CN110041221A - 一种神经酰胺类似物及其制备方法和应用 - Google Patents
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Abstract
本发明公开一种神经酰胺类似物及其制备方法和应用。本发明利用FA1‑5与9‑羟基‑十八烷酸反应合成了一类新型脂肪酸羟基脂肪酸酯(FA‑FAs)。用鞘氨醇分别与相对应的5个FA‑FAs反应合成了5个新型的神经酰胺类似物(FA‑FA‑CerA‑E)。通过体外细胞实验来考察了FA‑FA‑CerA‑E逆转乳腺癌他莫昔芬耐药的活性。研究发现本发明制备的神经酰胺类似物与与他莫昔芬合用时,逆转他莫昔芬的耐药产生;对减轻乳腺癌他莫昔芬耐药患者的痛苦和经济负担,提高治愈率和生存期具有重要临床意义,为今后抗肿瘤神经酰胺类化合物的设计和合成提供了有价值的参考依据。
Description
技术领域
本发明涉及化学合成及临床机制研究领域,特别涉及可逆转乳腺癌他莫昔芬耐药的新型神经酰胺的合成及其机制研究,具体涉及一种神经酰胺类似物及其制备方法和应用。
背景技术
鞘脂是细胞膜的主要组成成分,可作为第一或第二信使调控细胞的生命活动,如生长、分化、衰老和凋亡等许多重要的信号转导过程;近年来鞘脂组学可以与基因组或蛋白质组相媲美,甚至超过他们。调控肿瘤细胞生长及转移的活性鞘脂主要有神经酰胺(ceramide,Cer)、鞘氨醇(sphingosine,Sph)和鞘氨醇-1-磷脂(sphingosine-1-phosphate,S1P),它们构成了一个重要的代谢平衡体,被称为鞘脂变阻器(Cer-sph-S1P)。其中Cer和Sph具有抑制细胞生长、促进细胞凋亡的作用;而S1P则具有促进细胞增殖、分化的作用(1)。鞘脂变阻器的失衡(Cer减少而S1P升高)是肿瘤细胞增殖和迁移的关键决定因素。
神经酰胺是天然存在于细胞中的脂质分子家族。通常,神经酰胺由长链碱基(鞘氨醇或鞘氨醇类似物)和酰胺连接的脂肪酸(N-酰基链)组成。长链碱基的主要结构是鞘氨醇,并且N-酰基链长度为10~26个碳,在大多数哺乳动物细胞中饱和或单不饱和。
神经酰胺的抗肿瘤活性:神经酰胺是鞘脂代谢的主要中间体,与癌症发病机理的不同阶段密切相关。其具有显著的肿瘤抑制特性,并诱导癌细胞中的衰老,生长停滞和凋亡。越来越多的证据表明,通过提高体内外神经酰胺水平可以杀死各种类型的癌细胞或肿瘤。有研究表明,2'-羟基化神经酰胺可诱导C6胶质瘤细胞凋亡相关的蛋白质组学变化(2)。C6神经酰胺已被证明可增强姜黄素介导的黑色素瘤细胞死亡。制备C6神经酰胺和姜黄素的共包封并评估KHOS和MG-63细胞系中的抗癌功效时发现:与单独使用姜黄素相比,姜黄素与C6神经酰胺组合在KHOS和MG-63细胞系的情况下增强了1.5倍的抗癌功效。此外,C6神经酰胺-姜黄素组合对未转化的人细胞毒性较小。这些研究均为在临床水平上合成神经酰胺和神经酰胺类似物对特定抗药性癌症的进一步评估奠定了夯实基础。此外,神经酰胺在耐药产生中的作用逐渐被重视,前期研究表明Cer转化为S1P后可介导各种癌症产生耐药性(3)(4),例如:阻断S1P-S1PR1使抗性结直肠癌肿瘤对西妥昔单抗恢复敏感;外源性S1P促进乳腺肿瘤小鼠放疗抗性等(5)。研究者们在乳腺癌治疗中也发现:SPHK1在正常乳腺组织中无表达而在转移乳腺癌中高表达;高表达的SPHK1通过抑制乳腺癌转移抑制因子1(BRMS1)的信号诱导转移(6)。提示:Cer-Sph-S1P平衡所涉及的酶(SPHK1)、代谢物(S1P)和受体(S1PR1)可能与乳腺癌耐药相关。并且,他莫昔芬在靶向ERα治疗中会脱靶至鞘脂代谢,通过抑制Cer的水解从而促进其诱导的细胞凋亡;提示,他莫昔芬诱导的细胞凋亡与鞘脂代谢相关。
靶向鞘脂变阻器Cer-Sph-S1P,恢复神经酰胺功能并逆转肿瘤增殖迁移的抑制剂可以分为三类:①神经酰胺类制剂(Cer)。纳米脂质体Cer(NLC)以恢复Cer促进细胞凋亡功能,目前正在进行晚期实体瘤患者的I期临床试验。②鞘氨醇类似物。SPHK1(鞘氨醇激酶1)和S1PR1(鞘氨醇1磷酸酶受体1)抑制剂均属于这一类。比如:SK1-I(鞘氨醇激酶1抑制剂I),SPHK1的竞争性抑制剂,可减弱细胞系和异种移植模型中的胶质母细胞瘤生长和/或增殖;Fingolimod(FTY720)是一种鞘氨醇(Sph)类似物,进入细胞中被SPHK1磷酸化成为磷酸Fingolimod(p-FTY720);通过竞争性拮抗S1P与S1PR1结合从而阻断S1P-S1PR1细胞存活信号传导,已被FDA批准用于临床。③抗体类药物。Sphingomab已在II期临床试验中用于治疗对VEGF治疗难以治疗的肾透明细胞癌(RCC)(表1)。目前,神经酰胺类药物是临床新药密集开发的主题。
表1调控鞘脂代谢的抗癌药物目录
Name | Target or activity | Stage of development | Refs |
Fingolimod(FTY720) | S1PR1 | FDA批准用于多发硬化症 | (7) |
SK1-I | SPHK1 | Preclinical | (8) |
PF543 | SPHK1 | Preclinical | (9) |
