大黄酸-氨基酸缀合物的合成及初步抗肿瘤活性研究

引用本文: 周昌健, 谢建伟, 张洁, 代斌. 大黄酸-氨基酸缀合物的合成及初步抗肿瘤活性研究[J]. 有机化学, 2017, 37(1): 122-132. doi: 10.6023/cjoc201607020 shu

Citation: Zhou Changjian, Xie Jianwei, Zhang Jie, Dai Bin. Study on the Synthesis and Anti-tumor Activities of Rhein-Amino Acid Conjugates[J]. Chinese Journal of Organic Chemistry, 2017, 37(1): 122-132. doi: 10.6023/cjoc201607020 shu

大黄酸-氨基酸缀合物的合成及初步抗肿瘤活性研究

周昌健 ^a, ,
谢建伟 ^{a,*,
,} ,
张洁 ^{a,*,
,} ,
代斌 ^a,

a.
石河子大学化学化工学院新疆兵团化工绿色过程重点实验室-省部共建国家重点实验室培育基地石河子 832003

通讯作者: 谢建伟, E-mail: cesxjw@foxmail.com; 张洁, E-mail:zhangjie-xj@163.com

摘要: 以大黄酸为原料, 经过酯化、醚化、水解、缩合等步骤, 合成了20个大黄酸-氨基酸缀合物.所有目标化合物经¹H NMR, ¹³C NMR和HRMS进行结构确证.以顺铂和阿霉素为阳性对照药, 采用四甲基偶氮唑盐(MTT)法考察了目标化合物体外抗肿瘤(Hela, MCF-7, HepG2, KB和HEK293T等5株细胞)活性.结果显示2-(4, 5-双(苄氧基)-9, 10-蒽醌-2-甲酰氨基)-4-甲基戊酸钠(6eb)显示出较好的抗肿瘤活性, DNA对2-(4, 5-二丁氧基-9, 10-蒽醌-2-甲酰氨基)-4-甲基戊酸钠(6db)和6eb作用的荧光光谱均表现出荧光猝灭现象, 推测6db可能仅是静电结合或分子部分嵌入DNA.

关键词:

大黄酸
/ 氨基酸
/ 抗肿瘤
/ 构效关系

English

Study on the Synthesis and Anti-tumor Activities of Rhein-Amino Acid Conjugates

a.
School of Chemistry and Chemical Engineering and Key Laboratory for Green Processing of Chemical Engineering of Xinjiang Bingtuan, Shihezi University, Shihezi 832003

Corresponding author: Xie Jianwei, cesxjw@foxmail.com; Zhang Jie, zhangjie-xj@163.com

Received Date: 12 July 2016
Revised Date: 20 August 2016

Abstract: 20 novel rhein-amino acid conjugates were designed and synthesized from the rhein via esterification, etherification, hydrolysis, condensation and salification. Their structures were confirmed by ¹H NMR, ¹³C NMR and HRMS. All the target compounds were tested for cytotoxic activity against five cancer cell lines including Hela, MCF-7, HepG2, KB and HEK293T by methyl thiazolyl tetrazolium (MTT) method in vitro. The results showed that sodium (4, 5-bis (benzyloxy)-9, 10-dioxo-9, 10-dihydroanthracene-2-carbonyl) leucinate (6eb) demonstrated moderate cytotoxic activities (IC₅₀ < 50 µmol•L^-1). The fluorescence of compounds sodium (4, 5-dibutoxy-9, 10-dioxo-9, 10-dihydroanthracene-2-carbonyl) leucinate (6db) and 6eb could be both quenched by DNA. It was speculated that compound 6db interacted with DNA by the static attraction or was partially embedded into DNA.

Key words:

图1 阿霉素、米托蒽醌和大黄酸的化学结构

Figure 1. Chemical structures of doxorubicin, mitoxantrone and rhein

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目前, 对于大黄酸的研究, 主要集中在其药理活性以及3-位的结构衍生化^[21~26], 对其1, 8-位和3-位同时衍生化的研究很少.本文围绕大黄酸1, 8-位进行衍生化的同时, 将氨基酸片段引入到大黄酸的羧基位中, 重点研究不同氨基酸取代及不同长度的烷基取代衍生物的抗肿瘤活性及其与DNA作用能力, 期望能够增强该系列药物的溶解性并缓解药物的细胞毒性, 提高其生物利用率.

大黄酸是大黄的有效成分和标志性成分, 属于羟基蒽醌类衍生物, 具有抗炎、抗病毒、降糖调脂等广泛的药理活性^[3~9].更为重要的是, 大黄酸还具有优良的抗肿瘤活性^[10~14]及在协同抗肿瘤方面表现突出^{[15, 16]}, 并且其引起的泻下作用在人体忍受范围内.众多的文献报道表明, 蒽醌衍生物在抗肿瘤药物研究方面^[17~20]应用广泛, 以蒽醌结构作为药效团的药物已经成为一类重要的临床抗癌药物, 如阿霉素(Doxorubicin)、米托蒽醌(Mitoxantrone)等(图 1).阿霉素主要通过影响拓扑异构酶Ⅱ(topoisomerase, TOPO Ⅱ)的活性而发挥抗肿瘤作用; 而米托蒽醌是DNA嵌入剂, 它能嵌入到DNA碱基之间, 导致DNA链的胶连和链结构的破坏, 从而阻断DNA合成与转录; 它还能够抑制DNA拓扑异构酶Ⅱ, 导致基因组DNA的解旋.因此, 大黄酸可作为一种潜在的抗肿瘤药物先导物.

癌症是当今威胁人类生命健康的重要疾病之一.全球癌症研究所发布的报告表明, 整个亚洲死于癌症的人数约占癌症死亡总人数55%^[1].近年来, 植物来源的大量活性天然组分是发现抗肿瘤创新药的重要途径. Newman等^[2]调研发现20世纪80年代后26年间全球推出的药物小分子新化学实体中, 超过百分之六十的药物小分子可追溯到天然产物激发的灵感或者天然产物本身.

1 结果与讨论

1.1 化合物合成与结构

图图式2 目标化合物的合成路线

Figure 图式2. Synthetic route of the target compounds

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如Scheme 1所示, 本实验以大黄酸(1)为起始原料, SOCl₂作为催化剂, 先与乙醇在回流条件下发生酯化反应, 得到大黄酸乙酯2; 2与不同卤代烃在强碱NaH作用下生成醚, 再酯水解, 得到1, 8-二烷氧基大黄酸3a~3e; 在EDCI/DMAP条件下, 3a~3e与不同氨基酸酯发生缩合, 得到化合物4aa~4ed; 在氢氧化钠作用下, 氨基酸酯水解, 得化合物5aa~5ed; 为了增加化合物的水溶性, 最后将5aa~5ed与等物质的量的NaOH反应转化成相应的钠盐6aa~6ed.目标化合物结构经¹H NMR, ¹³C NMR和HRMS进行结构确证.

1.2 生物活性-结构分析

表1 化合物的体外抗肿瘤活性 Table1. Anti-tumor activity of derivativesin vitro

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Compd.	R¹	R²	IC₅₀^a/(µmol•L^-1)
Compd.	R¹	R²	Hela	MCF7	HepG2	KB	HEK293T
6aa	CH₃	H	＞100	＞100	＞100	＞100	＞100
6ab	CH₃	CH₂CH (CH₃)₂	＞100	＞100	＞100	＞100	＞100
6ac	CH₃	CH₂Ph	＞100	＞100	＞100	＞100	＞100
6ad	CH₃	CH₂CH₂SCH₃	＞100	＞100	＞100	＞100	＞100
6ba	CH₂CH₃	H	＞100	＞100	＞100	＞100	＞100
6bb	CH₂CH₃	CH₂CH (CH₃)₂	＞100	＞100	＞100	＞100	＞100
6bc	CH₂CH₃	CH₂Ph	＞100	＞100	＞100	＞100	＞100
6bd	CH₂CH₃	CH₂CH₂SCH₃	＞100	＞100	＞100	＞100	＞100
6ca	CH₂CH₂CH₃	H	＞100	＞100	＞100	＞100	＞100
6cb	CH₂CH₂CH₃	CH₂CH (CH₃)₂	75.8	22.8	79.1	62.6	＞100
6cc	CH₂CH₂CH₃	CH₂Ph	＞100	95.4	81.6	68.8	97.6
6cd	CH₂CH₂CH₃	CH₂CH₂SCH₃	＞100	＞100	＞100	＞100	＞100
6da	CH₂CH₂CH₂CH₃	H	＞100	＞100	＞100	64.5	28.4
6db	CH₂CH₂CH₂CH₃	CH₂CH (CH₃)₂	62.1	80.2	51.2	27.7	37.4
6dc	CH₂CH₂CH₂CH₃	CH₂Ph	70.2	＞100	42.3	43.4	52.2
6dd	CH₂CH₂CH₂CH₃	CH₂CH₂SCH₃	＞100	＞100	＞100	62.4	83.2
6ea	CH₂Ph	H	＞100	＞100	＞100	＞100	＞100
6eb	CH₂Ph	CH₂CH (CH₃)₂	41.9	44.4	31.7	26.7	24.1
6ec	CH₂Ph	CH₂Ph	50.1	＞100	64.6	64.3	＞100
6ed	CH₂Ph	CH₂CH₂SCH₃	＞100	＞100	＞100	99.8	＞100
顺铂	-	-	5.5	15.8	14.3	6.1	3.3
阿霉素	-	-	0.99	2.8	4.7	3.7	0.7
^aThe data represent the mean values of three independent determinations.

表1 化合物的体外抗肿瘤活性
Table1. Anti-tumor activity of derivativesin vitro

实验采用四甲基偶氮唑盐(MTT)比色法对目标化合物6aa~6ed进行了体外抗肿瘤活性测试, 以顺铂和阿霉素作为阳性对照.结果如表 1所示, 当C-1, 8位为甲氧基和乙氧基时, 所有衍生物整体上并未表现出明显的抗肿瘤活性.当C-1, 8位为丙氧基和丁氧基, C-3位氨基酸残基为亮氨酸和苯丙氨酸时, 所有衍生物的抗肿瘤活性与甲氧基和乙氧基系列相比都有所提高, 其中6cb和6db活性最好, 均优于同系列其他化合物.当C-1, 8位为苄氧基时, 所有衍生物的抗肿瘤活性进一步提高, 并且整个系列对所测的5个细胞株表现出普遍的抑制活性, 其中6eb对所测试的5个细胞株均表现出显著的抑制活性(IC₅₀＜50 µmol/L), 也是所有测试化合物中抗肿瘤活性最强的化合物.综上可知, C-1, 8位随着基团链的增长或体积增加, 抗肿瘤活性成递增趋势, 具体顺序为:苄氧基＞丁氧基＞丙氧基＞乙氧基＞甲氧基; C-3位氨基酸残基种类对活性的影响顺序为:亮氨酸＞苯丙氨酸＞蛋氨酸＞甘氨酸.

