠抗BrdU,1:2000;羊抗Dcx,1:200,再与合适的荧光二抗室温孵育1小时。DAPI对细胞核染色。之后用OlympusFV100i或LeicaSP-8对染色的脑片进行拍照。阳性或双染细胞的数量通过ImageProPlus软件分析。对于和BrdU共染,需要进行抗原修复的脑片,于血清封闭之前,在抗原修复液(10mM柠檬酸钠,pH6.5)中95℃处理20分钟。BrdU染色,脑片在血清封闭之前,用2M的盐酸在37℃处理30分钟,再用0.1M的硼酸缓冲液(pH8.5)清洗。
(8)实时定量PCR
用Trizol从细胞中提取得到总RNA,进一步用M-MLV逆转录酶反转录为cDNA。cDNA样品和SYBRGreenqPCRMix混匀,并用MX3000PStratagenePCR仪进行实时定量PCR分析。通过内参GAPDH来计算相对表达量。
(9)行为学实验
旷场实验、强迫游泳实验和悬尾实验参考文献中的方法进行双盲实验。在给药结束的后一天,小鼠先进行旷场实验。旷场反应箱长40cm,宽40cm,高40cm,箱内底面分割成16个10X10cm的方格。摄像机置于箱体正上方来记录箱内小鼠的活动。轻轻放置小鼠于箱内一角,任其探索15分钟。每只小鼠实验结束后,清理箱体。摄像机记录的影像利用自动追踪系统(EthovisionXTsoftware)进行分析。小鼠的运动表现为其行动的距离和速度。24小时后,小鼠进行悬尾实验或强迫游泳实验。在悬尾实验中,每只小鼠用胶带固定尾巴倒挂在横杆上。摄像机记录其在6分钟内的行为活动,统计最后4分钟内的不动时间。在强迫游泳实验中,小鼠逐个被放入装有水(水深15cm,水温23±1℃)的玻璃圆筒中(直径20cm,高30cm)。6分钟后取出小鼠,擦干,并放回笼中。每次实验更换水。利用摄像机记录小鼠的活动,统计最后4分钟内的不动时间。
(10)数据统计分析
所有实验数据表示为平均值±标准误差。不同处理组之间采用t检验进行比较。多组结果之间采用one-wayANOVA进行分析,并用Fisher’sprotectedleastsignificantdifferencetest进行事后检验。P<0.05时认为组间有显著性差异。
实验结果:
1.CG402促进体外和成体小鼠海马神经干细胞增殖
研究表明,细辛醚和阿魏酸有助于治疗神经系统疾病。因此,我们探究它们的连接产物能否促进神经发生。由于细辛醚的甲氧基不稳定,我们将阿魏酸和细辛醚通过之间连接。此外,我们将阿魏酸上的羟基改为乙酰基来增加脂溶性并促进其通过血脑屏障。为了探究CG402在体外对神经干细胞的作用,我们从成体小鼠海马中分离培养神经干细胞。成体海马神经干细胞在含有20ng/mlEGF和20ng/mlbFGF的细胞培液中快速增殖。研究表明,在收到长期温和压力的小鼠和抑郁症病人中,调控神经干细胞功能的生长因子和神经营养因子水平下降,从而可能导致神经发生水平下降。因此,我们采用了低EGF和bFGF的培养条件来模拟这种病例条件。如图1B所示,在1ng/mlEGF和1ng/mlbFGF的培养条件下,CG402呈剂量依赖性地增加神经干细胞中的EdU掺入。类似地,CG402也促进了胚胎小鼠皮层来源的神经干细胞的增殖(图1C)。另一方面,在分化条件下与对照组相比,CG402对Tuj1和GFAP阳性细胞的比例没有明显的影响,表明在体外CG402对神经干细胞的谱系分化(lineagecommitment)没有明显影响(图1D)。
为了探究CG402是否促进体内成体海马神经发生,8周大的C57BL/6小鼠口服给药14天,最后一天注射溴脱氧尿苷(bromodeoxyuridine,BrdU)以标记分裂的细胞。免疫荧光分析结果表明,与对照组相比,60mg/kgCG402给药组小鼠SGZ区BrdU阳性的增殖细胞数量增加;而200mg/kgCG402给药组没有明显变化(图1E)。
