CN106831383A - 二芳基庚烷类化合物 - Google Patents
二芳基庚烷类化合物 Download PDFInfo
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- CN106831383A CN106831383A CN201611230567.3A CN201611230567A CN106831383A CN 106831383 A CN106831383 A CN 106831383A CN 201611230567 A CN201611230567 A CN 201611230567A CN 106831383 A CN106831383 A CN 106831383A
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Abstract
本发明公开了一种式(A)所示的二芳基庚烷类化合物。本发明化合物可以显著地抑制iNOS、IL‑1β、IL‑6的转录和蛋白表达以及IκB蛋白的磷酸化水平,可以用于炎症及其他多种疾病的治疗,包括治疗败血症和治疗阿尔茨海默病。
Description
技术领域
本发明涉及二芳基庚烷类化合物。
背景技术
炎症(inflammation)是生命体被细菌、病毒感染,组织器官病理变化,免疫系统的异常或者有害物质刺激及物理损伤时引起的局部或者全身性反应。炎症与多种疾病的发生和发展相关。早在1983年,Rudolf Virchow就提出癌细胞来源炎症位点。机体损伤后,炎症的发生也可能会导致正常细胞的癌变。肿瘤中的炎性微环境会促进肿瘤细胞的恶化、增殖、生存、迁移,并且抑制生物体自身免疫反应。另外,炎症与动脉粥样硬化、肥胖、心脏病、糖尿病、阿尔茨海默病等疾病相关。在正常的生理情况下炎症反应对机体是有利的,能够杀伤、杀死入侵的病原微生物,排除外源性刺激,促进受损组织修复,愈合等;但是机体自生的调节能力失调时,将会导致炎症反应的失控,会导致许多疾病,如关节炎,感染性休克,二型糖尿病,支气管哮喘以及老年性痴呆等。
内毒素,是一种由脂多糖(LPS)、蛋白质和磷脂组成的复合体,是导致炎症反应产生的主要因素。脂多糖(LPS)是内毒素的活性成分,而蛋白质和磷脂与内毒素的活性无关。内毒素的致炎机制是一种多途径、多介质共同参与,并引发机体多器官系统反应及导致机体损伤的复杂过程。主要是通过LPS激活受体TLR4,TLR4可活化接头蛋白MyD88,活化的MyD88与丝/苏氨酸蛋白激酶IRAK相互作用,使IRAK自主磷酸化。游离的IRAK与另一衔接分子肿瘤坏死因子受体相关因子6(TRAF6)结合形成复合物,激活NF-κB,启动靶基因的转录。TRAF6还可与ECSIT相互作用,依次激活MAPKKK、MAPKK、ERK、p38和JNK/SAPK,最后导致转录因子AP-1家族成员Jun和Fos的活化。NF-κB和AP-1的活化均可导致炎症因子如TNF-α、IL-1、NO、PAF、PGs和粘附分子等的大量表达。
体内的NO由一氧化氮合酶(Nitric oxide synthases,NOS)催化L-精氨酸(L-Arg)产生。已知的NOS有不同基因编码的3种同工酶,即神经型(nNOS)、内皮型(eNOS)和诱导型(iNOS)。诱导型NO合酶iNOS可被一些外来刺激激活,如内毒素(LPS)、干扰素类、细菌、病毒及促炎症因子(TNF-α、IL-1、IL-6)等。NO在体内发挥双重作用,是免疫系统发挥作用不可缺少的调节因子。然而,NO的过度产生则会导致组织损伤。在炎症部位,NO作用于血管平滑肌细胞,升高其cGMP水平,使血管舒张,通透性增高,以利于炎症介质和致痛物质到达作用部位。同时,NO在高浓度状态下,可激活NF-κB,诱导促炎症细胞因子TNF-α、IL-1产生,进而导致iNOS的激活,促进机体产生更多的NO,从而导致NO自身的表达得以持续,使炎症反应更为持久剧烈。此外,在多种炎症性或免疫性疾病的动物模型中,NO浓度也显著增高,而抑制NO合成可使炎症症状得到明显改善。
乙酰胆碱(ACh)是中枢神经系统(CNS)中的主要神经递质之一。高亲和力的尼古丁乙酰胆碱受体(nAChR)由两个或三个α亚基与两个或三个β亚单位组成;而低亲和力受体大多是同源的亚基组成,主要包括α7亚基。大量的研究已经证明nAChR参与健康和疾病的调控。其中,α7 nAChR广泛表达于中枢神经系统、自主神经系统、外周血液系统。在神经系统中,α7 nAChR激动剂可以用于治疗神经性疾病,比如精神分裂症、阿尔茨海默症等。此外,一些临床前研究还表明,α7 nAChR激动剂可以用于治疗炎症及相关的疼痛。其产生抗炎作用的主要机制如下:在巨噬细胞中,α7 nAChR激动剂(如:尼古丁,乙酰胆碱,CAP55和GTS-21)可激活α7 nAChR受体,导致JAK2的磷酸化,促进STAT3的磷酸化;α7 nAChR受体也可通过激活Ras/Raf/MEK/ERK级联,进一步促进STAT3的磷酸化。当STAT3被磷酸化以后,一方面通过抑制NF-κB转录因子的转录活性,从而减少促炎症因子的产生(如TNF-α、IL-1、IL-6、HMGB-1等);另一方面,STAT3被激活以后,启动相关基因的表达,促进抗炎因子的产生(如IL-10、TGF-β等)。丝裂原活化蛋白激酶家族(MAPKs)包括细胞外信号调节激酶1/2(ERK 1/2)、c-Jun氨基端激酶1/2/3(JNK 1/2/3)、p38、ERK5、ERK3/4、ERK7、Nemo样激酶(NLK),这些激酶参与调控了基因表达、有丝分裂、代谢、存活、凋亡、分化、免疫应答等生理病理过程。目前已上市的某些抗炎药物主要是通过抑制TLR4或者MAPKs信号通路而发挥抗炎作用。然而,长期抑制MAPKs信号通路,会产生较大的副作用,可能会妨碍细胞正常的免疫应答等正常生理功能。然而α7 nAChR激动剂只是通过JAK2/STAT3信号通路负调控NF-κB或者促进抗炎因子的产生,并不会影响MAPKs信号通路。同时,α7 nAChR只是在神经系统和外周血液系统的细胞中高表达,所以相对于一些传统的抗炎药物,α7 nAChR激动剂的副作用可能更小,是一种极具潜力的抗炎药物靶标。
二芳基庚烷类化合物是一类具有1、7-二取代芳基并以庚烷骨架为母体结构的化合物的统称。根据其成环与否以及两个苯环连接方式的不同,分为3种类型,即直线型、大环联苯型和环二苯醚型。该类化合物主要存在于植物的根、茎、皮、花以及果皮等部位。
发明内容
本发明提供了式(A)所示的二芳基庚烷类化合物或其顺反异构体、或其外消旋混合物、或其对映异构体、或其药学上可接受的盐、或其溶剂合物:
其中,R1、R2分别独立地表示无或者苯环上的1~5个取代基,所述取代基分别独立地选自-OR3,R3表示氢、C1~C4的烷基或C1~C4的烷酰基;所述C1~C4的烷基任选进一步被苯基所取代;
L1表示4个碳原子的直链烷基或直链烯基;
L2表示-CH2CH2-或-CH=CH-;
X表示R4表示氢或C1~C4的烷酰基。
进一步地,所述式(A)化合物的结构如下式(Ⅰ)~(Ⅶ)中任一所示:
进一步地,所述C1~C4的烷基为甲基。
进一步地,所述C1~C4的烷酰基为乙酰基。
进一步地,R1表示无,R2表示苯环上的2个取代基,所述取代基选自-OR3,R3表示氢或乙酰基。
进一步地,所述式(A)化合物为如下化合物之一:
Me表示甲基,Ac表示乙酰基。
本发明还提供了式(A)所示的二芳基庚烷类化合物或其顺反异构体、或其对映异构体、或其药学上可接受的盐、或其溶剂合物、或其前药在制备一氧化氮生成抑制剂、一氧化氮合酶抑制剂、IL-1β抑制剂、IL-6抑制剂、α7尼古丁乙酰胆碱受体激动剂、抑制AKT和/或NF-κB的药物、促进JAK2和/或STAT3磷酸化的药物以及治疗炎症、败血症、关节炎、炎性肠病、过敏性肠综合症、偏头痛、头痛、腰痛、纤维肌痛、肌筋膜障碍、病毒感染、细菌感染、真菌感染、灼伤、外科治疗、恶性肿瘤、动脉粥样硬化、痛风、发热、脊柱炎、红斑狼疮、血管炎、胰腺炎、肾炎、结膜炎、虹膜炎、巩膜炎、葡萄膜炎、创伤、皮炎、湿疹、牛皮癣、中风、糖尿病、神经变性疾病、自身免疫性疾病、鼻炎、溃疡、心脏病、炎性痛、高前列腺素E综合症、典型巴特综合症、何杰金病或治疗阿尔茨海默病的药物中的用途。
进一步地,所述一氧化氮生成抑制剂是抑制巨噬细胞生成一氧化氮和/或抑制巨噬细胞增殖的药物。
进一步地,所述治疗炎症的药物是抑制巨噬细胞生成一氧化氮和/或抑制巨噬细胞增殖的药物。
进一步地,所述治疗败血症的药物是治疗由脂多糖引起的败血症的药物。
试验证明,本发明的化合物可以有效抑制一氧化氮生成、治疗炎症、治疗败血症和治疗阿尔茨海默病,大幅扩展了二芳基庚烷类化合物应用范围,极具临床应用前景。
本发明中,所述一氧化氮生成抑制剂指的是对生物体中一氧化氮生成具有抑制作用的药物。
本发明中,所述C1~C4的烷基是指C1、C2、C3、C4的烷基,即具有1~4个碳原子的直链或支链的烷基,例如甲基、乙基、丙基、异丙基、丁基、异丁基、叔丁基、仲丁基等等。
本发明中,所述C1~C4的烷氧基是指C1、C2、C3、C4的烷氧基,即具有1~4个碳原子的直链或支链的烷氧基,例如甲氧基、乙氧基等等。
本发明中,所述C1~C4的烷酰基是指C1、C2、C3、C4的烷酰基,即具有1~4个碳原子的直链或支链的烷酰基,例如甲酰基、乙酰基、丙酰基、异丙酰基、丁酰基等等。
在本发明的含义之内,“治疗”也包括复发性(relapse)预防或阶段性(phase)预防,以及急性或慢性体征、症状和/或功能失常的治疗。治疗可以是对症治疗,例如抑制症状。它可以在短期内实现,在中期内调整,或者可以说是长期治疗,例如在维持疗法里面。所述预防包括延迟和/或阻止病症、疾病或病况和/或其伴发症状的发作;防止对象染上病症、疾病或病况;或降低对象染上病症、疾病或病况的风险的方法。
实验证明,本发明化合物可以有效地抑制脂多糖(LPS)诱导小鼠RAW246.7巨噬细胞生成一氧化氮(NO)、白细胞介素1β(IL-1β)、白细胞介素6(IL-6),并且本发明化合物呈现浓度依赖性,显示良好的剂量关系;同时动物实验表明,本发明化合物可以有效地抑制脂多糖(LPS)刺激的小鼠血清中的白细胞介素1β(IL-1β)和白细胞介素6(IL-6)水平,并且显著抑制了LPS诱导的小鼠急性中毒死亡,表明本发明化合物可以用于炎症的治疗,具有潜在的临床使用价值。
本发明通过研究LPS诱导炎症信号通路的研究,发现本发明化合物可以显著地抑制iNOS、IL-1β、IL-6的转录和蛋白表达以及IκB蛋白的磷酸化水平,说明本发明化合物可以通过抑制NF-κB信号通路,从而抑制炎症的发生。进一步研究发现本发明化合物可以通过激活α7尼古丁乙酰胆碱受体,经JAK2/STAT3信号级联,从而抑制促炎症因子的表达,促进抑炎因子的表达,进而抑制炎症的发生。进一步说明本发明所示的二芳基庚烷类化合物可以作为炎症和/或与α7尼古丁乙酰胆碱信号通路和/或JAK2/STAT3信号通路和/或NF-κB信号通路和/或MAPKs信号通路相关的疾病的药物中的应用。
显然,根据本发明的上述内容,按照本领域的普通技术知识和惯用手段,在不脱离本发明上述基本技术思想前提下,还可以做出其它多种形式的修改、替换或变更。
以下通过实施例形式的具体实施方式,对本发明的上述内容再作进一步的详细说明。但不应将此理解为本发明上述主题的范围仅限于以下的实例。凡基于本发明上述内容所实现的技术均属于本发明的范围。
附图说明
图1为二芳基庚烷类化合物1-37,39-49。
图2为化合物1-37,39-49对LPS诱导RAW264.7巨噬细胞生成NO的影响。C(Curcumin)和D(Dexamethasone)为阳性药物。LPS,0.5μg/mL;Curcumin,5μM;Dexamethasone,10μM。
图3为化合物27-29,37,39-41抑制LPS诱导RAW264.7巨噬细胞生成NO的IC50值。
图4为化合物21,25,27-29,34-37,39,41,40细胞毒性活性的IC50值。
图5为化合物21,25,27-29,34-37,39,41,40抑制iNOS、IL-1β、IL-6mRNA和蛋白的表达。空白:DMSO,0.1%;模型:LPS,0.5μg/mL;阳性:BAY(BAY 11-7082),10μM;通过ANOVA方差分析于LPS组比较(*P<0.05,**P<0.01,***P<0.001)。