1.3 与DNA的相互作用

由于化合物6db和6eb含有共同的蒽醌环作为荧光发射基团, 我们比较了DNA对6db和6eb荧光光谱的影响(图 2).随着DNA浓度的增加, 两个体系均出现不同程度的荧光猝灭.这恰恰表明, 这两个化合物与DNA作用方式具有相似性, 即蒽环进入了DNA双螺旋结构的影响区域^[27].比较化合物6db和6eb的结构, 可以看出两个化合物C-3位具有相同的氨基酸残基, C-1, 8位为分别为丁氧基和苄氧基, 具有较高的结构相似性, 因而两个化合物与DNA相互作用的荧光光谱未显示明显差别. 6db与DNA的相互作用方式更接近于长链类脂化合物的表面活性作用, 推测它与DNA的作用可能仅是静电结合或分子部分嵌入DNA.

与DNA的相互作用是抗肿瘤药物设计中一个重要靶点, 我们合成的化合物初步抗肿瘤活性结果基本达到预期设计目标, 通过选择有代表性的化合物与DNA作用, 希望可以判断抗肿瘤活性的原因所在.

图2 化合物6db(a)和6eb(b)与DNA作用的荧光光谱图

Figure 2. Spectrofluorimetric tiration of 6db (a) and 6eb (b) interacting with DNA

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2 结论

本文以大黄酸为原料, 经过酯化、烷基化、水解、缩合等步骤, 设计合成了20个未见文献报道的大黄酸-氨基酸缀合物, 达到了预期目标.通过¹H NMR, ¹³C NMR和HRMS确证了该系列化合物的结构, 采用MTT法研究了化合物对子宫颈癌细胞(Hela)、人肝癌细胞(HepG-2)、人乳腺癌细胞(MCF-7)、人口腔表皮癌细胞(KB)和人胚肾细胞(HEK293T)的体外生长抑制活性, 部分化合物对肿瘤细胞具有一定的生长抑制作用.化合物6db和6eb与DNA的相互作用均出现不同程度的荧光猝灭, 表明该类化合物是DNA嵌入剂.本研究对大黄酸衍生物的研究提供了实验与理论基础, 具有一定的指导意义.

3 实验部分

3.1 仪器与试剂

大黄酸(分析纯, 多点化学); 碘甲烷、溴乙烷、溴丙烷、1-溴丁烷、溴化苄等烷基化试剂均为分析纯, 购自于阿拉丁试剂公司; 甘氨酸乙酯盐酸盐、苯丙氨酸乙酯盐酸盐、亮氨酸甲酯盐酸盐和蛋氨酸甲酯盐酸盐均为分析纯, 购自于Adamas试剂公司.

WRS-1B型数字熔点仪(上海精密科学仪器有限公司); FT/IR-360型红外光谱仪(美国Nicolet公司); ZF-2型紫外分析仪(上海安亭电子仪器厂); Bruker Avance Ⅲ HD 400核磁共振仪(CDCl₃或DMSO-d₆为溶剂, TMS为内标, 美国布鲁克公司); AL104型分析天平(瑞士梅特勒); Synergy 2多功能酶标仪(美国伯腾仪器有限公司(BioTek); F-2500型荧光光谱仪(日立高新技术公司); 全自动旋光仪(鲁道夫Autopol V型); 柱色谱用硅胶(200~300目, 青岛海洋化工有限公司); IC₅₀浓度计算软件为Origin 9.0.

3.2 化合物的合成

3.3 体外抗肿瘤活性测试

存活率(%)=样品组平均OD值/对照组平均OD值×100%

将细胞以约10⁵个/mL的密度接种于96孔板, 每孔接种100 μL, 置CO₂培养箱中培养至对数生长期.然后按预设的浓度梯度加入待测样品, 每一梯度3个重复.对照组加入等体积的溶解样品用的溶剂.培养48 h后, 每孔加入20 μL的MTT (5 mg/mL), 然后置于37 ℃温育4 h.除去上清后, 每孔加入100 μL的DMSO, 振荡10 min溶解沉淀, 随后用酶标仪检测OD值, 波长490 nm.用下式求出样品一定浓度下的细胞存活率:

以细胞存活率对药物浓度对数作图, 按作图法求出每个样品的IC₅₀值.

3.4 化合物与DNA相互作用的荧光光谱研究

辅助材料(Supporting Information) 化合物4aa~4ed的¹H NMR谱图, 5aa~5ed的¹H NMR和¹³C NMR谱图, 以及6aa~6ed的¹H NMR谱图.这些材料可以免费从本刊网站(http://sioc-journal.cn/)上下载.

用pH7.4的Tris-HCl缓冲溶液配制化合物的待测溶液, 浓度为1×10^-5mol/L, 向此化合物中加入不同体积的ct-DNA溶液, 使DNA浓度分别为0、8、16、24、32和40 mmol/L, 在激发波长为260 nm时, 依次测试溶液的荧光发射光谱, 得到化合物与DNA相互作用的荧光光谱图.

3.2.4 化合物5aa~5ed的合成

2-(3, 5-二丙氧基-9, 10-蒽醌-2-羧酰氨基)乙酸(5ca):黄色固体, 产率91%. m.p. 120.1~121.0℃; ¹H NMR (DMSO-d₆, 400 MHz) δ: 9.22 (t, J=6.0 Hz, 1H), 8.17 (s, 1H), 7.89 (s, 1H), 7.69~7.75 (m, 2H), 7.53 (d, J=8.0 Hz, 1H), 4.17 (t, J=6.4 Hz, 2H), 4.10 (t, J=6.4 Hz, 2H), 3.96 (d, J=5.6 Hz, 2H), 1.75~1.87 (m, 4H), 1.07 (q, J=7.2 Hz, 6H); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 183.0, 180.6, 171.1, 164.7, 158.3, 158.3, 138.3, 134.3, 134.3, 134.1, 125.4, 123.7, 120.1, 118.2, 117.9, 116.8, 70.7, 70.4, 41.6, 22.1, 10.3. HRMS calcd for C₂₃H₂₂NO₇ [M-H]^- 424.1402, found 424.1397.

2-(4, 5-二丙氧基-9, 10-蒽醌-2-甲酰氨基)-4-甲基戊酸(5cb):黄色固体, 产率94%. m.p. 118.0~118.9℃; [α]_D²⁵-2.8 (c0.1, MeOH); ¹H NMR (DMSO-d₆, 400 MHz) δ: 9.06 (d, J=3.6 Hz, 1H), 8.22 (d, J=1.2 Hz, 1H), 7.89 (d, J=1.2 Hz, 1H), 7.75 (t, J=7.6 Hz, 1H), 7.71 (dd, J=1.2, 7.6 Hz, 1H), 7.54 (dd, J=1.2, 8.0 Hz, 1H), 4.48~4.56 (m, 1H), 4.18 (t, J=6.4 Hz, 2H), 4.11 (t, J=6.4 Hz, 2H), 1.78~1.86 (m, 4H), 1.62~1.76 (m, 3H), 1.08 (q, J=7.2 Hz, 6H), 0.95 (d, J=6.4 Hz, 3H), 0.91 (d, J=6.4 Hz, 3H); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 183.1, 180.7, 174.0, 164.7, 158.3, 158.2, 138.4, 134.3, 134.2, 125.5, 123.7, 120.1, 118.2, 118.1, 117.0, 70.7, 70.4, 51.1, 24.6, 23.0, 22.1, 21.2, 10.4, 10.3. HRMS calcd for C₂₇H₃₀NO₇ [M-H]^- 480.2028, found 480.2030.

2-(4, 5-二丁氧基-9, 10-蒽醌-2-羧酰氨基)乙酸(5da):黄色固体, 产率73%. m.p. 167.8~168.7℃; ¹H NMR (DMSO-d₆, 400 MHz) δ: 9.27 (t, J=6.0 Hz, 1H), 8.17 (dd, J=1.2, 6.0 Hz, 1H), 7.89 (dd, J=1.2, 7.2 Hz, 1H), 7.67~7.75 (m, 2H), 7.53 (dd, J=1.2, 7.6 Hz, 1H), 4.20 (t, J=6.4 Hz, 2H), 4.14 (t, J=6.4 Hz, 2H), 4.00 (d, J=6.0 Hz, 2H), 1.73~1.82 (m, 4H), 1.52~1.63 (m, 4H), 0.96~1.00 (m, 6H); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 183.0, 180.6, 171.0, 164.8, 158.3, 158.2, 138.2, 134.3, 134.2, 134.1, 125.6, 123.8, 120.1, 118.1, 117.9, 116.8, 68.9, 68.7, 41.4, 40.2, 39.9, 39.7, 39.5, 39.3, 39.1, 38.9, 30.7, 18.6, 13.7. HRMS calcd for C₂₅H₂₆NO₇ [M-H]^- 452.1715, found 452.1710.

2-(4, 5-二乙氧基-9, 10-蒽醌-2-甲酰氨基)-4-(甲硫基)丁酸(5bd):黄色固体, 产率80%. m.p. 102.0~102.5℃; [α]_D²⁵-18.4 (c0.1, MeOH); ¹H NMR (DMSO-d₆, 400 MHz) δ: 12.79 (s, 1H), 9.10 (d, J=7.6 Hz, 1H), 8.23 (s, 1H), 7.89 (s, 1H), 7.71~7.78 (m, 2H), 7.55 (d, J=7.6 Hz, 1H), 4.59 (q, J=8.0 Hz, 1H), 4.30 (q, J=7.2 Hz, 2H), 4.23 (q, J=7.2 Hz, 2H), 2.54~2.66 (m, 2H), 2.09~2.14 (m, 2H), 2.08 (s, 3H), 1.40~1.46 (m, 6H); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 183.0, 180.6, 173.2, 164.9, 158.1, 158.1, 138.4, 134.3, 134.3, 134.2, 125.3, 123.5, 120.1, 118.2, 118.1, 117.0, 65.0, 64.7, 51.8, 30.1, 14.6. HRMS calcd for C₂₄H₂₄NO₇S [M-H]^- 470.1279, found 470.1282.

