CN111961107B - Ouabain 19-position primary hydroxyl derivative and preparation method and application thereof - Google Patents

Ouabain 19-position primary hydroxyl derivative and preparation method and application thereof Download PDF

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CN111961107B
CN111961107B CN202010873734.6A CN202010873734A CN111961107B CN 111961107 B CN111961107 B CN 111961107B CN 202010873734 A CN202010873734 A CN 202010873734A CN 111961107 B CN111961107 B CN 111961107B
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张霞
钮大文
左昊
杨金亮
崔红燕
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Abstract

本发明提供了一种乌本苷19位伯羟基衍生物及其制备方法及用途。该乌本苷19位伯羟基衍生物的结构如式I所示。该衍生物对正常细胞的毒性非常低,同时还具有优异的抑制肿瘤细胞的效果。此外,该衍生物还能有效的抑制肿瘤细胞迁移和侵袭。因此,本发明提供的乌本苷19位伯羟基衍生物在制备抗肿瘤、抑制肿瘤侵袭和/或迁移的药物中具有非常好的应用前景。

Figure DDA0002651962740000011

Figure 202010873734

The invention provides a 19-position primary hydroxyl derivative of ouabain and a preparation method and application thereof. The structure of the 19-position primary hydroxyl derivative of ouabain is shown in formula I. The derivative has very low toxicity to normal cells, and at the same time has excellent tumor cell inhibitory effect. In addition, the derivative can effectively inhibit tumor cell migration and invasion. Therefore, the 19-position primary hydroxyl derivatives of ouabain provided by the present invention have very good application prospects in the preparation of drugs for anti-tumor and inhibiting tumor invasion and/or migration.

Figure DDA0002651962740000011

Figure 202010873734

Description

乌本苷19位伯羟基衍生物及其制备方法及用途19-position primary hydroxyl derivative of ouabain and its preparation method and use

技术领域technical field

本发明属于制药领域,具体涉及一种乌本苷19位伯羟基衍生物及其制备方法及用途。The invention belongs to the field of pharmacy, and in particular relates to a 19-position primary hydroxyl derivative of ouabain and a preparation method and application thereof.

背景技术Background technique

在天然产物中,强心苷(Cardiac glycosides,CGs)是一类重要的化合物,它可以特异性地作用于Na+/K+-ATPase,具有强心的功能。根据17位不饱和内酯的不同,强心苷可划分为强心甾(五元不饱和内酯)和蟾蜍二烯羟酸内酯(六元不饱和内酯)。其中,蟾蜍二烯羟酸内酯主要从蟾蜍的毒液中分离得到;而强心甾主要存在于植物中,例如玄参科(洋地黄属)、夹竹桃科(黄花夹竹桃属、羊角拗属)、毛茛科(侧金盏花属)等,且在植物叶中含量最高。研究发现,在毛地黄叶中,含有30多种强心苷,其中包括洋地黄、地高辛和乌本苷等。Among natural products, Cardiac glycosides (CGs) are an important class of compounds, which can specifically act on Na + /K + -ATPase and have cardiotonic function. Cardiac glycosides can be divided into cardiac steroids (five-membered unsaturated lactone) and bufadienolide (six-membered unsaturated lactone) according to the 17-position unsaturated lactone. Among them, bufadienolide is mainly isolated from the venom of toad; and cardiac steroids are mainly found in plants, such as Scrophulariaceae (digitalis), Oleander (Oleander genus, shorn horns) genus), Ranunculaceae (Calendula genus), etc., and the highest content in plant leaves. Studies have found that there are more than 30 cardiac glycosides in foxglove leaves, including digitalis, digoxin and ouabain.

Figure BDA0002651962720000011
Figure BDA0002651962720000011

早在200多年前,强心苷就已经被人们用于治疗心衰;1785年,英国内科医师William Withering出版了《毛地黄及其医疗用途的记述》,之后强心苷以其组织特异性和强大的心肌细胞收缩功效,被广泛地用于治疗充血性心力衰竭、合用利尿药治疗心房颤动和一些心律失常。研究者们还发现,强心苷类化合物除了具有强心的功能,还存在抗肿瘤、抗病毒、抗菌、免疫调节、影响神经细胞分化、降压、抗抑郁等作用,具有很大的药用价值。其中,乌本苷(Ouabain)作为一种强心苷类化合物,已经被发现在乳腺癌、前列腺癌、胰腺癌、白血病、神经母细胞瘤等肿瘤的治疗中具有较好的潜力(P.Kometiani,L.Liu,A.Askari.Mol.Pharmacol.2005,67,929–936)。Cardiac glycosides have been used for the treatment of heart failure as early as more than 200 years ago; in 1785, British physician William Withering published "Description of foxglove and its medical uses", and then cardiac glycosides were used for their tissue-specific and medical uses. Strong cardiomyocyte contractile effect, is widely used in the treatment of congestive heart failure, combined with diuretics in the treatment of atrial fibrillation and some arrhythmias. The researchers also found that in addition to the function of strengthening the heart, cardiac glycosides also have antitumor, antiviral, antibacterial, immune regulation, influence on nerve cell differentiation, blood pressure, antidepressant and other effects, which have great medicinal properties. value. Among them, Ouabain, as a cardiac glycoside compound, has been found to have good potential in the treatment of breast cancer, prostate cancer, pancreatic cancer, leukemia, neuroblastoma and other tumors (P. , L. Liu, A. Askari. Mol. Pharmacol. 2005, 67, 929–936).

Figure BDA0002651962720000021
Figure BDA0002651962720000021

然而,乌本苷虽然药效显著,但稍一过量就会产生严重的不良反应,甚至危及生命。乌本苷药物的治疗浓度与中毒浓度接近(治疗窗狭窄),而且乌本苷的药效(疗效和毒性)因个人体质而大不相同,因而其不安全性极大地限制了乌本苷类药物的临床应用。研究还发现,乌本苷对人体正常细胞具有较大的毒性,服用时容易对正常组织产生毒副作用。人们试图对乌本苷类化合物进行修饰,以降低其对正常组织的毒副作用,但是所得衍生物往往在降低对正常组织毒性的同时,对肿瘤的抑制活性也大幅降低。而且,乌本苷类化合物上具有多个活性位点,如何高选择性地进行特定位点的修饰也是本领域的一个难题。However, although ouabain has a significant effect, a slight overdose can cause serious adverse reactions, even life-threatening. The therapeutic concentration of ouabain is close to the toxic concentration (narrow therapeutic window), and the efficacy (efficacy and toxicity) of ouabain varies greatly due to individual constitution, so its unsafety greatly limits ouabain. Clinical application of drugs. The study also found that ouabain has great toxicity to normal human cells, and it is easy to produce toxic side effects on normal tissues when taking it. People try to modify the ouabain compounds to reduce their toxic and side effects on normal tissues, but the obtained derivatives often reduce the toxicity to normal tissues, and at the same time, the inhibitory activity on tumors is also greatly reduced. Moreover, ouabain compounds have multiple active sites, and how to modify specific sites with high selectivity is also a difficult problem in the art.

因此,对乌本苷类化合物进行恰当的修饰,制备出能够显著降低对正常细胞的毒性、同时还能保持优异的药物活性的乌本苷衍生物对乌本苷类化合物的临床应用具有非常重要的意义。Therefore, proper modification of ouabain compounds to prepare ouabain derivatives that can significantly reduce the toxicity to normal cells while maintaining excellent pharmaceutical activity is very important for the clinical application of ouabain compounds meaning.

发明内容SUMMARY OF THE INVENTION

本发明的目的在于提供一种乌本苷19位伯羟基衍生物及其制备方法及用途。The purpose of the present invention is to provide a 19-position primary hydroxyl derivative of ouabain and its preparation method and use.

本发明提供了一种式I所示化合物、或其药学上可接受的盐、或其立体异构体、或其旋光异构体、或其同位素替换形式:The present invention provides a compound shown in formula I, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer thereof, or an isotopic substitution form thereof:

Figure BDA0002651962720000022
Figure BDA0002651962720000022

其中,n为0~4的整数;Among them, n is an integer from 0 to 4;

n个R0各自独立的选自氢、羟基、卤素、C1~3烷基或C1~3烷氧基;The n pieces of R 0 are each independently selected from hydrogen, hydroxyl, halogen, C 1-3 alkyl or C 1-3 alkoxy;

R1选自氢、取代或未取代的3~6元饱和环烷基、取代或未取代的3~6元饱和杂环基、取代或未取代的芳基、取代或未取代的杂芳基、羟基、卤素、C1~5烷基或C1~5烷氧基;R 1 is selected from hydrogen, substituted or unsubstituted 3-6 membered saturated cycloalkyl, substituted or unsubstituted 3-6 membered saturated heterocyclic group, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl , hydroxyl, halogen, C 1-5 alkyl or C 1-5 alkoxy;

所述取代基为1个或多个,且各自独立的选自卤代或未卤代的C1~5烷基、卤代或未卤代的C1~5烷氧基、卤代或未卤代的C2~6烯基、卤代或未卤代的C2~6炔基、卤素、N3、硝基或羟基。The substituents are one or more, and each is independently selected from halogenated or unhalogenated C 1-5 alkyl, halogenated or unhalogenated C 1-5 alkoxy, halogenated or unhalogenated C 1-5 alkoxy Halogenated C2-6 alkenyl, halogenated or unhalogenated C2-6 alkynyl, halogen, N3 , nitro or hydroxy.

进一步地,所述化合物的结构如式II所示:Further, the structure of the compound is shown in formula II:

Figure BDA0002651962720000031
Figure BDA0002651962720000031

其中,R1选自氢、取代或未取代的5~6元饱和环烷基、取代或未取代的5~6元饱和杂环基、取代或未取代的芳基、取代或未取代的杂芳基、羟基、卤素、C1~3烷基或C1~3烷氧基;Wherein, R 1 is selected from hydrogen, substituted or unsubstituted 5-6 membered saturated cycloalkyl, substituted or unsubstituted 5-6 membered saturated heterocyclic group, substituted or unsubstituted aryl, substituted or unsubstituted heterocyclic group Aryl, hydroxyl, halogen, C 1-3 alkyl or C 1-3 alkoxy;

所述取代基为1个或多个,且各自独立的选自卤代或未卤代的C1~3烷基、卤代或未卤代的C1~3烷氧基、卤代或未卤代的C2~4烯基、卤代或未卤代的C2~4炔基、卤素、N3、硝基或羟基。The substituents are one or more, and each is independently selected from halogenated or unhalogenated C 1-3 alkyl, halogenated or unhalogenated C 1-3 alkoxy, halogenated or unhalogenated C 1-3 alkoxy Halogenated C2-4 alkenyl, halogenated or unhalogenated C2-4 alkynyl, halogen, N3, nitro or hydroxy.

进一步地,所述化合物的结构如式III所示:Further, the structure of the compound is shown in formula III:

Figure BDA0002651962720000032
Figure BDA0002651962720000032

其中,m为1~5的整数,优选为1或2;m个R2各自独立的选自氢、卤代或未卤代的C1~3烷基、卤代或未卤代的C1~3烷氧基、卤代或未卤代的C2~4烯基、卤代或未卤代的C2~4炔基、卤素、N3或硝基;所述卤素优选为氟或氯。Wherein, m is an integer of 1-5, preferably 1 or 2; m R 2 are each independently selected from hydrogen, halogenated or unhalogenated C 1-3 alkyl, halogenated or unhalogenated C 1 ~3 alkoxy , halogenated or unhalogenated C2-4 alkenyl, halogenated or unhalogenated C2-4 alkynyl, halogen, N3 or nitro; the halogen is preferably fluorine or chlorine .

进一步地,所述化合物的结构如下所示:Further, the structure of the compound is as follows:

Figure BDA0002651962720000033
Figure BDA0002651962720000033

Figure BDA0002651962720000041
Figure BDA0002651962720000041

本发明还提供了上述化合物、或其药学上可接受的盐、或其立体异构体、或其旋光异构体、或其同位素替换形式的制备方法,所述方法包括以下步骤:The present invention also provides a method for preparing the above compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer, or an isotopic substitution form thereof, the method comprising the following steps:

(1)将乌本苷、有机硼试剂和有机溶剂在反应瓶中混合;(1) mix ouabain, organoboron reagent and organic solvent in reaction flask;

(2)将烯丙基化试剂、钯催化剂、磷配体和有机溶剂在另一反应瓶中混合,并搅拌活化;所述烯丙基化试剂的结构为

Figure BDA0002651962720000051
R3为保护基团,R1如上所述;(2) mix allylating reagent, palladium catalyst, phosphorus ligand and organic solvent in another reaction flask, and stir and activate; the structure of described allylating reagent is
Figure BDA0002651962720000051
R 3 is a protecting group, R 1 is as described above;

(3)将步骤(2)所得体系加入步骤(1)所得体系中,反应,即得。(3) The system obtained in step (2) is added to the system obtained in step (1), and the reaction is performed.

进一步地,所述步骤(1)~(3)是在惰性气体保护的环境下进行的;步骤(3)中,所述反应温度为20~30℃,优选为室温;反应时间为6~24小时,优选为16小时。Further, the steps (1)-(3) are carried out in an environment protected by an inert gas; in the step (3), the reaction temperature is 20-30°C, preferably room temperature; the reaction time is 6-24 hours, preferably 16 hours.

进一步地,步骤(1)中,所述有机硼试剂为甲基苯硼酸,所述有机溶剂为四氢呋喃、异丙醇中的一种或两种的混合物,优选为四氢呋喃与异丙醇的混合物;所述乌本苷、有机硼试剂的摩尔比为1:(0.5~1.5),优选为1:1;Further, in step (1), the organic boron reagent is methylphenylboronic acid, and the organic solvent is one or both of tetrahydrofuran and isopropanol, preferably a mixture of tetrahydrofuran and isopropanol; The molar ratio of ouabain and organoboron reagent is 1:(0.5-1.5), preferably 1:1;

步骤(2)中,所述钯催化剂为Pd(dba)3·CHCl3,所述磷配体为PPh3;所述有机溶剂为四氢呋喃、异丙醇中的一种或两种;所述乌本苷与烯丙基化试剂、钯催化剂、磷配体的摩尔比为1:(1.1~5.0):(0.01~0.2):(0.05~0.3),优选为1:1.5:0.025:0.1;和/或,所述搅拌活化时间为5~20分钟,优选为10分钟;和/或,所述R3为羟基保护基团,优选为Boc基团。In step (2), the palladium catalyst is Pd(dba) 3 ·CHCl 3 , the phosphorus ligand is PPh 3 ; the organic solvent is one or both of tetrahydrofuran and isopropanol; The molar ratio of this glycoside to allylation reagent, palladium catalyst and phosphorus ligand is 1:(1.1-5.0):(0.01-0.2):(0.05-0.3), preferably 1:1.5:0.025:0.1; and /or, the stirring activation time is 5-20 minutes, preferably 10 minutes; and/or, the R 3 is a hydroxyl protecting group, preferably a Boc group.

本发明还提供了一种药物,所述药物是以上述化合物、或其药学上可接受的盐、或其立体异构体、或其旋光异构体、或其同位素替换形式为活性成分,加上药学上可接受的辅料制得的制剂。The present invention also provides a medicament, the medicament is the above-mentioned compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer or an isotopic substitution form thereof as an active ingredient, plus Preparations prepared with pharmaceutically acceptable excipients.