图6为化合物28和40对MAPK信号通路的影响。GAPDH作为内参。实验结果通过灰度比矫正。空白:DMSO,0.1%;模型:LPS,0.5μg/mL;阳性:p38抑制剂,SB(SB203580),10μM;JNK抑制剂,SP(SP600125),20μM;ERK抑制剂,PD(PD98059),20μM。
图7为化合物28和40对NF-κB信号通路的影响。GAPDH作为内参。实验结果通过灰度比矫正。空白:DMSO,0.1%;模型:LPS,0.5μg/mL;阳性:BAY(BAY 11-7082),10μM。
图8为化合物28和40激活α7尼古丁乙酰胆碱信号通路。GAPDH作为内参。实验结果通过灰度比矫正。空白:DMSO,0.1%;模型:LPS,0.5μg/mL;α7尼古丁乙酰胆碱拮抗剂:α-BGT(α银环蛇毒素),100nM;阳性:GTS(GTS-21),20μM;Acetycholine(乙酰胆碱),1mM。
图9为化合物28和40抑制LPS诱导的小鼠急性中毒。
具体实施方式
本发明化合物可以从生物资源获得或者通过有机合成获得。
实施例1二芳基庚烷类化合物的提取、分离和鉴定
1.1仪器设备及材料
Perkin-Elmer FT-IR红外分光光度计,KBr压片;核磁共振实验采用BrukerAVANCE 400核磁共振仪;使用ES-TOFMS和ES-MS质谱分别使用Bruker microTOF and aFinnigan LC-Q II mass spectrometer;旋光测定采用JASCO-1020polarimeter;高效液相色谱:Agilent 1260HPLC,制备柱使用Kromasil 100-10-C18;Merck硅胶60(细于0.063毫米)和Pharmacia公司交联葡聚糖LH-20。Curcuma comosa根茎在泰国的Nakhon PathomSakon Nakhon和Prachin Buri,凭证标本(Apichart Suksamrarn,052号和074)存放在Faculty of Science,Ramkhamhaeng University。
1.2化合物分离提取
C.comosa的根茎(5.2kg)被切片,风干,研磨并用正己烷和EtOH依次浸渍,然后分别进行减压蒸馏,得到正己烷提取物(347.7g)和乙醇提取物(400.4g)。正己烷和乙醇萃取物通过柱层析(Merck硅胶60PF254,0.063-0.200mm,580g)分级分离,使用正己烷、正己烷-乙酸乙酯、乙酸乙酯、乙酸乙酯-甲醇和甲醇的梯度洗脱。洗脱液通过薄层色谱法检测,共分为7组馏分(E1-E7)。其中E3馏分(3.54g)进行柱层析,以正己烷、正己烷-乙酸乙酯进行洗脱,得馏分E31-E35。E33馏分随后进行等度条件下的柱层析,以正己烷-乙酸乙酯(80:20,逐步增加乙酸乙酯含量)进行洗脱,得到化合物1(15.1mg)、化合物2(15mg)。E32馏分进行等度条件下的柱层析,以正己烷-乙酸乙酯(80:20,逐步增加乙酸乙酯含量)进行洗脱,得到化合物4(13.9mg)。
C.comosa的根茎(5.8kg)被切片,风干,研磨并用正己烷和EtOH依次浸渍,然后分别进行减压蒸馏,得到正己烷提取物(335.07g)和乙醇提取物(322.04g)。乙醇萃取物(300g)通过柱层析(Merck硅胶60PF254,0.063-0.200mm,520g)分级分离,使用正己烷、正己烷-乙酸乙酯、乙酸乙酯、乙酸乙酯-甲醇和甲醇的梯度洗脱。洗脱液通过薄层色谱法检测,共分为11组馏分(E1-E11)。其中E9馏分(14.3g)进行柱层析,以正己烷-二氯甲烷(50:50)进行洗脱,得馏分E91-E95。E92馏分随后进行等度条件下的柱层析,以正己烷-乙酸乙酯(80:20)及SephadexLH-20进行柱层析脱,然后以甲醇洗脱,再以二氯甲烷-甲醇(100:1)在等度条件下进行洗脱,得化合物3(18.1mg)。馏分95以正己烷-丙酮(70:30)和Sephadex LH-20进行柱层析,以甲醇洗脱得到化合物5(130mg)。最终,5个新的二苯庚烷类化合物1-5和16个已知的二苯庚烷类化合物6-21[1,2]。
其中,化合物2、3、4为两种构型都存在的混合物,化合物5为S-构型的。
1.3部分化合物波谱数据
1-(4-Hydroxy-3-methoxyphenyl)-7-phenyl-(6E)-6-hepten-3-one(化合物1)
无色油状物;IR:νmax:3426,2923,2845,1711,1597,1514,1453,1373,1267,1233,1208,1026,967,811,748,699;1H NMR(400MHz,CDCl3):δ2.82(dd,7.32,7.36,H1),2.70(dd,7.36,7.03,H2),2.55(dd,8.15,7.72,H4),2.45(dd,7.72,7.26,H5),6.14(dt,7.26,15.8,H6),6.55(d,15.8,H7),6.65(s,H2′),6.79(d,8.0,H5′),6.64(d,8.0,H6′),7.18-7.28(m,H2″),7.18-7.28(m,H3″),7.17(m,H4″),7.18-7.28(m,H5″),7.18-7.28(m,H6″),3.82(s,OMe3″);13C NMR(100MHz,CDCl3):δ29.4(C1),44.7(C2),209.2(C3),37.0(C4),29.2(C5),128.7(C6),130.7(C7),132.9(C1′),111.1(C2′),146.3(C3′),143.7(C4′),114.2(C5′),120.9(C6′),137.3(C1″),125.9(C2″),128.4(C3″),127.0(C4″),128.4(C5″),125.9(C6″),55.8(OMe3′).
(3S)-and(3R)-1-(4-Hydroxy-3-methoxyphenyl)-7-phenyl-(6E)-6-hepten-3-ol(化合物2)
无色油状物;[α]D27.1=-1.59[EtOH,C=0.65w/v];IR:νmax:3359,3193,2923,2852,1659,1630,1515,1466,1426,1373,1272,1233,1152,1035,965,816,794,735,693;1HNMR(400MHz,CDCl3):δ2.54,2.66(m,H1),1.70(m,H2),3.63(m,H3),1.59(m,H4),2.26(m,H5),6.15(dt,6.8,15.8,H6),6.33(d,15.8,H7),6.63(s,H2′),6.75(d,6.4,H5′),6.62(d,6.4,H6′),7.18-7.28(m,H2″),7.18-7.28(m,H3″),7.12(t,7.0,H4″),7.18-7.28(m,H5″),7.18-7.28(m,H6″),3.78(s,OMe3);13C NMR(100MHz,CDCl3):δ31.7(C1),39.4(C2),70.9(C3),37.0(C4),29.2(C5),130.2(C6),130.3(C7),133.9(C1′),120.8(C2′),146.4(C3′),143.7(C4′),114.2(C5′),110.9(C6′),137.5(C1″),125.9(C2″),128.4(C3″),126.9(C4″),128.4(C5″),125.9(C6″),55.8(OMe3′).
2:1 Mixture of(3S)-and(3R)-1-(4-Hydroxy-3-methoxyphenyl)-7-phenyl-(4E,6E)-4,6-heptadien-3-ol(化合物3)
淡黄色油状物;(c 1.16,EtOH);IR:νmax:3398,3025,2933,2858,1613,1600,1515,1463,1451,1369,1270,1234,1153,1123,1033,991,749,693;1H NMR(400MHz,CDCl3):δ2.65(m,H1),1.88(m,H2),4.21(m,H3),5.83(dd,6.8,15.1,H4),6.38(dd,10.7,15.1,H5),6.76(dd,10.7,15.6,H6),6.54(d,15.6,H7),6.70(brs,H2′),6.82(brd,7.4,H5′),6.69(brd,7.4,H6′),7.38(brd,7.2,H2″),7.30(brt,7.6,H3″),7.24(m,H4″),7.30(brt,7.6,H5″),7.38(brd,7.2,H6″),3.86(brs,OMe3);13C NMR(100MHz,CDCl3):δ31.3(C1),39.0(C2),72.0(C3),136.2(C4),130.9(C5),128.1(C6),132.8(C7),133.7(C1′),111.0(C2′),146.4(C3′),143.7(C4′),114.2(C5′),120.9(C6′),137.1(C1″),126.3(C2″),128.6(C3″),127.5(C4″),128.5(C5″),126.3(C6″),55.8(OMe3).
(3S)-1-Phenyl-7-(4-hydroxyphenyl)heptan-3-ol(化合物4)
无色油状物;[α]D 28=+1.63[EtOH,C=0.58w/v];IR:νmax:3297,3025,2930,2856,1598,1513,1495,1453,1366,1228,1170,1028,824,745,697;1H NMR(400MHz,CDCl3):δ2.56,2.70(m,H1),1.67(m,H2),3.52(H3),1.41(m,H4),1.29,1.41(m,H5),1.50(m,H6),2.44(t,7.56,H7),7.08-7.15(m,H2′),7.18-7.24(m,H3′),7.08-7.15(m,H4′),7.18-7.24(m,H5′),7.08-7.15(m,H6′),6.92(d,8.30,H2″),6.66(d,8.30,H3″),6.66(d,8.30,H5″),6.92(d,8.30,H6″);13C NMR(100MHz,CDCl3):δ31.9(C1),38.8(C2),71.1(C3),37.2(C4),25.0(C5),31.6(C6),34.8(C7),142.2(C1′),128.3(C2′),125.6(C3′),128.3(C4′),125.6(C5′),128.3(C6′),133.8(C1″),129.2(C2″),115.0(C3″),154.2(C4″),115.0(C5″),129.2(C6″).
(3S)-1-(3,4-Dihydroxyphenyl)-7-phenyl-(4E,6E)-4,6-heptadien-3-ol(化合物5)
白色固体;m.p.105-106℃;(c 1.16,EtOH);IR:νmax:3396,3233,3022,2949,2924,2849,1612,1596,1515,1447,1254,1228,1175,1096,1044,990,825,748,692,513;1H NMR(400MHz,CDCl3):δ2.57(m,H1),1.77(m,H2),4.16(H3),5.19(dd,6.1,15.2,H4),6.41(dd,10.3,15.2,H5),6.89(dd,10.3,15.6,H6),6.56(d,15.6,H7),6.53(brs,H2′),6.70(brd,7.9,H5′),6.72(brd,7.9,H6′),7.44(brd,7.5,H2″),7.30(brd,7.5,H3″),7.20(brt,7.5,H4″),7.30(brd,7.5,H5″),7.44(brd,7.5,H6″),3.85(D,4.3,OH3),7.59(brs,OH3′4′);13C NMR(100MHz,CDCl3):δ30.85(C1),39.59(C2),70.65(C3),138.36(C4),129.40(C5),128.90(C6),131.42(C7),134.01(C1′),119.50(C2′),142.89(C3′),144.81(C4′),115.03(C5′),115.39(C6′),115.39(C1″),137.53(C2″),126.13(C3″),128.49(C4″),127.19(C5″),128.49(C6″).