VPC03090 | S1PR1;S1PR3 | Preclinical | (10) |
Sphingomab | S1P | PhaseⅡ | (11) |
这些药物的开发表明神经酰胺的诱导或S1P信号传导的抑制是临床抗癌疗法的创新策略。神经酰胺是鞘脂类新药合成的一个重要目标,通常是由长链碱基(鞘氨醇或鞘氨醇类似物)和酰胺连接的脂肪酸(N-酰基链)组成。酰胺连接部分都是一些普通类型的饱和或不饱和的16~20链长的脂肪酸。本发明的主要目的是合成含有新型结构脂肪酸的神经酰胺,并且筛选其生物活性。
参考文献:
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发明内容
本发明通过观测乳腺癌细胞基因表达谱显示:耐药细胞中鞘脂代谢酶基因特异性高表达,包括:神经酰胺水解酶(ASAH2)、SPHK1和神经酰胺激酶(CERK);并且从癌症基因组图谱(TCGA)、基因表达综合数据库(GEO)和Oncomine三种在线数据库的统计结果与表达谱数据一致。Kaplan-Meier生存分析表明:SPHK1的高表达与乳腺癌患者的预后不良正相关。
高表达的SPHK1究竟会导致Cer-Sph-S1P产生怎样的变化?申请人用高分辨QE质谱分析技术比较了他莫昔芬治疗失败10组乳腺癌组织标本的鞘脂代谢物差异。结果表明,Cer的含量在乳腺癌耐药肿瘤组织中比例明显下降,而S1P则明显增加这点与mRNA的结果相匹配。这种现象在他莫昔芬耐药的MCF-7乳腺癌细胞中也得到了验证。证明:他莫昔芬耐药乳腺癌组织及细胞中,鞘脂变阻器Cer-Sph-S1P的平衡被干扰。目前临床上对他莫昔芬耐药的乳腺癌并没有很好的治疗药物;并且可选的神经酰胺类药物较少。
为了克服现有药物的缺点与不足,本发明的目的在于提供一种神经酰胺类似物。
本发明的另一目的在于提供上述神经酰胺类似物的制备方法。
本发明的再一目的在于提供上述神经酰胺类似物的应用。
利用脂肪酸FA1-5与9位羟基十八烷酸反应合成了一类新型脂肪酸羟基脂肪酸酯(FA-FAs)。用鞘氨醇分别与相对应的5个FA-FAs反应合成了5个新型的神经酰胺类似物(FA-FA-CerA-E)。通过体外细胞实验来考察了FA-FA-CerA-E逆转乳腺癌他莫昔芬耐药的活性。研究发现FA-FA-CerA在MCF7细胞上显示出较好的抗耐药肿瘤细胞活性。并且,与他莫昔芬合用时,逆转他莫昔芬的耐药产生。
本文首次报导了此类新型神经酰胺类似物的合成以及逆转乳腺癌他莫昔芬耐药的活性评价,为今后抗肿瘤神经酰胺类化合物的设计和合成提供了有价值的参考依据。
本发明的目的通过下述技术方案实现:
一种神经酰胺类似物,其结构式如下所示:
其中,
一种神经酰胺类似物的制备方法,包括如下步骤:
1、9-羟基-十八烷酸的合成:将油酸在四氢呋喃中与硼烷·四氢呋喃-络合物反应;将溶液在室温下搅拌过夜,加入水中以破坏硼烷;然后,将溶液冷却至0℃并逐滴加入3NNaOH溶液和30%H2O2-溶液;在室温下反应,加入水和饱和NaCl,水相用乙酸乙酯萃取;将乙酸乙酯层干燥,过滤,并真空浓缩;通过快速色谱法纯化残余物,得到9-羟基-十八烷酸;
步骤1中所述的油酸、四氢呋喃、硼烷·四氢呋喃-络合物的比值为0.5mmol:3mL:0.4mL;
步骤1中所述的硼烷·四氢呋喃-络合物中四氢呋喃的浓度为0.9M,硼烷的浓度为0.3mmol/0.4mL(=0.75mmol/mL);
步骤1中,加入水的用量与油酸的比值为1mL:0.5mmol;
步骤1中,3N NaOH溶液、30%H2O2-溶液与油酸的比值为2mL:6mL:0.5mmol;
步骤1中,所述的室温下反应的时间为1小时;
步骤1中,所述的干燥为用MgSO4干燥;
步骤1中所述的快速色谱法所用的洗脱液为氯仿/甲醇=200:1;
2、FA-FA1-5的反应条件为:FA1-5和草酰氯溶解在二氯甲烷溶液,将反应混合物在室温下搅拌3~4小时,然后加入9-羟基-十八烷酸;在室温下搅拌过夜后,将反应混合物真空浓缩;通过快速色谱法纯化残余物,得到FA-FA1-5。
所述的FA1-5的结构式如下所示:
步骤2中,FA1-5、草酰氯、9-羟基-十八烷酸的摩尔比为1:1.2~1.5:1;优选为1:1.5:1;
步骤2中,9-羟基-十八烷酸在反应体系中的浓度为0.05~0.1mmol/mL;优选为0.1mmol/mL。
步骤2中所述的快速色谱法所用的洗脱液为氯仿/甲醇=100:1;
3、FA-FA-Cer A-E的合成条件为:鞘氨醇溶解在无水二氯乙烷溶液中,在0℃下加入1-羟基苯并三唑和1-乙基-(3-二甲基氨基丙基)碳酰二亚胺,然后加入三甲胺和FA-FA1-5,将混合物在0℃下搅拌4小时并在室温下搅拌过夜;将其用二氯甲烷稀释,并将有机层用2%HCl(3×10mL)和5%NaHCO3(3×10mL)洗涤;将合并的有机物真空蒸发,通过快速色谱法纯化残余物;得到产物FA-FA-Cer A-E,即神经酰胺类似物。
步骤3中,所述的鞘氨醇、FA-FA1-5的摩尔比值为1:1~1.2;
步骤3中,所述的鞘氨醇、1-羟基苯并三唑、1-乙基-(3-二甲基氨基丙基)碳酰二亚胺、三甲胺的比值为1mmol:1~1.2mmol:1~1.2mmol:2~4mL;