本发明还提供了上述化合物、或其药学上可接受的盐、或其立体异构体、或其旋光异构体、或其同位素替换形式在制备抗肿瘤或抑制肿瘤转移的药物中的用途;所述肿瘤优选为乳腺癌、宫颈癌、肝癌、前列腺癌、胰腺癌,更优选为乳腺癌、胰腺癌、肝癌、宫颈癌;所述肝癌优选为肝腹水腺癌。The present invention also provides the use of the above compound, or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or an optical isomer, or an isotopic substitution form thereof, in the preparation of a drug for anti-tumor or inhibiting tumor metastasis; The tumor is preferably breast cancer, cervical cancer, liver cancer, prostate cancer, and pancreatic cancer, more preferably breast cancer, pancreatic cancer, liver cancer, and cervical cancer; the liver cancer is preferably liver ascites adenocarcinoma.

进一步地,所述药物能够抑制肿瘤细胞增殖、侵袭和/或迁移。Further, the drug can inhibit tumor cell proliferation, invasion and/or migration.

关于本发明的使用术语的定义:除非另有说明,本文中基团或者术语提供的初始定义适用于整篇说明书的该基团或者术语;对于本文没有具体定义的术语,应该根据公开内容和上下文,给出本领域技术人员能够给予它们的含义。Definitions of terms used in the present invention: Unless otherwise specified, the initial definitions of groups or terms provided herein apply to the groups or terms throughout the specification; for terms that are not specifically defined herein, they should be based on the disclosure and context. , give their meanings that those skilled in the art can give them.

本发明中,“室温”指25±2℃。In the present invention, "room temperature" refers to 25±2°C.

“过夜”指8~16小时。"Overnight" refers to 8 to 16 hours.

“卤素”指氟、氯、溴或碘。"Halogen" refers to fluorine, chlorine, bromine or iodine.

“同位素替换形式”指是指化合物中的一个或两个以上的原子被其对应的同位素替换后得到的化合物,比如化合物中的氢被替换为氕、氘或氚。"Isotopic substitution form" refers to a compound obtained by replacing one or more than two atoms in the compound with its corresponding isotope, for example, hydrogen in the compound is replaced by protium, deuterium or tritium.

“药学上可接受的”是指某载体、运载物、稀释剂、辅料,和/或所形成的盐通常在化学上或物理上与构成某药物剂型的其它成分相兼容,并在生理上与受体相兼容。"Pharmaceutically acceptable" means that a carrier, vehicle, diluent, adjuvant, and/or salt formed is generally chemically or physically compatible with the other ingredients that make up a pharmaceutical dosage form and physiologically compatible with receptor compatible.

“盐”是将化合物与无机和/或有机酸和/或碱形成的酸式和/或碱式盐,也包括两性离子盐(内盐),还包括季铵盐,例如烷基铵盐。这些盐可以是在化合物的最后分离和纯化中直接得到。也可以是通过将化合物与一定数量的酸或碱适当(例如等当量)进行混合而得到。这些盐可能在溶液中形成沉淀而以过滤方法收集,或在溶剂蒸发后回收而得到,或在水介质中反应后冷冻干燥制得。"Salts" are acidic and/or basic salts of compounds formed with inorganic and/or organic acids and/or bases, and also include zwitterionic (inner) salts, as well as quaternary ammonium salts, such as alkylammonium salts. These salts can be obtained directly in the final isolation and purification of the compounds. It can also be obtained by mixing the compound with a certain amount of acid or base as appropriate (eg, equivalent). These salts may be precipitated in solution and collected by filtration, recovered after evaporation of the solvent, or obtained by lyophilization after reaction in an aqueous medium.

本发明中所述“药学上可接受的盐”可以是化合物的盐酸盐、硫酸盐、枸橼酸盐、苯磺酸盐、氢溴酸盐、氢氟酸盐、磷酸盐、乙酸盐、丙酸盐、丁二酸盐、草酸盐、苹果酸盐、琥珀酸盐、富马酸盐、马来酸盐、酒石酸盐或三氟乙酸盐。The "pharmaceutically acceptable salt" in the present invention can be the hydrochloride, sulfate, citrate, benzenesulfonate, hydrobromide, hydrofluoride, phosphate, acetate of the compound , propionate, succinate, oxalate, malate, succinate, fumarate, maleate, tartrate or trifluoroacetate.

本发明中,碳氢基团中碳原子含量的最小值和最大值通过前缀表示,例如,前缀(Ca~b)烷基表明任何含“a”至“b”个碳原子的烷基。例如,C1~3烷基是指包含1~3个碳原子的直链或支链的烷基。In the present invention, the minimum and maximum carbon content in a hydrocarbon group is indicated by a prefix, eg, the prefix (C a~b )alkyl denotes any alkyl group containing "a" to "b" carbon atoms. For example, a C 1-3 alkyl group refers to a straight or branched chain alkyl group containing 1 to 3 carbon atoms.

类似的,C1~3烷氧基是指包含1~3个碳原子的直链或支链的烷氧基。Similarly, C 1-3 alkoxy refers to a straight or branched alkoxy group containing 1 to 3 carbon atoms.

“环烷基”指饱和或不饱和的环状烃取代基;环状烃可以是单环也可以是多环。“饱和环烷基”指饱和的环烷基。例如,“3~6元饱和环烷基”指环碳原子数为3~6的饱和环烷基。"Cycloalkyl" refers to a saturated or unsaturated cyclic hydrocarbon substituent; the cyclic hydrocarbon may be monocyclic or polycyclic. "Saturated cycloalkyl" refers to a saturated cycloalkyl group. For example, "a 3- to 6-membered saturated cycloalkyl group" refers to a saturated cycloalkyl group having 3 to 6 ring carbon atoms.

“杂环基”指饱和或不饱和的环状烃取代基;环状烃可以是单环也可以是多环,且携带至少一个环杂原子(包括但不限于O、S或N)。“饱和杂环基”指饱和的杂环基。例如,“3~6元饱和杂环基”指环原子数为3~6的饱和杂环基。"Heterocyclyl" refers to a saturated or unsaturated cyclic hydrocarbon substituent; cyclic hydrocarbons may be monocyclic or polycyclic and carry at least one ring heteroatom (including but not limited to O, S or N). "Saturated heterocyclyl" refers to a saturated heterocyclyl group. For example, "a 3- to 6-membered saturated heterocyclic group" refers to a saturated heterocyclic group having 3 to 6 ring atoms.

“芳基”指具有共轭的π电子体系的全碳单环或稠合多环(也就是共享毗邻碳原子对的环)基团,例如苯基和萘基。所述芳基环可以稠合于其它环状基团(包括饱和和不饱和环),但不能含有杂原子如氮,氧,或硫,同时连接母体的点必须在具有共轭的π电子体系的环上的碳原子上。芳基可以是取代的或未取代的。"Aryl" refers to an all-carbon monocyclic or fused polycyclic (ie, rings that share adjacent pairs of carbon atoms) groups having a conjugated pi-electron system, such as phenyl and naphthyl. The aryl ring can be fused to other cyclic groups (including saturated and unsaturated rings), but cannot contain heteroatoms such as nitrogen, oxygen, or sulfur, and the point of attachment to the parent must be in a conjugated pi-electron system on the carbon atom of the ring. Aryl groups can be substituted or unsubstituted.

“杂芳基”指包含一个到多个杂原子的杂芳族基团。这里所指的杂原子包括氧、硫和氮。例如呋喃基、噻吩基、吡啶基、吡唑基、吡咯基、N-烷基吡咯基、嘧啶基、吡嗪基、咪唑基、四唑基等。所述杂芳基环可以稠合于芳基、杂环基或环烷基环上,其中与母体结构连接在一起的环为杂芳基环。杂芳基可以是任选取代的或未取代的。"Heteroaryl" refers to a heteroaromatic group containing one to more heteroatoms. The heteroatoms referred to herein include oxygen, sulfur and nitrogen. For example, furyl, thienyl, pyridyl, pyrazolyl, pyrrolyl, N-alkylpyrrolyl, pyrimidinyl, pyrazinyl, imidazolyl, tetrazolyl and the like. The heteroaryl ring can be fused to an aryl, heterocyclyl or cycloalkyl ring, wherein the ring attached to the parent structure is a heteroaryl ring. Heteroaryl groups can be optionally substituted or unsubstituted.

保护基团:在有机合成中,含有2个或多个官能团的分子,为使其中某个官能团免遭反应的破坏,常用某种试剂先将其保护,待反应完成后再脱去保护试剂中的基团,该基团即保护基团。Protecting group: In organic synthesis, a molecule containing two or more functional groups, in order to prevent one of the functional groups from being destroyed by the reaction, a certain reagent is commonly used to protect it first, and then remove the protective reagent after the reaction is completed. group, the group is the protecting group.

本发明以特定的烯丙基化试剂SI1~SI9为改性剂,在磷配体存在的情况下,利用金属钯试剂与有机硼试剂共催化,通过特定位置的烯丙基化修饰成功制得了一类乌本苷19位伯羟基衍生物,该乌本苷19位伯羟基衍生物的制备条件温和,催化效率高,反应选择性好,产物收率高,适合产业化生产。In the present invention, specific allylation reagents SI1-SI9 are used as modifiers, and in the presence of phosphorus ligands, metal palladium reagents and organoboron reagents are co-catalyzed to be successfully prepared by allylation modification at specific positions. A class of ouabain 19-position primary hydroxyl derivatives, the preparation conditions of the ouabain 19-position primary hydroxyl derivatives are mild, the catalytic efficiency is high, the reaction selectivity is good, the product yield is high, and it is suitable for industrial production.

与现有技术相比,本发明提供的乌本苷19位伯羟基衍生物取得了以下有益效果:Compared with the prior art, the 19-position primary hydroxyl derivative of ouabain provided by the invention has achieved the following beneficial effects:

(1)本发明提供的乌本苷19位伯羟基衍生物在降低对正常细胞的毒性的同时,还具有优异的抑制包括乳腺癌、宫颈癌、肝癌(特别是肝腹水腺癌)、前列腺癌、胰腺癌在内的多种肿瘤细胞的效果;(1) The 19-position primary hydroxyl derivatives of ouabain provided by the present invention can reduce the toxicity to normal cells, and also have excellent inhibition of breast cancer, cervical cancer, liver cancer (especially liver ascites adenocarcinoma), prostate cancer , the effect of various tumor cells including pancreatic cancer;

(2)该乌本苷19位伯羟基衍生物对肿瘤细胞的抑制作用具有较好的选择性,其对胰腺癌细胞、SK-HEP-1肝腹水腺癌细胞、宫颈癌细胞细胞的抑制效果明显优于前列腺癌细胞和Hep G2肝癌细胞;(2) The 19-position primary hydroxyl derivative of ouabain has good selectivity for the inhibitory effect on tumor cells, and its inhibitory effect on pancreatic cancer cells, SK-HEP-1 liver ascites adenocarcinoma cells, and cervical cancer cells Significantly better than prostate cancer cells and Hep G2 liver cancer cells;

(3)该乌本苷19位伯羟基衍生物(特别是化合物Ouabain-2、Ouabain-26)能够有效抑制胰腺癌细胞增殖;(3) The 19-position primary hydroxyl derivatives of ouabain (especially the compounds Ouabain-2 and Ouabain-26) can effectively inhibit the proliferation of pancreatic cancer cells;

(4)该乌本苷19位伯羟基衍生物还能有效的抑制肿瘤细胞迁移和/或侵袭。(4) The 19-position primary hydroxyl derivative of ouabain can also effectively inhibit tumor cell migration and/or invasion.

所以,本发明通过特定位置的烯丙基化修饰得到的乌本苷19位伯羟基衍生物在制备抗肿瘤、抑制肿瘤侵袭和/或迁移的药物中具有非常好的应用前景。Therefore, the 19-position primary hydroxyl derivative of ouabain obtained by the allylation modification at a specific position of the present invention has a very good application prospect in the preparation of anti-tumor drugs and inhibiting tumor invasion and/or migration.

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。Obviously, according to the above-mentioned content of the present invention, according to the common technical knowledge and conventional means in the field, without departing from the above-mentioned basic technical idea of the present invention, other various forms of modification, replacement or change can also be made.

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。The above content of the present invention will be further described in detail below through the specific implementation in the form of examples. However, this should not be construed as limiting the scope of the above-mentioned subject matter of the present invention to the following examples. All technologies implemented based on the above content of the present invention belong to the scope of the present invention.

附图说明Description of drawings

图1为烯丙基化试剂SI1~SI9的合成路线图。Fig. 1 is the synthetic route of allylation reagents SI1-SI9.

图2为浓度梯度(0.01μM、0.1μM、1.0μM、10μM、100μM)下各化合物对乳腺癌细胞(MDA-MB-231)迁移的影响。Figure 2 shows the effect of each compound on the migration of breast cancer cells (MDA-MB-231) under the concentration gradient (0.01 μM, 0.1 μM, 1.0 μM, 10 μM, 100 μM).

图3为0.1μM、10μM下各化合物的细胞侵袭实验结果。Figure 3 shows the results of cell invasion experiments of each compound at 0.1 μM and 10 μM.

具体实施方式Detailed ways

本发明所用原料与设备均为已知产品,通过购买市售产品所得。The raw materials and equipment used in the present invention are all known products, obtained by purchasing commercially available products.

实施例1:合成烯丙基化试剂SI1~SI9Example 1: Synthesis of allylation reagents SI1-SI9

参照已知文献(Tetrahedron Letters.2012,53,1319–1322;Jr.Eur.J.Org.Chem.2016,28,4800–4804;Org.Lett.2009,11,2944-2947;J.Am.Chem.Soc.2015,137,6335-6349;Org.Lett.2019,21,7424-7429)记载的方法,采用图1所示合成路线,制得以下9个烯丙基化试剂SI1~SI9。烯丙基化试剂SI-1~SI-9的表征数据如下:Reference to known literature (Tetrahedron Letters. 2012, 53, 1319-1322; Jr. Eur. J. Org. Chem. 2016, 28, 4800-4804; Org. Lett. 2009, 11, 2944-2947; Chem. Soc. 2015, 137, 6335-6349; Org. Lett. 2019, 21, 7424-7429), the following 9 allylation reagents SI1 to SI9 were prepared by the synthetic route shown in Fig. 1 . The characterization data of allylation reagents SI-1 to SI-9 are as follows:

Figure BDA0002651962720000071
Figure BDA0002651962720000071

SI-1:Allyl-tert-butylcarbonateSI-1: Allyl-tert-butylcarbonate

1H NMR(CDCl3,400MHz)δ:5.98–5.83(m,1H),5.38–5.14(m,2H),4.63–4.47(m,2H).13C NMR(CDCl3,101MHz)δ:153.4,132.1,118.2,82.1,67.6,28.0,27.8. 1 H NMR (CDCl 3 , 400MHz) δ: 5.98-5.83 (m, 1H), 5.38-5.14 (m, 2H), 4.63-4.47 (m, 2H). 13 C NMR (CDCl 3 , 101MHz) δ: 153.4 ,132.1,118.2,82.1,67.6,28.0,27.8.

