CN106117189A - 乙酰基白杨素Mannich碱衍生物及其用途 - Google Patents
乙酰基白杨素Mannich碱衍生物及其用途 Download PDFInfo
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- CN106117189A CN106117189A CN201610427354.3A CN201610427354A CN106117189A CN 106117189 A CN106117189 A CN 106117189A CN 201610427354 A CN201610427354 A CN 201610427354A CN 106117189 A CN106117189 A CN 106117189A
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- methylene
- acetyl group
- chrysin
- cell
- mannich base
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Abstract
乙酰基白杨素Mannich碱衍生物及其用途，所述衍生物具有通式I的结构，其中，R1和R2各自独立地选自乙酰基和有机仲胺亚甲基，R1与R2中有且只有一个是乙酰基。该类化合物是一种ROS调控的缺氧诱导转录因子(HIF‑1α)抑制剂，利用正常细胞和肿瘤细胞内氧化和还原的不同机制，通过捕获细胞内氧自由基，特异性提高肿瘤细胞内过氧化氢水平，靶向氧化降解HIF‑1α和氧化激活Caspases，激活细胞内凋亡通路，从而选择性诱导肿瘤细胞凋亡并抑制增殖和转移。该类化合物对实体瘤具有良好的选择性。并且药代动力学性质独特，高效低毒，作用机理明确。全合成原料来源丰富，工艺简捷，纯度高，成本低。
Description
技术领域
本发明涉及乙酰基白杨素Mannich碱衍生物、合成方法及应用，尤其涉及该类化合物在制备肿瘤治疗药物中的潜在用途。
背景技术
肿瘤细胞和正常细胞具有不同的氧化还原机制，肿瘤细胞增殖和转移需要大量耗氧，所以缺氧是实体瘤微环境的一个重要特征。实体瘤细胞生存必须依赖于低氧状态下缺氧诱导转录因子(HIF-1α)。HIF-1α与细胞增殖，血管生长，侵袭，转移，凋亡和耐药等密切相关。加州大学和斯坦福大学提出HIF-1α将成为实体瘤治疗的最有效靶点，已成为当前抗实体瘤药物研究的热点。(Nature，2015，524：298-300；Cell，2015，163：1288-1288.e1；NatureReviews Cancer，2012，12：9-22；Nature Reviews Cancer，2003，3：721-732；NatureMedicine，2003，9：667-84；Nature Reviews Drug Discovery，2003，2：1-13)
既往发现对HIF-1α有抑制活性的抗癌药物主要有拓扑异构酶I抑制剂(拓扑替康)，长春新碱，雷帕酶素衍生物(依维莫司)，酪氨酸激酶靶向药物等，但是毒副作用均较大。目前还没有一个真正有效的靶向HIF-1α靶抑制剂。因此，找高效低毒的新型HIF-1α抑制剂类抗实体瘤药物仍然是一个全新的挑战。(Molecular Cancer Therapeutics，2007，6：220-226)
天然黄酮类化合物广泛存在于许多水果，蔬菜和药材中，具有抗血栓，抗炎抗变态免疫，抗病毒，抗肿瘤等多种生物活性，是一类通过细胞内活性氧(ROS)调控的天然HIF-1α抑制剂。(Eur J Med Chem，2012，49：24-40)黄酮类化合物并不直接杀伤细胞，而是利用正常细胞和肿瘤细胞的氧化还原状态的不同机制，通过对ROS调控，激活细胞内凋亡通路，抑制HIF-1α和VEGF，从而选择性诱导肿瘤细胞凋亡并抑制转移。
然而，由于黄酮类化合物溶解性差，广泛的II相代谢，导致口服给药产生较强的“肝肠外排效应”，体内生物利用度较低。因此，通过对天然黄酮结构修饰，改善成药性，具有重要的实用价值。(Eur J Pharmacology，2010，630：121-130)
黄芩为唇形科黄芩属多年生草本植物Scutellaria baicalensis Georgi的干燥根，黄芩的有效成分为黄酮类化合物主要成分为黄芩苷，其水解产物黄芩素(BA)抗增殖活性更强。(Cancer Treatment Reviews，2009，35：57-68)通过ROS调控的HIF-1α抑制，从而选择性诱导肿瘤细胞凋亡，而不影响正常细胞(Eur J Pharmacology，2010，630：121-130；Neuroscience Letters，2008，444：264-269)。以BA为先导化合物进行结构修饰当属黄芩素Mannich碱衍生物。CN1427003A公开了具有抑制蛋白激酶C活性的黄芩素8-位取代的甲胺类衍生物及其制备方法。CN200910140275.4和US8377895B2公开了以数十种天然黄酮苷元为先导化合物，经过Mannich缩合反应半合成出百余种黄酮Mannich碱系列衍生物。其中，黄芩素哌啶醇(BA-j)最为优良。