步骤3中所述的快速色谱法所用的洗脱液为氯仿/甲醇=50:1;
上述神经酰胺类似物在制备逆转他莫昔芬耐药产生的产品中的应用。
具体的,所述的神经酰胺类似物与他莫昔芬合用时,逆转他莫昔芬的耐药产生。
本发明相对于现有技术具有如下的优点及效果:
本发明制备的神经酰胺类似物与他莫昔芬合用时,能够逆转他莫昔芬的耐药产生;对减轻乳腺癌他莫昔芬耐药患者的痛苦和经济负担,提高治愈率和生存期具有重要临床意义,为今后抗肿瘤神经酰胺类化合物的设计和合成提供了有价值的参考依据。
附图说明
图1是本发明FA-FA1-5的结构式。
图2是本发明神经酰胺类似物的合成路线图。
图3是神经酰胺类似物FA-FA-Cer A与神经酰胺的结构差异。
图4是FA-FA-Cer A对他莫昔芬耐药MCF-7细胞增殖和迁移的影响;其中,图中FA-FA-Cer表示FA-FA-CerA;Tamoxifen表示他莫昔芬。
图5是他莫昔芬耐药MCF-7细胞中加入神经酰胺类似物FA-FA-Cer A对磷酸化PI3K,Akt和p53蛋白表达量的免疫印迹实验。
具体实施方式
下面结合实施例及附图对本发明作进一步详细的描述,但本发明的实施方式不限于此。
下列实施例中未注明具体条件的实验方法,通常按照常规条件或按照厂商所建议的条件实施。
实施例中所涉及的结构表征涉及:旋光度测量:Rudolph Research AnalyticalAutopol I自动旋光仪(Rudolph Research Analytical,Hackettstown,NJ,USA)上。结构鉴定:核磁共振(NMR)光谱在Bruker Ascend 600NMR光谱仪(Bruker,Zurich,Switzerland)600MHz获得1H NMR,150MHz获得13C NMR。样品溶解:CD3OD或MeOD中(残留溶剂或TMS作为内部参考)。化学位移以δ(ppm)表示,偶合常数(J)以赫兹(Hz)给出。分子量测定在ThermoQExtaractive质谱仪(Thermo fisher scientific,USA)上以正负离子交替模式进行。用Davisil硅胶(粒径40-63微米)进行柱色谱分离。在硅胶60F254板和TLC硅胶60RP-18F254S板(200μm厚,Merck KGaA,Germany)上进行薄层色谱(TLC)。
实施例1
1、9-羟基-十八烷酸的合成。油酸(0.1g,0.5mmol)在四氢呋喃(3mL)中与硼烷·四氢呋喃-络合物(0.4mL,0.9M,0.3mmol硼烷)反应。将溶液在室温下搅拌过夜,加入水(1.0mL)中以破坏硼烷。然后,将溶液冷却至0℃并逐滴加入3N NaOH溶液(2mL)和30%H2O2-溶液(6mL)。在室温下反应1小时后,加入水(10mL)和饱和NaCl,水相用乙酸乙酯(3×25mL)萃取。将乙酸乙酯层干燥(MgSO4),过滤,并真空浓缩。通过快速色谱法(氯仿/甲醇200:1,v/v)纯化残余物,得到白色粉末9-羟基-十八烷酸(42.2mg,42%)。Rf=0.3(氯仿/甲醇10:1,v/v)。1H NMR(600MHz,CDCl3):δ3.58-3.60(m,1H,H-9),2.34(t,J=7.5Hz,2H),1.61-1.64(m,2H),1.26-1.46(m,26H),0.88(t,J=7.0Hz,3H);13C NMR(150MHz,CDCl3):δ179.4,72.1,37.4,37.3,34.0,31.9,29.7,29.6,29.6,29.4,29.3,29.2,29.0,25.7,25.5,24.7,22.70,14.1。ESI-MS(m/z):[M-H]-的测量值为299.2592。
2、FA-FAs 1-5的反应条件为:脂肪酸1-5(FA1-5)(1当量)(结构式如图1所示)和草酰氯(1.2~1.5当量)溶解在二氯甲烷(DCM)溶液(10~20mL),将反应混合物在室温下搅拌3~4小时,然后加入9-羟基-十八烷酸(1.0当量)。在室温下搅拌过夜后,将反应混合物真空浓缩。通过快速色谱法(氯仿/甲醇100:1,v/v)纯化残余物,得到白色固体。所得反应物的Rf=0.7(氯仿/甲醇10:1,v/v)。
(1)FA-FA 1的合成:(9-(((2Z,4E)-癸-2,4-二烯酰基)氧基)十八烷酸)的合成。FA1(0.2mmol),草酰氯(0.3mmol)和9-羟基-十八烷酸(0.2mmol)的无水DCM(2mL)溶液。通过柱色谱(氯仿/甲醇100:1,v/v)纯化粗产物,得到化合物FA-FA 1,为白色固体(产率约59%)。Rf=0.7(氯仿/甲醇10:1,v/v)。1H NMR(600MHz,CDCl3):δ7.37(dd,J=11.3,15.2Hz,1H,H-4'),6.54(t,J=11.3Hz,1H,H-3'),6.07(dt,J=15.1,7.3Hz,1H,H-5'),5.55(d,J=11.3Hz,1H,H-2'),4.89-4.93(m,1H,H-9),2.34(t,J=7.5Hz,2H),2.19(q,2H),1.60-1.65(m,2H),1.53-1.57(m,4H),1.42-1.47(m,2H),1.25-1.34(m,26H),0.86-0.90(m,6H);13CNMR(150MHz,CDCl3):δ178.2,166.5,145.6,145.2,126.9,115.9,73.9,34.2,34.2,33.7,33.0,31.9,31.5,29.7,29.6,29.3,29.1,29.0,28.5,25.4,25.3,24.6,22.7,22.5,14.1,14.0。ESI-MS(m/z):[M-H]-[C28H49O4]的测量值为499.3642。