Figure BDA0002651962720000081
Figure BDA0002651962720000081

SI-2:(E)-3-(4-Methoxyphenyl)allyl-tert-butyl carbonateSI-2: (E)-3-(4-Methoxyphenyl)allyl-tert-butyl carbonate

1H NMR(CDCl3,400MHz)δ:7.31(d,J=8.7Hz,2H),6.84(d,J=8.7Hz,2H),6.61(d,J=15.9Hz,1H),6.15(dt,J=15.9,6.6Hz,1H),4.69(dd,J=6.6,1.3Hz,2H),3.79(s,3H),1.49(s,5H).13C NMR(CDCl3,101MHz)δ:159.7,153.5,134.3,129.0,128.0,120.7,114.1,82.1,67.8,55.3,27.9. 1 H NMR (CDCl 3 , 400 MHz) δ: 7.31 (d, J=8.7 Hz, 2H), 6.84 (d, J=8.7 Hz, 2H), 6.61 (d, J=15.9 Hz, 1H), 6.15 (dt , J=15.9, 6.6Hz, 1H), 4.69(dd, J=6.6, 1.3Hz, 2H), 3.79(s, 3H), 1.49(s, 5H). 13 C NMR(CDCl 3 , 101MHz)δ: 159.7, 153.5, 134.3, 129.0, 128.0, 120.7, 114.1, 82.1, 67.8, 55.3, 27.9.

Figure BDA0002651962720000082
Figure BDA0002651962720000082

SI-3:(E)-3-(3-Azidophenyl)allyl-tert-butyl carbonateSI-3: (E)-3-(3-Azidophenyl)allyl-tert-butyl carbonate

1H NMR(CDCl3,400MHz)δ:7.29(t,J=7.8Hz,1H),7.14(d,J=7.7Hz,1H),7.01(t,J=2.0Hz,1H),6.92(dd,J=8.0,2.3Hz,1H),6.62(d,J=15.9Hz,1H),6.30(dt,J=15.9,6.3Hz,1H),4.71(dd,J=6.3,1.5Hz,2H),1.50(s,9H).13C NMR(CDCl3,101MHz)δ:153.4,140.5,138.1,133.1,130.0,124.5,123.4,118.6,117.1,82.4,67.1,27.9. 1 H NMR (CDCl 3 , 400 MHz) δ: 7.29 (t, J=7.8 Hz, 1H), 7.14 (d, J=7.7 Hz, 1H), 7.01 (t, J=2.0 Hz, 1H), 6.92 (dd , J=8.0, 2.3Hz, 1H), 6.62 (d, J=15.9Hz, 1H), 6.30 (dt, J=15.9, 6.3Hz, 1H), 4.71 (dd, J=6.3, 1.5Hz, 2H) , 1.50(s, 9H). 13 C NMR (CDCl 3 , 101MHz) δ: 153.4, 140.5, 138.1, 133.1, 130.0, 124.5, 123.4, 118.6, 117.1, 82.4, 67.1, 27.9.

Figure BDA0002651962720000083
Figure BDA0002651962720000083

SI-4:(E)-3-(4-Fluorophenyl)allyl-tert-butyl carbonateSI-4: (E)-3-(4-Fluorophenyl)allyl-tert-butyl carbonate

1H NMR(CDCl3,400MHz)δ:7.35(dd,J=8.5,5.5Hz,2H),7.00(t,J=8.7Hz,2H),6.63(d,J=15.9Hz,1H),6.21(dt,J=15.9,6.4Hz,1H),4.70(dd,J=6.4,1.3Hz,2H),1.50(s,9H).13C NMR(CDCl3,101MHz)δ:153.5,133.4,128.4,128.3,122.9,122.8,115.8,115.6,82.4,67.5,27.9. 1 H NMR (CDCl 3 , 400 MHz) δ: 7.35 (dd, J=8.5, 5.5 Hz, 2H), 7.00 (t, J=8.7 Hz, 2H), 6.63 (d, J=15.9 Hz, 1H), 6.21 (dt, J=15.9, 6.4 Hz, 1H), 4.70 (dd, J=6.4, 1.3 Hz, 2H), 1.50 (s, 9H). 13 C NMR (CDCl 3 , 101 MHz) δ: 153.5, 133.4, 128.4 ,128.3,122.9,122.8,115.8,115.6,82.4,67.5,27.9.

Figure BDA0002651962720000084
Figure BDA0002651962720000084

SI-5:(E)-3-(4-Ethynylphenyl)allyl-tert-butyl carbonateSI-5: (E)-3-(4-Ethynylphenyl)allyl-tert-butyl carbonate

1H NMR(CDCl3,400MHz)δ7.44(d,J=8.0Hz,2H),7.33(d,J=8.0Hz,2H),6.64(d,J=15.9Hz,1H),6.31(dt,J=16.0,6.3Hz,1H),4.72(dd,J=6.4,1.4Hz,2H),1.50(s,9H).13CNMR(CDCl3,101MHz)δ:153.4,136.8,133.5,132.5,126.7,124.4,121.8,83.7,82.5,78.1,67.3,28.0,27.9. 1 H NMR (CDCl 3 , 400MHz) δ 7.44 (d, J=8.0 Hz, 2H), 7.33 (d, J=8.0 Hz, 2H), 6.64 (d, J=15.9 Hz, 1H), 6.31 (dt , J=16.0, 6.3Hz, 1H), 4.72 (dd, J=6.4, 1.4Hz, 2H), 1.50 (s, 9H). 13 CNMR (CDCl 3 , 101MHz) δ: 153.4, 136.8, 133.5, 132.5, 126.7, 124.4, 121.8, 83.7, 82.5, 78.1, 67.3, 28.0, 27.9.

Figure BDA0002651962720000085
Figure BDA0002651962720000085

SI-6:(E)-3-(4-Trifluoromethylphenyl)allyl-tert-butyl carbonateSI-6: (E)-3-(4-Trifluoromethylphenyl)allyl-tert-butyl carbonate

1H NMR(CDCl3,400MHz)δ:7.57(d,J=8.1Hz,2H),7.47(d,J=8.1Hz,2H),6.69(d,J=15.9Hz,1H),6.38(dt,J=16.0,6.1Hz,1H),4.74(dd,J=6.2,1.5Hz,2H),1.51(s,9H).13CNMR(CDCl3,101MHz)δ:153.4,139.8,139.8,132.5,129.9(q,J=32.4Hz),126.9,125.9,125.7(q,J=3.9Hz),125.5(d,J=3.8Hz),82.5,81.0,67.0,28.0,27.9. 1 H NMR (CDCl 3 , 400MHz) δ: 7.57 (d, J=8.1 Hz, 2H), 7.47 (d, J=8.1 Hz, 2H), 6.69 (d, J=15.9 Hz, 1H), 6.38 (dt , J=16.0, 6.1Hz, 1H), 4.74 (dd, J=6.2, 1.5Hz, 2H), 1.51 (s, 9H). 13 CNMR (CDCl 3 , 101MHz) δ: 153.4, 139.8, 139.8, 132.5, 129.9 (q, J=32.4Hz), 126.9, 125.9, 125.7 (q, J=3.9Hz), 125.5 (d, J=3.8Hz), 82.5, 81.0, 67.0, 28.0, 27.9.

Figure BDA0002651962720000091
Figure BDA0002651962720000091

SI-7:(E)-Tert-butylcinnamyl carbonateSI-7: (E)-Tert-butylcinnamyl carbonate

1H NMR(CDCl3,400MHz)δ:7.40–7.35(m,2H),7.33–7.27(m,2H),7.27–7.21(m,1H),6.66(dd,J=15.9,1.5Hz,1H),6.28(dt,J=15.9,6.4Hz,1H),4.71(dd,J=6.5,1.4Hz,2H),1.50(s,9H).13C NMR(CDCl3,101MHz)δ:153.4,136.3,134.5,128.7,128.1,126.7,123.0,82.2,67.5,27.9. 1 H NMR (CDCl 3 , 400MHz) δ: 7.40-7.35 (m, 2H), 7.33-7.27 (m, 2H), 7.27-7.21 (m, 1H), 6.66 (dd, J=15.9, 1.5Hz, 1H ), 6.28 (dt, J=15.9, 6.4Hz, 1H), 4.71 (dd, J=6.5, 1.4Hz, 2H), 1.50 (s, 9H). 13 C NMR (CDCl 3 , 101MHz) δ: 153.4, 136.3, 134.5, 128.7, 128.1, 126.7, 123.0, 82.2, 67.5, 27.9.

Figure BDA0002651962720000092
Figure BDA0002651962720000092

SI-8:(E)-3-(3-Nitrophenyl)allyl-tert-butyl carbonateSI-8: (E)-3-(3-Nitrophenyl)allyl-tert-butyl carbonate

1H NMR(CDCl3,400MHz)δ:8.24(t,J=2.0Hz,1H),8.11(ddd,J=8.2,2.3,1.0Hz,1H),7.69(dt,J=7.7,1.4Hz,1H),7.50(t,J=8.0Hz,1H),6.73(dt,J=16.0,1.5Hz,1H),6.43(dt,J=16.0,6.0Hz,1H),4.76(dd,J=6.1,1.5Hz,2H),1.52(s,9H).13C NMR(CDCl3,101MHz)δ:153.4,138.2,132.5,131.5,129.7,126.6,122.7,121.4,82.7,66.8,27.9. 1 H NMR (CDCl 3 , 400MHz) δ: 8.24 (t, J=2.0Hz, 1H), 8.11 (ddd, J=8.2, 2.3, 1.0Hz, 1H), 7.69 (dt, J=7.7, 1.4Hz, 1H), 7.50 (t, J=8.0Hz, 1H), 6.73 (dt, J=16.0, 1.5Hz, 1H), 6.43 (dt, J=16.0, 6.0Hz, 1H), 4.76 (dd, J=6.1 , 1.5Hz, 2H), 1.52(s, 9H). 13 C NMR(CDCl 3 , 101MHz) δ: 153.4, 138.2, 132.5, 131.5, 129.7, 126.6, 122.7, 121.4, 82.7, 66.8, 27.9.

Figure BDA0002651962720000093
Figure BDA0002651962720000093

SI-9:(E)-3-(3,4-Dichlorophenyl)allyl-tert-butyl carbonateSI-9: (E)-3-(3,4-Dichlorophenyl)allyl-tert-butyl carbonate

1H NMR(CDCl3,400MHz)δ:7.45(d,J=2.1Hz,1H),7.38(dd,J=8.3,1.6Hz,1H),7.20(dd,J=8.3,2.1Hz,1H),6.57(dt,J=15.9,1.5Hz,1H),6.28(dt,J=15.9,6.2Hz,1H),4.71(dd,J=6.1,1.5Hz,2H),1.50(d,J=3.1Hz,9H).13C NMR(CDCl3,101MHz)δ:153.4,136.5,132.9,131.9,131.7,130.6,128.5,125.9,125.3,82.6,67.0,27.9. 1 H NMR (CDCl 3 , 400MHz) δ: 7.45 (d, J=2.1 Hz, 1H), 7.38 (dd, J=8.3, 1.6 Hz, 1H), 7.20 (dd, J=8.3, 2.1 Hz, 1H) ,6.57(dt,J=15.9,1.5Hz,1H),6.28(dt,J=15.9,6.2Hz,1H),4.71(dd,J=6.1,1.5Hz,2H),1.50(d,J=3.1 Hz, 9H). 13 C NMR (CDCl 3 , 101MHz) δ: 153.4, 136.5, 132.9, 131.9, 131.7, 130.6, 128.5, 125.9, 125.3, 82.6, 67.0, 27.9.

实施例2:合成本发明的乌本苷19位伯羟基衍生物Example 2: Synthesis of ouabain 19-position primary hydroxyl derivative of the present invention

采用以下合成路线,制备本发明的乌本苷19位伯羟基衍生物:The following synthetic route is adopted to prepare the 19-position primary hydroxyl derivative of ouabain of the present invention:

Figure BDA0002651962720000101
Figure BDA0002651962720000101

将乌本苷(29.2mg,0.04mmol,1.0equiv.)、甲基苯硼酸(2.4mg,0.04mmol,1.0equiv.)称入预先烘干的带有搅拌子的反应瓶A中,关上瓶盖;称取相应的烯丙基化试剂(SI1~SI9中的一种,0.06mmol)于预先烘干的带有搅拌子的反应瓶B中,关上瓶盖。将反应转移至氮气保护的手套箱。向反应瓶A加入300μL THF与200μL i-PrOH;再将Pd(dba)3·CHCl3(1.0mg,0.1%mmol,2.5%equiv.)、PPh3(1.0mg,0.4%mmol,10%equiv.)依次加入反应瓶B,随后加入超干THF(300μL),关上瓶盖,搅拌活化10min。用1mL的注射器量取300μL反应B中的液体于反应瓶A中,旋紧盖子,将反应移出手套箱,于25℃封闭反应。反应16小时。反应完成后,直接将反应体系减压旋干,得到粗品,再通过柱层析纯化,即得对应的乌本苷19位伯羟基衍生物。烯丙基化试剂与乌本苷19位伯羟基衍生物的对应关系如表1所示。Weigh ouabain (29.2mg, 0.04mmol, 1.0equiv.) and methylphenylboronic acid (2.4mg, 0.04mmol, 1.0equiv.) into a pre-dried reaction flask A with a stirring bar, close the cap ; Weigh the corresponding allylation reagent (one of SI1 to SI9, 0.06 mmol) in a pre-dried reaction bottle B with a stirrer, and close the bottle cap. The reaction was transferred to a nitrogen-protected glove box. 300 μL of THF and 200 μL of i-PrOH were added to reaction vial A; then Pd(dba) 3 ·CHCl 3 (1.0 mg, 0.1% mmol, 2.5% equiv.), PPh 3 (1.0 mg, 0.4% mmol, 10% equiv.) were added .) Add reaction bottle B in turn, then add ultra-dry THF (300 μL), close the bottle cap, and stir to activate for 10 min. Measure 300 μL of the liquid in reaction B into reaction bottle A with a 1 mL syringe, tighten the lid, remove the reaction from the glove box, and seal the reaction at 25°C. The reaction was carried out for 16 hours. After the reaction is completed, the reaction system is directly spin-dried under reduced pressure to obtain a crude product, which is then purified by column chromatography to obtain the corresponding ouabain 19-position primary hydroxyl derivative. The corresponding relationship between the allylation reagent and the 19-position primary hydroxyl derivative of ouabain is shown in Table 1.

表1原料烯丙基化试剂与产物乌本苷19位伯羟基衍生物的对应关系Table 1 Correspondence relationship between raw material allylation reagent and product ouabain 19-position primary hydroxyl derivative

Figure BDA0002651962720000102
Figure BDA0002651962720000102

以下为制得的各乌本苷19位伯羟基衍生物的结构和表征数据:The following are the structure and characterization data of each prepared ouabain 19-position primary hydroxyl derivative:

Figure BDA0002651962720000103
Figure BDA0002651962720000103

(E)-19-Allyl-ouabain(Ouabain-2)(E)-19-Allyl-ouabain (Ouabain-2)

柱层析纯化方法:硅胶色谱法(CH2Cl2:MeOH=20:1-15:1)纯化Ouabain-2粗品,得到固体产物Ouabain-2(18.5mg,纯度大于90%,总收率74%).Column chromatography purification method: Silica gel chromatography (CH 2 Cl 2 :MeOH=20:1-15:1) purifies the crude Ouabain-2 to obtain the solid product Ouabain-2 (18.5 mg, the purity is more than 90%, the total yield is 74%) %).