1,7-Diphenylhepta-l,3,5-trien(化合物21)
白色固体;mp 76.0-76.5℃;IR(KBr):3009.5-3101.9,2831.9-2925.9,1595.3,1490.6,1452.0,1427.3,1073.0,1027.7,992.9,742.6,697.3cm-1;1H NMR(300MHz,CDCl3):δ3.45(d,2H,J=7.1Hz),5.89(dt,1H,J=14.8,7.0Hz),6.14-6.34(m,3H),6.51(d,1H,J=15.6Hz),6.79(m,1H),7.17-7.39(m,10H,ArH);MS m/e(intensity):246(65.0,M+),155(100.0),142(30.5),129(18.2),I15(20.7),103(3.5),91(31.9).
2.1本发明涉及的二芳基庚烷类化合物可以根据以下路线制备
路线1:
备注:a)HCl.NH2OH,DMF,Pyridine
b)H2,Pd-C,EtOH
c)Ac2O,Pyridine
d)PhCH2Br,DMF,K2CO3e)PCC,CH2Cl2
路线2:
备注:(i)excess acetaldehyde,20%aq NaOH,EtOH,0–10℃;
(ii)MeI,K2CO3,acetone,rt.;(iii)20%aq NaOH,EtOH,0℃–rt;(iv)H2(balloon)/10%Pd-C,EtOH.
如路线1所示,对化合物6,12和20进行结构修饰,可以获得化合物22-25,26-29,30-33。如路线2所示,不饱和trienone类似物34-37分别根据文献[3]中的条件,碱催化条件下醛醇缩合,在20%的NaOH的存在下,使用乙醇作为溶剂,用cinnamaldehydes 2a和2b取代cinnamones 1a-d。三个新的二芳基庚烷类似物(39和40-41)和前面的合成方法类似。
化合物22的合成方法:化合物12的吡啶溶液(58mg,0.2mM)加入NH2OH.HCl(20.7mg,0.3mM),室温下混合搅拌4h。随后,加入20ml水,搅拌后,混合液以EtOAc提取3次(40ml/次)合并有机层,用水洗涤,以无水Na2SO4干燥,真空蒸发除去溶剂。残渣经柱层析纯化等度条件下用正己烷/乙酸乙酯(5:1)洗脱得到化合物23(66.7mg,89%)为白色固体。
化合物23的合成方法:化合物6的二甲基甲酰胺溶液(58mg,0.2mM)加入苄基溴(0.5ml,4.2mM),室温下混合搅拌1h。随后,加入20ml水,搅拌后,混合液以EtOAc提取3次(40ml/次)合并有机层,用水洗涤,以无水Na2SO4干燥,真空蒸发除去溶剂。残渣经柱层析纯化等度条件下用正己烷/乙酸乙酯(5:1)洗脱得到化合物23(66.7mg,89%)为白色固体。
化合物24的合成方法:化合物12的二氯甲烷溶液(55mg,0.195mM)加入氯铬酸吡啶,室温下混合搅拌0.5h。混合物通过硅胶过滤,固体以正己烷/乙酸乙酯(1:1)洗涤,滤液真空浓缩即为1-(4-Hydroxy)-7-phenyl-(6E)-6-hepten-3-one(35.5mg,65%)。然后,化合物1-(4-Hydroxy)-7-phenyl-(6E)-6-hepten-3-one的吡啶溶液(22mg,0.08mM)中加入乙酸酐(0.3ml,d=1.081g/ml,3.15mM)混合,室温下搅拌反应1h。混合液以EtOAc提取3次(40ml/次)合并有机层,用水洗涤,以无水Na2SO4干燥,真空蒸发除去溶剂。残渣经柱层析纯化等度条件下用正己烷/乙酸乙酯(1:1)洗脱得到化合物32(21mg,81%)。化合物32的乙醇溶液(15mg,0.05mM)在1个大气压力,以10%的Pa-C为催化剂,加氢反应30min。混合物以硅藻土过滤,固体以EtOAc洗涤,滤液在真空中浓缩,得到化合物24(14.5mg,90%),为无色油状物。
化合物25的合成方法:化合物20的吡啶溶液(20mg,0.07mM)加入乙酸酐(0.3ml,d=1.081g/ml,3.15mM)混合,室温下混合搅拌1h。随后,加入30ml水,搅拌后,混合液以EtOAc提取3次(40ml/次)合并有机层,用水洗涤,以无水Na2SO4干燥,真空蒸发除去溶剂。残渣经柱层析纯化等度条件下用正己烷/乙酸乙酯(1:1)洗脱得到化合物25(19.2mg,75%)为无色油状物。
化合物26、27、28、29的合成方法:化合物20的乙醇溶液(50mg,0.168mM)在1个大气压力,以10%的Pa-C(20mg,0.018mM)为催化剂,加氢反应30min。混合物以硅藻土过滤,固体以EtOAc洗涤,滤液在真空中浓缩,得到化合物26(46.7mg,93%),为无色油物体。化合物26的吡啶溶液(50mg,0.168mM)加入乙酸酐(0.3ml,d=1.081g/ml,3.15mM)混合,室温下混合搅拌0.5h。随后,加入30ml水,搅拌后,混合液以EtOAc提取3次(40ml/次)合并有机层,用水洗涤,以无水Na2SO4干燥,真空蒸发除去溶剂。残渣经柱层析纯化等度条件下用正己烷/乙酸乙酯(1:1)洗脱得到化合物27(30.7mg,57%)和28(24.4mg,41%),均为无色油状物。化合物27的二氯甲烷溶液(17.2mg,0.05mM)加入氯铬酸吡啶,室温下混合搅拌0.5h。混合物通过硅胶过滤,固体以正己烷/乙酸乙酯(1:1)洗涤,滤液真空浓缩,得到化合物29(10.1mg,52%)为无色油状物。
不饱和trienones化合物34-36和38是通过在碱催化条件下的肉桂酮(2a、b、d)和肉桂醛(3a、3b)羟醛缩合反应制备得到[3]。新的化合物37和39-41是采用与化合物34-36和38同样的方法得到的[3]。
取代的肉桂酮(2a-d)和肉桂醛(3a-d)的合成:肉桂酮(2a-d)和肉桂醛(3a-b)通过苯甲醛和丙酮或者乙醛在碱催化的条件下经羟醛缩合反应制备得到[3]。肉桂酮(3c、d)也采用与肉桂醛3a和b同样的方法进行合成[3]。
Cinnamaldehyde 3c((E)-3-(2,4-Dimethoxyphenyl)acrylaldehyde))的合成:2,4-dimethoxybenzaldehyde(1c)(2.4g,14.44mM)、20%的氢氧化钠水溶液(10ml)和乙醇(25ml)的混合溶液,在0-5℃环境中,滴加过量的乙醛(10ml)。随后在0-10℃环境中搅拌30-45min,并通过薄层色谱法进行监测。接着,将反应混合物以冷的3M HCl进行酸化反应。沉淀产物经真空过滤后,以水彻底冲洗。所产生的固体残渣经10%的二氯甲烷/正己烷柱层析所得混合物3c(500.8mg,18%),为淡黄色固体。
Cinnamaldehyde 3d((E)-3-(2,5-Dimethoxyphenyl)acrylaldehyde)的合成:参考化合物3c的合成方法,以2,5-dimethoxybenzaldehyde(1d)(3.38g,20.34mM)、20%的氢氧化钠水溶液(20ml)和乙醇(50ml)的混合溶液,滴加过量乙醛(10ml)。由此产生的固体残渣经10%乙酸乙酯/正己烷柱层析得化合物3d(2.94g,75%),为淡黄色固体。
不饱和的trienone二芳基庚烷类似物(34-41)的合成
不饱和trienones二芳基庚烷类似物(34-36和38)通过肉桂酮2a、b、d和肉桂醛3a和b在碱催化的条件下,通过羟醛缩合反应制备得到[3]。新化合物37和39-41采用和34-36及38类似的方法制备得到[3]。
化合物37(1-(4-Methoxyphenyl)-7-(3-hydroxyphenyl)-1,4,6-heptatrien-3-one)的合成:在0-5℃条件下,肉桂酮2c(178.4mg,1.03mM)、肉桂醛3a(100.0mg,0.67mM)的乙醇溶液(10ml)中加入20ml的氢氧化钠水溶液。混合物充分搅拌,深橙色的产生表明该反应已经发生。反应结束后,真空除去乙醇。残留溶液在冰水浴中以预冷的3M HCl中和。残渣经甲醇重结晶,所得固体经预冷甲醇洗涤后,得到化合物37(131.7mg,64%),位淡黄色固体。
化合物38(1-(3-Hydroxy-4-methoxyphenyl)-7-(3-hydroxyphenyl)-1,4,6-heptatrien-3-one)的合成:参考化合物38的合成程序,采用肉桂酮2d(126.2mg,0.66mM)和肉桂醛3a(81.2mg,0.55mM)的乙醇溶液(10ml),加入5ml的20%氢氧化钠水溶液,进行反应。所得残渣经柱层析,以4%丙酮/二氯甲烷进行洗脱,得到化合物38(57.8mg,33%),为橙色固体。
化合物39(1-(3-Hydroxyphenyl)-7-(2,4-dimethoxyphenyl)-1,4,6-heptatrien-3-one)的合成:参考化合物37的合成方法,采用肉桂酮2a(100mg,0.62mM)和肉桂醛3c(154mg,0.80mM)的乙醇溶液(10ml),加入2ml的20%氢氧化钠水溶液,进行反应。所得残渣经柱层析,以4%丙酮/二氯甲烷进行洗脱,得到化合物39(87.5mg,42%),为橙色固体。
化合物40(1-(3-Hydroxy-4-methoxyphenyl)-7-(2,4-dimethoxyphenyl)-1,4,6-heptatrien-3-one)的合成:合成程序参考化合物37的方法,采用肉桂酮2d(250mg,1.3mM)和肉桂醛3c(300mg,1.56mM)的乙醇溶液(10ml),加入3ml的20%氢氧化钠水溶液,进行反应。所得残渣经预冷的二氯甲烷和甲醇依次结晶。所得固体以预冷甲醇洗涤,得化合物40(206.3mg,43%),为橙色固体。
化合物41(1-(3-Hydroxy-4-methoxyphenyl)-7-(2,5-dimethoxyphenyl)-1,4,6-heptatrien-3-one)的合成:合成程序参考化合物37的方法,采用肉桂酮2d(1170mg,6.09mM)和肉桂醛3d(1180mg,6.14mM)的乙醇溶液(30ml),加入10ml的20%氢氧化钠水溶液,进行反应。所得残渣经预冷的二氯甲烷-正己烷结晶2次后,再以甲醇重结晶,得化合物41(909mg,41%),为黄色固体。
化合物42(1-(4-Methoxyphenyl)-7-(3-hydroxyphenyl)-heptan-3-one)的合成:化合物37(14mg,0.046mM)的乙醇溶液(5ml),在1个大气压下,以10%的Pa-C(10mg,0.009mM)为催化剂,进行加氢饱和反应10min。所得化合物经硅藻土过滤,固体与乙酸乙酯洗涤,滤液经真空浓缩。所得残渣经柱层析,以20%乙酸乙酯/正己烷进行洗脱,得化合物42(4.9mg,34%),为无色油状物。
化合物43(1-(3-Hydroxy-4-methoxyphenyl)-7-(3-hydroxyphenyl)-heptan-3-one)的合成:参考化合物42的合成方法,以化合物38(12.7mg,0.057mM)的乙醇溶液(5ml)在1个大气压下,以10%的Pa-C(10mg,0.009mM)为催化剂,进行加氢饱和反应10min。所得化合物经硅藻土过滤,固体与乙酸乙酯洗涤,滤液经真空浓缩。所得残渣经柱层析,以20%乙酸乙酯/正己烷进行洗脱,得化合物43(8.2mg,63%),为无色油状物。
化合物44(1-(3-Hydroxyphenyl)-7-(2,4-dimethoxyphenyl)-heptan-3-one)和化合物45(1-(3-Hydroxyphenyl)-7-(2,4-dimethoxyphenyl)-heptan-3-ol)的合成:参考化合物42的合成方法,以化合物39(14.1mg,0.042mM)的乙醇溶液(5ml)在1个大气压下,以10%的Pa-C(10mg,0.009mM)为催化剂,进行加氢饱和反应10min。所得化合物经硅藻土过滤,固体与乙酸乙酯洗涤,滤液经真空浓缩。所得残渣经柱层析,以20%乙酸乙酯/正己烷进行洗脱,得化合物44(7.8mg,54%)和化合物45(6.3mg,44%),为无色油状物。
化合物46(1-(3-Hydroxy-4-methoxyphenyl)-7-(2,4-dimethoxyphenyl)-heptan-3-one)和化合物47(1-(3-Hydroxy-4-methoxyphenyl)-7-(2,4-dimethoxyphenyl)-heptan-3-ol)的合成:参考化合物42的合成方法,以化合物40(21.2mg,0.063mM)的乙醇溶液(5ml)在1个大气压下,以10%的Pa-C(10mg,0.009mM)为催化剂,进行加氢饱和反应10min。所得化合物经硅藻土过滤,固体与乙酸乙酯洗涤,滤液经真空浓缩。所得残渣经柱层析,以20%乙酸乙酯/正己烷进行洗脱,得化合物46(12.5mg,58%)和化合物47(5.3mg,24%),为无色油状物。
化合物48(1-(3-Hydroxy-4-methoxyphenyl)-7-(2,5-dimethoxyphenyl)-heptan-3-one)和化合物49(1-(3-Hydroxy-4-methoxyphenyl)-7-(2,5-dimethoxyphenyl)-heptan-3-ol)的合成:参考化合物42的合成方法,以化合物41(20.8mg,0.057mM)的乙醇溶液(5ml)在1个大气压下,以10%的Pa-C(10mg,0.009mM)为催化剂,进行加氢饱和反应10min。所得化合物经硅藻土过滤,固体与乙酸乙酯洗涤,滤液经真空浓缩。所得残渣经柱层析,以20%乙酸乙酯/正己烷进行洗脱,得化合物48(8.9mg,42%)和化合物49(6.3mg,30%),为无色油状物。
2.2部分化合物波谱数据
3-Hydroxyoxime-1,7-diphenyl-(6E)-6-heptene(化合物22)
白色固体;m.p.55-56℃;IR:νmax:3240,3026,2924,1711,1656,1599,1495,1447,1333,966,941,737,691;1H NMR(400MHz,CDCl3):δ2.93(dd,J=8.3,7.6Hz,2H,H-1),2.63(dd,J=8.3,7.6Hz,2H,H-2),2.49(m,2H,H-4),2.42,2.75(m,2H,H-5),6.24(m,1H,H-6),6.46(dd,J=15.3,10.3Hz,1H,H-7),7.23-7.38(m,9H,overlapping signal of H2′,3′,5′,6′and H2″-6″);13C NMR(100MHz,CDCl3):δ29.55(C1),33.90(C2),161.6(C3),31.34(C4),27.95(C5),130.8(C6),131.0(C7),114.8(C1′),128.3(C2′,6′),126.2(C3′,5′),126.8(C4′),140.7(C1″),128.3(C2″,4″,6″),126(C3″,5″);ESMS:m/z 280.9[M+H]+(100),[2M+Na]+HR-TOFMS(ES+):280.1703[M+H]+calcd for C19H21NO+H,280.1696.