BA-j是一种CDK1选择性抑制剂。BA-j与细胞毒类药物截然不同，细胞毒类药物在杀伤肿瘤细胞同时也伤害正常细胞。BA-j是利用正常细胞和肿瘤细胞的氧化还原状态的不同机制，通过对ROS的调控，从而选择性诱导肿瘤细胞和被激活的淋巴细胞凋亡，而不影响正常细胞。(Scientific Reports，2015，5：13626；Anti-Cancer Agents in MedicinalChemistry，2016，16：914-924；Fitoterapia，2015，107：36-34；Bioorganic&MedicinalChemistry，2008，16：7127-7132；RSC Advances，2015，5：89818-89826)BA-j的log P值较低，蛋白结合率较高，亲合力较强，主要分布在血液中，因此对血液系统异常(如白血病，艾滋病以及肿瘤转移)引起的变态免疫症状有良好的消除作用。
白杨素也是一种具有广泛药理活性的天然黄酮类化合物，是蜂胶的主要成分之一，具有抗肿瘤、放疗增敏、抑制芳香酶活性、抗炎、抗氧化、防治心脑血管疾病等多种药理作用。白杨素可通过HIF-1α抑制导致VEGF抑制，阻止新生血管的生成，从而选择性地诱导肿瘤细胞的凋亡并抑制实体瘤转移。(Molecular Cancer Therapeutics，2007，6：220-226)因此，通过对白杨素结构进行有效修饰，以获得高效低毒的新型候选抗实体瘤药物具有重要意义。
Flavopiridol是由Aventis公司全合成了第一个泛CDKs抑制剂，属于白杨素有机胺衍生物，为氯代白杨素甲基哌啶醇，能显著提高化疗药物的敏感性，同时还发现可阻碍艾滋病病毒HIV-1在细胞内的增殖，显示了良好的抗癌和艾滋病应用前景。在化疗后续应用，能有效提高化疗药物的敏感性，给癌症和艾滋病的治疗带来了新的理念与希望。
然而，Flavopiridol是一种泛CDKs抑制剂。溶解度较差，静脉点滴稳态血浆浓度较低(0.27uM)，口服给药易被葡萄糖醛酸化，导致较强的“肝肠外排效应”，生物利用度低等问题。进入组织细胞内的药物转运困难，不容易被降解,可产生强烈的细胞毒效应，引起严重的分泌性腹泻等不良反应。因此，成药性的不够优良，使其在应用受到了限制。(Blood，2009，113：2637-2644；Life Sciences，1998，62：1861-1873；Invest New Drugs，2012，30:629-638)
P276-00是由Nicholas Piramal公司全合成了一种CDK 4，1，9的选择性抑制剂，属于 白杨素有机胺衍生物，为白杨素甲基脯氨醇。抗癌活性和成药性优于Flavopiridol，对正常人肺成纤维细胞WT-38和MRC-5几乎无影响，已进入III临床期。与Flavopiridol相比，几乎无腹泻等不良反应。但是也存在类似的成药性的不够优良等问题。(Molecular CancerTherapeutics，2007，6：918-925，918-925；Leukemia，2009，23：961-970)
中国科学院上海有机所半合成了一种白杨素Mannich碱衍生物，为6-吗啉亚甲基-白杨素。(Synlett，2006，(8)：1225-1229)江苏工业学院全合成了七种8-取代白杨素Mannich碱衍生物。(沈阳药科大学学报，2010，27：448-452)大连理工大学半合成了三种双取代白杨素Mannich碱衍生物，为双环烷仲胺醇亚甲基白杨素。(Anti-Cancer Agents inMedicinal Chemistry，2016，16(2)：doi:10.2174/1871520615666150928114425)上述白杨素衍生物的抗癌活性有所提高，但是成药性还不十分理想。
发明内容
本发明的目的是希望通过对白杨素的全新的结构修饰，寻找有别于现有技术的、高效低毒的新型HIF-1α抑制剂类抗实体瘤药物
本发明的解决其技术问题所采用的技术方案：
本发明所公开的乙酰基白杨素Mannich碱衍生物，具有通式I的结构：
通式I中，R1和R2各自独立地选自乙酰基和有机仲胺亚甲基，R1与R2中有且只有一个是乙酰基。
具体实施方式之一，所述的通式I中，R1是乙酰基；R2是有机仲胺亚甲基，选自二甲胺基亚甲基，吡咯烷亚甲基，哌啶亚甲基，吗啉亚甲基，硫吗啉亚甲基，4`-甲基哌嗪亚甲基，哌嗪亚甲基，4`-羟乙基哌嗪亚甲基，脯氨醇(+)亚甲基，4`-哌啶醇亚甲基，4`-哌啶酮亚甲基，噻吩并[3，2-c]吡咯烷亚甲基和脯氨醇(-)亚甲基。
R2进一步优选吗啉亚甲基，硫吗啉亚甲基，4`-甲基哌嗪亚甲基，4`-羟乙基哌嗪亚甲基，脯氨醇(+)亚甲基或4`-哌啶醇亚甲基。R2最优选4`-哌啶醇亚甲基。
另一具体的实施方式，所述的通式I中，R2是乙酰基；R1是有机仲胺亚甲基，选自二甲胺基亚甲基，吡咯烷亚甲基，哌啶亚甲基，吗啉亚甲基，硫吗啉亚甲基，4`-甲基哌嗪亚甲基，哌嗪亚甲基，4`-羟乙基哌嗪亚甲基，脯氨醇(+)亚甲基，4`-哌啶醇亚甲基，4`-哌啶酮亚甲 基，噻吩并[3，2-c]吡咯烷亚甲基和脯氨醇(-)亚甲基。R1进一步优选吗啉亚甲基，硫吗啉亚甲基，4`-甲基哌嗪亚甲基，4`-羟乙基哌嗪亚甲基，脯氨醇(+)亚甲基或4`-哌啶醇亚甲基。R1最优选4`-哌啶醇亚甲基。