1H NMR(MeOD,400MHz)δ:5.89–5.78(m,1H),5.78(d,J=3.9Hz,1H),5.18(dq,J=17.3,1.7Hz,1H),5.05(dq,J=10.4,1.4Hz,1H),5.00–4.93(m,1H),4.89(dd,J=18.4,1.8Hz,1H),4.78(dd,J=18.4,1.7Hz,1H),4.62(d,J=1.6Hz,1H),4.02(d,J=3.6Hz,1H),3.97(dd,J=10.8,4.2Hz,1H),3.89(q,J=1.5Hz,1H),3.87(q,J=1.5Hz,1H),3.83(d,J=10.7Hz,1H),3.61(dd,J=3.4,1.7Hz,1H),3.52(dd,J=9.6,3.5Hz,1H),3.52–3.44(m,1H),3.44(d,J=10.5Hz,1H),3.27–3.18(t,J=9.50,1H),2.79–2.70(t,J=8.00,1H),2.26(ddd,J=15.5,5.3,2.1Hz,1H),2.15(dd,J=15.7,4.8Hz,1H),2.10–2.07(m,1H),2.07–2.03(m,1H),1.98(d,J=15.8Hz,1H),1.93–1.73(m,3H),1.69–1.59(m,3H),1.56(dd,J=13.2,4.5Hz,1H),1.45–1.28(m,3H),1.26–1.13(m,3H),1.12(d,J=6.2Hz,3H),0.81(s,3H).13C NMR(MeOD,101MHz)δ:177.6,177.1,136.5,118.0,117.2,99.3,85.7,75.9,75.9,75.3,74.2,73.6,74.2–74.2(m),72.6,72.4,72.4,72.4,71.8,70.4,69.9,69.0,51.6,50.9,50.3,41.2,41.2,41.1,33.4,27.8,24.5,18.0,17.5.IR(thin film,cm-1):3393,2928,1731,1390,1341,1042,and 672.HRMS(DART-TOF)calculated for C32H48NaO12 +[M+Na]+m/z 647.3043,found 647.3038.[α]D 26=-25.1(c=0.51,MeOH). 1 H NMR (MeOD, 400MHz) δ: 5.89-5.78 (m, 1H), 5.78 (d, J=3.9Hz, 1H), 5.18 (dq, J=17.3, 1.7Hz, 1H), 5.05 (dq, J =10.4,1.4Hz,1H),5.00–4.93(m,1H),4.89(dd,J=18.4,1.8Hz,1H),4.78(dd,J=18.4,1.7Hz,1H),4.62(d, J=1.6Hz, 1H), 4.02(d, J=3.6Hz, 1H), 3.97(dd, J=10.8, 4.2Hz, 1H), 3.89(q, J=1.5Hz, 1H), 3.87(q, J=1.5Hz, 1H), 3.83 (d, J=10.7Hz, 1H), 3.61 (dd, J=3.4, 1.7Hz, 1H), 3.52 (dd, J=9.6, 3.5Hz, 1H), 3.52– 3.44 (m, 1H), 3.44 (d, J=10.5Hz, 1H), 3.27–3.18 (t, J=9.50, 1H), 2.79–2.70 (t, J=8.00, 1H), 2.26 (ddd, J = 15.5, 5.3, 2.1Hz, 1H), 2.15 (dd, J=15.7, 4.8Hz, 1H), 2.10–2.07 (m, 1H), 2.07–2.03 (m, 1H), 1.98 (d, J=15.8 Hz, 1H), 1.93–1.73 (m, 3H), 1.69–1.59 (m, 3H), 1.56 (dd, J=13.2, 4.5Hz, 1H), 1.45–1.28 (m, 3H), 1.26–1.13 ( m, 3H), 1.12 (d, J=6.2Hz, 3H), 0.81 (s, 3H). 13 C NMR (MeOD, 101MHz) δ: 177.6, 177.1, 136.5, 118.0, 117.2, 99.3, 85.7, 75.9, 75.9,75.3,74.2,73.6,74.2–74.2(m),72.6,72.4,72.4,72.4,71.8,70.4,69.9,69.0,51.6,50.9,50.3,41.2,41.2,41.1,33.4,27.8,24.5,18.0 ,17.5.IR(thin film,cm -1 ): 3393,2928,1731,1390,1341,1042,and 672.HRMS(DART-TOF) calculated for C 32 H 48 NaO 12 + [M+Na] + m /z 647.3043,found 647.3038.[α] D 26 =-25.1 (c=0.51, MeOH).

Figure BDA0002651962720000111
Figure BDA0002651962720000111

(E)-19-(4-Methoxycinnamyl)-ouabain(Ouabain-5)(E)-19-(4-Methoxycinnamyl)-ouabain(Ouabain-5)

柱层析纯化方法:硅胶色谱法(CH2Cl2:MeOH=20:1-15:1)纯化Ouabain-5粗品,得到固体产物Ouabain-5(26.3mg,纯度大于90%,总收率89.4%).Column chromatography purification method: Silica gel chromatography (CH 2 Cl 2 :MeOH=20:1-15:1) to purify the crude Ouabain-5 to obtain a solid product Ouabain-5 (26.3 mg, purity greater than 90%, total yield 89.4 %).

1H NMR(MeOD,400MHz)δ:7.26(d,J=8.7Hz,2H),6.78(d,J=8.7Hz,2H),6.51(d,J=15.9Hz,1H),6.12(dt,J=15.9,6.3Hz,1H),5.83(d,J=1.6Hz,1H),5.04(dd,J=3.8,2.0Hz,1H),4.93(dd,J=18.6,1.8Hz,1H),4.82(dd,J=18.4,1.8Hz,1H),4.66(d,J=1.7Hz,1H),4.13–4.02(m,4H),3.91(d,J=10.5Hz,1H),3.70(s,3H),3.66(dd,J=3.4,1.6Hz,1H),3.61–3.49(m,3H),3.27(t,J=9.5Hz,1H),2.78(t,J=7.1Hz,1H),2.31(ddd,J=15.5,5.1,1.8Hz,1H),2.20(dd,J=15.7,4.8Hz,1H),2.16–2.08(m,2H),2.07–2.00(m,1H),1.99–1.76(m,4H),1.71(d,J=8.0Hz,1H),1.69–1.58(m,3H),1.52–1.33(m,4H),1.29–1.22(m,4H),1.17(d,J=6.2Hz,3H),0.83(s,3H).13C NMR(MeOD,101MHz)δ:177.6,177.1,160.9,133.8,130.8,128.8,124.8,118.1,115.0,99.0,85.5,75.3,74.5,74.2,73.4,72.3,72.2,71.1,70.1,70.1,69.2,68.1,55.8,51.8,51.4,49.9,49.5,49.3,49.1,48.9,47.3,45.7,40.9,34.8,30.7,28.0,24.1,18.0,17.5.IR(thin film,cm-1):3006,2984,1490,1275,1261,761,752 and 749.HRMS(DART-TOF)calculated for C39H54NaO13 +[M+Na]+m/z753.3462,found 647.3463.[α]D 26=-20.0(c=0.05,MeOH). 1 H NMR (MeOD, 400MHz) δ: 7.26 (d, J=8.7Hz, 2H), 6.78 (d, J=8.7Hz, 2H), 6.51 (d, J=15.9Hz, 1H), 6.12 (dt, J=15.9, 6.3Hz, 1H), 5.83 (d, J=1.6Hz, 1H), 5.04 (dd, J=3.8, 2.0Hz, 1H), 4.93 (dd, J=18.6, 1.8Hz, 1H), 4.82(dd,J=18.4,1.8Hz,1H),4.66(d,J=1.7Hz,1H),4.13–4.02(m,4H),3.91(d,J=10.5Hz,1H),3.70(s ,3H),3.66(dd,J=3.4,1.6Hz,1H),3.61–3.49(m,3H),3.27(t,J=9.5Hz,1H),2.78(t,J=7.1Hz,1H) ,2.31(ddd,J=15.5,5.1,1.8Hz,1H),2.20(dd,J=15.7,4.8Hz,1H),2.16-2.08(m,2H),2.07-2.00(m,1H),1.99 –1.76(m,4H),1.71(d,J=8.0Hz,1H),1.69-1.58(m,3H),1.52-1.33(m,4H),1.29-1.22(m,4H),1.17(d , J=6.2Hz, 3H), 0.83(s, 3H). 13 C NMR (MeOD, 101MHz) δ: 177.6, 177.1, 160.9, 133.8, 130.8, 128.8, 124.8, 118.1, 115.0, 99.0, 85.5, 75.3, 74.5,74.2,73.4,72.3,72.2,71.1,70.1,70.1,69.2,68.1,55.8,51.8,51.4,49.9,49.5,49.3,49.1,48.9,47.3,45.7,40.9,34.8,30.7,28.0,24.1, 18.0,17.5.IR(thin film,cm -1 ): 3006,2984,1490,1275,1261,761,752 and 749.HRMS(DART-TOF)calculated for C 39 H 54 NaO 13 + [M+Na] + m /z753.3462, found 647.3463. [α] D 26 =-20.0 (c=0.05, MeOH).

Figure BDA0002651962720000121
Figure BDA0002651962720000121

(E)-19-(3-Azidocinnamyl)-ouabain(Ouabain-8)(E)-19-(3-Azidocinnamyl)-ouabain(Ouabain-8)

柱层析纯化方法:硅胶色谱法(CH2Cl2:MeOH=20:1-15:1)纯化Ouabain-8粗品,得到固体产物Ouabain-8(17.6mg,纯度大于90%,总收率60.0%).Column chromatography purification method: Silica gel chromatography (CH 2 Cl 2 :MeOH=20:1-15:1) to purify the crude Ouabain-8 to obtain a solid product Ouabain-8 (17.6 mg, purity greater than 90%, total yield 60.0 %).

1H NMR(MeOD,400MHz)δ:7.24(t,J=7.8Hz,1H),7.14(d,J=7.8Hz,1H),7.01(t,J=1.9Hz,1H),6.85(dd,J=7.9,2.2Hz,1H),6.57(d,J=16.0Hz,1H),6.31(dt,J=16.0,5.8Hz,1H),5.82(d,J=1.8Hz,1H),5.06(s,1H),4.93(dd,J=18.4,1.8Hz,1H),4.81(dd,J=18.4,1.8Hz,1H),4.66(d,J=1.6Hz,1H),4.15–3.99(m,4H),3.93(d,J=10.5Hz,1H),3.66(dd,J=3.5,1.7Hz,1H),3.61–3.49(m,3H),3.30(m,1H),2.77(d,J=8.5Hz,1H),2.32(ddd,J=15.6,5.6,2.1Hz,1H),2.25–1.99(m,5H),1.99–1.77(m,4H),1.75–1.57(m,6H),1.54–1.30(m,6H),1.17(d,J=6.3Hz,3H),0.84(s,3H).13C NMR(MeOD,101MHz)δ:177.5,177.0,141.7,140.1,132.5,131.1,128.9,124.3,119.1,118.1,117.8,98.9,85.4,75.3,74.5,74.2,72.9,72.3,72.2,71.1,70.1,69.1,51.8,51.3,49.9,47.2,45.7,40.9,34.8,33.1,30.7,30.5,27.9,24.1,23.7,18.0,17.5,14.4.IR(thin film,cm-1):3006,2984,1471,1275,1260,765,762 and 750.HRMS(DART-TOF)calculated for C38H51N3NaO12 +[M+Na]+m/z 764.3370,found 764.3375.[α]D 26=-21.7(c=0.30,MeOH). 1 H NMR (MeOD, 400MHz) δ: 7.24 (t, J=7.8Hz, 1H), 7.14 (d, J=7.8Hz, 1H), 7.01 (t, J=1.9Hz, 1H), 6.85 (dd, J=7.9, 2.2Hz, 1H), 6.57(d, J=16.0Hz, 1H), 6.31(dt, J=16.0, 5.8Hz, 1H), 5.82(d, J=1.8Hz, 1H), 5.06( s, 1H), 4.93 (dd, J=18.4, 1.8Hz, 1H), 4.81 (dd, J=18.4, 1.8Hz, 1H), 4.66 (d, J=1.6Hz, 1H), 4.15–3.99 (m ,4H),3.93(d,J=10.5Hz,1H),3.66(dd,J=3.5,1.7Hz,1H),3.61–3.49(m,3H),3.30(m,1H),2.77(d, J=8.5Hz, 1H), 2.32 (ddd, J=15.6, 5.6, 2.1Hz, 1H), 2.25–1.99 (m, 5H), 1.99–1.77 (m, 4H), 1.75–1.57 (m, 6H) , 1.54–1.30 (m, 6H), 1.17 (d, J=6.3Hz, 3H), 0.84 (s, 3H). 13 C NMR (MeOD, 101MHz) δ: 177.5, 177.0, 141.7, 140.1, 132.5, 131.1 ,128.9,124.3,119.1,118.1,117.8,98.9,85.4,75.3,74.5,74.2,72.9,72.3,72.2,71.1,70.1,69.1,51.8,51.3,49.9,47.2,45.7,40.9,34.8,33.1 ,30.5,27.9,24.1,23.7,18.0,17.5,14.4.IR(thin film,cm -1 ):3006,2984,1471,1275,1260,765,762 and 750.HRMS(DART-TOF)calculated for C 38 H 51 N 3 NaO 12 + [M+Na] + m/z 764.3370, found 764.3375. [α] D 26 =-21.7 (c=0.30, MeOH).

Figure BDA0002651962720000122
Figure BDA0002651962720000122

(E)-19-(4-Fluorocinnamyl)-ouabain(Ouabain-11)(E)-19-(4-Fluorocinnamyl)-ouabain (Ouabain-11)

柱层析纯化方法:硅胶色谱法(CH2Cl2:MeOH=20:1-15:1)纯化Ouabain-11粗品,得到固体产物Ouabain-11(18.1mg,纯度大于90%,总收率62.7%).Column chromatography purification method: Silica gel chromatography (CH 2 Cl 2 :MeOH=20:1-15:1) purifies the crude Ouabain-11 to obtain the solid product Ouabain-11 (18.1 mg, the purity is more than 90%, the total yield is 62.7 %).