2-(4, 5-二乙氧基-9, 10-蒽醌-2-羧酰氨基)-3-苯基丙酸(5bc):黄色固体, 产率74%. m.p. 213.6~214.0℃; [α]_D²⁵-78.8 (c0.1, MeOH); ¹H NMR (DMSO-d₆, 400 MHz) δ: 9.16 (d, J=8.0 Hz, 1H), 8.13 (s, 1H), 7.70-7.76 (m, 3H), 7.54 (d, J=8.0 Hz, 1H), 7.34 (d, J=7.2 Hz, 2H), 7.28 (t, J=7.6 Hz, 2H), 7.19 (t, J=7.2 Hz, 1H), 4.65~4.71 (m, 1H), 4.27 (q, J=6.8 Hz, 2H), 4.22 (q, J=6.8 Hz, 2H), 3.25 (dd, J=4.8, 13.6 Hz, 1H), 3.10 (q, J=10.8 Hz, 1H), 1.42 (q, J=7.2 Hz, 6H); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 183.0, 180.6, 172.9, 164.6, 158.1, 158.0, 138.4, 138.1, 134.3, 134.3, 134.1, 129.1, 128.2, 126.4, 125.3, 123.5, 120.1, 118.2, 118.0, 116.9, 64.9, 64.7, 54.4, 36.3, 14.6, 14.5. HRMS calcd for C₂₈H₂₄NO₇ [M-H]^- 486.1558, found 486.1561.

2-(4, 5-二丁氧基-9, 10-蒽醌-2-羧酰氨基)-3-苯基丙酸(5dc):黄色固体, 产率93%. m.p. 156.3~157.2℃; [α]_D²⁵-46.1 (c0.1, MeOH); ¹H NMR (DMSO-d₆, 400 MHz) δ: 9.11 (d, J=8.0 Hz, 1H), 8.10 (d, J=1.2 Hz, 1H), 7.68~7.76 (m, 3H), 7.53 (dd, J=1.2, 8.0 Hz, 1H), 7.33 (d, J=7.2 Hz, 2H), 7.27 (t, J=7.6 Hz, 2H), 7.18 (t, J=7.6 Hz, 1H), 4.64~4.69 (m, 1H), 4.18 (t, J=6.4 Hz, 2H), 4.13 (t, J=6.4 Hz, 2H), 3.25 (dd, J=4.4, 15.6 Hz, 1H), 3.10 (q, J=10.8 Hz, 1H), 1.72~1.82 (m, 4H), 1.51~1.62 (m, 4H), 0.95~1.00 (m, 6H); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 183.0, 180.6, 173.1, 164.6, 158.2, 158.1, 138.5, 138.3, 134.2, 134.1, 129.1, 128.2, 126.3, 125.5, 123.7, 120.1, 118.1, 118.0, 116.8, 68.9, 68.7, 54.7, 36.4, 30.7, 30.7, 18.7, 13.7. HRMS calcd for C₃₂H₃₂NO₇ [M-H]^- 542.2184, found 542.2185.

2-(4, 5-二甲氧基-9, 10-蒽醌-2-甲酰氨基)-4-(甲硫基)丁酸(5ad):黄色固体, 产率80%. m.p. 140.6~141.2 ℃; [α]_D²⁵-20.6 (c0.1, MeOH); ¹H NMR (DMSO-d₆, 400 MHz) δ: 8.90 (d, J=7.6 Hz, 1H), 8.18 (d, J=1.2 Hz, 1H), 7.89 (d, J=1.2 Hz, 1H), 7.77 (t, J=8.0 Hz, 1H), 7.71 (dd, J=1.2, 7.6 Hz, 1H), 7.55 (dd, J=1.2, 8.0 Hz, 1H), 4.49~4.54 (m, 1H), 3.99 (s, 3H), 3.93 (s, 3H), 2.54~2.60 (m, 2H), 2.07~2.14 (m, 2H), 2.06 (s, 3H); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 183.0, 181.0, 164.7, 158.8, 138.8, 134.5, 134.2, 134.1, 125.2, 123.4, 119.0, 118.2, 117.1, 117.0, 56.6, 56.4, 56.1, 52.5, 30.6, 30.2, 18.6, 14.6. HRMS calcd for C₂₂H₂₀NO₇S [M-H]^- 442.0966, found 442.0969.

2-(4, 5-二丙氧基-9, 10-蒽醌-2-羧酰氨基)-3-苯基丙酸(5cc):黄色固体, 产率96%. m.p. 115.6~116.3℃; [α]_D²⁵-13.0 (c0.1, MeOH); ¹H NMR (DMSO-d₆, 400 MHz) δ: 9.14 (d, J=8.4 Hz, 1H), 8.12 (d, J=1.6 Hz, 1H), 7.69~7.76 (m, 3H), 7.53 (dd, J=1.6, 8.0 Hz, 1H), 7.33 (d, J=6.8 Hz, 2H), 7.27 (t, J=7.6 Hz, 2H), 7.19 (t, J=7.2 Hz, 1H), 4.65~4.71 (m, 1H), 4.15 (t, J=6.4 Hz, 2H), 4.10 (t, J=6.4 Hz, 2H), 3.25 (dd, J=4.4, 14 Hz, 1H), 3.11 (q, J=10.8 Hz, 1H), 1.75~1.86 (m, 4H), 1.09 (d, J=7.6 Hz, 3H), 1.06 (d, J=7.2 Hz, 3H); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 183.0, 180.6, 173.0, 164.6, 158.3, 158.2, 138.4, 138.1, 134.3, 134.1, 129.1, 128.2, 126.4, 125.5, 123.6, 120.1, 118.1, 118.0, 116.9, 70.6, 70.4, 54.5, 36.3, 22.1, 22.0, 10.3. HRMS calcd for C₃₀H₂₈NO₇ [M-H]^- 514.1871, found 514.1873.

2-(4, 5-二甲氧基-9, 10-蒽醌-2-羧酰氨基)-3-苯基丙酸(5ac):黄色固体, 产率70%. m.p. 130.2~130.8℃; [α]_D²⁵-80.4 (c0.1, MeOH); ¹H NMR (DMSO-d₆, 400 MHz) δ: 12.85 (s, 1H), 9.19 (d, J=8.4 Hz, 1H), 8.14 (s, 1H), 7.70~7.78 (m, 3H), 7.54 (d, J=8.4 Hz, 1H), 7.34 (d, J=7.2 Hz, 2H), 7.29 (t, J=7.2 Hz 2H), 7.19 (t, J=7.2 Hz, 1H), 4.67~4.73 (m, 1H), 3.97 (s, 3H), 3.92 (s, 3H), 3.25 (dd, J=4.4, 13.6 Hz, 1H), 3.12 (t, J=10.8 Hz, 1H); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 182.9, 180.9, 172.9, 164.7, 158.7, 158.7, 138.5, 138.0, 134.4, 134.2, 134.1, 129.1, 128.2, 126.4, 125.2, 123.4, 119.0, 118.2, 117.0, 116.9, 56.5, 56.3, 54.3, 36.3. HRMS calcd for C₂₆H₂₀NO₇ [M-H]^- 458.1245, found 458.1249.

2-(4, 5-二丁氧基-9, 10-蒽醌-2-甲酰氨基)-4-甲基戊酸(5db):黄色固体, 产率89%. m.p. 73.5~74.1℃; [α]_D²⁵-3.1 (c0.1, MeOH); ¹H NMR (DMSO-d₆, 400 MHz) δ: 9.06 (d, J=8.0 Hz, 1H), 8.22 (d, J=1.6 Hz, 1H), 7.89 (d, J=1.2 Hz, 1H), 7.74 (t, J=8.0 Hz, 1H), 7.70 (dd, J=1.6, 8.0 Hz, 1H), 7.54 (dd, J=1.6, 8.0 Hz, 1H), 4.49~4.55 (m, 1H), 4.21 (t, J=6.4 Hz, 2H), 4.14 (t, J=6.4 Hz, 2H), 1.73~1.85 (m, 6H), 1.65~1.69 (m, 1H), 1.54~1.61 (m, 4H), 0.95~1.00 (m, 9H), 0.91 (d, J=6.0 Hz, 3H); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 183.1, 180.7, 173.9, 164.8, 158.2, 158.2, 138.4, 134.2, 134.1, 125.6, 123.8, 120.1, 118.2, 118.1, 117.0, 68.9, 68.7, 51.1, 30.8, 24.6, 22.9, 21.2, 18.7, 13.7. HRMS calcd for C₂₉H₃₄NO₇ [M-H]^- 508.2341, found 508.2343.

取化合物4 (1 mmol)溶于10 mL乙醇中, 滴加20 mL NaOH (1 mmol/L)溶液, 室温至70℃搅拌反应, TLC监测反应进程.待反应结束, 将反应液缓慢倒入20 mL HCl (1 mmol/L)溶液中, 搅拌的过程中会析出黄色固体, 直接将固体过滤, 并用大量的蒸馏水洗涤固体, 即得化合物5aa~5ed.

2-[4, 5-双(苄氧基)-9, 10-蒽醌-2-羧酰氨基]-3-苯基丙酸(5ec):黄色固体, 产率93%. m.p. 159.8~160.4℃; [α]_D²⁵-9.4 (c0.1, MeOH); ¹H NMR (DMSO-d₆, 400 MHz) δ: 8.79 (d, J=7.2 Hz, 1H), 8.07 (s, 1H), 7.95 (s, 1H), 7.69~7.76 (m, 2H), 7.60~7.66 (m, 5H), 7.33~7.42 (m, 6H), 7.29 (d, J=7.2 Hz, 2H), 7.20 (t, J=7.2 Hz, 2H), 7.12 (t, J=7.2 Hz, 1H), 5.34 (s, 2H), 5.31 (s, 2H), 4.57~4.63 (m, 1H), 3.11 (q, J=9.2 Hz, 2H); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 182.6, 180.7, 163.9, 157.6, 139.4, 139.0, 136.7, 136.6, 134.2, 134.1, 129.0, 128.2, 127.8, 127.5, 126.8, 126.7, 125.8, 125.3, 123.8, 120.5, 118.6, 117.1, 70.4, 70.2, 55.6, 37.1. HRMS calcd for C₃₈H₂₈NO₇ [M-H]^- 610.1871, found 610.1863.