(2)FA-FA 2的合成:9-(((2E,4E)-癸-2,4-二烯酰基)氧基)十八烷酸。FA 2(0.2mmol),草酰氯(0.3mmol)和9-羟基-十八烷酸(5)(0.2mmol)在无水二氯甲烷(2mL)中。通过柱色谱(氯仿/甲醇100:1,v/v)纯化粗产物,得到化合物FA-FA 2,为白色固体(产率71%)。Rf=0.7(氯仿/甲醇10:1,v/v)。1H NMR(600MHz,CDCl 3):δ7.24(dd,J=10.1,15.3Hz,1H,H-3'),6.10-6.19(m,2H,H-4'和H-5'),5.78(d,J=15.3Hz,1H,H-2'),4.91-4.95(m,1H,H-9),2.33(t,J=7.5Hz,2H),2.16(q,2H),1.59-1.63(m,2H),1.49-1.56(m,4H),1.40-1.45(m,2H),1.25-1.30(m,26H),0.86-0.90(m,6H);13C NMR(150MHz,CDCl3):δ179.1,167.2,144.9,144.5,128.4,119.7,74.1,34.3,34.2,33.9,33.0,31.9,31.4,29.6,29.6,29.3,29.3,29.1,29.0,25.3,25.3,24.7,22.7,22.5,14.1。ESI-MS(m/z):[M-H]-[C28H49O4]的测量值为499.3640。
(3)FA-FA 3的合成:9-(((2Z,4Z)-十二碳-2,4-二烯酰基)氧基)十八烷酸。FA 3(0.2mmol),草酰氯(0.3mmol)和9-羟基-十八烷酸(0.2mmol)在无水二氯甲烷(2mL)中。通过柱色谱(氯仿/甲醇100:1,v/v)纯化粗产物,得到化合物FA-FA 3,为白色固体(产率为52%)。Rf=0.7(氯仿/甲醇10:1,v/v)。1H NMR(600MHz,CDCl3):δ7.27(dd,J=11.5,11.5Hz,1H,H-4'),6.92(t,J=11.7Hz,1H,H-3'),5.90(dt,J=11.5,7.7Hz,1H,H-5'),5.66(d,J=11.7Hz,1H,H-2'),4.90-4.94(m,1H,H-9),2.34(t,J=7.5Hz,2H),2.26(q,2H),1.60-1.64(m,2H),1.51-1.56(m,4H),1.40-1.44(m,2H),1.25-1.31(m,26H),0.86-0.90(m,6H);13C NMR(150MHz,CDCl3):δ179.6,166.5,141.5,138.7,124.5,117.8,73.9,34.2,34.2,34.0,31.9,31.4,29.6,29.5,29.329.1,29.0,29.0,27.5,25.4,25.3,24.6,22.7,22.5,14.1,14.0。ESI-MS(m/z):[M-H]-[C28H49O4]的测量值为499.3637。
(4)FA-FA 4的合成:9-(((2E,4Z)-十二碳-2,4-二烯酰基)氧基)十八烷酸。FA 4(0.2mmol),草酰氯(0.3mmol)和9-羟基-十八烷酸(0.2mmol)在无水二氯甲烷(2mL)中。通过柱色谱(氯仿/甲醇100:1,v/v)纯化粗产物,得到化合物FA-FA 4,为白色固体(40.3mg,45%)。Rf=0.7(氯仿/甲醇10:1,v/v)。1H NMR(600MHz,CDCl3):δ7.60(dd,J=11.7,15.2Hz,1H,H-3'),6.12(t,J=11.1Hz,1H,H-4'),5.77-5.88(m,2H,H-2'和H-5'),4.92-4.97(m,1H,H-9),2.34(t,J=7.5Hz,2H),2.30(q,2H),1.60-1.63(m,2H),1.50-1.57(m,4H),1.39-1.43(m,2H),1.25-1.31(m,26H),0.86-0.90(m,6H);13C NMR(150MHz,CDCl3):δ177.8,167.3,141.6,139.4,126.4,121.5,74.2,34.3,34.2,33.6,31.9,31.4,29.6,29.5,29.3,29.1,29.1,29.0,28.2,25.3,25.3,24.6,22.7,22.5,14.1,14.0。ESI-MS(m/z):[M-H]-[C28H49O4]的测量值为499.3631。