1H NMR(MeOD,400MHz)δ:7.35(dd,J=8.6,5.5Hz,2H),6.95(t,J=8.8Hz,2H),6.57(d,J=15.9Hz,1H),6.22(dt,J=16.0,6.0Hz,1H),5.84(d,J=1.8Hz,1H),5.06(dd,J=3.7,1.9Hz,1H),4.94(dd,J=18.4,1.8Hz,1H),4.82(dd,J=18.4,1.8Hz,1H),4.67(d,J=1.6Hz,1H),4.14–3.99(m,4H),3.93(d,J=10.5Hz,1H),3.67(dd,J=3.4,1.6Hz,1H),3.62–3.45(m,4H),3.34–3.25(m,1H),2.78(d,J=9.2Hz,1H),2.32(ddd,J=15.6,5.4,2.1Hz,1H),2.23–2.00(m,4H),1.98–1.80(m,3H),1.75–1.56(m,4H),1.42(ddt,J=16.3,11.1,4.1Hz,3H),1.33–1.19(m,4H),1.18(d,J=6.2Hz,3H),1.10(t,J=7.0Hz,1H),0.85(s,3H).13C NMR(MeOD,101MHz)δ:177.6,177.0,164.9,162.5,134.5,134.5,132.5,129.3,129.2,127.2,127.2,118.1,116.4,116.1,98.9,85.4,75.2,74.4,74.2,73.1,72.3,72.2,71.0,70.1,70.0,69.1,51.7,51.3,50.5,47.2,45.7,40.9,34.8,33.3,33.2,27.9,24.1,18.0,17.5.IR(thin film,cm-1):3006,2992,1467,1275,1261,761,751 and 767.HRMS(DART-TOF)calculated for C38H51FNaO12 +[M+Na]+m/z 741.3262,found 741.3257.[α]D 26=-33.3(c=0.41,MeOH). 1 H NMR (MeOD, 400 MHz) δ: 7.35 (dd, J=8.6, 5.5 Hz, 2H), 6.95 (t, J=8.8 Hz, 2H), 6.57 (d, J=15.9 Hz, 1H), 6.22 ( dt,J=16.0,6.0Hz,1H),5.84(d,J=1.8Hz,1H),5.06(dd,J=3.7,1.9Hz,1H),4.94(dd,J=18.4,1.8Hz,1H) ),4.82(dd,J=18.4,1.8Hz,1H),4.67(d,J=1.6Hz,1H),4.14–3.99(m,4H),3.93(d,J=10.5Hz,1H),3.67 (dd, J=3.4, 1.6Hz, 1H), 3.62–3.45 (m, 4H), 3.34–3.25 (m, 1H), 2.78 (d, J=9.2Hz, 1H), 2.32 (ddd, J=15.6 , 5.4, 2.1Hz, 1H), 2.23–2.00 (m, 4H), 1.98–1.80 (m, 3H), 1.75–1.56 (m, 4H), 1.42 (ddt, J=16.3, 11.1, 4.1Hz, 3H ), 1.33–1.19 (m, 4H), 1.18 (d, J=6.2Hz, 3H), 1.10 (t, J=7.0Hz, 1H), 0.85 (s, 3H). 13 C NMR (MeOD, 101MHz) δ:177.6,177.0,164.9,162.5,134.5,134.5,132.5,129.3,129.2,127.2,127.2,118.1,116.4,116.1,98.9,85.4,75.2,74.4,74.2,73.1,7,72.3,72.2 70.0,69.1,51.7,51.3,50.5,47.2,45.7,40.9,34.8,33.3,33.2,27.9,24.1,18.0,17.5.IR(thin film,cm -1 ):3006,2992,1467,1275,1261, 761,751 and 767.HRMS(DART-TOF)calculated for C 38 H 51 FNaO 12 + [M+Na] + m/z 741.3262,found 741.3257.[α] D 26 =-33.3(c=0.41,MeOH).

Figure BDA0002651962720000131
Figure BDA0002651962720000131

(E)-19-(4-Ethynylcinnamyl)-ouabain(Ouabain-14)(E)-19-(4-Ethynylcinnamyl)-ouabain (Ouabain-14)

柱层析纯化方法:硅胶色谱法(CH2Cl2:MeOH=20:1-15:1)纯化Ouabain-14粗品,得到固体产物Ouabain-14(18.1mg,纯度大于90%,总收率62.7%).Column chromatography purification method: Silica gel chromatography (CH 2 Cl 2 :MeOH=20:1-15:1) purifies the crude Ouabain-14 to obtain the solid product Ouabain-14 (18.1 mg, the purity is more than 90%, the total yield is 62.7 %).

1H NMR(400MHz,MeOD)δ:7.32(s,4H),6.59(d,J=16.0Hz,1H),6.32(dt,J=16.0,5.9Hz,1H),5.84(s,1H),5.07(s,1H),4.94(d,J=18.4Hz,1H),4.82(d,J=18.3Hz,1H),4.67(s,1H),4.10(dh,J=9.7,5.9Hz,4H),3.94(d,J=10.6Hz,1H),3.67(d,J=3.3Hz,1H),3.57(tt,J=9.4,4.9Hz,3H),3.30(d,J=10.0Hz,1H),2.79(t,J=6.9Hz,1H),2.39–2.28(m,1H),2.23–2.02(m,4H),1.98–1.78(m,4H),1.76–1.57(m,4H),1.55–1.34(m,4H),1.32–1.22(m,6H),1.18(d,J=6.3Hz,3H),0.85(s,3H).13C NMR(101MHz,MeOD)δ:177.5,177.1,138.6,133.2,132.7,128.9,127.5,118.1,99.0,85.4,79.2,75.3,74.4,74.2,73.0,72.3,72.2,71.1,70.1,69.1,68.4,51.8,51.3,50.5,49.9,47.2,45.7,40.9,34.8,33.3,33.3,33.1,30.7,30.5,28.0,24.1,23.7,18.0,17.5,14.4.IR(thin film,cm-1):3006,2988,1478,1275,1261,764,760,756 and 749.HRMS(DART-TOF)calculated for C40H52NaO12 +[M+Na]+m/z 747.3356,found 747.3354.[α]D 26=-14.1(c=0.48,MeOH). 1 H NMR (400MHz, MeOD)δ: 7.32(s, 4H), 6.59(d, J=16.0Hz, 1H), 6.32(dt, J=16.0, 5.9Hz, 1H), 5.84(s, 1H), 5.07(s, 1H), 4.94(d, J=18.4Hz, 1H), 4.82(d, J=18.3Hz, 1H), 4.67(s, 1H), 4.10(dh, J=9.7, 5.9Hz, 4H) ),3.94(d,J=10.6Hz,1H),3.67(d,J=3.3Hz,1H),3.57(tt,J=9.4,4.9Hz,3H),3.30(d,J=10.0Hz,1H) ), 2.79(t, J=6.9Hz, 1H), 2.39-2.28(m, 1H), 2.23-2.02(m, 4H), 1.98-1.78(m, 4H), 1.76-1.57(m, 4H), 1.55–1.34 (m, 4H), 1.32–1.22 (m, 6H), 1.18 (d, J=6.3 Hz, 3H), 0.85 (s, 3H). 13 C NMR (101 MHz, MeOD) δ: 177.5, 177.1 ,138.6,133.2,132.7,128.9,127.5,118.1,99.0,85.4,79.2,75.3,74.4,74.2,73.0,72.3,72.2,71.1,70.1,69.1,68.4,51.8,51.3,50.5,49.9,47. ,40.9,34.8,33.3,33.3,33.1,30.7,30.5,28.0,24.1,23.7,18.0,17.5,14.4.IR(thin film,cm -1 ):3006,2988,1478,1275,1261,764,760,756 and 749 .HRMS(DART-TOF) calculated for C 40 H 52 NaO 12 + [M+Na] + m/z 747.3356, found 747.3354. [α] D 26 = -14.1 (c=0.48, MeOH).

Figure BDA0002651962720000141
Figure BDA0002651962720000141

(E)-19-(4-Trifluoromethylcinnamyl)-ouabain(Ouabain-17)(E)-19-(4-Trifluoromethylcinnamyl)-ouabain (Ouabain-17)

柱层析纯化方法:硅胶色谱法(CH2Cl2:MeOH=20:1-15:1)纯化Ouabain-17粗品,得到固体产物Ouabain-17(16.0mg,纯度大于90%,总收率52.3%).Column chromatography purification method: Silica gel chromatography (CH 2 Cl 2 :MeOH=20:1-15:1) to purify the crude Ouabain-17 to obtain a solid product Ouabain-17 (16.0 mg, purity greater than 90%, total yield 52.3 %).

1H NMR(400MHz,MeOD)δ:7.52(s,4H),6.68(d,J=16.0Hz,1H),6.43(dt,J=16.0,5.7Hz,1H),5.83(s,1H),5.09(d,J=3.4Hz,0H),4.94(dd,J=18.4,1.8Hz,1H),4.82(dd,J=18.3,1.7Hz,1H),4.67(s,1H),4.18–4.03(m,4H),3.96(d,J=10.6Hz,1H),3.66(dd,J=3.3,1.7Hz,1H),3.58(dd,J=10.3,2.3Hz,3H),3.32–3.25(m,1H),2.79(dd,J=8.9,5.4Hz,1H),2.38–2.29(m,1H),2.24–2.00(m,5H),1.97–1.77(m,4H),1.76–1.58(m,5H),1.51–1.34(m,5H),1.21(s,5H),1.18(d,J=6.3Hz,3H),0.85(s,3H).13C NMR(101MHz,MeOD)δ:177.5,177.0,142.1,131.7,130.7,128.0,126.5,126.4,118.1,99.0,85.4,75.3,74.4,74.2,72.8,72.3,72.2,71.1,70.1,70.1,69.1,68.6,51.8,51.3,50.5,47.2,45.8,40.9,34.8,33.4,33.1,30.7,30.5,27.9,24.1,23.7,18.0,17.5,14.4.IR(thin film,cm-1):3007,2988,1455,1275,1261,766,756,and 749.HRMS(DART-TOF)calculated for C39H51F3NaO12 +[M+Na]+m/z 791.3230,found 791.3232.[α]D 26=-24.3(c=0.40,MeOH). 1 H NMR (400MHz, MeOD)δ: 7.52(s, 4H), 6.68(d, J=16.0Hz, 1H), 6.43(dt, J=16.0, 5.7Hz, 1H), 5.83(s, 1H), 5.09(d,J=3.4Hz,0H),4.94(dd,J=18.4,1.8Hz,1H),4.82(dd,J=18.3,1.7Hz,1H),4.67(s,1H),4.18–4.03 (m,4H),3.96(d,J=10.6Hz,1H),3.66(dd,J=3.3,1.7Hz,1H),3.58(dd,J=10.3,2.3Hz,3H),3.32–3.25( m, 1H), 2.79 (dd, J=8.9, 5.4Hz, 1H), 2.38–2.29 (m, 1H), 2.24–2.00 (m, 5H), 1.97–1.77 (m, 4H), 1.76–1.58 ( m, 5H), 1.51–1.34 (m, 5H), 1.21 (s, 5H), 1.18 (d, J=6.3 Hz, 3H), 0.85 (s, 3H). 13 C NMR (101 MHz, MeOD) δ: 177.5,177.0,142.1,131.7,130.7,128.0,126.5,126.4,118.1,99.0,85.4,75.3,74.4,74.2,72.8,72.3,72.2,71.1,70.1,70.1,69.1,68.6,50.5,51.8,5 47.2,45.8,40.9,34.8,33.4,33.1,30.7,30.5,27.9,24.1,23.7,18.0,17.5,14.4.IR(thin film,cm -1 ):3007,2988,1455,1275,1261,766,756, and 749.HRMS(DART-TOF) calculated for C 39 H 51 F 3 NaO 12 + [M+Na] + m/z 791.3230,found 791.3232.[α] D 26 =-24.3(c=0.40,MeOH).

Figure BDA0002651962720000142
Figure BDA0002651962720000142

(E)-19-Cinnamyl-ouabain(Ouabain-20)(E)-19-Cinnamyl-ouabain (Ouabain-20)

柱层析纯化方法:硅胶色谱法(CH2Cl2:MeOH=20:1-15:1)纯化Ouabain-20粗品,得到固体产物Ouabain-20(23.9mg,纯度大于90%,总收率85.6%).Column chromatography purification method: Silica gel chromatography (CH 2 Cl 2 :MeOH=20:1-15:1) to purify the crude Ouabain-20 to obtain a solid product Ouabain-20 (23.9 mg, purity greater than 90%, total yield 85.6 %).

1H NMR(400MHz,MeOD)δ:7.36–7.29(m,2H),7.21(t,J=7.5Hz,2H),7.13(dd,J=8.3,6.3Hz,1H),6.57(d,J=15.9Hz,1H),6.27(dt,J=15.9,6.0Hz,1H),5.82(d,J=2.0Hz,1H),5.06(s,1H),4.92(dd,J=18.4,1.8Hz,1H),4.81(dd,J=18.4,1.7Hz,1H),4.66(s,1H),4.16–4.01(m,4H),3.93(d,J=10.6Hz,1H),3.66(dd,J=3.3,1.6Hz,1H),3.62–3.49(m,3H),3.27(t,J=9.5Hz,1H),2.77(d,J=8.5Hz,1H),2.32(ddd,J=15.9,5.1,1.8Hz,1H),2.19(dd,J=15.6,4.8Hz,1H),2.15–2.00(m,2H),1.98–1.77(m,4H),1.71(s,1H),1.69–1.58(m,3H),1.52–1.33(m,4H),1.21(dq,J=3.5,1.9Hz,4H),1.17(d,J=6.2Hz,3H),0.83(s,3H).13C NMR(101MHz,MeOD)δ:177.6,177.1,138.1,133.9,129.5,128.7,127.6,127.2,118.1,98.9,85.4,75.3,74.5,74.2,73.2,72.3,72.2,71.1,70.1,70.1,69.2,68.3,51.8,51.3,50.5,47.2,45.7,40.9,34.8,33.3,33.3,32.1,31.9,30.7,28.0,24.1,18.0,17.5.IR(thin film,cm-1):3006,2983,1455,1275,1261,763,757,and 748.HRMS(DART-TOF)calculated for C38H52NaO12 +[M+Na]+m/z 723.3356,found 723.3352.[α]D 26=-59.0(c=0.25,MeOH). 1 H NMR (400MHz, MeOD) δ: 7.36-7.29 (m, 2H), 7.21 (t, J=7.5Hz, 2H), 7.13 (dd, J=8.3, 6.3Hz, 1H), 6.57 (d, J =15.9Hz,1H),6.27(dt,J=15.9,6.0Hz,1H),5.82(d,J=2.0Hz,1H),5.06(s,1H),4.92(dd,J=18.4,1.8Hz ,1H),4.81(dd,J=18.4,1.7Hz,1H),4.66(s,1H),4.16–4.01(m,4H),3.93(d,J=10.6Hz,1H),3.66(dd, J=3.3, 1.6Hz, 1H), 3.62–3.49 (m, 3H), 3.27 (t, J=9.5Hz, 1H), 2.77 (d, J=8.5Hz, 1H), 2.32 (ddd, J=15.9 ,5.1,1.8Hz,1H),2.19(dd,J=15.6,4.8Hz,1H),2.15–2.00(m,2H),1.98–1.77(m,4H),1.71(s,1H),1.69– 1.58(m,3H),1.52–1.33(m,4H),1.21(dq,J=3.5,1.9Hz,4H),1.17(d,J=6.2Hz,3H),0.83(s,3H). 13 C NMR (101MHz, MeOD) δ: 177.6, 177.1, 138.1, 133.9, 129.5, 128.7, 127.6, 127.2, 118.1, 98.9, 85.4, 75.3, 74.5, 24.2, 73.2, 72.3, 72.2, 71.1, 70.1, 70.1, 69. ,68.3,51.8,51.3,50.5,47.2,45.7,40.9,34.8,33.3,33.3,32.1,31.9,30.7,28.0,24.1,18.0,17.5.IR(thin film,cm -1 ):3006,2983,1455 ,1275,1261,763,757,and 748.HRMS(DART-TOF)calculated for C 38 H 52 NaO 12 + [M+Na] + m/z 723.3356,found 723.3352.[α] D 26 =-59.0(c= 0.25, MeOH).

Figure BDA0002651962720000151
Figure BDA0002651962720000151

(E)-19-(3-Nitrocinnamyl)-ouabain(Ouabain-23)(E)-19-(3-Nitrocinnamyl)-ouabain(Ouabain-23)

柱层析纯化方法:硅胶色谱法(CH2Cl2:MeOH=20:1-15:1)纯化Ouabain-23粗品,得到固体产物Ouabain-23(18.6mg,纯度大于90%,总收率62.7%).Column chromatography purification method: Silica gel chromatography (CH 2 Cl 2 :MeOH=20:1-15:1) purifies the crude Ouabain-23 to obtain the solid product Ouabain-23 (18.6 mg, the purity is more than 90%, the total yield is 62.7 %).