2-[4, 5-双(苄氧基)-9, 10-蒽醌-2-羧酰氨基]乙酸(5ea):黄色固体, 产率85%. m.p. 152.8~153.6℃; ¹H NMR (DMSO-d₆, 400 MHz) δ: 9.25 (t, J=5.6 Hz, 1H), 8.22 (d, J=1.6 Hz, 1H), 8.04 (d, J=1.6 Hz, 1H), 7.73~7.79 (m 2H), 7.62~7.68 (m, 5H), 7.36~7.44 (m, 6H), 5.39 (s, 2H), 5.32 (s, 2H), 3.98 (d, J=5.6 Hz, 2H); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 182.8, 180.9, 171.3, 164.6, 157.8, 157.7, 138.5, 136.8, 136.7, 134.4, 134.4, 134.2, 128.5, 128.3, 128.3, 127.6, 127.6, 127.0, 126.8, 126.8, 125.6, 123.8, 120.5, 118.7, 118.4, 117.2, 70.4, 70.1, 41.7. HRMS calcd for C₃₁H₂₂NO₇ [M-H]^- 520.1402, found 520.1395.

2-[4, 5-双(苄氧基)-9, 10-蒽醌-2-甲酰氨基]-4-(甲硫基)丁酸(5ed):黄色固体, 产率90%. m.p. 113.2~113.7 ℃; [α]_D²⁵-13 (c0.1, MeOH); ¹H NMR (DMSO-d₆, 400 MHz) δ: 8.98 (d, J=7.6 Hz, 1H), 8.25 (d, J=1.2 Hz, 1H), 8.03 (d, J=1.2 Hz, 1H), 7.75~7.80 (m, 2H), 7.63~7.68 (m, 5H), 7.35~7.43 (m, 6H), 5.40 (s, 2H), 5.34 (s, 2H), 4.53~4.58 (m, 1H), 2.54~2.65 (m, 2H), 2.10~2.15 (m, 2H), 2.07 (s, 3H); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 182.9, 180.9, 173.4, 164.6, 157.8, 157.7, 138.7, 136.8, 136.7, 134.4, 134.3, 134.2, 128.3, 127.6, 127.6, 126.9, 126.8, 125.6, 123.9, 120.5, 118.7, 117.4, 70.4, 70.2, 52.3, 30.5, 30.2, 14.6. HRMS calcd for C₃₄H₂₈NO₇S [M-H]^- 594.1592, found 594.1588.

2-(4, 5-二甲氧基-9, 10-蒽醌-2-羧酰氨基)乙酸(5aa):黄色固体, 产率78%. m.p. 203.5~204.5 ℃; ¹H NMR (DMSO-d₆, 400 MHz) δ: 9.28 (t, J=6.0 Hz, 1H), 8.19 (d, J=1.6 Hz, 1H), 7.91 (d, J=1.6 Hz, 1H), 7.78 (t, J=8.0 Hz, 1H), 7.72 (dd, J=1.2, 8.0 Hz, 1H), 7.56 (dd, J=1.2, 8.4 Hz, 1H), 3.99 (s, 3H), 3.98(s, 2H), 3.93 (s, 3H); ¹³C NMR (DMSO-d₆, 100 MHz)δ: 182.7, 180.8, 172.2, 163.5, 158.7, 158.7, 139.6, 134.2, 133.9, 133.8, 124.5, 123.4, 119.0, 118.1, 116.8, 116.7, 56.5, 56.3, 44.4. HRMS calcd for C₁₉H₁₄NO₇ [M-H]^- 368.0776, found 368.0778.

2-[4, 5-双(苄氧基)-9, 10-蒽醌-2-甲酰氨基]-4-甲基戊酸(5eb):黄色固体, 产率80%. m.p. 175.9~176.3℃; [α]_D²⁵-3.1 (c0.1, MeOH); ¹H NMR (DMSO-d₆, 400 MHz) δ: 8.93 (d, J=7.6 Hz, 1H), 8.25 (d, J=1.6 Hz, 1H), 8.04 (d, J=1.2 Hz, 1H), 7.74~7.80 (m, 2H), 7.63~7.68 (m, 5H), 7.34~7.43 (m, 6H), 5.40 (s, 2H), 5.34 (s, 2H), 4.46~4.52 (m, 1H), 1.65~1.81 (m, 3H), 0.95 (d, J=6.0 Hz, 3H), 0.91 (d, J=6.0 Hz, 3H); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 182.9, 180.9, 164.4, 157.7, 157.6, 138.8, 136.8, 136.7, 134.4, 134.3, 134.2, 128.3, 127.6, 126.9, 126.8, 125.5, 123.9, 120.5, 118.7, 118.6, 117.4, 70.4, 70.2, 51.7, 24.6, 23.0, 21.3. HRMS calcd for C₃₅H₃₀NO₇ [M-H]^- 576.2028, found 576.2020.

2-(4, 5-二丁氧基-9, 10-蒽醌-2-甲酰氨基-4-甲硫基)丁酸(5dd):黄色固体, 产率92%. m.p. 147.0~147.9℃; [α]_D²⁵-19.1 (c0.1, MeOH); ¹H NMR (DMSO-d₆, 400 MHz) δ: 9.09 (d, J=7.6 Hz, 1H), 8.21 (d, J=0.8 Hz, 1H), 7.89 (s, 1H), 7.75 (t, J=7.6 Hz, 1H), 7.71 (dd, J=1.2, 7.6 Hz, 1H), 7.55 (dd, J=0.8, 8.0 Hz, 1H), 4.56~4.61 (m, 1H), 4.21 (t, J=6.4 Hz, 2H), 4.14 (t, J=6.4 Hz, 2H), 2.55~2.66 (m, 2H), 2.09~2.16 (m, 2H), 2.08 (s, 3H), 1.73~1.83 (m, 4H), 1.52~1.63 (m, 4H), 0.95~1.00 (m, 6H); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 183.1, 180.7, 173.2, 164.9, 158.2, 138.3, 134.3, 134.2, 125.6, 123.8, 120.1, 118.2, 118.1, 117.0, 68.9, 68.7, 51.9, 30.7, 30.1, 18.7, 14.6, 13.7. HRMS calcd for C₂₈H₃₂NO₇S [M-H]^- 526.1905, found 526.1908.

2-(3, 5-二丙氧基-9, 10-蒽醌-2-羧酰氨基)-4-(甲硫基)丁酸(5cd):黄色固体, 产率63%. m.p. 96.3~97.3℃; [α]_D²⁵-19.6 (c0.1, MeOH); ¹H NMR (DMSO-d₆, 400 MHz) δ: 9.08 (d, J=7.6 Hz, 1H), 8.22 (d, J=1.6 Hz, 1H), 7.89 (d, J=1.2 Hz, 1H), 7.75 (t, J=7.6 Hz, 1H), 7.71 (dd, J=1.2, 7.6 Hz, 1H), 7.55 (dd, J=1.2, 8.0 Hz, 1H), 4.55~4.61 (m, 1H), 4.18 (t, J=6.4 Hz, 2H), 4.11 (t, J=6.4 Hz, 2H), 2.54~2.64 (m, 2H), 2.09~2.14 (m, 2H), 2.08 (s, 3H), 1.76~1.87 (m, 4H), 1.10 (d, J=7.2 Hz, 3H), 1.06 (d, J=7.6 Hz, 3H); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 183.1, 180.7, 173.2, 164.9, 158.3, 158.2, 138.4, 134.3, 134.2, 125.5, 123.7, 120.1, 118.2, 118.1, 117.0, 70.7, 70.4, 51.9, 30.1, 22.1, 14.6, 10.4, 10.3. HRMS calcd for C₂₆H₂₈NO₇S [M-H]^- 498.1592, found 498.1595.

2-(4, 5-二乙氧基-9, 10-蒽醌-2-羧酰氨基)乙酸(5ba):黄色固体, 产率85%. m.p. 230.9~231.7℃; ¹H NMR (DMSO-d₆, 400 MHz) δ: 12.70 (s, 1H), 9.27 (t, J=5.6 Hz, 1H), 8.18 (d, J=1.6 Hz, 1H), 7.89 (d, J=1.6 Hz, 1H), 7.74 (t, J=7.6 Hz, 1H), 7.71 (dd, J=1.6, 7.6 Hz, 1H), 7.54 (dd, J=1.6, 7.6 Hz, 1H), 4.28 (q, J=7.2 Hz, 2H), 4.22 (q, J=7.2 Hz, 2H), 3.98 (d, J=6.0 Hz, 2H), 1.44 (t, J=7.2 Hz, 3H), 1.41 (t, J=7.2 Hz, 3H); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 183.0, 180.6, 171.0, 164.8, 158.2, 158.1, 138.2, 134.3, 134.1, 125.3, 123.5, 120.1, 118.2, 117.9, 116.8, 65.0, 64.7, 41.4, 14.6. HRMS calcd for C₂₁H₁₈NO₇ [M-H]^- 396.1089, found 396.1085.

2-(4, 5-二乙氧基-9, 10-蒽醌-2-甲酰氨基)-4-甲基戊酸(5bb):黄色固体, 产率87%. m.p. 108.5~109.5℃; [α]_D²⁵-0.9 (c0.1, MeOH); ¹H NMR (DMSO-d₆, 400 MHz) δ: 12.68 (s, 1H), 9.07 (d, J=7.2 Hz, 1H), 8.23 (s, 1H), 7.89 (s, 1H), 7.70~7.76 (m, 2H), 7.54 (d, J=7.6 Hz, 1H), 4.51 (t, J=10.8 Hz, 1H), 4.29 (q, J=7.2 Hz, 2H), 4.22 (q, J=7.2 Hz, 2H), 1.81 (t, J=10.8 Hz, 1H), 1.64~1.72 (m, 2H), 1.42 (q, J=6.4 Hz, 6H), 0.95 (d, J=5.6 Hz, 3H), 0.90 (d, J=5.6 Hz, 3H); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 183.0, 180.6, 173.9, 164.8, 158.2, 158.1, 138.4, 134.3, 134.3, 134.2, 125.3, 123.5, 120.1, 118.2, 117.1, 65.0, 64.7, 51.1, 24.5, 22.9, 21.2, 14.6. HRMS calcd for C₂₅H₂₆NO₇ [M-H]^- 452.1715, found 452.1709.