(5)FA-FA 5的合成:(E)-9-(癸-2-烯酰氧基)十八烷酸。FA 5(0.2mmol),草酰氯(0.3mmol)和9-羟基-十八烷酸(0.2mmol)的无水二氯甲烷(2mL)。通过柱色谱(氯仿/甲醇100:1,v/v)纯化粗产物,得到化合物FA-FA 4,为白色固体(46.1mg,50%)。Rf=0.7(氯仿/甲醇10:1,v/v)。1H NMR(600MHz,CDCl3):δ6.95(dt,J=15.5,6.9Hz,1H,H-3'),5.80(d,J=15.5Hz,1H,H-2'),4.89-4.94(m,1H,H-9),2.34(t,J=7.6Hz,2H),2.19(q,2H),1.60-1.63(m,2H),1.50-1.55(m,4H),1.43-1.47(m,2H),1.25-1.30(m,30H),0.86-0.89(m,6H);13CNMR(150MHz,CDCl3):δ179.1,166.8,149.3,121.5,74.1,34.2,34.2,33.9,32.2,31.9,31.7,29.6,29.5,29.3,29.2,29.1,29.0,28.9,28.0,25.3,25.2,24.6,22.7,22.6,14.1,14.1。ESI-MS(m/z):[M-H]-[C28H49O4]的测量值为451.3795。
3、FA-FA-Cers A-E的合成条件为:鞘氨醇(1.0当量)溶解在无水二氯乙烷溶液中,在0℃下加入1-羟基苯并三唑(1~1.2当量)和1-乙基-(3-二甲基氨基丙基)碳酰二亚胺(1~1.2当量),然后加入三甲胺(100~200μL)和FA-FA 1-5(1~1.2当量),将混合物在0℃下搅拌4小时并在室温下搅拌过夜。将其用二氯甲烷稀释,并将有机层用2%HCl(3×10mL)和5%NaHCO3(3×10mL)洗涤。将合并的有机物真空蒸发,得到产物。通过硅胶快速色谱法纯化残余物,用氯仿/甲醇洗脱。(合成线路图如图2所示)
(1)1-(((3R,E)-1,3-二羟基十八碳-4-烯-2-基)氨基)-1-氧代十八烷-9-基(2Z,4E)-癸-2,4-二烯酸酯(FA-FA-CerA)的合成。鞘氨醇(0.05mmol),1-羟基苯并三唑(0.05mmol)、1-乙基-(3-二甲基氨基丙基)碳酰二亚胺(0.05mmol)、三乙胺(100μL)和FA-FA1(0.05mmol)的无水DCM(二氯甲烷)(2mL)溶液。通过柱色谱(氯仿/甲醇50:1,v/v)纯化粗产物,得到化合物FA-FA-CerA,为白色固体(15.1mg,86%)。Rf=0.5(氯仿/甲醇10:1,v/v)。[α]20D=-42.3(c=0.1,DMSO)。1H NMR(600MHz,CDCl3):δ7.35(dd,J=11.3,15.2Hz,1H,H-4”),6.54(t,J=11.3Hz,1H,H-3”),6.37(dd,J=2.5,7.6Hz,1H,NH),6.07(dt,J=15.3,7.3Hz,1H,H-5”),5.78(dt,J=14.3,7.2Hz,1H,H-5),5.55(d,J=11.1Hz,1H,H-2”),5.53(dd,J=6.4,15.3Hz,1H,H-4),4.89-4.92(m,1H),4.31-4.33(m,1H),3.94-3.97(m,1H),3.90-3.91(m,1H),3.70(dd,J=2.4,11.3Hz,1H),2.17-2.24(m,4H),2.03-2.07(m,2H),1.60-1.65(m,2H),1.54-1.56(m,4H),1.42-1.47(m,2H),1.35-1.38(m,2H),1.25-1.30(m,46H),0.86-0.90(m,9H);13C NMR(150MHz,CDCl3):δ174.0,166.7,145.8,145.3,134.2,128.8,126.9,115.8,74.6,73.9,62.4,54.4,36.7,34.3,34.2,33.0,32.3,31.9,31.9,31.5,29.7,29.7,29.6,29.6,29.6,29.5,29.4,29.3,29.3,29.2,29.1,29.1,29.0,29.0,28.5,25.7,25.4,25.3,22.7,22.9,22.5,18.2,14.1,14.0。ESI-MS(m/z):[M+H]+[C46H8NO5]+的实测值732.6507。其中,神经酰胺衍生物FA-FA-CerA与神经酰胺Ceramide的结构差异,如图3所示。
(2)1-(((3R,E)-1,3-二羟基十八碳-4-烯-2-基)氨基)-1-氧代十八烷-9-基(2E,4E)-癸-2,4-二烯酸酯(FA-FA-CerB)的合成。鞘氨醇(0.05mmol),1-羟基苯并三唑(0.05mmol)、1-乙基-(3-二甲基氨基丙基)碳酰二亚胺(0.05mmol)、三乙胺(100μL)和FA-FA2(0.05mmol)的无水二氯甲烷(2mL)溶液。通过柱色谱(氯仿/甲醇50:1,v/v)纯化粗产物,得到化合物FA-FA-CerB,为白色固体(32.1mg,88%)。Rf=0.5(氯仿/甲醇10:1,v/v)。[α]20D=16.4(c=0.1,DMSO)。