本发明进一步提供上述乙酰基白杨素Mannich碱衍生物的合成方法，是乙酰基白杨素、有机仲胺类化合物和甲醛溶液在有机溶剂中经Mannich缩合反应而得；其中：所述的有机仲胺类化合物选自二甲胺，吡咯烷，哌啶，吗啉，硫吗啉，4-甲基哌嗪，哌嗪，4-羟乙基哌嗪，脯氨醇(+)，4-羟基哌啶，4`-哌啶酮，噻吩并[3，2-c]吡咯烷和脯氨醇(-)。所述的有机溶剂选自乙醇，乙腈，四氢呋喃，二氧六环，乙酸乙酯，乙酸丁酯，乙酰丙酮，乙酰乙酸乙酯和甲基异丁酮。
具体实施方式之一，上述方法中的乙酰基白杨素是以2，4，6-三羟基苯乙酮为起始原料，与苯甲酰乙酸乙酯热缩合得到。(J.Chem.Soc.Perkin Trans I，1973，503-505)经分离后分别的得到6-乙酰基白杨素和8-乙酰基白杨素。
具体地，以6-乙酰基白杨素哌啶醇(CH-j)和8-乙酰基白杨素哌啶醇(CH8-j)为代表，上述合成方法的合成路线可表示为：
上述本发明的乙酰基白杨素Mannich碱衍生物是一种细胞内活性氧(ROS)调控的缺氧诱导因子(HIF-1α)抑制剂，利用正常细胞和肿瘤细胞内氧化和还原的不同机制，通过捕获细胞内氧自由基，特异性提高肿瘤细胞内过氧化氢水平，靶向氧化降解HIF-1α和氧化激活Caspases，激活细胞内凋亡通路，从而选择性诱导肿瘤细胞凋亡并抑制增殖和转移。经实验证明，其中CH-j的成药性最为优良，具有更加优异体内分布特性，对实体瘤具有良好的选择性。CH-j选择性诱导肿瘤凋亡活性比BA-j强一倍，对实体瘤选择性比BA-j更强，对血液系统几乎不无影响。CH-j的药代动力学性质独特，高效低毒，作用机理明确。CH-j的且全合成原料来源丰富，工艺简捷，纯度高，成本低，有望开发成为对实体瘤更有效的ROS调控的HIF-1α抑制剂类新型抗癌药物。
基于此，本发明再一方面提供上述乙酰基白杨素Mannich碱衍生物在制备抗肿瘤药物中 的应用。尤其是在抗实体瘤药物制备中的应用。
技术效果
本发明说明书所用缩写：6-乙酰基-8-哌啶醇亚甲基-白杨素(CH-j)；对照品8-哌啶醇亚甲基-黄芩素(BA-j)。
本实施例中化合物编号的规则与本说明书中其余部分保持一致，如无特别说明，当述及“参测化合物”时，是指按照本发明的方法合成、确定结构的参与测试的乙酰基白杨素Mannich碱衍生物。对照品是8-哌啶醇亚甲基-黄芩素(BA-j)。
(1)溶解度与log P筛选
参测化合物溶解度与log P筛查结果见表1。其中，CH-j在37。C正辛醇/水中log P为1.63(约是BA-j0.69的2倍)，在37。C正辛醇层中的溶解度2421μ421度(约是BA-j 377μ377度的6倍多)，水层中的溶解度56μ6层中的(BA-j 77μ7-j)。
由于CH-j属于酸碱两性化合物，因此在pKa 5.3。在37。C的0.1M盐酸溶液(pH1.2)，0.05M醋酸钠-醋酸缓冲溶液(pH4.5)，磷酸盐缓冲溶液(PBS pH6.8)中稳定。溶解度分别为574，9800，30(73μ300别为冲溶。溶出度分别为100％，100％，6.8％，属于pH依赖快速释放型。提示本品可制成固体剂型口服给药。
综合分析，在参测化合物中，CH-j的溶解度与log P比较合适，推测CH-j更容易进入组织细胞内。
表1.参测的乙酰基白杨素Mannich碱衍生物溶解度与log P
(2)CH-j稳定性
按国家药品食品药品监督管理局药物稳定性试验指导原则试验，CH-j原料药对高温试验，高湿度试验，强光照射试验下均稳定。CH-j原料药加速试验和长期稳定性试验均稳定。在模 拟人工胃液，肠液和细胞内液中稳定。
(3)CH-j对细胞内H2O2水平的影响筛选
体外培养细胞给CH-j 3.5h，加入H2O2选择性PF1荧光探针0.5h后检测细胞内H2O2浓度，并进行荧光呈像，结果见图1。体外培养细胞给药3.5h，加入NaOCl选择性BODTPY荧光探针0.5h后检测细胞内NaOCl浓度，并进行荧光呈像，结果CH-j未呈现荧光，而给羟基喜树碱对照细胞呈现荧光。(未附图)
图1-1结果显示，在参测化合物中，4-哌啶醇取代基对肿瘤细胞内H2O2水平的影响最为显著。据析这是由于4-哌啶醇是一种强效抗氧化剂，可捕获3个以上的.O2 -，并被氧化释放3个H2O2。
图1-2和4结果显示，CH-j可提高肿瘤BGC-823和Hep G2细胞内H2O2的水平，在低剂量范围呈现明显的浓度依赖关系，当CH-j达到5-10μM后，释放出约4倍量H2O2，达到最大值35-45μM，继续提高CH-j剂量H2O2反而呈现明显的浓度负依赖关系。推测这是因为此水平的H2O2与Fe++结合，产生羟基自由基(.OH-)，氧化降解HIF-1羟以解除凋亡抑制，细胞开始大量凋亡。
图1-3和4结果显示，CH-j对人正常PBMCs和被激活的淋巴细胞内H2O2的水平均在10μM以下，无明显的浓度依赖关系，细胞均未凋亡和坏死。(BA-j对人正常人肝细胞Liver和PBMCs细胞内H2O2的水平均在10μM以下，无明显的浓度依赖关系，细胞均未凋亡和坏死。但是对被激活的淋巴细胞内H2O2的水平呈现明显的浓度依赖关系，细胞大量凋亡而未坏死。)结果显示，在人正常PBMCs和被激活的淋巴细胞内10μM的H2O2水平是可以耐受的。结果表明，CH-j可选择性提高实体瘤细胞内H2O2的水平，几乎不影响人正常PBMCs和被激活的淋巴细胞，因此，CH-j对实体肿瘤细胞的选择性比血液细胞更强。这是由于CH-j的log P大，与血浆蛋白结合率和结合力小，导致与血液细胞的结合力较弱，而与组织细胞的结合力较强。因此，CH-j更适用于实体瘤。(BA-j的log P小，与血浆蛋白结合率和结合力大，导致与血液细胞的结合力较强，而与组织细胞的结合力较弱，因此，相对而言BA-j更适用于血液系统。