1-(4-Benzyloxy-7-phenyl-(6E)-6-hepten-3-ol(化合物23)
白色固体;m.p.70-71℃;IR:νmax:3305,3028,2939,2868,1607,1583,1509,1453,1380,1296,1237,1221,1175,1084,1007,963,896,819,737,691;1H NMR(400MHz,CDCl3):δ2.69,2.77(m,2H,H-1),1.81(m,2H,H-2),3.74(m,1H,H-3),1.69(m,2H,H-4),2.37(m,2H,H-5),6.24(dt,J=15.8,6.8Hz,1H,H-6),6.45(d,J=15.8Hz,1H,H-7),7.16(d,J=8.3Hz,2H,H-2′,6′),6.94(d,J=8.3Hz,2H,H-3′,5′),7.24-7.48(m,10H,H-2″-5″and H-2″′-5″′)5.07(s,2H,-CH2-C6H5);13C NMR(100MHz,CDCl3):δ31.1(C1),39.2(C2),70.3(C3),37.0(C4),29.2(C5),130.2(C6,C7),137.2(C1′),129.2(C2′,6′),114.8(C3′,5′),157.0(C4′),134.3(C1″),125.9(C2″,6″),126.9(C4″),127.4(C3″,5″),137.6(C1″′),127.4(C2″′,6″′),127.8(C4″′),128.5(C3″′,5″′),70.0(-CH2-C6H5);ESMS:m/z 378.3[M+H]+;HR-TOFMS(ES+):m/z 395.1983[M+Na]+calcd for C26H28O2+Na,m/z 395.1982
1-(4-Acethoxy)-7-phenylheptan-3-one(化合物24)
无色油状物;IR:νmax:3027,2931,2862,1760,1710,1507,1453,1368,1191,1165,1017,910,747,699;1H NMR(400MHz,CDCl3):δ2.94(t,J=7.50Hz,2H,H-1),2.77(t,J=7.50HZ,2H,H-2),2.47(br t,J=7.56Hz,2H,H-4),1.67(br s,4H,H-5,6),2.67(br t,J=6.38Hz,2H,H-7),7.35(d,J=8.0Hz,2H,H-2′,6′),7.05(d,J=8.0Hz,2H,H-3′,5′),7.22-7.33(m,5H,H-2″-6″,2.36(s,3H,-CH3-C=O);13C NMR(100MHz,CDCl3):δ29.0(C1),44.1(C2),209.7(C3),42.8(C4),23.3(C5),30.9(C6),35.6(C7),142.1(C1′),129.2(C2′,6′),121.4(C3′,5′),148.9(C4′),138.6(C1″),128.2(C2″,6″),125.7(4″),128.3(C3″,5″)21.0,169.5(-CH3-C=O);ESMS:m/z 347.9[M+Na]+;HR-TOFMS(ES+):m/z 347.1637[M+Na]+calcd for C21H24O3+Na,347.1618.
1-(3,4-Diacetoxyphenyl)-7-phenyl-(6E)-6-hepten-3-ol(化合物25)
无色油状物;IR:νmax:3417,3027,2933,2862,1765,1597,1504,1425,1369,1257,1205,1179,1109,1011,964,898,831,743,693;1H NMR(400MHz,CDCl3):δ2.65,2.77(m,2H,H-1),1.76(m,2H,H-2),3.67(m,2H,H-3),1.63(m,2H,H-4),2.30(m,2H,H-5),6.20(dt,J=15.8,8.6Hz,1H,H-6),6.39(d,J=15.8Hz,1H,H-7),7.00(s,2H,H-2′),7.05(s,2H,H-5′,6′),7.17-7.29(m,5H,H-2″-6″),2.25(s,3H,-CH 3-C=O);13C NMR(100MHz,CDCl3):δ31.3(C1),38.7(C2),70.7(C3),37.0(C4),29.2(C5),130.1(C6,7),130.3(C1′),123.1(C2′),140.9(C3′),137.5(C4′),123.2(C5′),126.5(C6′),130.3(C1″),125.9(C2″,6″),126.9(4″),128.4(C3″,5″),20.6,168.4(-CH3-C=O);ESMS:m/z 405.8[M+Na]+,HR-TOFMS(ES+):m/z 405.1689[M+Na]+calcd for C23H26O5+Na,405.1672.
1-(3,4-Dihydroxyphenyl)-7-phenylheptan-3-ol(化合物26)
无色油状物;IR:νmax:3319,2931,2856,1603,1518,1495,1451,1361,1279,1191,1111,1058,954,866,811,786,743,697,631;1H NMR(400MHz,CDCl3):δ2.58(m,2H,H-1),1.69(m,2H,H-2),3.60(m,1H,H-3),1.61(m,2H,H-4),1.31,1.47(m,2H,H-5),1.47(m,2H,H-6),2.58(t,J=7.65Hz,2H,H-7),6.66(s,2H,H-2′,6′),6.74(d,J=8.0Hz,1H,H-5′),6.59(d,J=8.0Hz,1H,H-6′),7.13-7.17,7.20-7.27(m,5H,H-2″-6″);13C NMR(100MHz,CDCl3):δ31.4(C1),38.9(C2),71.5(C3),31.3(C4),25.2(C5),37.3(C6),35.8(C7),141.6(C1′),115.3(C2′,6′),143.5(C3′),142.4(C4′),115.4(C5′),120.6(6′),134.9(C1″),128.2(C2″,6″),125.6(C4″),128.3(C3″,5″);ESMS:m/z 599.8[2M-H]-HR-TOFMS(ES+):m/z323.1632[M+Na]+calcd for C19H24O3+Na,323.1618.
1-(3,4-Diacetoxyphenyl)-7-phenylheptan-3-ol(化合物27)
无色油状物;IR:νmax:3423,3026,2932,2857,1766,1603,1504,1425,1369,1257,1206,1180,1109,1011,899,830,746,699;1H NMR(400MHz,CDCl3):δ2.64,2.76(m,2H,H-1),1.77(m,2H,H-2),3.59(m,1H,H-3),1.62(m,2H,H-4),1.36,1.47(m,2H,H-5),1.47(m,2H,H-6),2.59(t,J=7.5Hz,2H,H-7),6.99(s,1H,H-2′),7.05(dd,J=8.2,8.2),2H,H-5′,6′),7.14-7.16,7.24-7.27(m,5H,H-2″-6″),2.26(s,6H,-CH 3-C=O);13C NMR(100MHz,CDCl3):δ31.4(C1),38.9(C2),71.0(C3),31.3(C4),25.2(C5),37.4(C6),35.8(C7),141.0(C1′),123.1(C2′),142.5(C3′),141.8(C4′),123.2(C5′),125.6(6′),140.0(C1″),128.2(C2″,6″),128.2(C4″),128.3(C3″,5″),20.6(-CH3-),168.4(C=O);ESMS:m/z 791.0[2M+Na]+HR-TOFMS(ES+):m/z 407.1847[M+Na]+calcd for C23H28O5+Na,407.1829.