2-(4, 5-二甲氧基-9, 10-蒽醌-2-甲酰氨基)-4-甲基戊酸(5ab):黄色固体, 产率79%. m.p. 127.5~128.5℃; [α]_D²⁵-5.1 (c0.1, MeOH); ¹H NMR (DMSO-d₆, 400 MHz) δ: 12.72 (s, 1H), 9.08 (d, J=8.0 Hz, 1H), 8.23 (s, 1H), 7.90 (s, 1H), 7.78 (t, J=7.6 Hz, 1H), 7.72 (d, J=7.2 Hz, 1H), 7.56 (d, J=8.0 Hz, 1H), 4.49~4.55 (m, 1H), 4.00 (s, 3H), 3.93 (s, 3H), 1.79~1.85 (m, 1H), 1.65~1.76 (m, 2H), 0.95 (d, J=6.0 Hz, 3H), 0.91 (d, J=6.0 Hz, 3H); ¹³C NMR (DMSO-d₆, 100 MHz) δ: 182.9, 180.9, 173.9, 164.7, 158.8, 138.6, 134.4, 134.2, 134.1, 125.3, 123.4, 119.0, 118.2, 117.1, 117.0, 109.6, 56.5, 56.4, 51.1, 24.5, 22.9, 21.2. HRMS calcd for C₂₃H₂₂NO₇ [M-H]^- 424.1402, found 424.1404.

3.2.3 中间体4aa~4ed的合成

2-(3, 5-二丙氧基-9, 10-蒽醌-2-羧酰氨基)-4-(甲硫基)丁酸甲酯(4cd):黄色固体, 产率77%. m.p. 143.6~144.5℃; ¹H NMR (CDCl₃, 400 MHz) δ: 8.09 (d, J=1.6 Hz, 1H), 7.85 (d, J=1.6 Hz, 1H), 7.82 (dd, J=0.8, 7.6 Hz, 1H), 7.62 (t, J=8.0 Hz, 1H), 7.30 (dd, J=0.8, 8.4 Hz, 1H), 7.25 (d, J=7.6 Hz, 1H), 4.94~4.99 (m, 1H), 4.16 (t, J=6.4 Hz, 2H), 4.11 (t, J=6.4 Hz, 2H), 3.82 (s, 3H), 2.63 (t, J=7.2 Hz, 2H), 2.17~2.35 (m, 2H), 2.15 (s, 3H), 1.90~1.99 (m, 4H), 1.10~1.15 (m, 6H).

2-(4, 5-二甲氧基-9, 10-蒽醌-2-甲酰氨基)-3-苯基丙酸乙酯(4ac):黄色固体, 产率73%. m.p. 171.6~172.5℃; ¹H NMR (CDCl₃, 400 MHz) δ: 8.00 (d, J=1.6 Hz, 1H), 7.83 (dd, J=1.2, 8.0 Hz, 1H), 7.80 (d, J=1.6 Hz, 1H), 7.66 (t, J=8.0 Hz, 1H), 7.25~7.35 (m, 4H), 7.18~7.21 (m, 2H), 6.89 (d, J=7.6 Hz, 1H), 5.05~5.10 (m, 1H), 4.24 (q, J=7.2 Hz, 2H), 4.04 (s, 3H), 4.01 (s, 3H), 3.23~3.34 (m, 2H), 1.29 (t, J=7.2 Hz, 3H).

2-(4, 5-二丁氧基-9, 10-蒽醌-2-甲酰氨基)-4-甲基戊酸甲酯(4db):黄色固体, 产率82%. m.p. 73.1~73.9℃; ¹H NMR (CDCl₃, 400 MHz) δ: 8.05 (d, J=1.6 Hz, 1H), 7.86 (d, J=1.6 Hz, 1H), 7.82 (dd, J=0.8, 7.6 Hz, 1H), 7.62 (t, J=8.0 Hz, 1H), 7.29 (d, J=8.4 Hz, 1H), 6.77 (d, J=8.4 Hz, 1H), 4.85~4.90 (m, 1H), 4.20 (t, J=6.4 Hz, 2H), 4.15 (t, J=6.4 Hz, 2H), 3.79 (s, 3H), 1.87~1.94 (m, 4H), 1.70~1.81 (m, 3H), 1.55~1.66 (m, 4H), 0.99~1.04 (m, 12H).

2-(4, 5-二乙氧基-9, 10-蒽醌-2-甲酰氨基)-3-苯基丙酸乙酯(4bc):黄色固体, 产率92%. m.p. 137.5~138.1℃; ¹H NMR (CDCl₃, 400 MHz) δ: 7.99 (d, J=1.6 Hz, 1H), 7.83 (dd, J=1.2, 7.6 Hz, 1H), 7.78 (d, J=1.6 Hz, 1H), 7.62 (t, J=8.0 Hz, 1H), 7.27~7.35 (m, 4H), 7.18~7.20 (m, 2H), 6.81 (d, J=7.6 Hz, 1H), 5.04~5.09 (m, 1H), 4.21~4.31 (m, 6H), 3.22~3.33 (m, 2H), 1.55 (t, J=7.2 Hz, 6H), 1.28 (t, J=7.2 Hz, 3H).

取化合物3 (1.56 g, 5 mmol)、4-N, N-二甲氨基吡啶(DMAP, 1.22 g, 10 mmol)、1-(3-二甲氨基丙基)-3-乙基碳二亚胺盐酸盐(EDCI, 1.44 g, 7.5 mmol)和L-氨基酸酯盐酸盐(6 mmol)混合, 加入100 mL二氯甲烷, 室温搅拌反应, TLC监测反应进程.待反应结束后, 将反应液缓慢倒入150 mL HCl (0.1 mol/L)溶液中, 并用二氯甲烷萃取三次, 合并有机相, 无水硫酸钠干燥, 浓缩.经柱层析分离[V(石油醚):V(乙酸乙酯)=2:1]纯化得黄色液体, 减压浓缩至干得目标产物.

2-(4, 5-二甲氧基-9, 10-蒽醌-2-甲酰氨基)-4-甲基戊酸甲酯(4ab):黄色固体, 产率89%. m.p. 106.3~107.3℃; ¹H NMR (CDCl₃, 400 MHz) δ: 8.09 (d, J=1.6 Hz, 1H), 7.89 (d, J=1.6 Hz, 1H), 7.85 (dd, J=1.2, 7.6 Hz, 1H), 7.67 (t, J=8.0 Hz, 1H), 7.33 (dd, J=0.8, 8.8 Hz, 1H), 6.92 (d, J=8.4 Hz, 1H), 4.85~4.91 (m, 1H), 4.05 (s, 3H), 4.02 (s, 3H), 3.80 (s, 3H), 1.72~1.82 (m, 3H), 1.02 (d, J=2.8 Hz, 3H), 1.01 (d, J=3.2 Hz, 3H).

2-(4, 5-二乙氧基-9, 10-蒽醌-2-甲酰氨基)-4-甲基戊酸甲酯(4bb):黄色固体, 产率83%. m.p. 149.6~150.4℃; ¹H NMR (CDCl₃, 400 MHz) δ: 8.07 (d, J=1.6 Hz, 1H), 7.87 (d, J=1.6 Hz, 1H), 7.84 (dd, J=0.8, 7.6 Hz, 1H), 7.63 (t, J=8.0 Hz, 1H), 7.32 (dd, J=0.8, 8.4 Hz, 1H), 6.83 (d, J=8.0 Hz, 1H), 4.85~4.90 (m, 1H), 4.30 (q, J=6.8 Hz, 2H), 4.26 (q, J=7.2 Hz, 2H), 3.79 (s, 3H), 1.71~1.81 (m, 3H), 1.56 (t, J=7.2 Hz, 6H), 1.02 (d, J=3.2 Hz, 3H), 1.00 (d, J=2.8 Hz, 3H).

2-(3, 5-二丙氧基-9, 10-蒽醌-2-羧酰氨基)-4-甲基戊酸甲酯(4cb):黄色固体, 产率91%. m.p. 141.5~142.1℃; ¹H NMR (CDCl₃, 400 MHz) δ: 8.06 (s, 1H), 7.86 (s, 1H), 7.82 (d, J=7.2 Hz, 1H), 7.62 (t, J=8.0 Hz, 1H), 7.30 (d, J=8.0 Hz, 1H), 6.80 (d, J=8.0 Hz, 1H), 4.85~4.90 (m, 1H), 4.16 (t, J=6.4 Hz, 2H), 4.11 (t, J=6.4 Hz, 2H), 3.79 (s, 3H), 1.90~1.99 (m, 4H), 1.71~1.81 (m, 3H), 1.13 (q, J=7.2 Hz, 6H), 1.02 (d, J=3.2 Hz, 3H), 1.00 (d, J=2.8 Hz, 3H).

2-[4, 5-双(苄氧基)-9, 10-蒽醌-2-甲酰氨基]-4-甲基戊酸甲酯(4eb):黄色固体, 产率82%. m.p. 85.7~86.5℃; ¹H NMR (CDCl₃, 400 MHz) δ: 8.11 (d, J=1.6 Hz, 1H), 7.94 (d, J=1.6 Hz, 1H), 7.87 (dd, J=1.2, 7.6 Hz, 1H), 7.60~7.66 (m, 5H), 7.33~7.42 (m, 7H), 6.82 (d, J=8.4 Hz, 1H), 5.35 (s, 2H), 5.31 (s, 2H), 4.85~4.90 (m, 1H), 3.80 (s, 3H), 1.71~1.81 (m, 3H), 1.02 (d, J=2.0 Hz, 3H), 1.00 (d, J=2.0 Hz, 3H).

2-[4, 5-双(苄氧基)-9, 10-蒽醌-2-甲酰氨基]-4-(甲硫基)丁酸甲酯(4ed):黄色固体, 产率63%. m.p. 125.4~126.2℃; ¹H NMR (CDCl₃, 400 MHz) δ: 8.14 (d, J=1.6 Hz, 1H), 7.95 (d, J=1.6 Hz, 1H), 7.87 (dd, J=0.8, 7.6 Hz, 1H), 7.61~7.67 (m, 5H), 7.33~7.42 (m, 7H), 7.23 (d, J=7.6 Hz, 1H), 5.37 (s, 2H), 5.32 (s, 2H), 4.94~4.99 (m, 1H), 3.83 (s, 3H), 2.63 (t, J=7.6 Hz, 2H), 2.28~2.36 (m, 1H), 2.16~2.22 (m, 1H), 2.15 (s, 3H).