(4)体外抗肿瘤细胞增殖活性筛选
体外人胃BGC-823抗肿瘤活性初筛(MTT，48h)结果见图2。结果显示，6种参测化合物在1.25-20μM范围内，均呈现明显的浓度依赖关系，抗增殖活性强度类似，无显著差异。体外CH-j对5种肿瘤细胞复筛结果见表2。CH-j抗增殖活性强度IC506.1μM(2.5μg/m1)。比白杨素IC5025μM)强4倍，比BA-jIC5012.3μM，4.7μg/m1强1倍。依据中国国家食品药品监督管理局《抗肿瘤药物药效学指导原则》规定，当合成化合物或植物提取纯品的IC50＜10μg/m1或植物粗提物的IC50＜20μg/m1时，视为体外具有抗肿瘤活性。
表2.CH-j体外抗肿瘤细胞活性复筛(MTT，48h)
| IC50μM | BA-j | CH-j |
| MCF-7 | 10.6±0.30 | 5.7±1.24 |
| H-460 | 13.7±1.83 | 6.5±1.13 |
| BGC-823 | 12.3±1.73 | 6.3±1.13 |
| PC-3 | 12.8±2.06 | 6.2±1.16 |
| Hep G2 | 15.7±1.30 | 9.0±1.50 |
(5)CH-j诱导肿瘤细胞凋亡
Hoechst33258是与DNA特异结合的活性荧光染料，主要结合在DNA的A-T碱基区，紫外光激发时发射蓝色荧光。活细胞发出均匀荧光，凋亡细胞由于染色质固缩，细胞核致密浓染而呈明亮的蓝色荧光。
用CH-j于0，2.5，5，10μM条件下处理人胃BGC-823肿瘤细胞24h，Hoechst33258荧光染色，细胞形态变化如图3-1所示。随着药物浓度的增加，细胞数量明显减少，而且出现细胞核发出致密荧光，核膜边缘可见凋亡小体的凋亡细胞，凋亡细胞比例呈剂量依赖性增长。CH-j与细胞毒类药物羟基喜树碱完全不同，后者在低剂量时呈现凋亡状态，而在高剂量时肿瘤细胞呈现坏死状态。结果表明，CH-j能够显著诱导BGC-823肿瘤细胞凋亡。
PI特异性结合于DNA，荧光强度与PI的结合量呈良好的线性关系。细胞固定后用PI染色，采用流式细胞仪对细胞中的DNA含量进行定量分析。由于不同细胞周期中DNA的含量不同，可以由此判断药物作用于细胞后细胞周期的变化。细胞凋亡时在细胞和分子水平上发生了许多的特征性改变，其中细胞核的改变最具特征性。由于凋亡细胞核酸内切酶活化，DNA降解，细胞DNA减少，因而可在GO/G1峰前出现一个亚二倍体凋亡峰(sub-G1)，为185Da整数倍DNA裂解峰，无小于185Da的裂解峰。用CH-j处理BGC-823肿瘤细胞24h，PI荧光染色结果如图3-2所示。CH-j能剂量依赖性地使BGC-823细胞阻滞于G1,G2期，使凋亡峰的比例呈剂量依赖性增长。另外，高浓度的药物还可以使S期细胞比例有所减少。结果表明，CH-j够使肿瘤细胞周期被阻滞于G1和G2/M期，并使S期的细胞数目减少，能够显著诱导BGC-823细胞凋亡。
细胞发生凋亡时会有一系列的形态学特征性改变，其中，质膜的改变是早期凋亡的特征之一。细胞发生凋亡时，胞膜磷脂酞丝氨酸(PS)从胞膜内侧翻转到外侧，暴露于细胞外表面。Annexin V是一个Ca2+依赖性的磷脂结合蛋白，它与磷脂酞丝氨酸有高度亲和力，可以与细胞外暴露的磷脂酞丝氨酸特异性结合。然而，PS转移到细胞膜外并不是凋亡所独特的，也可发生在细胞坏死中。两种细胞死亡方式间的差别是在凋亡的初始阶段细胞膜是完好的，而细胞 坏死在其早期阶段细胞膜的完整性就破坏了。PI是一种核酸染料，它不能透过完整的细胞膜，但在凋亡中晚期的细胞和坏死细胞，PI能够透过细胞膜而使细胞核染色。将Annexin V-FITC与PI匹配使用，在流式细胞仪上灵敏地检测凋亡和坏死细胞的比率。
用CH-j处理BGC-823肿瘤细胞24h，Annexin V-FITC与PI双荧光染色结果如图3-3所示。CH-j能剂量依赖性地使BGC-823细胞凋亡。当用CH-j处理24h后，早期坏死细胞无明显变化，早期凋亡细胞的比例和晚期凋亡细胞比例明显上升。由此可见，早期凋亡的细胞随着药物浓度的增加而显著增加。晚期凋亡细胞增加的相对较少。而细胞毒类药物HCPT是在低剂量时可诱导细胞凋亡，高剂量可使早期细胞发炎坏死。结果表明，与细胞毒类药物不同，CH-j抗增殖活性主要是通过诱导BGC-823肿瘤细胞凋亡来实现的，而并不直接杀伤细胞导致细胞发炎坏死。