1H NMR(400MHz,MeOD)δ:8.20(t,J=2.0Hz,1H),8.00(ddd,J=8.2,2.3,1.0Hz,1H),7.74(dt,J=7.8,1.3Hz,1H),7.47(t,J=8.0Hz,1H),6.78–6.64(m,1H),6.47(dt,J=16.0,5.6Hz,1H),5.83(s,1H),5.10(dd,J=3.6,2.0Hz,1H),4.94(dd,J=18.4,1.8Hz,1H),4.82(dd,J=18.4,1.7Hz,1H),4.67(d,J=1.6Hz,1H),4.17(tdd,J=12.2,5.6,1.6Hz,2H),4.07(d,J=10.2Hz,2H),3.96(d,J=10.5Hz,1H),3.66(dd,J=3.5,1.6Hz,1H),3.62–3.48(m,3H),3.29(d,J=9.6Hz,1H),2.79(t,J=7.1Hz,1H),2.34(ddd,J=15.6,5.4,2.1Hz,1H),2.21(dd,J=15.8,4.9Hz,1H),2.17–2.01(m,3H),1.99–1.78(m,3H),1.78–1.59(m,4H),1.43(ddt,J=19.0,12.3,5.4Hz,3H),1.29–1.20(m,3H),1.17(d,J=6.2Hz,3H),0.85(s,3H).13C NMR(101MHz,MeOD)δ:177.5,177.0,150.0,140.2,133.4,131.0,130.8,123.0,121.9,118.1,98.9,85.4,75.2,74.4,74.2,72.6,72.3,72.2,71.1,70.0,69.1,68.7,54.8,51.8,51.3,50.5,49.8,47.2,45.8,40.9,34.8,34.8,33.3,33.2,33.0,30.7,27.9,24.1,18.0,17.5.IR(thin film,cm-1):3006,2982,1478,1275,1261,763,760,751,and 747.HRMS(DART-TOF)calculated for C38H51NNaO14 +[M+Na]+m/z 768.3207,found 768.3212.[α]D 26=-27.0(c=0.49,MeOH). 1 H NMR (400MHz, MeOD) δ: 8.20 (t, J=2.0Hz, 1H), 8.00 (ddd, J=8.2, 2.3, 1.0Hz, 1H), 7.74 (dt, J=7.8, 1.3Hz, 1H) ),7.47(t,J=8.0Hz,1H),6.78–6.64(m,1H),6.47(dt,J=16.0,5.6Hz,1H),5.83(s,1H),5.10(dd,J= 3.6,2.0Hz,1H),4.94(dd,J=18.4,1.8Hz,1H),4.82(dd,J=18.4,1.7Hz,1H),4.67(d,J=1.6Hz,1H),4.17( tdd,J=12.2,5.6,1.6Hz,2H),4.07(d,J=10.2Hz,2H),3.96(d,J=10.5Hz,1H),3.66(dd,J=3.5,1.6Hz,1H) ), 3.62–3.48 (m, 3H), 3.29 (d, J=9.6Hz, 1H), 2.79 (t, J=7.1Hz, 1H), 2.34 (ddd, J=15.6, 5.4, 2.1Hz, 1H) , 2.21 (dd, J=15.8, 4.9Hz, 1H), 2.17–2.01 (m, 3H), 1.99–1.78 (m, 3H), 1.78–1.59 (m, 4H), 1.43 (ddt, J=19.0, 12.3, 5.4Hz, 3H), 1.29–1.20 (m, 3H), 1.17 (d, J=6.2Hz, 3H), 0.85 (s, 3H). 13 C NMR (101MHz, MeOD) δ: 177.5, 177.0, 150.0,140.2,133.4,131.0,130.8,123.0,121.9,118.1,98.9,85.4,75.2,74.4,74.2,72.6,72.3,72.2,71.1,70.0,69.1,68.7,54.8,51.8.,51.3,50 47.2, 45.8, 40.9, 34.8, 34.8, 33.3, 33.2, 33.0, 30.7, 27.9, 24.1, 18.0, 17.5. IR (thin film, cm -1 ): 3006, 2982, 1478, 1275, 1261, 763, 760, 751, and 747 .HRMS(DART-TOF) calculated for C 38 H 51 NNaO 14 + [M+Na] + m/z 768.3207,found 768.3212.[α] D 26 =-27.0(c=0.49,MeOH) .

Figure BDA0002651962720000161
Figure BDA0002651962720000161

(E)-19-(3,4-Dichlorocinnamyl)-ouabain(Ouabain-26)(E)-19-(3,4-Dichlorocinnamyl)-ouabain (Ouabain-26)

柱层析纯化方法:硅胶色谱法(CH2Cl2:MeOH=20:1-15:1)纯化Ouabain-26粗品,得到固体产物Ouabain-26(27.1mg,纯度大于90%,总收率88.2%).Column chromatography purification method: Silica gel chromatography (CH 2 Cl 2 :MeOH=20:1-15:1) purifies the crude Ouabain-26 to obtain the solid product Ouabain-26 (27.1 mg, the purity is more than 90%, the total yield is 88.2 %).

1H NMR(400MHz,MeOD)δ:7.49(d,J=2.0Hz,1H),7.35(d,J=8.4Hz,1H),7.26(dd,J=8.4,2.1Hz,1H),6.55(d,J=16.0Hz,1H),6.33(dt,J=16.0,5.7Hz,1H),5.84(d,J=1.8Hz,1H),5.07(dd,J=3.8,2.0Hz,1H),4.94(dd,J=18.4,1.8Hz,1H),4.82(dd,J=18.4,1.7Hz,1H),4.67(d,J=1.6Hz,1H),4.18–4.04(m,4H),3.93(d,J=10.5Hz,1H),3.66(dd,J=3.4,1.6Hz,1H),3.61–3.48(m,3H),3.28(t,J=9.5Hz,1H),2.79(t,J=7.1Hz,1H),2.33(ddd,J=15.5,5.4,2.1Hz,1H),2.20(dd,J=15.7,5.0Hz,1H),2.15–2.05(m,2H),2.03(s,1H),1.87–1.79(m,1H),1.72(q,J=4.1,3.1Hz,1H),1.67(d,J=8.7Hz,1H),1.65–1.57(m,1H),1.42(dtd,J=15.6,8.6,3.7Hz,3H),1.27(d,J=7.6Hz,1H),1.22(d,J=3.5Hz,2H),1.19(d,J=6.8Hz,4H),1.16(d,J=4.5Hz,3H),0.83(d,J=11.7Hz,3H).13C NMR(101MHz,MeOD)δ:177.0,138.9,133.6,132.0,131.6,130.7,130.1,129.3,127.1,118.1,98.9,85.4,74.4,74.2,72.7,72.3,72.2,70.1,70.1,69.1,51.8,51.3,50.5,49.9,49.7,47.2,45.8,40.9,34.8,34.8,33.3,33.0,30.8,27.9,24.1,18.01,17.5.IR(thin film,cm-1):3006,2995,1475,1275,1261,764,and 751.HRMS(DART-TOF)calculated for C38H50Cl2NaO12 +[M+Na]+m/z 791.2577,found 791.2570.[α]D 26=-31.6(c=0.34,MeOH). 1 H NMR (400 MHz, MeOD) δ: 7.49 (d, J=2.0 Hz, 1H), 7.35 (d, J=8.4 Hz, 1H), 7.26 (dd, J=8.4, 2.1 Hz, 1H), 6.55 ( d, J=16.0Hz, 1H), 6.33 (dt, J=16.0, 5.7Hz, 1H), 5.84 (d, J=1.8Hz, 1H), 5.07 (dd, J=3.8, 2.0Hz, 1H), 4.94(dd,J=18.4,1.8Hz,1H),4.82(dd,J=18.4,1.7Hz,1H),4.67(d,J=1.6Hz,1H),4.18–4.04(m,4H),3.93 (d, J=10.5Hz, 1H), 3.66 (dd, J=3.4, 1.6Hz, 1H), 3.61–3.48 (m, 3H), 3.28 (t, J=9.5Hz, 1H), 2.79 (t, J=7.1Hz, 1H), 2.33(ddd, J=15.5, 5.4, 2.1Hz, 1H), 2.20(dd, J=15.7, 5.0Hz, 1H), 2.15–2.05(m, 2H), 2.03(s) ,1H),1.87–1.79(m,1H),1.72(q,J=4.1,3.1Hz,1H),1.67(d,J=8.7Hz,1H),1.65–1.57(m,1H),1.42( dtd, J=15.6, 8.6, 3.7Hz, 3H), 1.27 (d, J=7.6Hz, 1H), 1.22 (d, J=3.5Hz, 2H), 1.19 (d, J=6.8Hz, 4H), 1.16 (d, J=4.5Hz, 3H), 0.83 (d, J=11.7Hz, 3H). 13 C NMR (101MHz, MeOD) δ: 177.0, 138.9, 133.6, 132.0, 131.6, 130.7, 130.1, 129.3, 127.1,118.1,98.9,85.4,74.4,74.2,72.7,72.3,72.2,70.1,70.1,69.1,51.8,51.3,50.5,49.9,49.7,47.2,45.8,40.9,34.8,34.8,33.3,33.0,30.8 27.9, 24.1, 18.01, 17.5. IR(thin film, cm -1 ): 3006, 2995, 1475, 1275, 1261, 764, and 751. HRMS(DART-TOF) calculated for C 38 H 50 Cl 2 NaO 12 + [M+Na] + m/z 791.2577, found 791. 2570. [α] D 26 =-31.6 (c=0.34, MeOH).

以下通过实验例证明本发明的有益效果。The beneficial effects of the present invention are demonstrated below through experimental examples.

实验例1:本发明化合物对肿瘤细胞的迁移能力的影响Experimental Example 1: Effects of the compounds of the present invention on the migration ability of tumor cells

在本实验例中,选取MAB-MD-231(乳腺癌细胞)细胞株对各乌本苷及其衍生物进行细胞划痕实验。In this experimental example, the MAB-MD-231 (breast cancer cell) cell line was selected to carry out cell scratch experiments on each ouabain and its derivatives.

(1)实验材料:(1) Experimental materials:

MAB-MD-231细胞(乳腺癌细胞)、含有20%FBS完全培养基DMEM(已预加L-谷氨酰胺,青霉素,100μg/mL和链霉素,100μg/mL,10nM HEPES)、胰酶、无菌PBS灭菌包布(大中小枪尖各一盒)、6孔板。MAB-MD-231 cells (breast cancer cells), complete medium DMEM containing 20% FBS (pre-added with L-glutamine, penicillin, 100 μg/mL and streptomycin, 100 μg/mL, 10 nM HEPES), trypsin , Sterile PBS sterilization cloth (one box for each of the large, medium and small gun tips), and a 6-well plate.

(2)实验步骤:(2) Experimental steps:

1)培养板划线:首先在6孔板背后,用直尺比着,均匀地划横线,大约每隔0.5cm-1cm一道,横穿过孔。每孔至少穿过5条线。划线时注意线不要太粗。1) Scribing the culture plate: First, draw a horizontal line evenly on the back of the 6-well plate with a ruler, about every 0.5cm-1cm, across the hole. Pass at least 5 lines through each hole. Be careful not to make the lines too thick when you draw the lines.

2)铺细胞:在孔中加入约5×105个细胞(不同的细胞数量有所不同,根据细胞的生长快慢调整),接种原则为过夜后融合率达到100%。2) Plating cells: add about 5×10 5 cells into the well (different cells have different numbers and adjust according to the growth speed of the cells). The principle of seeding is that the fusion rate reaches 100% after overnight.

3)细胞划线:第二天用20μL枪头(灭菌)或者无菌牙签,垂直与细胞平面,沿着前一天划在平板背面的线在细胞层上进行划痕。3) Cell streaking: On the second day, use a 20 μL pipette tip (sterilized) or a sterile toothpick, perpendicular to the cell plane, and scratch the cell layer along the line drawn on the back of the plate the day before.

4)洗细胞:划痕完成后,使用无菌PBS洗细胞3次,洗去不贴壁的细胞,使得划线后留下的间隙清晰可见,然后更换新鲜无血清培养基或低血清(<2%)的培养基。4) Wash the cells: After the scratch is completed, wash the cells 3 times with sterile PBS to remove the non-adherent cells, so that the gap left after the streak is clearly visible, and then replace with fresh serum-free medium or low serum (< 2%) medium.

5)加药作用:每一种化合物分别在10nM、100nM、1μM、10μM、100μM浓度下进行实验;以加入相同体积的无血清DMEM作为空白组。5) Dosing effect: each compound was tested at concentrations of 10 nM, 100 nM, 1 μM, 10 μM and 100 μM respectively; the same volume of serum-free DMEM was added as blank group.

6)细胞培养、观察:将细胞放入37℃、5%CO2培养箱培养。24h后取出细胞,显微镜下观察并测量划痕的宽度,拍照记录。6) Cell culture and observation: The cells were cultured in a 37°C, 5% CO 2 incubator. After 24 hours, the cells were taken out, and the width of the scratch was observed and measured under a microscope, and photographed and recorded.

7)结果分析:使用Image J软件打开图片后,随机划取6至8条水平线,测量细胞间距离的均值,并计算面积,得到覆盖率统计图。采用细胞覆盖率来表示细胞迁移的效果,细胞覆盖率越大,说明化合物抑制细胞迁移的作用越弱。7) Result analysis: After opening the picture with Image J software, randomly draw 6 to 8 horizontal lines, measure the mean value of the distance between cells, and calculate the area to obtain a coverage statistics map. The cell coverage rate was used to represent the effect of cell migration. The larger the cell coverage rate, the weaker the effect of the compound in inhibiting cell migration.

(3)实验结果(3) Experimental results

结果如图2A~C所示。可以看出,与空白组相比,本发明制得的乌本苷19位伯羟基衍生物均具有抑制肿瘤细胞迁移的作用。The results are shown in Figures 2A-C. It can be seen that, compared with the blank group, the 19-position primary hydroxyl derivatives of ouabain prepared by the present invention all have the effect of inhibiting tumor cell migration.

实验结果表明,本发明通过特定位置的烯丙基化修饰制得的乌本苷19位伯羟基衍生物能够有效抑制肿瘤细胞迁移。The experimental results show that the 19-position primary hydroxyl derivative of ouabain prepared by the allylation modification at a specific position of the present invention can effectively inhibit the migration of tumor cells.

实验例2:本发明化合物对肿瘤细胞的侵袭能力的影响Experimental Example 2: Influence of the compounds of the present invention on the invasive ability of tumor cells

在本实验例中,选取细胞划痕实验中表现良好的化合物Ouabain-14,进行Transwell细胞侵袭实验,进一步确定化合物抑制肿瘤细胞侵袭的效果。In this experimental example, the compound Ouabain-14, which performed well in the cell scratch test, was selected, and the Transwell cell invasion test was performed to further determine the effect of the compound on inhibiting tumor cell invasion.