3-Acetoxy-1-(3,4-diacetoxyphenyl)-7-phenylheptane(化合物28)
无色油状物;IR:νmax:2935,2868,1769,1730,1601,1505,1426,1369,1241,1204,1178,1110,1011,896,831,747,699;1H NMR(400MHz,CDCl3):δ2.65(m,2H,H-1),1.91(m,2H,H-2),4.98(m,1H,H-3),1.66(m,4H,H-4,5),1.40(br s,2H,H-6),2.65(m,2H,H-7),6.99(s,1H,H-2′),7.13(d,J=8.3Hz,1H,H-5′),7.08(d,J=8.3Hz,1H,H-6′),7.20-7.25,7.30-7.34(m,5H,H-2″-6″),2.06,2.32(s,9H,-CH 3-C=O);13C NMR(100MHz,CDCl3):δ35.3(C1),35.6(C2),73.5(C3),31.1(C4),24.7(C5),33.8(C6),31.1(C7),140.4(C1′),123.0(C2′),142.3(C3′),141.8(C4′),123.0(C5′),125.6(6′),140.0(C1″),128.1(C2″,6″),126.3(C4″),128.3(C3″,5″),20.5,21.0,(-CH3),168.2,168.3,170.8(C=O);ESMS:m/z449.80[M+Na]+HR-TOFMS(ES+):m/z 449.1936[M+Na]+calcd for C25H30O6+Na,449.1935
1-(3,4-Diacetoxyphenyl)-7-phenylheptan-3-one(化合物29)
无色油状物;IR:νmax:2934,2868,1767,1710,1602,1505,1425,1369,1257,1204,1178,1109,1010,899,831,747,699;1H NMR(400MHz,CDCl3):δ2.84(t,J=7.5Hz,2H,H-1),2.68(t,J=7.5Hz,2H,H-2),2.36(t,J=6.0Hz,2H,H-4),1.58(m,2H,H-5),1.55(m,2H,H-6),2.58(t,J=6.5Hz,1H,H-7),6.97(s,1H,H-2′),7.04(dd,J=12.0Hz,8.2Hz,2H,H-5′,6′),7.13-7.16,7.24-7.26(m,5H,H-2″-6″),2.25(s,-CH 3-C=O);13C NMR(100MHz,CDCl3):δ28.9(C1),43.8(C2),209.4(C3),42.8(C4),23.3(C5),30.9(C6),35.6(C7),140.0(C1′),123.2(C2′,5′),142.1(C3′),141.8(C4′),123.2(C5′),125.7(C6′),140.2(C1″),128.2(C2″,6″),126.5(4″),128.3(C3″,5″),20.6,168.3(-CH3-C=O);ESMS:m/z 405.8[M+Na]+;HR-TOFMS(ES+):m/z 405.1691[M+Na]+calcd for C23H226O5+Na,405.1672
Cinnamaldehyde(or(E)-3-(2,4-Dimethoxyphenyl)acrylaldehyde)).(化合物3c)
淡黄色固体;IR:νmax 2840,2767,1660,1606,1570,1502,1425,1324,1296,1275,1202,1166,1023,969,921,835,783cm-1;1H NMR(400MHz,CDCl3):δ9.60(d,J=7.9Hz,1H),7.71(d,J=15.9Hz,1H),7.46(d,J=8.6Hz,1H),6.68(dd,J=15.9,7.9Hz,1H),6.51(dd,J=8.6,2.3Hz,1H),6.44(d,J=2.3Hz,1H),3.86(s,3H),3.83(s,1H).;13C NMR(100MHz,CDCl3):δ194.6,163.7,159.9,148.4,130.5,126.8,116.2,105.6,98.3,55.53,55.51;ESMS(+ve):m/z 407[2M+Na]+.
Cinnamaldehyde 2d((E)-3-(2,5-Dimethoxyphenyl)acrylaldehyde).(化合物3d)
淡黄色固体;IR:νmax 2921,2839,2810,2738,2712,1668,1610,1493,1424,1224,1134,1040,1022,973,846,804,711cm-1;1H NMR(400MHz,CDCl3):δ9.67(d,J=7.8Hz,1H),7.81(d,J=16.1Hz,1H),7.05(d,J=3.0Hz,1H),6.96(dd,J=9.0,3.0Hz,1H),6.87(d,J=9.0Hz,1H),6.73(dd,J=16.1,7.8Hz,1H),3.85(s,3H),3.78(s,3H).;13C NMR(100MHz,CDCl3):δ194.3,153.6,152.8,147.7,129.2,123.5,118.5,113.0,112.6,56.1,55.8;ESMS(+ve):m/z 193[M+H]+.
1,7-Bis(3-hydroxyphenyl)-1,4,6-heptatrien-3-one(化合物34).
橙黄色固体;m.p.191-194℃;ESMS(+ve):m/z 607[2M+Na]+;HR-TOFMS(ES–):m/z291.1019[M–H]–(Calcd.for C19H16O3–H,291.1021).
1-(3-Hydroxyphenyl)-7-(3-methoxyphenyl)-1,4,6-heptatrien-3-one(化合物35).
亮黄非晶固体;m.p.120-122℃;ESMS(+ve):m/z 307[M+H]+;HR-TOFMS(ES+):m/z329.1161[M+Na]+(Calcd.for C20H18O3+Na,329.1154).
1-(3-Methoxyphenyl)-7-(3-hydroxyphenyl)-1,4,6-heptatrien-3-one(化合物36).
亮黄非晶固体;m.p.120-121℃;ESMS(+ve):m/z 307[M+H]+;HR-TOFMS(ES+):m/z329.1157[M+Na]+(Calcd.for C20H18O3+Na,329.1154).
1-(4-Methoxyphenyl)-7-(3-hydroxyphenyl)-1,4,6-heptatrien-3-one(化合物37).
淡黄色固体;IR:νmax 3261,2930,1655,1591,1547,1509,1445,1423,1255,1167,1089,870,779cm-1;1H NMR(400MHz,CDCl3):δ7.65(d,J=15.8,1H),7.54(d,J=8.7,2H),7.48(dt,J=15.1,5.1,1H),7.22(t,J=7.8,1H),7.04(d,J=7.8,1H),6.84-6.94(m,6H),6.79(dd,J=7.8,1.9,1H),6.61(d,J=15.1,1H),5.40(br s,1H),3.83(s,3H);13C NMR(100MHz,CDCl3):δ189.0,161.7,156.0,143.0,142.9,141.0,137.8,130.1(2C),130.0,129.3,127.5,127.4,123.3,120.0,116.3,114.4(2C),113.7,55.4.;ESMS(+ve):m/z 307[M+H]+;HR-TOFMS(ES–):m/z 305.1180[M-H]–(Calcd.for C20H17O3-H(C20H18O3-H),305.1178).
1-(3-Hydroxy-4-methoxyphenyl)-7-(3-hydroxyphenyl)-1,4,6-heptatrien-3-one(化合物38).
橙黄色固体;IR:νmax 3465,3185,3011,2848,1648,1578,1551,1514,1447,1347,1265,1226,1125,1090,1014,992,870,789,678cm-1;1H NMR(400MHz,acetone-d6):δ8.66(s,1H),7.97(s,1H),7.58(d,J=15.9Hz,1H)7.55(dd,J=15.3,8.7Hz,1H),7.25(br d,J=1.6Hz,1H),7.14-7.23(m,2H),7.02-7.13(m,5H),7.00(d,J=8.3Hz,1H),6.82(d,J=8.1Hz,1H),6.71(d,J=15.3Hz,1H),3.89(s,3H).;13C NMR(100MHz,acetone-d6):δ188.8,158.7,150.8,147.9,143.3(2C),141.8,138.8,130.7,130.5,129.2,128.2,124.1,122.7,119.6,117.1,114.6,114.5,112.4,56.3.;ESMS(-ve):m/z 643[2M–H]–;HR-TOFMS(ES–):m/z321.1124[M-H]–(Calcd.for C20H18O4-H,321.1127).
(1-(3-Hydroxyphenyl)-7-(2,4-dimethoxyphenyl)-1,4,6-heptatrien-3-one(化合物39).
橙色固体;IR:νmax 3281,2940,2837,1659,1594,1559,1446,1267,1206,1088,1024,996,976,860,828,779,672cm-1;1H NMR(400MHz,acetone-d6):δ8.61(s,1H),7.53-7.65(m,3H),7.16-7.34(m,5H),7.06(dd,J=15.6,11.2Hz,1H),6.90(m,1H),6.54-6.64(m,3H),3.91(s,3H),3.84(s,3H);13CNMR(100MHz,acetone-d6):δ188.8,163.1,160.0,158.7,145.4,142.5,137.6,137.3,130.8,129.5,128.8,126.2,126.1,120.7,118.9,118.2,115.5,106.8,99.1,56.0,55.8.;ESMS(+ve):m/z 359[M+Na]+;HR-TOFMS(ES+):m/z359.1250[M+Na]+(Calcd.for C21H20O4+Na,359.1254).
1-(3-Hydroxy-4-methoxyphenyl)-7-(2,4-dimethoxyphenyl)-1,4,6-heptatrien-3-one(化合物40).
橙色固体;IR:νmax 3298,3005,2938,2838,1639,1571,1505,1438,1261,1199,1022,984,836cm-1;1H NMR(400MHz,CDCl3):δ7.57(d,J=15.8Hz,1H),7.51(dd,J=15.2,11.2Hz,1H),7.44(d,J=8.6Hz,1H),7.25(d,J=15.6Hz,1H),7.20(d,J=1.5Hz,1H),7.08(dd,J=8.3,1.5Hz,1H),6.91(dd,J=15.6,11.2Hz,1H),6.89(d,J=15.8Hz,1H),6.84(d,J=8.3Hz,1H),6.50(d,J=15.2Hz,1H),6.49(dd,J=8.6,2.0Hz,1H),6.43(d,J=2.0Hz,1H),5.68(s,1H),3.91(s,3H),3.85(s,3H),3.81(s,3H);13C NMR(100MHz,CDCl3):δ189.0,161.9,158.9,148.6,145.9,144.8,142.3,136.8,128.7(2C),127.7,125.4,123.8,122.3,118.4,113.0,110.6,105.3,98.5,56.0,55.5,55.4;ESMS(-ve):m/z 356[M-H]-;HR-TOFMS(ES+):m/z 389.1367[M+Na]+(Calcd.for C22H22O5+Na,389.1365)
(1-(3-Hydroxy-4-methoxyphenyl)-7-(2,5-dimethoxyphenyl)-1,4,6-heptatrien-3-one(化合物41).
黄色固体;IR:νmax 3228,3001,2945,2839,1635,1630,1557,1505,1233,1133,1016,874,803cm-1;1H NMR(400MHz,CDCl3):δ7.58(d,J=15.8Hz,1H),7.51(dd,J=15.2,11.1Hz,1H),7.30(d,J=15.6Hz,1H),7.20(d,J=1.8Hz,1H),7.08(dd,J=8.2,1.8Hz,1H),7.05(d,J=2.2Hz,1H),6.96(dd,J=15.6,11.1Hz,1H),6.88(d,J=15.8Hz,1H),6.84(d,J=8.2Hz,1H),6.82(overlapped,1H),6.81(overlapped,1H),6.56(d,J=15.2Hz,1H),5.73(br s,1H),3.91(s,3H),3.82(s,3H),3.79(s,3H);13CNMR(100MHz,CDCl3):δ189.0,153.6,152.1,148.7,145.9,143.9,142.7,136.3,128.9,128.5,127.7,125.8,123.6,122.4,115.7,113.1,112.4,112.1,110.6,56.2,56.0,55.8;ESMS(+ve):m/z 367[M+H]+;HR-TOFMS(ES+):m/z 389.1365[M+Na]+(Calcd.for C22H22O5+Na,389.1365)
1-(4-Methoxyphenyl)-7-(3-hydroxyphenyl)-heptan-3-one(化合物42).
无色油状物;IR:νmax 3384,2931,2859,1698,1511,1454,1242,1177,1154,1033,825,781,695cm-1;1H NMR(400MHz,CDCl3):δ7.10(distorted t,J=8.5Hz,1H),7.07(d,J=8.5Hz,2H),6.80(d,J=8.5Hz,2H),6.69(distorted d,J=7.5Hz,1H),6.63(distorted d,J=8.1Hz,1H),6.56(br s,1H),3.77(s,3H),2.81(distorted t,J=7.4Hz,2H),2.66(distorted t,J=7.4Hz,2H),2.50(distorted t,J=7.1Hz,2H),2.36(distorted t,J=6.8Hz,2H),1.44-1.69(m,4H);13C NMR(100MHz,CDCl3):δ211.0,158.0,155.8,144.0,133.1,129.4,129.3(2C),120.6,115.3,113.9(2C),112.7,55.3,44.5,42.9,35.5,30.6,28.9,23.3;ESMS(-ve):m/z 311[M-H]-.
1-(3-Hydroxy-4-methoxyphenyl)-7-(3-hydroxyphenyl)-heptan-3-one(化合物43).
无色油状;IR:νmax 3465,3326,2917,2853,1693,1594,1514,1446,1377,1275,1237,1198,1131,1025,999,924,780,691cm-1;1H NMR(400MHz,CDCl3):δ7.10(t,J=7.7Hz,1H),6.57-6.77(m,6H),3.83(s,3H),2.77(distorted t,J=7.1Hz,2H),2.65(distortedt,J=7.0Hz,2H),2.50(distorted t,J=6.7Hz,2H),2.36(distorted t,J=6.5Hz,2H),1.44-1.63(m,4H);13C NMR(100MHz,CDCl3):δ211.0,155.8,145.4,144.9,144.0,134.3,129.4,120.7,119.8,115.3,114.5,112.8,110.8,56.0,44.2,42.8,35.4,30.5,29.2,23.3;ESMS(-ve):m/z 327[M-H]-.
1-(3-Hydroxyphenyl)-7-(2,4-dimethoxyphenyl)-heptan-3-one(化合物44).