1H NMR(600MHz,CDCl3):δ7.35(dd,J=11.3,15.1Hz,1H,H-4”),6.54(t,J=11.31Hz,1H,H-3”),6.29-6.32(m,1H,NH),6.07(dt,J=15.1,7.29Hz,1H,H-5”),5.78(dt,J=14.5,7.2Hz,1H,H-5),5.55(d,J=11.1Hz,1H,H-2”,5.53(dd,J=6.4,15.4Hz,1H,H-4),4.88-4.93(m,1H),4.31-4.33(m,1H),3.96(dd,J=3.7,11.3Hz,1H),3.96(dt,J=10.9,3.6Hz,1H),3.70(dd,J=2.2,11.5Hz,1H),2.88(b,2H),2.17-2.24(m,4H),2.04-2.07(m,2H),1.61-1.66(m,4H),1.54-1.56(m,4H),1.42-1.47(m,2H),1.35-1.38(m,2H),1.26-1.30(m,44H),0.86-0.90(m,9H);13C NMR(150MHz,CDCl3):δ173.9,166.6,145.7,145.2,134.2,128.8,126.9,115.9,74.8,73.9,62.5,54.3,36.8,34.2,34.2,33.0,32.3,31.9,31.9,31.9,31.5,29.7,29.7,29.6,29.6,29.6,29.5,29.4,29.3,29.3,29.2,29.2,29.1,28.5,25.7,25.4,25.3,22.7,22.7,22.7,22.5,14.1,14.0。ESI-MS(m/z):[M+H]+[C46H8NO5]+的实测值为732.6501。
(3)1-(((3R,E)-1,3-二羟基十八碳-4-烯-2-基)氨基)-1-氧代十八烷-9-基(2Z,4Z)-癸-2,4-二烯酸酯(FA-FA-CerC)的合成。鞘氨醇(0.05mmol),1-羟基苯并三唑(0.05mmol)、1-乙基-(3-二甲基氨基丙基)碳酰二亚胺(0.05mmol)、三乙胺(100μL)和FA-FA3(0.05mmol)的无水二氯甲烷(2mL)溶液。通过柱色谱(氯仿/甲醇50:1,v/v)纯化粗产物,得到化合物FA-FA-CerC,为白色固体(31.7mg,87%)。Rf=0.5(氯仿/甲醇10:1,v/v)。[α]20D=-18.7(c=0.1,DMSO)。1H NMR(600MHz,CDCl3):δ7.21-7.26(m,1H),6.45(d,J=7.6Hz,1H),6.10-6.19(m,2H),5.76-5.79(m,2H),5.51-5.55(m,1H,H-4),4.91-4.96(m,1H),4.30-4.32(m,1H),3.95(dd,J=4.1,11.2Hz,1H),3.89-3.92(m,1H),3.70(dd,J=3.3,11.2Hz,1H),2.22(t,J=7.5Hz,2H),2.16(q,2H),2.03-2.07(m,2H),1.60-1.66(m(2H),1.51-1.56(m,4H),1.40-1.45(m,2H),1.34-1.38(m,2H),1.25-1.30(m,46H),0.86-0.90(m,9H);13CNMR(150MHz,CDCl3):δ174.1,167.5,145.1,144.9,134.0,128.8,128.3,119.5,74.5,74.2,62.4,54.4,36.7,34.4,34.3,34.3,33.0,32.3,31.9,31.9,31.4,29.7,29.7,29.7,29.6,29.5,29.4,29.3,29.3,29.2,29.1,29.1,29.0,29.0,28.9,28.4,25.6,25.4,25.2,22.7,22.7,22.5,14.1,14.0。ESI-MS(m/z):[M+H]+[C46H8NO5]+的实测值为732.6501。
(4)1-(((3R,E)-1,3-二羟基十八碳-4-烯-2-基)氨基)-1-氧代十八烷-9-基(2E,4Z)-癸-2,4-二烯酸酯(FA-FA-CerD)的合成。鞘氨醇(0.05mmol),1-羟基苯并三唑(0.05mmol)、1-乙基-(3-二甲基氨基丙基)碳酰二亚胺(0.05mmol)、三乙胺(100μL)和FA-FA4(0.05mmol)的无水二氯甲烷(2mL)溶液。通过柱色谱(氯仿/甲醇50:1,v/v)纯化粗产物,得到化合物FA-FA-CerD,为白色固体(29.5mg,81%)。Rf=0.5(氯仿/甲醇10:1,v/v)。[α]20D=-9.7(c=0.1,DMSO)。1H NMR(600MHz,CDCl3):δ7.21-7.26(m,1H),6.22(d,J=7.6Hz,1H,NH),6.10-6.19(m,2H),5.77-5.81(m,2H),5.52-5.55(m,1H,H-4),4.90-4.95(m,1H),4.32-4.33(m,1H),3.97(dd,J=2.5,11.3Hz,1H),3.90-3.93(m,1H),3.71(dd,J=2.2,11.0Hz,1H),2.23(t,J=7.5Hz,2H),2.16(q,2H),2.05(q,2H),1.61-1.66(m(2H),1.51-1.56(m,4H),1.40-1.45(m,2H),1.34-1.38(m,2H),1.25-1.30(m,46H),0.86-0.90(m,9H);13C NMR(150MHz,CDCl3):δ173.9,167.4,145.0,144.8,134.2,128.8,128.3,119.5,74.8,74.1,62.5,54.3,36.8,34.3,34.2,33.0,32.3,31.9,31.9,31.4,29.7,29.7,29.6,29.6,29.5,29.4,29.3,29.2,29.1,29.0,28.4,25.7,25.3,25.2,22.7,22.7,22.5,14.1,14.0。ESI-MS(m/z):[M+H]+[C46H8NO5]+的实测值为732.6500。