Caspases为含半胱氨酸的天冬氨酸蛋白水解酶(cysteinyl aspartate specificproteinase)，是一组存在于细胞质中具有类似结构的蛋白酶，与细胞凋亡密切相关，并参与细胞的生长、分化与凋亡调节。Caspases是细胞凋亡过程中的关键酶，在介导细胞凋亡中，几乎所有的凋亡信号转导通路上都有Caspase酶的参与，即共有途径。Caspases活性位点为半胱氨酸巯基，是以非活性pro-Caspases状态存在的，它对于H2O2比较敏感，当被H2O2氧化为-S-S-二聚体形式时，就被激活转变成有活性的Caspase-S-S-Caspase，诱导凋亡活性被连续激活放大。Caspase-8为诱导细胞凋亡的早期启动者，caspase-3为最终晚期执行者。如pro-caspase-9巯基游离，与磷酸化survivin结合，抑制细胞凋亡，促进分裂。当pro-caspase-9巯基被H2O2氧化成-S-S-二聚体形式，与磷酸化survivin分离，抑制细胞分裂，诱导细胞凋亡。H2O2可直接氧化激活Caspas，从而选择性诱导肿瘤细胞凋亡，减少损害正常细胞。
用CH-j处理BGC-823肿瘤细胞24h后，Caspase-3，8，9检测结果见图3-4，C。CH-j在0-10μM在范围内激活Caspase-3，8，9均呈剂量依赖性的上升趋势，继续提高CH-j剂量活性不再呈上升趋势。结果表明，CH-j选择性诱导BGC-823肿瘤凋亡与H2O2氧化激活caspase-3，8，9有关。
(6)CH-j对肿瘤细胞内HIF-1影响
HIF-1α抑制主要位点有：C800Cys-SH被H2O2和.OH-氧化；P402和P564Pro-NH被PHD羟基化，或被.OH-氧化；K532和T796被磷酸化。K532被乙酰化。HIF-1被第一活性位点位于C800半胱氨酸巯基(Cys-SH)，第二不可逆以活性位点位于P402和P564脯氨酸氨基(Pro-NH)。
在低氧状态下，脯氨酸羟化酶(PHD)失活，富半胱氨酸巯基蛋白(p300-SH/CBP使HIF-1S具有活性。在正常氧状态下，脯氨酸羟化酶将HIF-1状P402和P564羟基化为Pro-NOH而失活。HIF-1OC800Cys-SH对于H2O2比较敏感，H2O2将Cys-SH被氧化为Cys-SOH暂时失去活性。在缺氧状态下，Cys-SOH又可被p300-SH还原为活性Cys-SH。在高水平H2O2状态下，H2O2与Fe2+形成的羟基自由基(.OH-)，将Pro-NH化为Pro-NOH，将Cys-SOH氧化为Cys-SO2H，形成的 HON＝HIF-1α-SO2H迅速被泛素-蛋白酶途径降解。HIF-1β不以半胱氨酸为活性中心，因此不受H2O2和.OH-影响。
将人胃BGC-823肿瘤细胞用0，2.5，5，10μMCH-j处理培养1小时，再继续加入0.2μM胰岛素，培养24小时，收集细胞，裂解。用免疫印迹法测定HIF-1α，HIF-1β结果见图4。结果显示，CH-j以剂量依赖的方式抑制HIF-1α的表达，而不影响HIF-1β的表达。
结果表明，CH-j是一种HIF-1α选择性抑制剂，从而可选择性诱导实体瘤细胞凋亡并抑制转移。
(7)CH-j特殊安全性
按国家药品食品药品监督管理局药物特殊安全性试验研究指导原则试验，0.2％CH-j的葡萄糖注射液，按刺激性，过敏性和溶血性特殊安全性评价方法试验，除对眼角膜有发热刺激性外，其他均为呈阴性。符合注射或外用给药要求。
(8)CH-j蛋白结合率与可逆结合率
药物与血浆蛋白结合一般是可逆的，亲和力强度是由药物-蛋白复合物可逆结合率决定的，只有游离的药物才能够通过脂膜向组织扩散。通过对药物结构的修饰，适当提高药物-蛋白复合物可逆结合率，可提高靶部位药物的作用强度。
采用平衡透析法测定CH-j蛋白结合率为86％(BA-j 92％)，均呈现非浓度依赖性。目前，药物蛋白复合物解离常数的评价还没有统一的方法。本发明用80％甲醇溶液从药物与蛋白混合溶液中提取药物，用提取率评价可逆结合率。将药物血浆与甲醇按体积比1:4混合，离心，分取上清液，测定药物提取率。测定结果如表3:CH-j药物提取率(可逆结合率)为90％，而BA-j提取率仅为20％。
表3.CH-j和BA-j蛋白结合率与可逆结合率比较
多酚羟基构成的酸性部与则白蛋白结构中赖氨酸碱性位点结合。由于CH-jC6乙酰基代替了BA-j C6位酚羟基，削弱了酸性部分与白蛋白碱性位点的亲和力，使CH-j蛋白复合物可逆结合率明显大于BA-j。表明CH-j细胞内外渗透性良好，主要分布于全身体，因此，更容 易进入组织细胞内，将对实体瘤将更加有效。作为对比的BA-j则主要分布于血液，对血液系统癌症以及免疫系统更加有效。
(9)CH-j药物动力学
色谱条件与系统适用：以十八烷基键和硅胶为填充剂(耐酸性高柱效C18色谱柱，5谱柱，4.6*150mm)，以乙腈-甲醇-水-甲磺酸(体积比25：20：55：0.15)为流动相。紫外检测波长为283nm，流速为1ml/min，进样体积为20μl。理论塔板数按CH-j色谱峰计算不少于2000。
样品溶液：将猕猴用氟烷麻醉后，安插导尿管。口服CH-j为5mg/kg，在不同时间采集尿液，冰冻保存。取尿液，按1:4(v:v)加入甲醇，离心后分取上清液测定。对照品溶液：CH-j甲醇溶液5μg/ml。结果显示：猕猴口服CH-j(5mg/kg)尿药浓度-时间曲线测定结果见图5。CH-j的log P较大，脂溶性强，体内吸收分布很快，尿药浓度-时间曲线符合典型的二室模型。tmax1 1.3h，Cmax126.4μM；再分布期tmax2 9h，Cmax213.2μM(>IC50 6.1μM)，随后尿液中的CH-j浓度随时间缓慢下降，24h已低于3μM。消除相半衰期t1/2(β)为6.2h。曲线下面积AUC0-∞为20.4μM*h。表观分布容积(Vd)为42L，主要分布于全身体液(BA-j Vd为12.6L，主要分布于血液，见Fitoterapia，2015，107：36-34)。猕猴口服CH-j与BA-j药物动力学参数比较结果见表5。结果表明：CH-j比BA-j更容易分布于组织细胞内，推测对实体瘤将更加有效。