无色油状物;IR:νmax 3436,2934,2836,1702,1610,1585,1502,1459,1209,1156,1124,1030,844,778,687cm-1;1H NMR(400MHz,CDCl3):δ7.11(t,J=7.8Hz,1H),6.97(d,J=8.1Hz,1H),6.71(d,J=7.6Hz,1H),6.59-6.67(m,2H),6.36-6.44(m,2H),5.13(br s,1H),3.77(s,3H),3.75(s,3H),2.80(distorted t,J=7.5Hz,2H),2.66(distorted t,J=7.5Hz,2H),2.50(distorted t,J=7.3Hz,2H),2.38(distorted t,J=7.2Hz,2H),1.43-1.65(m,4H);13C NMR(100MHz,CDCl3):δ211.1,159.0,158.2,155.9,142.9,129.9,129.6,123.0,120.4,115.2,113.1,103.9,98.5,55.3,55.2,43.9,43.0,29.6,29.5,29.1,23.6.;ESMS(-ve):m/z 683[2M-H]-.
1-(3-Hydroxyphenyl)-7-(2,4-dimethoxyphenyl)-heptan-3-ol(化合物45).
无色油状物;IR:νmax 3317,2933,2857,1705,1611,1587,1505,1455,1285,1258,1206,1153,1035,934,832,783,696cm-1;1H NMR(400MHz,CDCl3):δ7.12(t,J=8.2Hz,1H),6.98(d,J=8.1Hz,1H),6.74(d,J=7.5Hz,1H),6.64(m,2H),6.37-6.44(m,2H),3.77(s,3H),3.76(s,3H),3.60(m,1H),2.70(m,1H),2.60(m,1H),2.52(t,J=7.5Hz,2H),1.70(m,2H),1.27-1.60(m,6H);13C NMR(100MHz,CDCl3):δ158.9,158.3,155.9,144.0,129.9,129.6,123.4,120.6,115.4,112.8,103.9,98.6,71.5,55.4,55.3,38.6,37.3,31.9,30.0,29.4,25.3;ESMS(-ve):m/z 687[2M-H]-.
1-(3-Hydroxy-4-methoxyphenyl)-7-(2,4-dimethoxyphenyl)-heptan-3-one(化合物46).
无色油状物;IR:νmax 3429,2935,2841,1707,1612,1587,1505,1270,1206,1153,1125,1030,832,801,760cm-1;1H NMR(400MHz,CDCl3):δ6.97(d,J=8.0Hz,1H),6.73(m,2H),6.62(d,J=8.1Hz,1H),6.39(m,2H),5.56(br s,1H),3.83(s,3H),3.76(s,3H),3.75(s,3H),2.77(distorted t,J=7.4Hz,2H),2.65(distorted t,J=7.5Hz,2H),2.50(distorted t,J=7.3Hz,2H),2.37(distorted t,J=7.1Hz,2H),1.47-1.60(m,4H);13CNMR(100MHz,CDCl3):δ210.4,159.0,158.2,145.5,144.9,134.4,129.9,123.0,119.6,114.4,110.7,103.8,98.5,56.0,55.3,55.2,44.3,42.9,29.6,29.2(2C),23.6;ESMS(+ve):m/z 767.9[2M+Na]+.
1-(3-Hydroxy-4-methoxyphenyl)-7-(2,4-dimethoxyphenyl)-heptan-3-ol(化合物47).
无色油状物;IR:νmax 3428,2932,2856,2835,1611,1587,1505,1454,1438,1271,1205,1153,1128,957,920.832,802,760,631cm-1;1H NMR(400MHz,CDCl3):δ6.98(d,J=8.1Hz,1H),6.76(br s,1H),6.74(distorted d,J=8.4Hz,1H),6.65(d,J=8.0Hz,1H),6.41(br s,1H),6.39(distorted d,J=8.3Hz,1H),5.55(br s,1H),3.84(s,3H),3.77(s,6H),3.59(m,1H),2.67(m,1H),2.57(m,1H),2.51(t,J=7.5Hz,2H),1.69(m,2H),1.26-1.60(m,6H);13C NMR(100MHz,CDCl3):δ159.0,158.3,145.5,144.7,135.6,129.9,123.4,119.7,114.6,110.7,103.8,98.5,71.3,56.0,55.3,55.2,39.1,37.4,31.4,30.1,29.4,25.4.;ESMS(+ve):m/z 771.8[2M+Na]+.
1-(3-Hydroxy-4-methoxyphenyl)-7-(2,5-dimethoxyphenyl)-heptan-3-one(化合物48).
无色油状物;IR:νmax 3406,2932,2835,1589,1497,1440,1272,1218,1128,1044,1025,957,867,800,760cm-1;1H NMR(400MHz,CDCl3):δ6.60-6.76(m,6H),5.54(br s,1H),3.83(s,3H),3.74(s,6H),2.77(distorted t,J=7.5Hz,2H),2.65(distorted t,J=7.4Hz,2H),2.54(distorted t,J=7.3Hz,2H),2.38(distorted t,J=7.1Hz,2H),1.55(m,4H);13C NMR(100MHz,CDCl3):δ210.3,153.4,151.7,145.5,144.9,134.4,131.9,119.6,116.2,114.4,111.2,110.8,110.7,56.0,55.9,55.6,44.3,42.9,29.9,29.4,29.2,23.6;ESMS(+ve):m/z 767[2M+Na]+.
1-(3-Hydroxy-4-methoxyphenyl)-7-(2,5-dimethoxyphenyl)-heptan-3-ol(化合物49).
无色油状物;IR:νmax 3406,2932,2857,2835,1589,1497,1440,1272,1218,1128,1044,1025,957,867,800,760,710cm-1;1H NMR(400MHz,CDCl3):δ6.60-6.79(m,6H),5.56(br s,1H),3.84(s,3H),3.75(s,3H),3.74(s,3H),3.59(m,1H),2.67(m,1H),2.56(m,3H),1.27-1.80(m,8H);13C NMR(100MHz,CDCl3):δ153.4,151.7,145.4,144.7,135.5,132.3,119.6,116.2,114.6,111.2,110.6(2C),71.2,55.9(2C),55.3,39.0,37.3,31.3,30.1,29.4,25.4;ESMS(+ve):m/z 771[2M+Na]+.
2.3参考文献:
1.Suksamrarn,A.,Ponglikitmongkol,M.,Wongkrajang,K.,Chindaduang,A.,Kittidanairak,S.,Jankam,A.,Yingyongnarongkul,B.,Kittipanumat,N.,Chokchaisiri,R.,Khetkam,P.,Piyachaturawat,P.Diarylheptanoids,new phytoestrogens from therhizomes of Curcuma comosa:Isolation,chemical modification and estrogenicactivity evaluation.Bioorg.Med.Chem.2008,16,6891-6902.
2.Sornkaew,N.,Lin,Y.,Wang,F.,Zhang,G.,Chokchaisiri,R.,Zhang,A.,Wongkrajang,K.,Suebsakwong,P.,Piyachaturawat,P.and Suksamrarn,A.Diarylheptanoids of Curcuma comosa with Inhibitory Effects on Nitric OxideProduction in Macrophage RAW 264.7Cells.Natural Product Communications.2015,10(1),89-93.
3.Chuprajob,T.;Changtam,C.;Chokchaisiri,R.;Chunglok,W.;Sornkaew N.;Suksamrarn,A.Synthesis,cytotoxicity against human oral cancer KB cells andstructure-activity relationship studies of trinone analogues ofcurcuminoids.Bioorg.Med.Chem.lett.2014,16,2839-2844.
4.Sodsai,A.;Piyachaturawat,P.;Sophasan,S.;Suksamrarn,A.;Vongsakul,M.Suppression by Curcuma comosa Roxb.of pro-inflammatory cytokine secretionin phorbol-12-myristate-13-acetate stimulated human mononuclearcells.Int.Immunopharmacol.2007,7,524-531.
实施例2二芳基庚烷类化合物抑制NO产生的活性作用。
1材料
化合物(1-37,39-49);小鼠RAW264.7巨噬细胞;内毒素(LPS,0.5μg/mL);BAY(10μM,阳性对照)NO检测试剂盒(Beyotime,Haimen,China)。
2主要仪器
荧光酶标仪(Thermo Scienti fic,Waltham,MA,USA)
3方法
3.1 LPS刺激RAW264.7巨噬细胞生成NO的测定
RAW264.7巨噬细胞接种于96-孔板,每孔接种量为1×104细胞,待接种细胞贴壁以后,加入待测化合物(1-37,39-49)孵育2h,然后加入LPS(终浓度为0.5μg/mL),继续培养24h。最后取96孔板培养基,用NO试剂盒进行检测,NO的含量通过测定其OD550来表示。每次实验取3个复孔,重复3次实验。
3.2 NO抑制率的计算
NO的抑制率(%),根据以下公式进行计算:
抑制率(%)=(A-B)/(A-C)×100,
A:LPS(+),化合物(-);B:LPS(+),化合物(+),C:LPS(-),化合物(-)。
3.3统计方法
所有的统计方法均使用GraphPad Prism 5.01软件进行处理。数据以平均数±标准误差表示,数据使用单因素方差分析(ANOVA)。p<0.05
4结果
结果如图2所示,结果表明化合物27-29,37,39-41在初筛浓度为5μM条件下,可明显抑制LPS诱导的NO生成,其抑制率在80%以上。
进一步测定活性较好的化合物27-29,37,39-41的IC50值,结果如图3所示,它们的IC50值在0.32-1.23μM。
实施例3二芳基庚烷类化合物对巨噬细胞增殖的抑制作用
1材料
化合物(1-37,39-49);小鼠RAW264.7巨噬细胞;Alamar-Blue试剂。
2主要仪器
荧光酶标仪(Thermo Scienti fic,Waltham,MA,USA)
3方法
3.1化合物(1-37,39-49)对RAW264.7巨噬细胞增殖的影响