(5)1-(((3R,E)-1,3-二羟基十八碳-4-烯-2-基)氨基)-1-氧代十八烷-9-基(E)-癸-2-烯酸酯(FA-FA-CerE)的合成。鞘氨醇(0.05mmol),1-羟基苯并三唑(0.05mmol)、1-乙基-(3-二甲基氨基丙基)碳酰二亚胺(0.05mmol)、三乙胺(100μL)和FA-FA 5(0.05mmol)的无水二氯甲烷(2mL)溶液。通过柱色谱(氯仿/甲醇50:1,v/v)纯化粗产物,得到化合物FA-FA-CerE,为白色固体(30.7mg,84%)。Rf=0.5(氯仿/甲醇10:1,v/v)。[α]20D=30.5(c=0.1,DMSO)。1H NMR(600MHz,CDCl3):δ7.25(t,J=11.0Hz,1H),6.92(t,J=11.7Hz,1H),6.31(d,J=7.6Hz,1H,NH),5.88-5.92(m,1H),5.79(dt,J=14.7,7.1Hz,1H,H-5),5.65(d,J=11.5Hz,1H),5.53(dd,J=6.1,15.7Hz,1H,H-4),4.89-4.93(m,1H),4.32-4.33(m,1H),3.95-3.97(m,1H),3.90-3.92(m,1H),3.70(d,J=10.7Hz,1H),2.89(b,2H),2.21-2.28(m,4H),2.05(q,2H),1.62-1.64(m,4H),1.51-1.56(m,4H),1.40-1.47(m,2H),1.35-1.37(m,2H),1.26-1.30(m,44H),0.86-0.90(m,9H);13C NMR(150MHz,CDCl3):δ173.9,166.7,141.6,138.8,134.2,128.8,124.4,117.7,74.8,74.0,62.5,54.3,36.7,34.3,34.2,32.3,31.9,31.9,31.4,29.7,29.7,29.6,29.5,29.5,29.4,29.3,29.2,29.2,29.1,29.0,29.0,28.9,27.5,25.7,25.4,25.2,22.7,22.7,22.5,14.1,14.0。ESI-MS(m/z):[M+H]+[C46H8NO5]+的实测值为732.6505。
4、他莫昔芬耐药MCF-7细胞株的构建。野生型MCF-7细胞株,体外低浓度梯度递增联合大剂量间断冲击方法:10nM连续培养4星期;100nM培养3天;20nM连续培养4星期;100nM培养3天;两个月后获得他莫昔芬耐药MCF-7细胞株。用MTT法测定野生型和他莫昔芬耐药细胞的IC50。结果证明经过耐药处理过的细胞,在加入了1000nM他莫昔芬之后,细胞并未产生凋亡;以此验证了耐药株的构建成功。
5、体外实验证明神经酰胺类似物FA-FA-CerA的抗肿瘤活性研究。FA-FA-CerA组(20nM FA-FA-CerA+50nM他莫昔芬)预处理的他莫昔芬耐药株(MCF-7TR),与对照组(50nM他莫昔芬)相比,细胞重新获得对他莫昔芬的敏感性,促凋亡增加,细胞死亡率明显大于未处理的(图4)。细胞划痕实验证明:与单独使用FA-FA-Cer A相比,MCF-7TR细胞加入FA-FA-CerA+他莫昔芬后,促迁移作用被抑制的更为明显;因此间接证明了:FA-FA-Cer A是MCF-7TR细胞重新获得对他莫昔芬的敏感性,促凋亡增加,细胞死亡率明显大于未处理的(图4)。申请人进一步通过Transwell细胞迁移实验(上室种MCF-7TR,下室加入20nM FA-FA-Cer A-/+50nM他莫昔芬)验证:加入FA-FA-CerA后,他莫昔芬抑制的细胞迁移作用明显增加(图4),并且发现其与他莫昔芬联用对细胞迁移的抑制作用比单用更强。提示:FA-FA-CerA可能会逆转乳腺癌他莫昔芬耐药相关。
6、体内实验证明神经酰胺类似物FA-FA-CerA的抗肿瘤活性研究。将他莫昔芬耐药的MCF-7细胞原位注射至NCG小鼠(人源免疫重建小鼠模型:即使用基因编辑技术敲出了NOD/ShiltNju小鼠的prkdc(Protein kinase,DNA activated,catalytic polypeptide)及Il2rg(common gamma receptor);购自江苏集萃药康生物科技有限公司,雌鼠)中,成瘤后,分为4组。A组空白;B组注射他莫昔芬(用量0.5mg/只小鼠);C组注射FA-FA-CerA(每只小鼠用量20μg/只小鼠);D组注射他莫昔芬+FA-FA-CerA(他莫昔芬10mg/mL,注射0.5mg/只小鼠;FA-FA-CerA 1mg/mL,注射20μg/只小鼠),观察肿瘤大小及肺和淋巴结转移。结果发现,FA-FA-CerA与他莫昔芬合用之后,明显减少了耐药肿瘤的大小和肺及淋巴结转移。