表5.猕猴口服CH-j与BA-j药物动力学参数比较
| 药物 | CH-j | BA-j |
| 口服剂量mg/kg | 5 | 5 |
| 药物浓度达峰时间Tmax h | 1.3 | 2.0 |
| 消除相半率期t1/2βh | 6.2 | 4.2 |
| 药物最大浓度CmaxμM | 26.4 | 25.4 |
| 曲线下面积AUC0-∞μM*h | 20.4 | 71.5 |
| 容量因子Vd L | 42.0 | 12.6 |
(10)CH-j药物代谢学
CH-j在猕猴主要代谢物为M179.06和M152，其次M264.03，其他微量(<10％)。
M179：将尿液加4倍量甲醇，离心，去上清液，以甲醇-水-甲酸(35：65：0.2)为流动相，用C18柱色谱HPLC-UV分离制备得到M179组分，用氨水中和，蒸出溶剂，80℃减压干燥，得到无色结晶。MSm/z180[M+H]+,152[M+H-CO]+,136[M+H-CO2]+,178[M-H]-,187[M+Cl-HCN]-。HR-MS(API-ESI)m/z:[M+H]+180.0653(计算值180.0660)，分子式C9H9NO3，平均分子量179.06。IR(KBr,cm-1)：3146(CH3),1401(νC＝N),1239,1206,1195(νC-O),1059(νC-N),784(δ PH-H),562,536。1H NMR(DMSO-d6,400MHz)δ:7.46(s，Ph-H)，3.86(d，J＝5.2Hz,N-CH2),2.33(CH3)；此外，7.87-7.85(d，N＝CH),7.54-7.52(d，N-CH＝),7.49-7.15(d,Ph-H),2.00(m，NH)为分离后形成的烯醇互变异构体共振峰。推测结构为2-Methyl-3-aza-bicyclo[3.3.1]nona-1(8),2,5(9),6-tetraene-6,8,9-triol。
M264：LC-MSm/z265[M+H]+,249[M+H-O]+，271[[M-O+Na]+，263[M-H]-,283[M-O+Cl]-；MS m/z 263[M-H]-，167[M-H-SO4]-为M 264丢失的96Da(SO4)，表明结构中存在硫酸酯基(-OSO3H)。推测结构为3,5-dihydroxy-2-(1-hydroxyethyl)-6-methylphenyl hydrogensulfate。
M152：LC-MS m/z153[M+H]+；MS m/z187和189[M+Cl]-。推测结构为2,6-二羟基苯乙酮。
主要代谢途径：
(I)碱式降解：CH-j捕获2分子.O2 -，脱去环二烯丁酮，被氧化降解为被氧化降解为氨甲基黄烷酮醇类活性中间代谢物M343，同时释放2分之H2O2。C8-CH2NH2与CH3C＝O发生分子内加成为希夫氏碱，再捕获3分子.O2 -，再被水解氧化重排脱去苯甲酸，同时释放2分子H2O2和2分子CO2，降解为代谢物M179。
(II)中性降解：CH-j捕获3分子.O2 -，同时释放3分子H2O2，脱去吡啶酮-4，其中脱下的1分子H2O加成到C2-C3间的双键上，被氧化降解为黄烷酮醇类活性中间代谢物M344。再捕获4分子.O2 -，再被水解氧化重排脱去苯甲酸，同时释放2分子H2O2和3分子CO2，降解为代谢物M152。
(III)酸性降解：CH-j C7位羟基成硫酸酯，并捕获2分子.O2 -，同时释放2分子H2O2，脱去吡啶酮-4，其中脱下的1分子H2O加成到C2-C3间的双键上，被降解为硫酸化甲基黄烷酮醇类活性中间代谢物M408。再捕获2分子.O2 -，再被水解氧化重排脱去苯甲酸，同时释放1分子H2O2和2分子CO2，降解为代谢物M264。
白杨素口服给药，主要为C7-葡萄糖醛酸化代谢物和硫酸化代谢物，“肝肠外排效应”大于95％，仅有微量进入细胞内捕获.O2 -和释放H2O2。白杨素有机胺衍生物Flavopiridol口服给药，主要为C7-葡萄糖醛酸化代谢物，其次为C5-葡萄糖醛酸化代谢物，仅有少量进入组织细胞内。而进入组织细胞内部分由于有机胺取代基不能被代谢，因而产生强细胞毒性。CH-j是在白杨素结构C6引入吸电子基团CH3C＝O，C8引入哌啶醇亚甲基形成的乙酰基白杨素Mannich碱衍生物，通过乙酰基共扼以及哌啶醇亚甲基对白杨素的活化效应，使口服给药葡萄糖醛酸化“肝肠外排效应”被显著抑制(<1％)。
白杨素是一种抗氧化剂，可捕获.O2 -，同时被氧化释放H2O2。哌啶醇是一种强效抗氧化剂，可捕获多分子.O2 -，同时被氧化释放多分子H2O2。因此，CH-j可捕获多分子.O2 -并同时释放多分子H2O2的被氧化分解代谢，或被氧化硫酸化降解。由此可见，CH-j，通过增强I相代谢，抑制II相无效代谢，从而可抑制“肝肠外排效应”，有效地提高生物利用度。CH-j的代谢途径与CYP3A4无关。这种捕获.O2 -和释放H2O2的代谢过程，可选择性提高肿瘤细胞内H2O2浓度，与诱导其凋亡密切相关。(Scientific Reports，2015，5：13626；Fitoterapia，2015，107：36-34)