(1)实验材料:(1) Experimental materials:

MAB-MD-231细胞(乳腺癌细胞)、基质胶Matrigel Basement Membrane Matrix,LDEV-free(BD Biocoat 354234)、无血清培养基(已预加L-谷氨酰胺\10nM HEPES、无血清培养基的抗生素可能会抑制细胞生长,不含或减为1/10,Transwell用)、含有20%FBS完全培养基(已预加L-谷氨酰胺,100μg/mL青霉素和100μg/mL链霉素,10nM HEPES,正常培养细胞用)、Transwell-24膜嵌套,6.5mm直径,8.0μm孔径PC膜(聚碳酸酯)、胰酶、甲醇(成都科隆化学,CAS67561)、结晶紫染液(碧云天,C0121)、无菌PBS(pH 7.2-7.4)。装置:Transwell实验装置。MAB-MD-231 cells (breast cancer cells), Matrigel Basement Membrane Matrix, LDEV-free (BD Biocoat 354234), serum-free medium (pre-supplemented with L-glutamine\10nM HEPES, serum-free medium Antibiotics may inhibit cell growth, without or reduced to 1/10, for Transwell), complete medium with 20% FBS (pre-supplemented with L-glutamine, 100 μg/mL penicillin and 100 μg/mL streptomycin, 10 nM HEPES, for normal cultured cells), Transwell-24 membrane nest, 6.5mm diameter, 8.0μm pore size PC membrane (polycarbonate), trypsin, methanol (Chengdu Cologne Chemical, CAS67561), crystal violet staining solution (Biyuntian, CO121), sterile PBS (pH 7.2-7.4). Apparatus: Transwell experimental apparatus.

(2)实验操作:(2) Experimental operation:

1)提前2个小时在小室中铺基质胶:首先用双无DMEM稀释基质胶,浓度(2μg/μL),体积50μL,加入小室中;1) Lay matrigel in the chamber 2 hours in advance: first dilute the matrigel with double-free DMEM, the concentration (2 μg/μL), and the volume of 50 μL, and add it to the chamber;

2)37℃孵育30min.-60min.,可在显微镜下观察,待基质胶凝固即可;2) Incubate at 37°C for 30min.-60min., which can be observed under a microscope until the matrigel solidifies;

3)分组:实验组(乌本苷衍生物1μg/mL,无血清DMEM配制,600μL)、空白组(无血清DMEM 600μL);3) Grouping: experimental group (ouabain derivative 1 μg/mL, prepared in serum-free DMEM, 600 μL), blank group (serum-free DMEM 600 μL);

4)制备细胞悬液:消化MAB-MD-231细胞,终止消化后离心(800g,3min)弃去培养液,用5mL含20%血清培养基重悬,调整细胞密度至5×104/mL。4) Preparation of cell suspension: Digest MAB-MD-231 cells, stop the digestion, centrifuge (800g, 3min) to discard the culture medium, resuspend in 5mL medium containing 20% serum, adjust the cell density to 5×10 4 /mL .

5)接种细胞:用完全培养基调整细胞密度至5×104/mL,取细胞悬液200μL加入Transwell小室(1×104/well),下层加500μL无血清培养基。5) Seeding cells: adjust the cell density to 5×10 4 /mL with complete medium, add 200 μL of cell suspension to a Transwell chamber (1×10 4 /well), and add 500 μL serum-free medium to the lower layer.

6)待自然贴壁12h后,上层置换层无血清培养基,下层置换成分组样品。6) After 12 hours of natural adherence, the upper layer was replaced with serum-free medium, and the lower layer was replaced with group samples.

7)培养细胞8-12h(根据不同细胞摸索时间)。培养结束后,用湿棉签小心擦去上室底部膜表面上的细胞。7) Culture the cells for 8-12h (exploration time according to different cells). After the incubation period, the cells on the membrane surface on the bottom of the upper chamber were carefully wiped off with a damp cotton swab.

8)吸干上室培养基(残留细胞可以用无血清培养基洗掉),用镊子小心取出小室,移到预先加入约800μL 100%甲醇的孔中,室温固定5-10min.。8) Aspirate the upper chamber medium (residual cells can be washed with serum-free medium), carefully remove the chamber with tweezers, move it to the well pre-added with about 800 μL of 100% methanol, and fix it at room temperature for 5-10 min.

9)取出小室,移到预先加入约800μL结晶紫染液的孔中,室温染色15min.左右(期间注意观察细胞染色情况,调整具体时间)。9) Take out the chamber, move it to the well pre-added with about 800 μL of crystal violet staining solution, and stain at room temperature for about 15 minutes.

10)取出小室,用去离子水润洗2次,底面朝上轻轻移至载玻片上,在倒置显微镜下取9个随机视野计数,统计结果。10) Take out the chamber, rinse it twice with deionized water, gently move the bottom side up to a glass slide, take 9 random fields of view under an inverted microscope and count the results.

(3)实验结果(3) Experimental results

结果如图3所示。与空白实验对照,本发明提供的乌本苷19位伯羟基衍生物Ouabain-14显示出良好的抑制肿瘤细胞侵袭的效果,且抑制效果与化合物的作用浓度有关系,随着浓度的增大,其抑制效果有所提高。The results are shown in Figure 3. Compared with the blank experiment, Ouabain-14, the 19-position primary hydroxyl derivative of ouabain provided by the present invention, shows a good effect of inhibiting tumor cell invasion, and the inhibitory effect is related to the concentration of the compound. Its inhibitory effect is improved.

实验例3:本发明化合物对正常细胞的毒性实验Experimental Example 3: Toxicity test of the compounds of the present invention on normal cells

(1)实验方法(1) Experimental method

选取正常人肾上皮质细胞293T,对本发明制得的化合物进行细胞毒性实验。将细胞制成单细胞悬浊液,细胞个数3000个/孔,接种于96孔板中,放置细胞培养箱过夜,使其附壁。用待测化合物处理孵化72小时后,于37℃加入20μL MTT溶液,放置2-4小时。线粒体内即形成暗紫色结晶,弃上清,加入100μL DMSO进行溶解,摇床孵育10min.后。用酶标仪(全波长酶标仪,赛默飞世尔科技公司)在570nm波长下测定吸光度。基于测定孔与控制孔吸光度的相关关系计算抑制率并采用Bliss法根据抑制率曲线计算出相应的IC50值。Select normal human renal epithelial cells 293T to carry out cytotoxicity experiments on the compounds prepared by the present invention. The cells were made into a single cell suspension with 3000 cells/well, seeded in a 96-well plate, and placed in a cell culture incubator overnight to allow them to attach to the wall. After 72 hours of incubation with the compounds to be tested, 20 μL of MTT solution was added at 37° C. and left for 2-4 hours. Dark purple crystals formed in the mitochondria, discard the supernatant, add 100 μL DMSO to dissolve, and incubate on a shaker for 10 min. The absorbance was measured at a wavelength of 570 nm with a microplate reader (full wavelength microplate reader, Thermo Fisher Scientific). The inhibition rate was calculated based on the correlation between the absorbance of the assay well and the control well, and the corresponding IC 50 value was calculated according to the inhibition rate curve using the Bliss method.

(2)实验结果(2) Experimental results

表2各化合物对正常人肾上皮质细胞293T和乳腺癌细胞MAB-MD-231的IC50Table 2 IC 50 values of each compound on normal human adrenal cortex cells 293T and breast cancer cells MAB-MD-231

Figure BDA0002651962720000181
Figure BDA0002651962720000181

Figure BDA0002651962720000191
Figure BDA0002651962720000191

化合物对正常人肾上皮质细胞293T的IC50值如表2所示。可以看出,与修饰前的Ouabain相比,本发明制得的乌本苷19位伯羟基衍生物对正常细胞的细胞毒性均显著降低,特别是化合物Ouabain-2、Ouabain-26。说明本发明制得的通过特定位置的烯丙基化修饰制得的乌本苷19位伯羟基衍生物可能具有更小的毒副作用。Table 2 shows the IC50 values of the compounds on normal human renal epithelial cells 293T. It can be seen that compared with Ouabain before modification, the 19-position primary hydroxyl derivatives of ouabain prepared by the present invention have significantly reduced cytotoxicity to normal cells, especially compounds Ouabain-2 and Ouabain-26. It is indicated that the 19-position primary hydroxyl derivative of ouabain prepared by the allylation modification at a specific position prepared by the present invention may have less toxic and side effects.

实验例4:本发明化合物对肿瘤细胞的毒性实验Experimental Example 4: Toxicity test of the compounds of the present invention on tumor cells

(1)实验方法(1) Experimental method

选取乳腺癌细胞MAB-MD-231以及其他肿瘤细胞(hela:宫颈癌细胞;Hep G2:人肝癌细胞;SK-HEP-1:人肝腹水腺癌细胞;Du 145:前列腺癌细胞;Panc 1:人胰腺癌细胞),对本发明制得的化合物进行细胞毒性实验。将细胞制成单细胞悬浊液,细胞个数3000个/孔,接种于96孔板中,放置细胞培养箱过夜,使其附壁。用待测化合物处理孵化72小时后,于37℃加入20μL MTT溶液,放置2-4小时。线粒体内即形成暗紫色结晶,弃上清,加入100μL DMSO进行溶解,摇床孵育10min.后。用酶标仪(全波长酶标仪,赛默飞世尔科技公司)在570nm波长下测定吸光度。基于测定孔与控制孔吸光度的相关关系计算抑制率并采用Bliss法根据抑制率曲线计算出相应的IC50值。Select breast cancer cells MAB-MD-231 and other tumor cells (hela: cervical cancer cells; Hep G2: human liver cancer cells; SK-HEP-1: human liver ascites adenocarcinoma cells; Du 145: prostate cancer cells; Panc 1: human pancreatic cancer cells), and the cytotoxicity experiments were carried out on the compounds prepared in the present invention. The cells were made into a single cell suspension with 3000 cells/well, seeded in a 96-well plate, and placed in a cell culture incubator overnight to allow them to attach to the wall. After 72 hours of incubation with the compounds to be tested, 20 μL of MTT solution was added at 37° C. and left for 2-4 hours. Dark purple crystals formed in the mitochondria, discard the supernatant, add 100 μL DMSO to dissolve, and incubate on a shaker for 10 min. The absorbance was measured at a wavelength of 570 nm with a microplate reader (full wavelength microplate reader, Thermo Fisher Scientific). The inhibition rate was calculated based on the correlation between the absorbance of the assay well and the control well, and the corresponding IC 50 value was calculated according to the inhibition rate curve using the Bliss method.

(2)实验结果(2) Experimental results

(2.1)化合物对乳腺癌细胞MAB-MD-231的毒性(2.1) Toxicity of compounds to breast cancer cells MAB-MD-231

化合物对乳腺癌细胞MAB-MD-231的IC50值如表2所示。可以看出,与修饰前的Ouabain相比,本发明制得的乌本苷19位伯羟基衍生物对乳腺癌细胞MAB-MD-231的细胞毒性出现了不同程度的降低。The IC50 values of the compounds against breast cancer cells MAB-MD-231 are shown in Table 2. It can be seen that, compared with Ouabain before modification, the 19-position primary hydroxyl derivatives of ouabain prepared by the present invention have reduced cytotoxicity to breast cancer cell MAB-MD-231 to different degrees.

从表2还可以看出,Ouabain对正常细胞毒性的IC50是其对乳腺癌细胞的IC50的1/4,说明Ouabain对正常细胞的毒性显著大于对乳腺癌细胞的毒性。与Ouabain相比,只有本发明Ouabain-20对正常细胞的细胞毒性的降低程度小于对乳腺癌细胞的细胞毒性的降低程度,而Ouabain-8、Ouabain-11、Ouabain-14、Ouabain-17和Ouabain-26对正常细胞的细胞毒性的降低程度均明显大于对乳腺癌细胞的细胞毒性的降低程度。It can also be seen from Table 2 that the IC 50 of Ouabain's toxicity to normal cells is 1/4 of its IC 50 to breast cancer cells, indicating that Ouabain's toxicity to normal cells is significantly greater than that to breast cancer cells. Compared with Ouabain, only Ouabain-20 of the present invention reduces the cytotoxicity to normal cells to a lesser degree than to breast cancer cells, while Ouabain-8, Ouabain-11, Ouabain-14, Ouabain-17 and Ouabain The cytotoxicity of -26 to normal cells was significantly greater than that to breast cancer cells.

所以,综合考虑化合物对正常细胞和对乳腺癌细胞的抑制效果,本发明通过特定位置的烯丙基化修饰制得的乌本苷19位伯羟基衍生物Ouabain-8、Ouabain-11、Ouabain-14、Ouabain-17和Ouabain-26作为抗肿瘤药物比Ouabain具有更好的应用前景。Therefore, considering the inhibitory effect of the compound on normal cells and breast cancer cells, the 19-position primary hydroxyl derivatives of ouabain-8, Ouabain-11, Ouabain- 14. Ouabain-17 and Ouabain-26 have better application prospects as antitumor drugs than Ouabain.

(2.2)化合物对其它肿瘤细胞的毒性(2.2) Toxicity of compounds to other tumor cells

表3化合物Ouabain-2对其它肿瘤细胞的IC50Table 3 IC 50 values of compound Ouabain-2 on other tumor cells

Figure BDA0002651962720000201
Figure BDA0002651962720000201

化合物Ouabain-2对其他肿瘤细胞(hela:宫颈癌细胞;Hep G2:人肝癌细胞;SK-HEP-1:人肝腹水腺癌细胞;Du 145:前列腺癌细胞;Panc 1:人胰腺癌细胞)的IC50值如表3所示。可以看出,Ouabain-2对人肾上皮质细胞293T的抑制能力最弱,对Panc 1的抑制能力最强,对Panc 1的作用浓度约是293T的1/30;Ouabain-2对hela和SK-HEP-1的抑制效果也很好,作用浓度分别约是293T的1/7、1/17;Ouabain-2对Hep G2和Du 145也具有一定的抑制效果,但是抑制效果比Panc 1、hela和SK-HEP-1差。Ouabain-2对上述肿瘤细胞的抑制作用为Panc 1>SK-HEP-1>hela>Du 145>Hep G2。Compound Ouabain-2 on other tumor cells (hela: cervical cancer cells; Hep G2: human hepatoma cells; SK-HEP-1: human liver ascites adenocarcinoma cells; Du 145: prostate cancer cells; Panc 1: human pancreatic cancer cells) The IC50 values are shown in Table 3. It can be seen that Ouabain-2 has the weakest inhibitory ability on 293T in human renal epithelial cells, and the strongest inhibitory ability on Panc 1. The concentration of Ouabain-2 on Panc 1 is about 1/30 of that of 293T; The inhibitory effect of -HEP-1 is also very good, and the concentration is about 1/7 and 1/17 of that of 293T, respectively; Ouabain-2 also has a certain inhibitory effect on Hep G2 and Du 145, but the inhibitory effect is stronger than that of Panc 1 and hela. Worse than SK-HEP-1. The inhibitory effect of Ouabain-2 on the above tumor cells was Panc 1>SK-HEP-1>hela>Du 145>Hep G2.

上述结果表明,本发明制得的乌本苷19位伯羟基衍生物在显著降低对正常细胞的毒性的同时,还能具有良好的抑制包括乳腺癌细胞在内的多种肿瘤细胞的效果。此外,本发明制得的乌本苷19位伯羟基衍生物对肿瘤细胞的抑制作用具有较好的选择性,在抑制胰腺癌细胞、SK-HEP-1肝腹水腺癌细胞、宫颈癌细胞细胞中具有更好的效果。The above results show that the ouabain 19-position primary hydroxyl derivative prepared by the present invention can significantly reduce the toxicity to normal cells, and at the same time, it can also have a good effect of inhibiting various tumor cells including breast cancer cells. In addition, the 19-position primary hydroxyl derivative of ouabain prepared in the present invention has good selectivity for the inhibitory effect on tumor cells, and can inhibit pancreatic cancer cells, SK-HEP-1 liver ascites adenocarcinoma cells, and cervical cancer cells. has better effect.