接种RAW264.7巨噬细胞于96-孔板,每孔接种1×104细胞,待接种细胞贴壁以后,加入待测化合物(1-37,39-49)孵育24h。然后每个每孔加入Am-Blue细胞增殖与活性检测试剂(SunBioTM)10μL并于培养箱中孵育2~6小时。当培养基的颜色由靛青蓝变成粉红色后用荧光酶标仪测定各孔相对荧光单位(RFU值),激发光波长560nm,发射光波长590nm。每次实验取3个复孔,重复3次实验。
3.2统计方法
所有的统计方法均使用GraphPad Prism 5.01软件进行处理。数据以平均数±标准误差表示,数据使用单因素方差分析(ANOVA),当p<0.05时有统计学意义。
4结果
结果如图4所示,结果显示,化合物37,39在50μM对细胞增殖有明显的抑制作用,IC50值分别为48.53μM,31.25μM。
实施例4本发明化合物抑制iNOS、IL-1β、IL-6的mRNA和蛋白表达
1材料
化合物28,化合物40;小鼠RAW264.7巨噬细胞;BAY(10μM,NF-κB抑制剂);LPS(0.5μg/mL)。
2主要仪器
荧光酶标仪(Thermo Scienti fic,Waltham,MA,USA)
增强化学发光检测系统(Amersham Bioscience,Piscataway,NJ,USA)
3方法
3.1 Western Immunoblotting检测
接种RAW264.7巨噬细胞于6孔板,过夜培养,加入不同浓度的化合物28和化合物40(0.2、1、5μM)孵育2h,然后加入LPS分别刺激。去掉6孔板中的培养基,加入细胞裂解液,蛋白酶和磷酸酶抑制剂,获得细胞总蛋白。蛋白含量采用BCA方法进行定量。
取等量的细胞裂解液(50μg)加入loading buffer,沸煮5分钟,再进行SDS-PAGE(10%)凝胶电泳。电泳以后,将蛋白质转移到硝酸纤维素膜,用5%牛血清白蛋白封闭2h;再加入anti-iNOS抗体4℃孵育过夜。然后将膜与辣根过氧化物酶标记二抗孵育2h,再用增强化学发光检测系统成像。每条蛋白带的信号强度使用计算机图像分析系统处理(QuantityOne,Bio-Rad,Hercules,CA,USA)。
3.2 BCA蛋白定量方法
(1)BCA工作液配制。根据样品数量,按照50:1的体积比将BCA试剂A与试剂B混合,配制适量BCA工作液,充分混匀。
(2)蛋白标准工作液配置:根据样品数量配置相应体积的蛋白标准工作液。将蛋白质标准储备液用双蒸水5倍稀释,配成1mg/mL的工作液。
(3)标准曲线绘制。取一块酶标板,按以下表格数据加入试剂:
(4)振荡混匀后,37℃放置30分钟。
(5)用酶标仪测定A562,以不含BCA的光吸收值为空白对照。
(6)以蛋白含量(g)为横坐标,吸光值为纵坐标,绘出标准曲线。
(7)样品测定:取待测蛋白样品(细胞裂解液)4μL用去离子水补足20μL,加入200μL的BCA工作液,混匀后37℃放置30分钟,然后以0号孔为对照,测定样品吸收值A562。
(8)根据测得的吸收值,在标准曲线上即可查得样品的蛋白含量。
(9)计算蛋白浓度:以查得的蛋白含量除以样品体积4μL即可得到待测样品的实际浓度(μg/μL)。
3.3 ELISA
采用R&D SystemsMouse IL-1β/IL-1F2 Immunoassay试剂盒测定IL-1β的含量。这个试剂盒采用定量夹心酶免疫测定技术。与小鼠IL-1β单克隆抗体预先涂布到微孔板,然后在各微孔中加入的标准品,待测样品与预先固定的单抗结合。洗去未结合的物质后,将小鼠IL-1β的多克隆抗体加入到各孔中,并且洗去多余未结合的抗体,然后加入底物反应。当酶促底物反应由蓝色变为黄色是,加入终止液。反应后颜色的深浅与IL-1β含量成正比,根据标准曲线读出的IL-1β含量。
3.3.1样品制备
接种适量的RAW264.7细胞于24孔板中,用含有10%新生小牛血清和1%青链霉素无酚红的DMEM高糖培养基进行培养(500μL/well)。待细胞贴壁以后(1-4小时),加入待测化合物和阳性药物,预先孵育2小时,然后加入LPS(终浓度为0.5μg/mL),继续培养22小时。每孔取200μL培养基13000r离心5分钟,去上清于-20℃或者-80℃保存备用。
3.3.2检测试剂准备
Mouse IL-1βKit Control(IL-1β对照品):用1mL蒸馏水溶解L-1β对照品,备用;
Wash Buffer(洗涤缓冲液):将浓缩物液加热至室温并轻轻搅拌,使其完全溶解;然后20mL Wash Buffer浓缩液用蒸馏水稀释为500mL备用;
Substrate Solution(底物溶液):将显示试剂A和B在使用前15分钟等体积混合,每孔100μL;
Mouse IL-1βStandard(IL-1β标准品):将用IL-1β标准品粉末用15mLCalibratorDiluent RD5-16稀释,该原液浓度为800pg/mL;待溶液混匀以后,按照说明书的方法,分别稀释为400pg/mL、200pg/mL、100pg/mL、50pg/mL、25pg/mL、12.5pg/mL备用。
3.3.3检测步骤
a.按照上面的方法,确保检测试剂准备就绪;
b.从96孔板架上取出足够数量的微孔板条,然后将多余的微孔板放回铝箔袋中,加干燥剂,重新密封;
c.没有孔加入50μL Assay Diluent RD1N;
d.在相应的微孔中分别加入50μL标准品,对照品和待测样品,做好对应标记,并且用胶带封好,在室温下孵育2小时;
e.吸出每孔的溶液,并且用400μL的wash buffer洗涤5次。每次洗涤需要尽可能的去除液体,最后一次洗涤时应该倒置用滤纸吸收完液体;
f.加入100μL Mouse IL-1βConjugate于各个孔中,用胶带封好,在室温下孵育2小时;
g.重复步骤e;
h.每孔加入100μL的底物溶液,避光反应30分钟;
i.每孔加入100μL的终止溶液,轻轻摇匀;
j.在30分钟内,在波长为450nm处检测其吸光度,用540nm或570nm吸光度进行矫正。
3.4实时荧光定量PCR(RT-PCR)
3.4.1细胞mRNA的提取
本实验所用枪头,EP管,镊子,PCR管子都应该是无核酸酶商业产品或者用DEPC浸泡后,高压灭菌产品。
a.接种RAW264.7细胞于6孔板中,用含有10%新生小牛血清和1%青链霉素的DMEM高糖培养基进行培养(2mL/well)。待细胞贴壁以后(1-4小时),加入待测化合物和阳性药物,预先孵育2小时,然后加入LPS(终浓度为0.5μg/mL),继续培养22小时。
b.去除每孔的培养基,用事先预冷的PBS洗2遍;
c.每孔加入1mL的TRIzol试剂(Invitrogen),在室温下静置5min;
d.用枪头反复的吹打,将裂解也吸入相应的EP管中,做好标记;
e.加入200μL的CHCl3,涡旋30秒(需保证完全混合),冰置3分钟;
f.然后13000r/min离心15分钟,将上层水相吸入新的EP管中(约为400μL),不可吸入其它杂质;
g.加入500μL的异丙醇,反复混匀,食物静置10分钟;13000r/min离心10分钟,弃掉上清,保留RNA沉淀;
h.再用现配的75%的乙醇,洗涤RNA沉淀,7500r/min离心10分钟,弃上清,室温干燥5-10分钟;
i.用25μL的DEPC水溶解RNA沉淀,用分光光度计测量其在OD260和OD280的吸光度。一般按照(4μL RNA原溶液+96μL DEPC水)的方式稀释测量。一般1.8<OD260/OD280<2.0时进行下一步实验。RNA浓度=OD260×稀释倍数×0.04(μg/μL)。
3.4.2总mRNA逆转录为cDNA
按照Promega的M-MLV逆转录酶说明书,根据以下步骤将总mRNA逆转录为cDNA。
a.对于总反应体系为25μL,按照如下体系配置成为引物合成混合体系:
b.将上述混合引物体系在70℃孵育5分钟,冰浴2分钟;
c.按照如下组分配置成cDNA合成体系:
d.将配置好的cDNA合成体系加入引物混合体系,涡旋混匀,适当离心;
e.按照如下条件进行PCR反应,37℃,50分钟,然后70℃反应15分钟。
3.4.3荧光定量PCR
设计相关引物,使用Maxima SYBR Green/ROX qPCR Master Mix(2X)(ThermoScientific)进行荧光定量PCR反应,GAPDH为内参。
a.按照如下组分配置反应体系(20μL):
b.iNOS mRNA荧光定量PCR按照如下体系进行反应:95℃热激活酶,5min;95℃变性,5s,60℃退火和延伸,60s,然后荧光读板,共计39个循环;70℃再延伸,5min;溶解曲线50℃-95℃,每0.5℃读一次。
c.IL-1β和IL-6mRNA荧光定量PCR按照如下体系进行反应:95℃热激活酶,5min;95℃变性,30s,50℃退火,30s,72℃延伸,30s,然后荧光读板,共计45个循环;70℃再延伸,5min;溶解曲线55℃-95℃,每0.5℃读一次。
d.采用2-Δ(ΔCt)对RT-qPCR结果进行计算。
5.4结果
结果如图5所示,化合物28和40(0.2、1、5μM)处理RAW264.7细胞以后,显著抑制了iNOS,IL-1β,IL-6的表达,并且呈现较好的剂量依赖性。并且,与LPS模型组相比较,iNOS、IL-6、IL-1β的mRNA表达显著降低,并且呈现一定的剂量依赖性。
证明本发明化合物可以作为一氧化氮合酶抑制剂、IL-1β抑制剂和IL-6抑制剂。
实施例6本发明化合物对AKT和NF-κB磷酸化的抑制作用
1材料
化合物28,化合物40;小鼠RAW264.7巨噬细胞;BAY(10μM,NF-κB抑制剂);SB203580(10μM,p38抑制剂),PD98059(20μM,ERK抑制剂),SP600125(20μM,JNK抑制剂),LY294006(20μM,AKT抑制剂),BAY(10μM,NF-κB抑制剂);LPS(0.5μg/mL)。
2主要仪器
荧光酶标仪(Thermo Scienti fic,Waltham,MA,USA)
增强化学发光检测系统(Amersham Bioscience,Piscataway,NJ,USA)
3方法
3.1Western Immunoblotting检测
接种RAW264.7巨噬细胞于6孔板,过夜培养,加入不同浓度的化合物28和化合物40(1、10、50μM)孵育2h,然后加入LPS分别刺激30min。去掉6孔板中的培养基,加入细胞裂解液,蛋白酶和磷酸酶抑制剂,获得细胞总蛋白。蛋白含量采用BCA方法进行定量。
取等量的细胞裂解液(50μg)加入loading buffer,沸煮5分钟,再进行SDS-PAGE(10%)凝胶电泳。电泳以后,将蛋白质转移到硝酸纤维素膜,用5%牛血清白蛋白封闭2h;再加入相应的抗体,4℃孵育过夜。然后将膜与辣根过氧化物酶标记二抗于室温下孵育2h,再用增强化学发光检测系统成像。每条蛋白带的信号强度使用计算机图像分析系统处理(Quantity One,Bio-Rad,Hercules,CA,USA)。
3.2BCA蛋白定量方法
蛋白定量方法参考实施例4中的方法。
4结果
结果如图6和图7所示。
(1)图6A结果显示,LPS可以激活p38,显著升高其磷酸化水平;当用p38抑制剂(SB203580)处理时,可以抑制LPS诱导p38磷酸化,而化合物28和40对p38的磷酸化影响很小,只有在高浓度时,有一定的抑制作用。在图7B结果中,LPS可以显著增加JNK的磷酸化水平,JNK抑制剂(SP600125)显著抑制LPS诱导JNK磷酸化,化合物28和40对JNK的磷酸化水平基本没有影响。
(2)图6C结果显示,LPS可以显著增加ERK1/2的磷酸化水平,ERK1/2抑制剂(PD98059)显著抑制LPS诱导ERK磷酸化,化合物28和40显著促进了ERK1/2的磷酸化水平,并且有一定的浓度依赖性。
(3)通过对化合物对NF-κB信号通路中IκBα的作用表明,LPS促进IκBα的磷酸化,抑制了IκBα的泛素化降解;而化合物28和40与阳性药物BAY一样,可以抑制IκBα的磷酸化及其泛素化降解。
(4)进一步研究表明,LPS可以显著增加AKT的磷酸化水平,AKT抑制剂(LY294006)显著抑制LPS诱导AKT磷酸化,化合物28和40预处理RAW264.7细胞后,显著抑制了AKT的磷酸化水平,且该作用呈现出良好的浓度依赖性(图7C)。以上结果表明,这两个化合物可以通过抑制AKT和NF-κB的激活,进而抑制促炎症因子的转录。
实施例7本发明化合物作为α7尼古丁乙酰胆碱受体的激动剂
1材料