(11)CH-j组织分布
CH-j 100mg/kg大鼠静脉给药(i.v.)或口服给药(p.o.)2h时CH-j组织分布结果见图6。CH-j在血液，尿液，胆汁和各组织中均可检测到原型药物和代谢物M179.06。CH-j口服给药在胃中浓度教大，约比其他组织高1倍。表明CH-j主要在胃部吸收，并且有较高的药物浓度，对各组织均无特殊的亲和力，能够通过血脑屏障。与静脉给药比较，口服给药吸收完全，生物利用度接近100％。药物进入血液系统后，由于log P较大，血浆蛋白结合率适中，渗透性较强，原形药物很快进入各组织细胞内，而对血液中被激活的淋巴细胞几乎无亲和性。而作为对比的BA-j仅在血液和尿液中可检测到原型药物，而在各组织中均检测不到原型药物，仅可检测到代谢物M179。表明主要在胃肠道吸收，通过肝脏门静脉吸收入血液循环，由于log P较小，血浆蛋白结合率太高，结合力太强，原形药物主要分布于血液系统，对被激活的淋巴细胞有特殊的亲和性，除吸收部位外很少进入组织细胞内。
(12)体内抗肿瘤活性
按文献(Scientific Reports，2015，5：13626)方法测定CH-j对异种移植模型的人非小细胞肺癌(H-460)的BALB/c裸鼠模型的抗肿瘤作用效果，结果见图7。CH-j5，10mg/kg，每日分两次灌胃给药，连续10天。与对照组相比较，CH-j能够明显抑制H-460肿瘤异种移植在体内的生长。与给药前比较，30天时对H-460肿瘤缩小率分别为90.5±9.2％和95.2±11.6％(P<0.05)。结果表明，CH-j不但能够明显抑制H-460肿瘤异种移植在体内的生长，而且可使瘤体显著缩小。
(13)CH-j作用机制分析
体内细胞通过消耗氧气来产生能量。但是，如果氧含量下降，细胞进入缺氧状态，不再生成能量，而是转化生成氧自由基(.O2 -)，激活HIF-1量，转而关闭H2O2生成，并向炎性细胞发送信号,让其迁移到缺氧区域(Cell，2015，163：1288-1288.e1)。
在正常细胞内95％以上.O2 -可被SOD歧化为H2O2，构成机体的第一道天然防线。过量的H2O2主要被谷胱苷肽酶(GPX)介导催化将还原型谷胱苷肽(GSH)氧化为氧化型GSH。部分H2O2被过氧化氢酶(CAT)介导催化分解为H2O和O2。因此，.O2 -和H2O2被SOD和GPX控制维持在一个很低的水平，使细胞处于非增殖状态。
肿瘤细胞和正常细胞具有不同的氧化还原机制。肿瘤细胞内.O2 -水平高于正常细胞，而H2O2，SOD，GSH和CAT低于正常细胞(European Journal of Pharmacology,2010,630:121-130.)。肿瘤细胞增殖和转移需要大量耗氧，所以缺氧是实体瘤微环境的一个重要特征。在低氧状态下肿瘤细胞生存依赖于HIF-1α，HIF-1α积累促进VEGF等生长因子表达。VEGF在肿瘤血管生成中起着至关重要的作用，是肿瘤的生长和转移所必须的。因此，抗HIF-1长/VEGF血管新生疗法系统是一个很有前景的治疗癌症的策略。(Molecular CancerTherapeutics，2007，6：220-226)
在低氧状态下肿瘤细胞HIF-1α过表达，使细胞处于增殖状态，同时对H2O2敏感性增加。因此，在相同浓度的H2O2作用下，肿瘤细胞比正常细胞更容易发生凋亡。
法国第五大学Carole Nicco课题组已证实在消除H2O2的抗氧化酶方面，GSH通路在非转化细胞中对H2O2的产生控制起着主要作用，但是GSH在肿瘤细胞中对H2O2的产生控制是微不足道的。因此，在正常细胞中，ROS处于一个很低的水平，来自于NADPH氧化酶，H2O2的浓度 由谷胱甘肽系统所调控。相反地，在肿瘤细胞中，更接近于细胞毒性的高水平的H2O2是通过线粒体呼吸链所产生的，H2O2浓度由CAT控制。因此，该课题组首次提出任何可以提高细胞内H2O2水平的物质都可以降低肿瘤的增殖并最终导致细胞凋亡。相反地，任何可以降低细胞内H2O2水平的物质都可以促进肿瘤的生长。所以，能特异性提高肿瘤细胞内H2O2的药物具有选择性诱导肿瘤细胞凋亡作用。(Biomedicine&Pharmacotherapy，2005，59：169-174)
西班牙Seville大学Miguel课题组提出细胞内H2O2水平的增加在癌症治疗中是一个重要的事件。细胞内高水平H2O2不但抑制癌细胞生存，而且诱导癌细胞凋亡比正常细胞更敏感。任何增加细胞内H2O2到足够水平的药物或策略都可选择性诱导肿瘤细胞凋亡并产生疗效。提高细胞内H2O2水平重要的是要依据药物性质和达到细胞内的浓度，这是抗癌药物疗效的关键。(Cancer Letters，2007，252：1-8)
CH-j通过对通过ROS调控，捕获细胞内.O2 -，特异性提高实体瘤细胞内的H2O2水平,靶向性氧化以半胱氨酸为活性中心的特定酶。如高水平的H2O2氧化Caspases，激活细胞内凋亡通路，从而选择性诱导实体瘤细胞凋亡。高水平的H2O2氧化降解HIF-1内，从而抑制实体瘤细胞增殖和转移。