实验例5:CCK-8法检测本发明化合物抑制胰腺癌细胞增殖的作用Experimental Example 5: CCK-8 assay to detect the effect of the compounds of the present invention on inhibiting the proliferation of pancreatic cancer cells

本实验例进一步利用CCK-8法检测本发明制得的化合物抑制人胰腺癌细胞株(BXPC3、PANC 1)增殖的作用。In this experimental example, the CCK-8 method was further used to detect the effect of the compounds prepared in the present invention on inhibiting the proliferation of human pancreatic cancer cell lines (BXPC3, PANC 1).

(1)实验材料(1) Experimental materials

DMEM、1640培养基购自Corning公司(Corning,USA);DMSO、CCK-8试剂盒购自Dojindo公司;BXPC3、PANC 1细胞系等(购自中国科学院典型培养物保藏委员会细胞库中国科学院上海生命科学研究院细胞资源中心);其它试剂为国产分析纯等。DMEM and 1640 medium were purchased from Corning Company (Corning, USA); DMSO and CCK-8 kits were purchased from Dojindo Company; BXPC3, PANC 1 cell lines, etc. Cell Resource Center of the Academy of Sciences); other reagents are of domestic analytical grade, etc.

(2)实验方法(2) Experimental method

(a)将处于对数生长期的BXPC3细胞系细胞50μl/孔接种于96孔微量培养板内,4-5×103细胞/孔(BXPC3),隔天加入以培养基稀释的化合物样品(化合物样品用DMSO溶解制备成20mM储备液),使得终浓度在100uM-0.002uM之间,终体积为100μl/孔;设置3个浓度梯度(1.111uM、3.333uM、10uM),每个浓度均为复孔,另设空白对照孔,即不加样品,仅加相应体积的样品溶解液。细胞在37℃、5%CO2条件下培养72小时后,加CCK-8试剂10μl/孔;继续培养0.5小时后检测。实验观察指标为:用酶标仪在450nm波长处检测96孔平板中每孔细胞的光密度值(OD值),计算细胞存活率(viability),用AVERAGE±SD表示。(a) Inoculate 50 μl/well of BXPC3 cell line cells in logarithmic growth phase in a 96-well microplate, 4-5×10 3 cells/well (BXPC3), and add compound samples diluted in culture medium every other day ( Compound samples were dissolved in DMSO to prepare a 20mM stock solution), so that the final concentration was between 100uM-0.002uM, and the final volume was 100μl/well; 3 concentration gradients (1.111uM, 3.333uM, 10uM) were set, and each concentration was Duplicate wells, and set up blank control wells, that is, no sample is added, only the corresponding volume of sample dissolving solution is added. After the cells were cultured at 37°C and 5% CO 2 for 72 hours, 10 μl/well of CCK-8 reagent was added; the cells were further cultured for 0.5 hours and detected. The experimental observation indicators are: detect the optical density value (OD value) of each well of cells in a 96-well plate with a microplate reader at a wavelength of 450 nm, calculate the cell viability, and express it as AVERAGE±SD.

(b)采用与(a)相同的方法,将浓度梯度增加为9个(0.014uM、0.041uM、0.123uM、1.111uM、3.333uM、10uM、30uM),重新测试,计算化合物的IC50值。(b) Using the same method as (a), increase the concentration gradient to 9 (0.014uM, 0.041uM, 0.123uM, 1.111uM, 3.333uM, 10uM, 30uM), retest, and calculate the IC50 value of the compound.

(3)实验结果(3) Experimental results

表4 BXPC3、PANC 1细胞与各化合物共培养下的存活率数据Table 4 Survival data of BXPC3, PANC 1 cells co-cultured with each compound

Figure BDA0002651962720000211
Figure BDA0002651962720000211

表5 Ouabain-26对BXPC3细胞的IC50Table 5 IC 50 values of Ouabain-26 on BXPC3 cells

Figure BDA0002651962720000221
Figure BDA0002651962720000221

表6 Ouabain-26对PANC 1细胞的IC50Table 6 IC 50 values of Ouabain-26 on PANC 1 cells

Figure BDA0002651962720000222
Figure BDA0002651962720000222

从表4可以看出,本发明制得的乌本苷19位伯羟基衍生物能够有效抑制胰腺癌细胞增殖,其中,化合物Ouabain-2、Ouabain-26的抑制效果均明显优于其余化合物。进一步从表5和表6可知,本发明化合物Ouabain-26对BXPC3细胞的IC50值低至24.46μmol/L,对PANC 1细胞的IC50值低至9.26μmol/L。It can be seen from Table 4 that the 19-position primary hydroxyl derivatives of ouabain prepared by the present invention can effectively inhibit the proliferation of pancreatic cancer cells, and the compounds Ouabain-2 and Ouabain-26 have significantly better inhibitory effects than other compounds. Further from Table 5 and Table 6, it can be seen that the IC 50 value of the compound Ouabain-26 of the present invention on BXPC3 cells is as low as 24.46 μmol/L, and the IC 50 value on PANC 1 cells is as low as 9.26 μmol/L.

实验结果表明,本发明化合物(特别是化合物Ouabain-2、Ouabain-26)能够有效抑制胰腺癌细胞增殖。The experimental results show that the compounds of the present invention (especially the compounds Ouabain-2 and Ouabain-26) can effectively inhibit the proliferation of pancreatic cancer cells.

综上,本发明以特定的烯丙基化试剂为改性剂,通过特定位置的烯丙基化修饰成功制得了一类乌本苷19位伯羟基衍生物,该乌本苷19位伯羟基衍生物在降低对正常细胞的毒性的同时,还具有优异的抑制包括乳腺癌、宫颈癌细胞、肝癌、前列腺癌、胰腺癌在内的多种肿瘤细胞的效果。而且,该乌本苷19位伯羟基衍生物对肿瘤细胞的抑制作用具有良好的选择性,其对胰腺癌细胞、SK-HEP-1肝腹水腺癌、宫颈癌细胞细胞的抑制效果明显优于前列腺癌细胞和Hep G2肝癌细胞。此外,该乌本苷19位伯羟基衍生物还能有效的抑制肿瘤细胞迁移和/或侵袭。所以,本发明提供的乌本苷19位伯羟基衍生物在制备抗肿瘤、抑制肿瘤侵袭和/或迁移的药物中具有非常好的应用前景。In summary, the present invention uses a specific allylation reagent as a modifier, and successfully prepares a class of ouabain 19-position primary hydroxyl derivatives through allylation modification at a specific position. The 19-position primary hydroxyl group of ouabain While reducing the toxicity to normal cells, the derivatives also have excellent inhibitory effects on various tumor cells including breast cancer, cervical cancer cells, liver cancer, prostate cancer, and pancreatic cancer. Moreover, the 19-position primary hydroxyl derivative of ouabain has good selectivity for the inhibitory effect on tumor cells, and its inhibitory effect on pancreatic cancer cells, SK-HEP-1 liver ascites adenocarcinoma, and cervical cancer cells is significantly better than Prostate cancer cells and Hep G2 liver cancer cells. In addition, the 19-position primary hydroxyl derivative of ouabain can effectively inhibit tumor cell migration and/or invasion. Therefore, the 19-position primary hydroxyl derivatives of ouabain provided by the present invention have very good application prospects in the preparation of drugs for anti-tumor and inhibiting tumor invasion and/or migration.

Claims (16)

1. A compound of formula I, or a pharmaceutically acceptable salt thereof:
Figure FDA0003570977890000011
wherein n is 4;
4 of R0Each independently selected from hydroxy or methyl;
R1selected from hydrogen, substituted or unsubstituted aryl;
the substituent is 1 or more and is selected from halogenated or non-halogenated C1~5Alkyl, halogenated or non-halogenated C1~5Alkoxy, halogenated or non-halogenated C2~6Alkynyl, halogen, N3And a nitro group.
2. The compound of claim 1, or a pharmaceutically acceptable salt thereof, wherein: the structure of the compound is shown as formula II:
Figure FDA0003570977890000012
wherein R is1Selected from hydrogen, substituted or unsubstituted aryl;
the substituent is 1 or more and is selected from halogenated or non-halogenated C1~3Alkyl, halogenated or non-halogenated C1~3Alkoxy, halogenated or non-halogenated C2~4Alkynyl, halogen, N3And a nitro group.
3. The compound of claim 2, or a pharmaceutically acceptable salt thereof, wherein: the structure of the compound is shown as formula III:
Figure FDA0003570977890000013
wherein m is an integer of 1-5; m R2Each independently selected from hydrogen, halogenated or non-halogenated C1~3Alkyl, halogenated or non-halogenated C1~3Alkoxy, halogenated or non-halogenated C2~4Alkenyl, halogenated or non-halogenated C2~4Alkynyl, halogen, N3Or a nitro group.
4. The compound of claim 3, or a pharmaceutically acceptable salt thereof, wherein: m is 1 or 2; the halogen is fluorine or chlorine.
5. The compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt thereof, wherein: the structure of the compound is shown as follows:
Figure FDA0003570977890000021
Figure FDA0003570977890000031
6. a process for the preparation of a compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, characterized in that: the method comprises the following steps:
(1) mixing ouabain, organic boron reagent and organic solvent in a reaction bottle;
(2) mixing an allylation reagent, a palladium catalyst, a phosphorus ligand and an organic solvent in another reaction bottle, and stirring and activating; the allylation reagent has the structure of
Figure FDA0003570977890000032
R3Is a protecting group, R1The method according to any one of claims 1 to 5;
(3) and (3) adding the system obtained in the step (2) into the system obtained in the step (1) for reaction to obtain the catalyst.
7. The method of claim 6, wherein: the steps (1) to (3) are carried out in an inert gas protection environment; in the step (3), the reaction temperature is 20-30 ℃; the reaction time is 6-24 hours.
8. The method of claim 7, wherein: in the step (3), the reaction temperature is room temperature; the reaction time was 16 hours.
9. The method according to any one of claims 6-8, wherein: in the step (1), the organoboron reagent is methyl phenylboronic acid, and the organic solvent is one or a mixture of tetrahydrofuran and isopropanol; the molar ratio of the ouabain to the organic boron reagent is 1: (0.5 to 1.5);
in the step (2), the palladium catalyst is Pd (dba)3·CHCl3The phosphorus ligand is PPh3(ii) a The organic solvent is one or two of tetrahydrofuran and isopropanol; the mol ratio of the ouabain to the allylation reagent, the palladium catalyst and the phosphorus ligand is 1: (1.1-5.0): (0.01-0.2): (0.05-0.3); and/or the stirring activation time is5-20 minutes; and/or, said R3Is a hydroxyl protecting group.
10. The method of claim 9, wherein: in the step (1), the organic solvent is a mixture of tetrahydrofuran and isopropanol; the molar ratio of the ouabain to the organic boron reagent is 1: 1;
in the step (2), the stirring activation time is 10 minutes; and/or, said R3Is Boc group.
11. A medicament, characterized by: the medicine is a preparation prepared by taking the compound or the pharmaceutically acceptable salt thereof as an active ingredient and adding pharmaceutically acceptable auxiliary materials.
12. Use of a compound according to any one of claims 1 to 5, or a pharmaceutically acceptable salt thereof, in the manufacture of a medicament for the treatment of an anti-tumour or inhibition of tumour metastasis.
13. Use according to claim 12, characterized in that: the tumor is breast cancer, cervical cancer, liver cancer, prostatic cancer and pancreatic cancer.
14. Use according to claim 12, characterized in that: the tumor is breast cancer, pancreatic cancer, liver cancer, and cervical cancer.
15. Use according to claim 14, characterized in that: the liver cancer is liver ascites adenocarcinoma.
16. Use according to any one of claims 12 to 15, characterized in that: the drug is capable of inhibiting tumor cell proliferation, invasion and/or migration.
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Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1075251A (en) * 1962-10-04 1967-07-12 Wellcombe Foundation Ltd Cardenolide derivatives
GB2034353A (en) * 1978-10-30 1980-06-04 Richter Gedeon Vegyeszet A process for increasing the substrate concentration of fermentation broths in the microbiological conversion of cardenolide derivatives
WO1998007739A1 (en) * 1996-08-21 1998-02-26 The General Hospital Corporation Novel ouabain analogs
US5872103A (en) * 1986-11-26 1999-02-16 Belletti; Dino A. Prevention of mammary tumors by treatment with cardiac glycosides
US5874423A (en) * 1992-09-10 1999-02-23 Yissum Research Development Co. Of The Hebrew University Of Jerusalem Digitalis-like compounds
CN103570792A (en) * 2012-08-10 2014-02-12 中国科学院上海药物研究所 Bufalin derivative as well as preparation method, pharmaceutical composition and application thereof
CN105037474A (en) * 2015-07-13 2015-11-11 中国科学院上海药物研究所 4'-amino-4'-dehydroxyl-oleandrin and 4'-amino-4'-dehydroxyl-odoroside A and use thereof
CN107473982A (en) * 2016-06-08 2017-12-15 四川大学 Hold position substitution high allyl amine derivative and its production and use
CN109021058A (en) * 2018-09-12 2018-12-18 烟台大学 With active ocotillol type sapogenin derivative of tumor drug resistance reversal and its preparation method and application

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9670247B2 (en) * 2012-07-02 2017-06-06 The University Of Kansas Contraceptive agents
WO2014164730A2 (en) * 2013-03-12 2014-10-09 The Board Of Trustees Of The Leland Stanford Junior University Modulation of cellular dna repair activity to intercept malignancy

Patent Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB1075251A (en) * 1962-10-04 1967-07-12 Wellcombe Foundation Ltd Cardenolide derivatives
GB2034353A (en) * 1978-10-30 1980-06-04 Richter Gedeon Vegyeszet A process for increasing the substrate concentration of fermentation broths in the microbiological conversion of cardenolide derivatives
US5872103A (en) * 1986-11-26 1999-02-16 Belletti; Dino A. Prevention of mammary tumors by treatment with cardiac glycosides
US5874423A (en) * 1992-09-10 1999-02-23 Yissum Research Development Co. Of The Hebrew University Of Jerusalem Digitalis-like compounds
WO1998007739A1 (en) * 1996-08-21 1998-02-26 The General Hospital Corporation Novel ouabain analogs
CN103570792A (en) * 2012-08-10 2014-02-12 中国科学院上海药物研究所 Bufalin derivative as well as preparation method, pharmaceutical composition and application thereof
CN105037474A (en) * 2015-07-13 2015-11-11 中国科学院上海药物研究所 4'-amino-4'-dehydroxyl-oleandrin and 4'-amino-4'-dehydroxyl-odoroside A and use thereof
CN107473982A (en) * 2016-06-08 2017-12-15 四川大学 Hold position substitution high allyl amine derivative and its production and use
CN109021058A (en) * 2018-09-12 2018-12-18 烟台大学 With active ocotillol type sapogenin derivative of tumor drug resistance reversal and its preparation method and application

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
"Preparation of an analogue of orbicuside A, an unusual cardiac glycoside";John T. Dixon等;《TETRAHEDRON-ASYMMETRY》;20051231;第16卷(第2期);第393-401页 *
"强心甙类药物抗肿瘤机制研究进展";王燕,等;《中国新药杂志》;20141231;第23卷(第6期);第677-681,691页 *
"强心甾的合成研究进展";祁小云,等;《中国医药工业杂志》;20031231;第34卷(第4期);第192-198页 *
"选择性化学修饰用于基于天然产物的探针合成";钮大文;《第十四届中国药学会青年药学论坛报告集》;20181231;第41页 *

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