2-(4, 5-二丁氧基-9, 10-蒽醌-2-羧酰氨基)乙酸乙酯(4da):黄色固体, 产率87%. m.p. 139.1~140.0℃; ¹H NMR (CDCl₃, 400 MHz) δ: 8.09 (d, J=1.6 Hz, 1H), 7.84 (d, J=1.6 Hz, 1H), 7.78 (dd, J=1.2, 7.6 Hz, 1H), 7.59 (t, J=8.0 Hz, 1H), 7.28 (dd, J=1.2, 8.0 Hz, 1H), 7.15 (t, J=5.2 Hz, 1H), 4.24~4.30 (m, 4H), 4.18 (t, J=6.4 Hz, 2H), 4.13 (t, J=6.4 Hz, 2H), 1.86~1.93 (m, 4H), 1.57~1.63 (m, 4H), 1.32 (t, J=7.2 Hz, 3H), 0.99~1.03 (m, 6H).

2-(4, 5-二乙氧基-9, 10-蒽醌-2-甲酰氨基)-4-(甲硫基)丁酸甲酯(4bd):黄色固体, 产率84%. m.p. 168.6~169.2℃; ¹H NMR (CDCl₃, 400 MHz) δ: 8.11 (d, J=1.6 Hz, 1H), 7.86 (d, J=1.6 Hz, 1H), 7.83 (dd, J=0.8, 7.6 Hz, 1H), 7.62 (t, J=8.0 Hz, 1H), 7.31 (dd, J=0.8, 8.4 Hz, 1H), 7.25 (s, 1H), 4.94~4.99 (m, 1H), 4.31 (q, J=7.2 Hz, 2H), 4.25 (q, J=7.2 Hz, 2H), 3.82 (s, 3H), 2.63 (t, J=7.2 Hz, 2H), 2.17~2.36 (m, 2H), 2.15 (s, 3H), 1.56 (t, J=6.8 Hz, 3H), 1.55 (t, J=6.8 Hz, 3H).

2-(4, 5-二丁氧基-9, 10-蒽醌-2-甲酰氨基)-3-苯基丙酸乙酯(4dc):黄色固体, 产率86%. m.p. 135.4~136.3℃; ¹H NMR (CDCl₃, 400 MHz) δ: 7.97 (d, J=1.6 Hz, 1H), 7.81 (dd, J=1.2, 7.6 Hz, 1H), 7.77 (d, J=1.6 Hz, 1H), 7.61 (t, J=8.0 Hz, 1H), 7.27~7.35 (m, 4H), 7.17~7.20 (m, 2H), 6.79 (d, J=7.6 Hz, 1H), 5.07 (q, J=6.0 Hz, 1H), 4.23 (q, J=7.2 Hz, 2H), 4.18 (t, J=6.4 Hz, 2H), 4.14 (t, J=6.4 Hz, 2H), 3.22~3.33 (m, 2H), 1.86~1.94 (m, 4H), 1.57~1.64 (m, 4H), 1.28 (t, J=7.2 Hz, 3H), 0.99~1.03 (m, 6H).

2-(4, 5-二丙氧基-9, 10-蒽醌-2-羧酰氨基)乙酸乙酯(4ca):黄色固体, 产率88%. m.p. 130.8~131.7℃; ¹H NMR (CDCl₃, 400 MHz) δ: 8.09 (d, J=1.2 Hz, 1H), 7.84 (d, J=1.2 Hz, 1H), 7.80 (dd, J=0.8, 7.6 Hz, 1H), 7.61 (t, J=8.0 Hz, 1H), 7.29 (d, J=8.0 Hz, 1H), 7.03 (t, J=4.8 Hz, 1H), 4.25~4.31 (m, 4H), 4.15 (t, J=6.4 Hz, 2H), 4.10 (t, J=6.4 Hz, 2H), 1.90~1.98 (m, 4H), 1.33 (t, J=7.2 Hz, 3H), 1.10~1.15 (m, 6H).

2-(4, 5-二甲氧基-9, 10-蒽醌-2-甲酰氨基)-4-(甲硫基)丁酸甲酯(4ad):黄色固体, 产率78%. m.p. 68.8~69.6℃; ¹H NMR (CDCl₃, 400 MHz) δ: 8.11 (d, J=1.6 Hz, 1H), 7.86 (d, J=1.6 Hz, 1H), 7.83 (dd, J=1.2, 7.6 Hz, 1H), 7.66 (t, J=8.0 Hz, 1H), 7.38 (d, J=7.6, Hz, 1H), 7.32 (dd, J=0.8, 8.4 Hz, 1H), 4.95~5.00 (m, 1H), 4.05 (s, 3H), 4.01 (s, 3H), 3.83 (s, 3H), 2.65 (t, J=7.2 Hz, 2H), 2.17~2.35 (m, 2H), 2.15 (s, 3H).

2-(4, 5-二乙氧基-9, 10-蒽醌-2-羧酰氨基)乙酸乙酯(4ba):黄色固体, 产率84%. m.p. 174.8~175.3℃; ¹H NMR (CDCl₃, 400 MHz) δ: 8.10 (d, J=1.6 Hz, 1H), 7.86 (d, J=1.6 Hz, 1H), 7.82 (dd, J=1.2, 7.6 Hz, 1H), 7.62 (t, J=8.0 Hz, 1H), 7.31 (dd, J=0.8, 8.4 Hz, 1H), 7.02 (t, J=4.8 Hz, 1H), 4.22~4.33 (m, 8H), 1.55 (t, J=7.2 Hz, 6H), 1.33 (t, J=7.2 Hz, 3H).

2-[4, 5-双(苄氧基)-9, 10-蒽醌-2-甲酰氨基]-3-苯基丙酸乙酯(4ec):黄色固体, 产率90%. m.p. 92.5~93.3℃; ¹H NMR (CDCl₃, 400 MHz) δ: 8.03 (d, J=1.6 Hz, 1H), 7.87 (dd, J=1.2, 8.0 Hz, 2H), 7.60~7.66 (m, 5H), 7.28~7.42 (m, 10H), 7.17~7.19 (m, 1H), 6.77 (d, J=8.0 Hz, 1H), 5.36 (s, 2H), 5.32 (s, 2H), 5.04~5.09 (m, 1H), 4.24 (q, J=7.2 Hz, 2H), 3.23~3.34 (m, 2H), 1.29 (t, J=7.2 Hz, 3H).

2-(3, 5-二丙氧基-9, 10-蒽醌-2-羧酰氨基)-3-苯基丙酸乙酯(4cc):黄色固体, 产率89%. m.p. 138.2~139.2℃; ¹H NMR (CDCl₃, 400 MHz) δ: 7.97 (d, J=1.6 Hz, 1H), 7.81 (dd, J=0.8, 7.6 Hz, 1H), 7.77 (d, J=1.6 Hz, 1H), 7.61 (t, J=8.0 Hz, 1H), 7.27~7.35 (m, 4H), 7.17~7.20 (m, 2H), 6.79 (d, J=7.6 Hz, 1H), 5.04~5.09 (m, 1H), 4.24 (q, J=7.2 Hz, 2H), 4.14 (t, J=6.4 Hz, 2H), 4.10 (t, J=1.6 Hz, 2H), 3.22~3.33 (m, 2H), 1.90~1.99 (m, 4H), 1.28 (t, J=7.2 Hz, 3H), 1.10~1.15 (m, 6H).

2-[4, 5-双(苄氧基)-9, 10-蒽醌-2-羧酰氨基]乙酸乙酯(4ea):黄色固体, 产率88%. m.p. 144.6~145.3℃; ¹H NMR (CDCl₃, 400 MHz) δ: 8.11 (d, J=1.6 Hz, 1H), 7.92 (d, J=1.6 Hz, 1H), 7.83 (dd, J=1.2, 7.6 Hz, 1H), 7.57~7.65 (m, 5H), 7.37~7.41 (m, 4H), 7.32~7.35 (m, 3H), 7.04 (t, J=5.2 Hz, 1H), 5.33 (s, 2H), 5.29 (s, 2H), 4.24~4.30 (m, 4H), 1.33 (t, J=7.2 Hz, 3H).

2-(4, 5-二甲氧基-9, 10-蒽醌-2-羧酰氨基)乙酸乙酯(4aa):黄色固体, 产率84%. m.p. 235.8~236.7℃; ¹H NMR (CDCl₃, 400 MHz) δ: 8.10 (d, J=1.6 Hz, 1H), 7.87 (d, J=1.6 Hz, 1H), 7.82 (dd, J=7.6, 1.2 Hz, 1H), 7.65 (t, J=8.0 Hz, 1H), 7.31 (dd, J=0.8, 8.4 Hz, 1H), 7.07 (t, J=4.8 Hz, 1H), 4.25~4.30 (m, 4H), 4.06 (s, 3H), 4.01 (s, 3H), 1.33 (t, J=7.2 Hz, 3H).

2-(4, 5-二丁氧基-9, 10-蒽醌-2-甲酰氨基)-4-(甲硫基)丁酸甲酯(4dd):黄色固体, 产率75%. m.p. 123.6~124.5℃; ¹H NMR (CDCl₃, 400 MHz) δ: 8.09 (d, J=1.2 Hz, 1H), 7.85 (d, J=1.2 Hz, 1H), 7.82 (d, J=7.2 Hz, 1H), 7.62 (t, J=8.0 Hz, 1H), 7.30 (d, J=8.0 Hz, 1H), 7.23 (d, J=7.6 Hz, 1H), 4.94~4.99 (m, 1H), 4.20 (t, J=6.4 Hz, 2H), 4.15 (t, J=6.4 Hz, 2H), 3.82 (s, 3H), 2.63 (t, J=7.2 Hz, 2H), 2.17~2.34 (m, 2H), 2.15 (s, 3H), 1.87~1.94 (m, 3H), 1.58~1.64 (m, 4H), 0.99~1.04 (m, 6H).