化合物28,化合物40;小鼠RAW264.7巨噬细胞;BAY(10μM,NF-κB抑制剂);α-BGT(α银环蛇毒素),100nM;GTS(GTS-21),20μM;Acetycholine(乙酰胆碱),1mM;LPS(0.5μg/mL)。
2主要仪器
荧光酶标仪(Thermo Scienti fic,Waltham,MA,USA)
增强化学发光检测系统(Amersham Bioscience,Piscataway,NJ,USA)
3方法
3.1Western Immunoblotting检测
参考实施例3中3.1的方法。即图8A预先用α-BGT处理RAW264.7细胞1h,然后再用GTS和Acetycholine处理RAW264.7细胞2h,最后用LPS处理细胞30min。细胞裂解液通过westernblot用anti-phosphor-JAK2(Tyr1007)和anti-phosphor-STAT3(Tyr705)抗体分别检测JAK2和STAT3的磷酸化。GAPDH作为内参。图8B和C,预先用α-BGT处理RAW264.7细胞1h,然后再用28和40处理RAW264.7细胞2h,最后用LPS处理细胞30min。细胞裂解液通过westernblot用anti-phosphor-JAK2(Tyr1007)和anti-phosphor-STAT3(Tyr705)抗体分别检测JAK2和STAT3的磷酸化。每条蛋白带的信号强度使用计算机图像分析系统处理(QuantityOne,Bio-Rad,Hercules,CA,USA)。
3.2BCA蛋白定量方法
蛋白定量方法参考实施例4中的方法。
4结果
(1)GTS-21和乙酰胆碱是α7尼古丁受体的激动剂,可以通过激活α7 nAChR促进STAT3的磷酸化。α-银环蛇毒素对人的nAChRs(α1、α7和α9)敏感,但是在单核细胞和巨噬细胞只表达α7受体,所以在α-银环蛇毒素只能阻断RAW264.7细胞的α7尼古丁受体,从而抑制STAT3的激活。图8A和B结果表明,GTS-21和乙酰胆碱可以促进JAK2和STAT3的磷酸化;当RAW264.7细胞预处理α7 nAChR抑制剂α-银环蛇毒素以后,可以阻断GTS-21和乙酰胆碱对α7nAChR的激活,抑制了JAK2和STAT3的磷酸化水平。
(2)进一步研究发现,用α-银环蛇毒素阻断α7 nAChR信号通路以后,化合物28和40对JAK2和STAT3的激活作用被显著的抑制(图8B和C)。说明化合物28和40是α7 nAChR激动剂,通过激活α7 nAChR促进JAK2和STAT3的磷酸化,进而抑制炎症的发生。
实施例8本发明化合物抑制LPS诱导的小鼠急性中毒
1材料
化合物28,化合物40;健康ICR小鼠,体重22~28g,每组10只;地塞米松;LPS。
2主要仪器
荧光酶标仪(Thermo Scienti fic,Waltham,MA,USA)
3方法
3.1动物实验
(1)小鼠male分成6组(control、LPS、dexamethasone、待测化合物高剂量、待测化合物中剂量、待测化合物低剂量),体重30~35g,每组10只。
(2)小鼠静脉注射阳性药物地塞米松(20mg/kg/day),小鼠灌胃给药化合物28和化合物40(低剂量:6.25mg/kg/day;中剂量:50mg/kg/day;高剂量:100mg/kg/day)五天,在第五天灌胃给药2小时以后,腹腔注射LPS(30mg/kg)。
(3)连续观察7天,记录小鼠的存活率和体重的变化。
(4)第七天老鼠处死,眼眶取血检测。
3.2 ELISA检测化合物28和化合物40对小鼠血清中IL-1β和IL-6含量的影响
方法参考实施例4中的方法。
4结果
结果如图9所示。
(1)如图9B和F所示,LPS会导致小鼠在体重的下降;当用阳性药物地塞米松和化合物28和40(中剂量和高剂量)预处理5天以后,抑制了LPS所致的小鼠体重降低。
(2)图9A和E结果表明,LPS会导致小鼠急性中毒死亡;当用阳性药物地塞米松和化合物28和40预处理5天以后,可显著抑制小鼠LPS中毒死亡,特别是化合物31、48在中剂量和高剂量给药时,小鼠的存活率大于90%。
(3)进一步研究发现(图9C,D,G和H),LPS升高了小鼠血清中IL-1β,IL-6的含量,而用阳性药物地塞米松、化合物28和40预处理以后,显著抑制了LPS所致的IL-1β、IL-6的生成。这些结果表明,化合物28和40可以显著的抑制LPS诱导的小鼠急性中毒。
实施例9本发明化合物抑制LPS诱导的大鼠败血症
1材料
化合物28、化合物29、化合物21、化合物40、化合物34、化合物36;健康SD雄性大鼠,体重260~310g;LPS。
2方法
2.1动物实验
(1)SD雄性大鼠分成8组(control、LPS、化合物28、化合物29、化合物21、化合物40、化合物34、化合物36),体重260~310g,每组10只。
(2)用化合物(化合物28、化合物29、化合物21、化合物40、化合物34、化合物36,浓度均为25mg/kg)预处理组于尾静脉予,然后立即腹腔注射LPS(10mg/kg);LPS模型组尾静脉予以生理盐水后,立即腹腔注射LPS(10mg/kg);Control组仅尾静脉予以生理盐水。
(3)各组均于4h后予3%戊巴比妥钠(10ml/100g)麻醉,将充满肝素生理盐水的动脉插管插入右颈总动脉描记血压10min,10min后于左颈总动脉取血。
4结果
结果如表1。腹腔大剂量注射LPS后,LPS模型组血小板1min聚集度(PAG1)、5min聚集度(PAG5)、最大聚集度(MA)、最大聚集时间(TMA)与Control组对比均显著下降;在化合物28、化合物29、化合物21、化合物40、化合物34、化合物36预处理组血小板聚集性各项指标较LPS模型组明显升高。表明化合物28、化合物29、化合物21、化合物40、化合物34、化合物36显著抑制LPS诱导的大鼠败血症。
表1化合物对败血症大鼠血小板聚集性的影响
备注:5min聚集度(PAG1)、5min聚集度(PAG5)、最大聚集度(MA)、最大聚集时间(TMA);**P<0.01各个化合物组vs LPS组。
实施例10本发明化合物抑制小鼠老年痴呆(AD)
1材料
化合物28、化合物29、化合物40、化合物36,健康昆明种小鼠雌雄各半,体重25~30g,随机分为6组,即正常对照组、模型组、化合物28实验组、化合物29实验组、化合物40实验组、化合物36实验组,每组10只。
2方法
2.1动物实验
(1)昆明种小鼠雌雄各半,体重25~30g,随机分为6组,正常对照组、模型组、化合物28实验组、化合物29实验组、化合物40实验组、化合物36实验组,每组10只。
(2)模型组及各试验药物组小鼠每天颈背部皮下给予1%D-半乳糖(150mg/kg)注射,共6周,在2周对化合物28实验组、化合物29实验组、化合物40实验组、化合物36实验组进行灌胃给药实验(25mg/kg),模型组生理盐水处理,共4周。
2.2 Morris水迷宫系列试验
隐蔽平台试验水迷宫为直径100cm,高50cm的圆形水池,水池内壁被漆为黑色,池内水深25cm,水温保持在25度,房间内光照恒定,无光线直射在水池内。池壁上以4个等距离点将水池分为4个象限,任选其中1个象限,正中放置1个圆形黑色安全平台,直径为12cm,高24cm,位于水面下1cm。迷宫上方安置连接显示系统的摄像机,同步记录大鼠运动轨迹。实验历时5d,每天上下午各1次,入池位置为平台所邻2个象限的池壁中点。将小鼠面向池壁分别从入水点放入水中,记录其在100s内寻找到平台的时间(逃避潜伏期)。如果小鼠在100s未找到平台,则由实验者用手牵引其至平台上,停留10s,逃避潜伏期记成100s。每天以两次潜伏期算术均值作为这一天的成绩进行统计分析。
探索试验撤除平台,任选1个入水点将小鼠放入水中,使小鼠在无平台情况下寻找记忆中平台,记录其100s内的游泳轨迹,计算其在原平台象限游泳时间和穿越原平台次数。
3结果
表2的结果表明,隐蔽平台试验从第1天起,模型组小鼠的逃避潜伏期明显长于正常组;化合物28、化合物29、化合物40、化合物36实验组显著增加痴呆小鼠逃避潜伏期。因此表明在用化合物28、化合物29、化合物40、化合物36后能改善痴呆小鼠的学习记忆能力。
表2各实验组隐蔽平台试验逃避潜伏期比较
备注:**P<0.01各个化合物组VS AD模型组;*P<0.05各个化合物组vs AD模型组。
表3结果表明,观察小鼠的空间探索能力,发现模型组小鼠基本围绕池壁游泳,其运动轨迹呈随机分布于各象限之中;化合物28、化合物29、化合物40、化合物36实验组停留原平台象限时间和穿越原平台次数均明显多于模型组,再次表明这些化合物可以提高痴呆小鼠的记忆能力。
表3各实验组小鼠探索试验比较
备注:**P<0.01各个化合物组VS AD模型组;*P<0.05各个化合物组vs AD模型组。
综上所述,本发明化合物可以有效地抑制脂多糖(LPS)诱导小鼠RAW246.7巨噬细胞生成一氧化氮(NO)、白细胞介素1β(IL-1β)、白细胞介素6(IL-6),并且本发明化合物呈现浓度依赖性,显示良好的剂量关系;同时动物实验表明,本发明化合物可以有效地抑制脂多糖(LPS)刺激的小鼠血清中的白细胞介素1β(IL-1β)和白细胞介素6(IL-6)水平,并且显著抑制了LPS诱导的小鼠急性中毒死亡,表明本发明化合物可以用于炎症的治疗,具有潜在的临床使用价值。
本发明通过研究LPS诱导炎症信号通路的研究,发现本发明化合物可以显著地抑制iNOS、IL-1β、IL-6的转录和蛋白表达以及IκB蛋白的磷酸化水平,说明本发明化合物可以通过抑制NF-κB信号通路,从而抑制炎症的发生。进一步研究发现本发明化合物可以通过激活α7尼古丁乙酰胆碱受体,经JAK2/STAT3信号级联,从而抑制促炎症因子的表达,促进抑炎因子的表达,进而抑制炎症的发生。进一步说明本发明所示的二芳基庚烷类化合物可以作为炎症和/或与α7尼古丁乙酰胆碱信号通路和/或JAK2/STAT3信号通路和/或NF-κB信号通路和/或MAPKs信号通路相关的疾病的药物中的应用。
Claims (10)
1.式(A)所示的二芳基庚烷类化合物或其顺反异构体、或其外消旋混合物、或其对映异构体、或其药学上可接受的盐、或其溶剂合物:
其中,R1、R2分别独立地表示无或者苯环上的1~5个取代基,所述取代基分别独立地选自-OR3,R3表示氢、C1~C4的烷基或C1~C4的烷酰基;所述C1~C4的烷基任选进一步被苯基所取代;
L1表示4个碳原子的直链烷基或直链烯基;
L2表示-CH2CH2-或-CH=CH-;
X表示R4表示氢或C1~C4的烷酰基。
2.根据权利要求1所述的化合物,其特征在于:所述式(A)化合物的结构如下式(Ⅰ)~(Ⅶ)中任一所示:
3.根据权利要求1或2所述的化合物,其特征在于:所述C1~C4的烷基为甲基。
4.根据权利要求1或2所述的化合物,其特征在于:所述C1~C4的烷酰基为乙酰基。
5.根据权利要求1-4任一项所述的化合物,其特征在于:R1表示无,R2表示苯环上的2个取代基,所述取代基选自-OR3,R3表示氢或乙酰基。
6.根据权利要求1所述的化合物,其特征在于:所述式(A)化合物为如下化合物之一:
Me表示甲基,Ac表示乙酰基。
7.式(A)所示的二芳基庚烷类化合物或其顺反异构体、或其对映异构体、或其药学上可接受的盐、或其溶剂合物、或其前药在制备一氧化氮生成抑制剂、一氧化氮合酶抑制剂、IL-1β抑制剂、IL-6抑制剂、α7尼古丁乙酰胆碱受体激动剂、抑制AKT和/或NF-κB的药物、促进JAK2和/或STAT3磷酸化的药物以及治疗炎症、败血症、关节炎、炎性肠病、过敏性肠综合症、偏头痛、头痛、腰痛、纤维肌痛、肌筋膜障碍、病毒感染、细菌感染、真菌感染、灼伤、外科治疗、恶性肿瘤、动脉粥样硬化、痛风、发热、脊柱炎、红斑狼疮、血管炎、胰腺炎、肾炎、结膜炎、虹膜炎、巩膜炎、葡萄膜炎、创伤、皮炎、湿疹、牛皮癣、中风、糖尿病、神经变性疾病、自身免疫性疾病、鼻炎、溃疡、心脏病、炎性痛、高前列腺素E综合症、典型巴特综合症、何杰金病或治疗阿尔茨海默病的药物中的用途。
8.根据权利要求7所述的用途,其特征在于:所述一氧化氮生成抑制剂是抑制巨噬细胞生成一氧化氮和/或抑制巨噬细胞增殖的药物。
9.根据权利要求7所述的用途,其特征在于:所述治疗炎症的药物是抑制巨噬细胞生成一氧化氮和/或抑制巨噬细胞增殖的药物。
10.根据权利要求7所述的用途,其特征在于:所述治疗败血症的药物是治疗由脂多糖引起的败血症的药物。
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