为了探究CG402给药是否能够影响行为学表现,我们采用了小鼠强迫游泳实验和悬尾实验来检测CG402的抗抑郁活性。氟西汀是一种常用的临床用抗抑郁药,我们采用氟西汀作为阳性对照。在强迫游泳实验中,CG402和氟西汀都能够减少小鼠的不动时间(图1F)。有趣的是,把CG402的剂量降低到20mg/kg后,其具有更显著的抗抑郁作用。在悬尾实验中,20mg/kgCG402和氟西汀都能减少小鼠的不动时间,而60mg/kgCG402与对照组相比没有显著差异(图1G)。
我们同时检测了这些小鼠海马中的神经发生。在给药的最后5天,我们通过注射BrdU标记增殖的神经干细胞,7天之后行为学实验结束,处死小鼠。我们检测了成神经细胞(neuroblast)和非成熟神经元的分子标记物Dcx在BrdU阳性细胞中的表达,发现与对照组小鼠相比,20mg/kgCG402给药小鼠的DG区中,BrdU/Dcx双阳性细胞数明显增加(图1H),表明向神经元分化和成熟的细胞数目增加。Pearson相关性分析进一步表明小鼠在悬尾实验中的不动时间与BrdU/Dcx双阳性细胞的数量呈负相关,表明抑郁样表现的减少和神经发生水平的增强可能具有一定的联系(图1J)。然而,CG402给药没有改变BrdU/Dcx双阳性细胞占所有BrdU阳性细胞的比例(图1I),表明CG402给药并不影响神经元谱系分化。
2.在慢性温和应激模型中CG402产生抗抑郁作用
为了进一步检测CG402的抗抑郁作用,我们采用了慢性温和应激的抑郁症模型。研究表明,慢性温和应激能够导致神经发生水平下降、毛色状态受损以及产生抑郁样行为。除无应激组小鼠外,其余小鼠受到6周的慢性温和应激,并于第3周开始口服给药溶剂对照、CG402和氟西汀(图2A)。慢性温和应激开始之前,小鼠毛发处于良好的状态,而在3周的慢性温和应激之后,毛发状态明显受损(图2B)。我们发现,慢性给药CG402和氟西汀改善了毛发状态(图2B)。我们进一步通过强迫游泳实验来检测这些小鼠的抑郁样行为。与之前报道的现象一致,受到慢性温和应激的小鼠不动时间明显增加;而CG402和氟西汀给药减少了不动时间(图2C)。另一方面,旷场实验中,CG402不影响小鼠行动的距离和速度,表明运动不受影响。综上所述,在慢性温和应激模型中,CG402改善了小鼠的抑郁样行为表现。
进一步地,我们在这些小鼠给药的最后5天注射BrdU,并于7天后检测了小鼠的神经发生水平(图2A)。慢性温和应激大幅降低了BrdU/Dcx双阳性细胞的数量,表明应激刺激抑制神经发生(图2D)。有趣的是,我们发现CG402显著增加了BrdU/Dcx双阳性细胞,而BrdU/Dcx双阳性细胞所占所有BrdU阳性细胞的比例并没有明显变化(图2D-E)。这些数据表明,CG402改善了由应激刺激损伤的神经发生,并产生抗抑郁作用。
3.CG402激活ERK级联反应并增加EGF水平
研究表明,ERK和/或Akt级联反应的激活对于神经干细胞增殖和自我更新非常关键。因此,我们通过免疫印迹的方法来检测这些级联反应的激活。如图3A所示,CG402加药后,细胞内ERK磷酸化水平增加,在2-10分钟到达峰值,然后在30分钟内缓慢下降到基础水平。然而,Akt和GSK3磷酸化水平则没有明显变化(图3A)。为了进一步检测CG402促进神经干细胞增殖的效果是否依赖于ERK级联反应的激活,我们在神经干细胞中加入MEK抑制剂U0126、ERK抑制剂FR180204、PI3K抑制剂LY294002或者Akt抑制剂MK-2206。如图3B所示,用U0126和FR180204处理细胞能够阻断G402促进神经干细胞增殖的作用,表明MEK/ERK级联反应是CG402促进神经干细胞增殖所必需的。有趣的是,LY294002和MK-2206也能在一定程度上阻断G402促进神经干细胞增殖的作用,提示可能同时存在其它机制。