3.2.2 中间体3a~3e的合成

在100 mL的圆底烧瓶中加入化合物2 (1 mmol, 312 mg)、DMF 50 mL、NaH (5 mmol, 200 mg)和碘/溴代烷烃12 mmol.反应液加热到90~130 ℃下反应至原料点消失(TLC监测).反应结束后, 将反应液倒入1 mol/L的30 mL HCl水溶液中, 并用二氯甲烷萃取, 收集有机相, 减压旋蒸得到的粗产物, 经柱层析分离[洗脱剂为V(石油醚):V(乙酸乙酯)=5:1]得到粗产物.将粗产物直接加入到20 mL 1 mol/L的NaOH水溶液中, 室温至60 ℃搅拌, TLC监测反应进程待水解完毕后, 用1 mol/L HCl水溶液调节pH至5~6, 待固体析出, 抽滤, 并用大量的去离子水洗涤固体, 将固体充分干燥即得到黄色固体3a~3e, 产率46~86%.直接投入下步反应.

3.2.6 化合物6aa~6ed的合成

2-(4, 5-二丁氧基-9, 10-蒽醌-2-甲酰氨基-4-甲硫基)丁酸钠(6dd):黄色固体. ¹H NMR (DMSO-d₆, 400 MHz, 40 ℃) δ: 8.48 (dd, J=3.2, 7.2 Hz, 1H), 8.02~8.04 (m, 1H), 7.87~7.88 (m, 1H), 7.66~7.74 (m, 2H), 7.52 (d, J=8.0 Hz, 1H), 4.09~4.22 (m, 5H), 2.62~2.89 (m, 2H), 2.49 (s, 3H), 2.04~2.25 (m, 2H), 1.74~1.82 (m, 4H), 1.54~1.60 (m, 4H), 0.96~1.00 (m, 6H).

2-(4, 5-二丙氧基-9, 10-蒽醌-2-甲酰氨基)-4-甲基戊酸钠(6cb):黄色固体. ¹H NMR (DMSO-d₆, 400 MHz, 30 ℃) δ: 8.39 (d, J=8.0 Hz, 1H), 8.05 (s, 1H), 7.87 (s, 1H), 7.72 (t, J=8.0 Hz, 1H), 7.67 (d, J=6.8 Hz, 1H), 7.52 (d, J=8.0 Hz, 1H), 4.15~4.17 (m, 3H), 4.10 (t, J=6.0 Hz, 2H), 1.77~1.83 (m, 4H), 1.56~1.68 (m, 3H), 1.05~1.09 (m, 6H), 0.88~0.90 (m, 6H).

2-(4, 5-二丁氧基-9, 10-蒽醌-2-羧酰氨基)-3-苯基丙酸钠(6dc):黄色固体. ¹H NMR (DMSO-d₆, 400 MHz, 40 ℃) δ: 8.19 (d, J=6.4 Hz, 1H), 7.88 (s, 1H), 7.66~7.74 (m, 3H), 7.52 (d, J=7.6 Hz, 1H), 7.08~7.20 (m, 5H), 4.12~4.22 (m, 5H), 3.22 (d, J=4.8 Hz, 1H), 3.08 (dd, J=6.4, 13.2 Hz, 1H), 1.73~1.81 (m, 4H), 1.53~1.59 (m, 4H), 0.95~0.99 (m, 6H).

2-(4, 5-二乙氧基-9, 10-蒽醌-2-羧酰氨基)乙酸钠(6ba):黄色固体. ¹H NMR (DMSO-d₆, 400 MHz) δ: 8.65 (t, J=4.8 Hz, 1H), 8.01 (d, J=1.6 Hz, 1H), 7.90 (d, J=1.2 Hz, 1H), 7.72 (t, J=8.0 Hz, 1H), 7.64 (d, J=8.0 Hz, 1H), 7.53 (d, J=8.0 Hz, 1H), 4.20-4.29 (m, 4H), 3.57 (d, J=4.8 Hz, 2H), 1.41~1.44 (m, 6H).

2-(4, 5-二乙氧基-9, 10-蒽醌-2-甲酰氨基)-4-(甲硫基)丁酸钠(6bd):黄色固体. ¹H NMR (DMSO-d₆, 400 MHz, 30 ℃) δ: 8.46 (d, J=6.8 Hz, 1H), 8.02 (d, J=1.2 Hz, 1H), 7.86 (d, J=1.2 Hz, 1H), 7.73 (t, J=8.0 Hz, 1H), 7.67 (dd, J=1.2, 7.6 Hz, 1H), 7.53 (dd, J=5.2, 8.4 Hz, 1H), 4.28 (q, J=6.8 Hz, 2H), 4.22 (q, J=6.8 Hz, 2H), 4.07~4.11 (m, 1H), 2.42~2.48 (m, 2H), 1.93~2.12 (m, 2H), 2.02 (s, 3H), 1.40~1.45 (m, 6H).

2-(4, 5-二乙氧基-9, 10-蒽醌-2-羧酰氨基)-3-苯基丙酸钠(6bc):黄色固体. ¹H NMR (DMSO-d₆, 400 MHz, 40 ℃) δ: 8.25 (d, J=6.8 Hz, 1H), 7.88 (d, J=1.6 Hz, 1H), 7.66~7.43 (m, 3H), 7.52 (d, J=8.0 Hz, 1H), 7.15~7.22 (m, 4H), 7.10 (t, J=7.2 Hz, 1H), 4.20~4.29 (m, 5H), 3.22 (d, J=4.8 Hz, 1H), 3.07 (dd, J=6.8, 13.2 Hz, 1H), 1.40~1.44 (m, 6H).

2-(4, 5-双(苄氧基)-9, 10-蒽醌-2-羧酰氨基)-3-苯基丙酸钠(6ec):黄色固体. ¹H NMR (DMSO-d₆, 400 MHz, 40 ℃) δ: 8.41 (d, J=5.2 Hz, 1H), 7.95 (d, J=1.2 Hz, 1H), 7.90 (d, J=1.2 Hz, 1H), 7.63~7.77 (m, 7H), 7.36~7.44 (m, 6H), 7.09~7.22 (m, 5H), 5.37 (s, 2H), 5.34 (s, 2H), 4.22~4.27 (m, 1H), 3.23 (d, J=4.4 Hz, 1H), 3.10 (dd, J=6.4, 13.2 Hz, 1H).

2-(4, 5-双(苄氧基)-9, 10-蒽醌-2-羧酰氨基)乙酸钠(6ea):黄色固体. ¹H NMR (DMSO-d₆, 400 MHz, 30 ℃) δ: 8.91 (t, J=5.2 Hz, 1H), 8.11 (d, J=1.6 Hz, 1H), 8.02 (d, J=1.6 Hz, 1H), 7.61~7.74 (m, 7H), 7.37~7.45 (m, 6H), 5.37 (s, 2H), 5.34 (s, 2H), 3.61 (d, J=5.2 Hz, 2H).

2-(4, 5-二丁氧基-9, 10-蒽醌-2-羧酰氨基)乙酸钠(6da):黄色固体. ¹H NMR (DMSO-d₆, 400 MHz, 40 ℃) δ: 8.37 (t, J=4.8 Hz, 1H), 8.03 (d, J=1.6 Hz, 1H), 7.86 (d, J=1.6 Hz, 1H), 7.72 (t, J=8.0 Hz, 1H), 7.67 (dd, J=1.2, 8.0 Hz, 1H), 7.52 (dd, J=1.2, 8.0, Hz, 1H), 4.20 (t, J=6.4 Hz, 2H), 4.14 (t, J=6.4 Hz, 2H), 3.56 (d, J=4.8 Hz, 2H), 1.74~1.82 (m, 4H), 1.52~1.62 (m, 4H), 0.98 (t, J=7.2 Hz, 3H), 0.97 (t, J=7.2 Hz, 3H).

2-(4, 5-双(苄氧基)-9, 10-蒽醌-2-甲酰氨基)-4-甲基戊酸钠(6eb):黄色固体. ¹H NMR (DMSO-d₆, 400 MHz, 40 ℃) δ: 8.51 (d, J=5.2 Hz, 1H), 8.06 (s, 2H), 7.63~7.78 (m, 7H), 7.36~7.44 (m, 6H), 5.40 (s, 2H), 5.34 (s, 2H), 4.14~4.19 (m, 1H), 1.60~1.74 (m, 3H), 0.91 (d, J=6.0 Hz, 6H).

2-(4, 5-二甲氧基-9, 10-蒽醌-2-羧酰氨基)乙酸钠(6aa):黄色固体. ¹H NMR (DMSO-d₆, 400 MHz) δ: 8.91 (t, J=5.2 Hz, 1H), 7.95 (dd, J=1.6, 8.0 Hz, 2H), 7.70 (t, J=8.0 Hz, 1H), 7.58 (dd, J=1.2, 8.0 Hz, 1H), 7.52 (dd, J=1.2, 8.0 Hz, 1H), 3.94 (s, 3H), 3.93 (s, 3H), 3.58 (d, J=5.2 Hz, 2H).

2-(3, 5-二丙氧基-9, 10-蒽醌-2-羧酰氨基)乙酸钠(6ca):黄色固体. ¹H NMR (DMSO-d₆, 400 MHz, 40 ℃) δ: 8.32 (t, J=4.8 Hz, 1H), 8.04 (d, J=1.2 Hz, 1H), 7.86 (d, J=1.6 Hz, 1H), 7.73 (t, J=8.0 Hz, 1H), 7.68 (dd, J=1.2, 7.2 Hz, 1H), 7.53 (dd, J=1.2, 8.0 Hz, 1H), 4.17 (t, J=6.4 Hz, 2H), 4.11 (t, J=6.4 Hz, 2H), 3.55 (d, J=6.4 Hz, 2H), 1.78~1.85 (m, 4H), 1.06~1.10 (m, 6H).

2-(4, 5-二甲氧基-9, 10-蒽醌-2-甲酰氨基)-4-(甲硫基)丁酸钠(6ad):黄色固体. ¹H NMR (DMSO-d₆, 400 MHz, 40 ℃) δ: 8.47 (d, J=6.8 Hz, 1H), 8.01 (d, J=1.2 Hz, 1H), 7.88 (d, J=1.2 Hz, 1H), 7.75 (t, J=8.0 Hz, 1H), 7.67 (d, J=7.2 Hz, 1H), 7.54 (d, J=8.4 Hz, 1H), 4.08~4.13 (m, 1H), 3.98 (s, 3H), 3.93 (s, 3H), 2.44~2.57 (m, 2H), 1.93~2.12 (m, 2H), 2.00 (s, 3H).