7、FA-FA-CerA抑制肿瘤生长机制研究。高达70%的乳腺癌中会出现PI3K/Akt通路的异常,经PI3K/Akt传递的信号可促使细胞在没有雌激素的情况下增殖,因此PI3K/Akt通路中成分的突变和高度激活与乳腺癌内分泌治疗耐药相关。本发明通过细胞实验表明:加入了FA-FA-CerA(50nM)之后PI3K,Akt和p53蛋白的磷酸化水平降低(图5),说明FA-FA-CerA与PI3K/Akt/P53通路抑制相关。
上述实施例为本发明较佳的实施方式,但本发明的实施方式并不受上述实施例的限制,其他的任何未背离本发明的精神实质与原理下所作的改变、修饰、替代、组合、简化,均应为等效的置换方式,都包含在本发明的保护范围之内。
Claims (10)
1.一种神经酰胺类似物,其特征在于:所述神经酰胺类似物的结构式如下所示:
其中,
2.权利要求1所述的神经酰胺类似物的制备方法,其特征在于:包括如下步骤:
(1)9-羟基-十八烷酸的合成:将油酸在四氢呋喃中与硼烷·四氢呋喃-络合物反应;将溶液在室温下搅拌过夜,加入水中以破坏硼烷;然后,将溶液冷却至0℃并逐滴加入3N NaOH溶液和30%H2O2-溶液;在室温下反应,加入水和饱和NaCl,水相用乙酸乙酯萃取;将乙酸乙酯层干燥,过滤,并真空浓缩;通过快速色谱法纯化残余物,得到9-羟基-十八烷酸;
(2)FA-FA1-5的反应条件为:FA1-5和草酰氯溶解在二氯甲烷溶液,将反应混合物在室温下搅拌3~4小时,然后加入9-羟基-十八烷酸;在室温下搅拌过夜后,将反应混合物真空浓缩;通过快速色谱法纯化残余物,得到FA-FA1-5;
所述的FA1-5的结构式如下所示:
(3)FA-FA-Cer A-E的合成条件为:鞘氨醇溶解在无水二氯乙烷溶液中,在0℃下加入1-羟基苯并三唑和1-乙基-(3-二甲基氨基丙基)碳酰二亚胺,然后加入三甲胺和FA-FA1-5,将混合物在0℃下搅拌4小时并在室温下搅拌过夜;将其用二氯甲烷稀释,并将有机层用3×10mL的2%HCl和3×10mL的5%NaHCO3洗涤;将合并的有机物真空蒸发,通过快速色谱法纯化残余物;得到产物FA-FA-Cer A-E,即神经酰胺类似物。
3.根据权利要求2所述的制备方法,其特征在于:
步骤(1)中所述的油酸、四氢呋喃、硼烷·四氢呋喃-络合物的比值为0.5mmol:3mL:0.4mL;
步骤(1)中所述的硼烷·四氢呋喃-络合物中四氢呋喃的浓度为0.9M,硼烷的浓度为0.75mmol/mL。
4.根据权利要求2所述的制备方法,其特征在于:
步骤(1)中,加入水的用量与油酸的比值为1mL:0.5mmol;
步骤(1)中,3N NaOH溶液、30%H2O2-溶液与油酸的比值为2mL:6mL:0.5mmol;
步骤(1)中,所述的室温下反应的时间为1小时;
步骤(1)中,所述的干燥为用MgSO4干燥。
5.根据权利要求2所述的制备方法,其特征在于:
步骤(2)中,FA1-5、草酰氯、9-羟基-十八烷酸的摩尔比为1:1.2~1.5:1;
步骤(2)中,9-羟基-十八烷酸在反应体系中的浓度为0.05~0.1mmol/mL。
6.根据权利要求2或5所述的制备方法,其特征在于:
步骤(2)中,FA1-5、草酰氯、9-羟基-十八烷酸的摩尔比为1:1.5:1;
步骤(2)中,9-羟基-十八烷酸在反应体系中的浓度为0.1mmol/mL。
7.根据权利要求2所述的制备方法,其特征在于:
步骤(3)中,所述的鞘氨醇、FA-FA1-5的摩尔比值为1:1~1.2;
步骤(3)中,所述的鞘氨醇、1-羟基苯并三唑、1-乙基-(3-二甲基氨基丙基)碳酰二亚胺、三甲胺的比值为1mmol:1~1.2mmol:1~1.2mmol:2~4mL。
8.根据权利要求2所述的制备方法,其特征在于:
步骤(1)中所述的快速色谱法所用的洗脱液为氯仿/甲醇=200:1;
步骤(2)中所述的快速色谱法所用的洗脱液为氯仿/甲醇=100:1;
步骤(3)中所述的快速色谱法所用的洗脱液为氯仿/甲醇=50:1。
9.权利要求1所述的神经酰胺类似物在制备逆转他莫昔芬耐药产生的产品中的应用。
10.根据权利要求9所述的应用,其特征在于:所述的神经酰胺类似物与他莫昔芬合用时,逆转他莫昔芬的耐药产生。
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CN117737146A (zh) * | 2023-12-20 | 2024-03-22 | 广东省农业科学院蚕业与农产品加工研究所 | 甘油酯型脂肪酸羟基脂肪酸酯及其制备方法 |
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CN117737146A (zh) * | 2023-12-20 | 2024-03-22 | 广东省农业科学院蚕业与农产品加工研究所 | 甘油酯型脂肪酸羟基脂肪酸酯及其制备方法 |
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