神经干细胞分泌生长因子和神经营养因子,这些因子通过ERK和Akt级联反应等反过来调控神经干细胞的功能。因此,我们用实时定量PCR检测了体外培养的神经干细胞中的Egf、Fgf2和Bdnf的基因表达。CG402处理后的24小时,基因Egf的表达增加到对照组的2倍;而基因Fgf2和Bdnf的表达没有明显差异(图3C)。为了进一步验证EGFR信号通路在CG402促进体内神经干细胞增殖中的作用,我们检测了CG402给药的应激小鼠海马内的EGF水平。有趣的是,我们发现慢性应激显著地降低了小鼠海马内的EGF水平,而CG402和氟西汀部分恢复了EGF水平(图3D)。综上所述,CG402激活ERK级联反应并增加EGF水平来促进NPC增殖,而不影响Akt级联反应。
Claims (10)
1.阿魏酸与细辛醚类似物拼合的化合物,其特征在于,结构式如(Ⅰ):
。
2.一种药物组合物,包含权利要求1所述的阿魏酸与细辛醚类似物拼合的化合物和药学上可接受的载体。
3.权利要求1所述的阿魏酸与细辛醚类似物拼合的化合物或权利要求2所述的组合物在制备神经退行性疾病治疗药物中的应用。
4.一种如权利要求1所述的阿魏酸与细辛醚类似物拼合的化合物的制备方法,其特征在于:
(1)阿魏酸酚羟基乙酰化
阿魏酸与乙酸酐,在合适的溶剂和催化剂作用下反应得乙酰化阿魏酸;
(2)(E)-3-(4-乙酰氧基-3-甲氧基苯基)丙烯酰氯合成
乙酰化的阿魏酸在合适的溶剂中,在催化剂作用下,与氯化试剂反应制得酰
氯;
(3)细辛醚类似物制备
1,3,4-三甲氧基苯与丙酰氯、无水三氯化铝,无溶剂或在合适溶剂中反应,反应液倾入到冰水中,分取有机相,有机相再用碱液萃取,合并碱液再调酸性,析出固体,抽滤,即得;
(4)缩合成酯
将步骤(2)制得的乙酰化阿魏酸酰氯与步骤(3)制得的细辛醚类似物在合适溶剂和碱作用下反应,反应液倾入冰水中,分取有机相,无水硫酸钠干燥后,蒸去有机溶剂,即得粗品,乙醇重结晶得精品。
5.如权利要求4所述的制备方法,其特征在于,步骤(1)、(2)或(4)中,所述的溶剂为二氯甲烷、氯仿、甲苯、二甲苯、四氢呋喃、2-甲基四氢呋喃、甲基叔丁基醚、乙酸乙酯或乙酸丁酯;步骤(3)中所述的溶剂为二氯甲烷、氯仿或甲基叔丁基醚。
6.如权利要求4或5所述的制备方法,其特征在于,步骤(1)中反应温度为-10-80℃,反应0.5-10小时,反应温度优选20-35℃,反应时间优选1-2小时。
7.如权利要求4-6任何一项所述的制备方法,其特征在于,步骤(2)中所述的催化剂为碱,优选吡啶、三乙胺反应温度为15-110℃,反应时间为1-10小时。
8.如权利要求4-7任何一项所述的制备方法,其特征在于,所述的氯化试剂为二氯亚砜、三氯化磷、五氯化磷、三氯氧磷或草酰氯。
9.如权利要求4-8任何一项所述的制备方法,其特征在于,步骤(3)中萃取碱液为氢氧化钠、氢氧化钾、碳酸钠、碳酸钾的水溶液;反应温度0-80℃,反应时间为1-12h。
10.如权利要求4-9任何一项所述的制备方法,其特征在于,步骤(4)中所述的碱为有机碱或无机碱,所述的有机碱为三乙胺或吡啶,无机碱为碳酸氢钠、碳酸酸氢钾、碳酸钠、碳酸钾,优选三乙胺;反应温度-10-50℃,反应时间0.5-5小时。
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