2-(4, 5-双(苄氧基)-9, 10-蒽醌-2-甲酰氨基)-4-(甲硫基)丁酸钠(6ed):黄色固体. ¹H NMR (CD₃OD, 400 MHz) δ: 8.26 (s, 1H), 8.03 (s, 1H), 7.79 (d, J=7.6 Hz, 1H), 7.65~7.69 (m, 3H), 7.62 (d, J=7.2 Hz, 2H), 7.52 (d, J=8.4 Hz, 1H), 7.32~7.41 (m, 6H), 5.38 (s, 2H), 5.30 (s, 2H), 4.63 (dd, J=4.4, 7.6 Hz, 1H), 2.61~2.68 (m, 2H), 2.28~2.34 (m, 1H), 2.15~2.19 (m, 1H), 2.13 (s, 1H).

2-(4, 5-二丁氧基-9, 10-蒽醌-2-甲酰氨基)-4-甲基戊酸钠(6db):黄色固体. ¹H NMR (DMSO-d₆, 400 MHz, 30 ℃) δ: 8.38 (d, J=7.6 Hz, 1H), 8.04 (d, J=1.2 Hz, 1H), 7.87 (d, J=1.6 Hz, 1H), 7.72 (t, J=8.0 Hz, 1H), 7.67 (dd, J=1.2, 7.6 Hz, 1H), 7.52 (dd, J=1.2, 8.0 Hz, 1H), 4.19 (t, J=6.0 Hz, 2H), 4.12~4.15 (m, 3H), 1.75~1.79 (m, 4H), 1.64~1.70 (m, 2H), 1.53~1.59 (m, 5H), 0.95~0.99 (m, 6H), 0.88~0.90 (m, 6H).

取1 mmol 5aa~5ed、1 mol/L的NaOH水溶液(1 mL)和5 mL无水乙醇加入到25 mL圆底烧瓶中, 室温搅拌过夜, 反应完成后, 减压蒸馏即可得到大黄酸-氨基酸缀合物钠盐6aa~6ed.

2-(4, 5-二乙氧基-9, 10-蒽醌-2-甲酰氨基)-4-甲基戊酸钠(6bb):黄色固体. ¹H NMR (DMSO-d₆, 400 MHz, 30 ℃) δ: 8.39 (d, J=7.6 Hz, 1H), 8.04 (s, 1H), 7.88 (s, 1H), 7.73 (t, J=8.0 Hz, 1H), 7.68 (d, J=6.8 Hz, 1H), 7.53 (d, J=8.0 Hz, 1H), 4.27 (q, J=6.8 Hz, 2H), 4.22 (q, J=6.8 Hz, 2H), 4.11-4.16 (m, 1H), 1.55~1.72 (m, 3H), 1.42 (q, J=6.8 Hz, 6H), 0.88~0.90 (m, 6H).

2-(4, 5-二甲氧基-9, 10-蒽醌-2-羧酰氨基)-3-苯基丙酸钠(6ac):黄色固体. ¹H NMR (DMSO-d₆, 400 MHz, 40 ℃) δ: 8.43 (d, J=6.8 Hz, 1H), 7.86 (d, J=1.6 Hz, 1H), 7.80 (d, J=1.2 Hz, 1H), 7.30 (t, J=8.0 Hz, 1H), 7.63 (d, J=7.6 Hz, 1H), 7.52 (d, J=8.0 Hz, 1H), 7.23 (d, J=6.8 Hz, 2H), 7.17 (t, J=7.2 Hz, 2H), 7.07~7.11 (m, 1H), 4.24~4.28 (m, 1H), 3.95 (s, 3H), 3.92 (s, 3H), 3.23 (d, J=4.4 Hz, 1H), 3.08 (dd, J=7.2, 13.2 Hz, 1H).

2-(4, 5-二甲氧基-9, 10-蒽醌-2-甲酰氨基)-4-甲基戊酸钠(6ab):黄色固体. ¹H NMR (DMSO-d₆, 400 MHz, 40 ℃) δ: 8.36 (d, J=7.6 Hz, 1H), 8.04 (d, J=1.6 Hz, 1H), 7.88 (d, J=1.6 Hz, 1H), 7.76 (t, J=8.0 Hz, 1H), 7.69 (dd, J=1.2, 7.6 Hz, 1H), 7.56 (dd, J=1.2, 8.4 Hz, 1H), 4.11~4.15 (m, 1H), 3.98 (s, 3H), 3.93 (s, 3H), 1.54~1.75 (m, 3H), 0.87~0.92 (m, 6H).

2-(4, 5-二丙氧基-9, 10-蒽醌-2-羧酰氨基)-3-苯基丙酸钠(6cc):黄色固体. ¹H NMR (DMSO-d₆, 400 MHz) δ: 8.35 (d, J=6.4 Hz, 1H), 7.87 (s, 1H), 7.75 (s, 1H), 7.71 (t, J=8.0 Hz, 1H), 7.64 (d, J=7.2 Hz, 1H), 7.52 (d, J=7.2 Hz, 1H), 7.15~7.21 (m, 4H), 7.09 (t, J=7.2 Hz, 1H), 4.22 (q, J=7.2 Hz, 1H), 4.08~4.15 (m, 4H), 3.22 (dd, J=4.4, 13.2 Hz, 1H), 3.05 (dd, J=7.2, 13.2 Hz, 1H), 1.77~1.83 (m, 4H), 1.04~1.09 (m, 6H).

2-(3, 5-二丙氧基-9, 10-蒽醌-2-羧酰氨基)-4-(甲硫基)丁酸钠(6cd):黄色固体. ¹H NMR (DMSO-d₆, 400 MHz, 40 ℃) δ: 8.38 (d, J=6.8 Hz, 0.5H), 8.32 (d, J=6.8 Hz, 0.5H), 8.03~8.04 (m, 1H), 7.84~7.86 (m, 1H), 7.70~7.76 (m, 2H), 7.54 (dd, J=1.6, 7.6 Hz, 1H), 4.18 (t, J=6.4 Hz, 2H), 4.12 (t, J=6.4 Hz, 2H), 4.02~4.07 (m, 1H), 2.71~2.88 (m, 2H), 2.10~2.43 (m, 2H), 2.02 (s, 3H), 1.78~1.85 (m, 4H), 1.06~1.10 (m, 6H).

3.2.1 中间体2的合成

将大黄酸2.84 g (10 mmol)、无水乙醇40 mL和SOCl₂10 mL加入到250 mL圆底烧瓶中, 加热至回流反应24 h, 待原料完全转化完毕, 直接将反应液倒入1 L的蒸馏水中, 抽滤, 并用大量的蒸馏水洗涤固体, 固体在真空干燥箱中充分干燥, 得到2.934 g化合物2, 产率为94%.直接投入下步反应.

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图 1 阿霉素、米托蒽醌和大黄酸的化学结构

Figure 1 Chemical structures of doxorubicin, mitoxantrone and rhein

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图式2 目标化合物的合成路线

Scheme 2 Synthetic route of the target compounds

R¹＝methyl, ethyl, propyl, n-butyl, benzyl; R²＝H, CH₂CH(CH₃)₂, CH₂Ph, CH₂CH₂SCH₃
Reagents and conditions: (i) SOCl₂, EtOH, reflux; (ii) R¹X, NaH, DMF, 90～130 ℃; (iii) 1 mol/L NaOH (aq.), r.t. ～60 ℃; then 1 mol/L HCl (aq.), r.t.; (iv) NH₂CH(R²)COOMe/Et, EDCI, DMAP, CH₂Cl₂, r.t.; (v) 1 mol/L NaOH EtOH, r.t.; then 1 mol/L HCl (aq.), r.t.; (vi) 1 mol/L NaOH (aq.), EtOH, r.t.

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图 2 化合物6db(a)和6eb(b)与DNA作用的荧光光谱图

Figure 2 Spectrofluorimetric tiration of 6db (a) and 6eb (b) interacting with DNA

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表 1 化合物的体外抗肿瘤活性

Table 1. Anti-tumor activity of derivativesin vitro

Compd.	R¹	R²	IC₅₀^a/(µmol•L^-1)
Compd.	R¹	R²	Hela	MCF7	HepG2	KB	HEK293T
6aa	CH₃	H	＞100	＞100	＞100	＞100	＞100
6ab	CH₃	CH₂CH (CH₃)₂	＞100	＞100	＞100	＞100	＞100
6ac	CH₃	CH₂Ph	＞100	＞100	＞100	＞100	＞100
6ad	CH₃	CH₂CH₂SCH₃	＞100	＞100	＞100	＞100	＞100
6ba	CH₂CH₃	H	＞100	＞100	＞100	＞100	＞100
6bb	CH₂CH₃	CH₂CH (CH₃)₂	＞100	＞100	＞100	＞100	＞100
6bc	CH₂CH₃	CH₂Ph	＞100	＞100	＞100	＞100	＞100
6bd	CH₂CH₃	CH₂CH₂SCH₃	＞100	＞100	＞100	＞100	＞100
6ca	CH₂CH₂CH₃	H	＞100	＞100	＞100	＞100	＞100
6cb	CH₂CH₂CH₃	CH₂CH (CH₃)₂	75.8	22.8	79.1	62.6	＞100
6cc	CH₂CH₂CH₃	CH₂Ph	＞100	95.4	81.6	68.8	97.6
6cd	CH₂CH₂CH₃	CH₂CH₂SCH₃	＞100	＞100	＞100	＞100	＞100
6da	CH₂CH₂CH₂CH₃	H	＞100	＞100	＞100	64.5	28.4
6db	CH₂CH₂CH₂CH₃	CH₂CH (CH₃)₂	62.1	80.2	51.2	27.7	37.4
6dc	CH₂CH₂CH₂CH₃	CH₂Ph	70.2	＞100	42.3	43.4	52.2
6dd	CH₂CH₂CH₂CH₃	CH₂CH₂SCH₃	＞100	＞100	＞100	62.4	83.2
6ea	CH₂Ph	H	＞100	＞100	＞100	＞100	＞100
6eb	CH₂Ph	CH₂CH (CH₃)₂	41.9	44.4	31.7	26.7	24.1
6ec	CH₂Ph	CH₂Ph	50.1	＞100	64.6	64.3	＞100
6ed	CH₂Ph	CH₂CH₂SCH₃	＞100	＞100	＞100	99.8	＞100
顺铂	-	-	5.5	15.8	14.3	6.1	3.3
阿霉素	-	-	0.99	2.8	4.7	3.7	0.7
^aThe data represent the mean values of three independent determinations.

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沈阳化工大学材料科学与工程学院沈阳 110142