综上所述，CH-j利用正常细胞和肿瘤细胞内氧化和还原的不同机制，通过ROS调控，捕获细胞内.O2 -，特异性提高肿瘤细胞内的H2O2水平，靶向氧化降解HIF-1肿和氧化激活Caspases，激活细胞内凋亡通路，从而选择性诱导实体瘤细胞凋亡并抑制增殖和转移。CH-j还可对抗NaOCl的氧化，抑制激活细胞膜死亡受体通路，从而抑制细胞发炎和坏死。CH-j的作用机制与细胞毒类药物截然不同，细胞毒类药物通过提高细胞内ROS(H2O2，.OH-，NaOCl)的水平，不但氧化激活细胞内线粒体凋亡通路，也激活死亡受体通路，因此，在杀伤肿瘤细胞同时也 伤害正常细胞。
与现有技术相比，本发明突出的优点在于：
本发明提供了一种ROS调控的HIF-1α抑制剂乙酰基白杨素Mannich碱衍生物。其中，CH-j选择性诱导肿瘤凋亡活性比BA-j强一倍，对实体瘤选择性比BA-j更强，对血液系统几乎不无影响。(BA-j对血液系统的选择性强)CH-j成药性优良，药代动力学性质独特，高效低毒，作用机理明确。CH-j全合成原料来源丰富，工艺简捷，纯度高，成本低，有望开发成为对实体瘤更有效的ROS调控的HIF-1α抑制剂类新型抗癌药物。
附图说明
本发明附图8幅，分别是：
图1：PF1选择性荧光探针测定乙酰基白杨素Mannich碱衍生物对细胞内H2O2水平的影响，其中：
图1-1：乙酰基白杨素Mannich碱衍生物对Hep G2肿瘤细胞内H2O2水平的影响：横坐标为参测化合物，测试浓度均为10μM，纵坐标为细胞内产生H2O2水平(μM)；
图1-2：CH-j/BA-j对肿瘤细胞内H2O2水平的影响，图中，横坐标为CH-j/BA-j浓度(μM)，纵坐标为细胞内产生H2O2水平(μM)；
图1-3：CH-j/BA-j对血液细胞内H2O2水平影响，图中，横坐标为CH-j/BA-j浓度(μM)，纵坐标为细胞内产生H2O2水平(μM)；
图1-4：CH-j对细胞内H2O2水平的影响PF1荧光呈像：上图为可见光下观察结果，下图为紫外光下观察结果。
图2：乙酰基白杨素Mannich碱衍生物体外对人胃BGC-823的抗肿瘤活性初筛结果(MTT，48h)，横坐标为给药浓度(μM)，纵坐标为细胞存活率(％)。
图3：以0、2.5、5、10或20μM CH-j处理人胃BGC-823肿瘤细胞24h凋亡检测结果:
图3-1是Hoechst33258染色结果；
图3-2是PI染色结果；
图3-3是Annexin V-FITC/PI染色结果；
图3-1、3-2和3-3中，A、B、C、D分别表示用0、2.5、5、10μM CH-j处理；
图3-4：Caspases：横坐标为CH-j浓度(μM)，纵坐标为Caspases活性(％)。
图4：CH-j对人胃BGC-823肿瘤细胞HIF影响，分别以0、2.5、5和10μMCH-j处理BGC-823肿瘤细胞1h，继而加入0.2μM胰岛素培养24h，用免疫印迹法测定HIF-1α和HIF-1β。
图5：CH-j猕猴口服给药(5mg/kg)的尿药浓度-时间曲线：横坐标为给药时间(h)，纵坐标为尿液中CH-j浓度(μM)。
图6：CH-j大鼠给药(100mg/kg)2小时组织分布：纵坐标为CH-j浓度(μg/g)，静脉给药(i.v.)，口服给药(p.o.)。
图7：CH-j体内抗肿瘤活：雄性BALB/c裸鼠H-460肿瘤异种移植模型，每天分两次灌胃给药CH-j 5，10mg/kg，连续10天，横坐标为实验时间(天)，纵坐标为H-460肿瘤体积(mm3)。
图8：CH-j作用机制图示。
具体实施方式
下述非限制性实施例用于进一步说明本发明的乙酰基白杨素Mannich碱衍生物、其制备方法和应用，但应当明确，属可替换方案的取代基并不局限于实施例中所述及的这些有机胺亚甲基，并且，这些实例也不应理解为对本发明任何形式的限定。
实施例1. 6-乙酰基-8哌啶醇亚甲基-白杨素(CH-j)
6-乙酰基白杨素(含量95.3％)50g，加入乙酸乙酯2000ml，加热回流搅拌。按摩尔比1:1.5加入哌啶醇和甲醛溶液，继续加热回流搅拌3h。将析出的结晶滤出，洗涤，60℃减压干燥，得到黄色结晶56.5g。HPLC-UV含量99.1％。重结晶，得到黄色柱状结晶，HPLC-UV含量99.5％。
本品与三氯化铁溶液呈紫红色。在25℃甲醇溶液中的比旋度为+2450。MS(API-ESI)m/z:[M+H]+410,[M+H-101]+309。HR-MS(API-ESI)m/z:[M-H]-408.1444(计算值408.1447),分子式C23H23NO6,平均分子量409.43。UV：1ml含5μg的甲醇溶液E1％ 1cm(λmax283nm)约为1044。IR(KBr,cm-1)：3513(弱νOH),2934(νCH3),1658(νC＝O),1625(C＝O),1587,1449,1379,777和687(γPh单取代)。1H-NMR(DMSO-d6,500MHz)δ:8.03(d,2H,Ph-2',6'-H),7.57(m,3H,Ph-3',4',5'-H),6.83(s,1H,3-H),3.97(s,2H,C8-CH2),3.63m,1H,CH-OH),3.06-2.64(m,4H,2”,6”-CH2),2.50(COCH3),1.83-1.49(m，4H，3”,5”-CH2)。 13C-NMR(DMSO-d6,500MHz)δ:204.6(C＝O),182.8(C-4),168.3(C-7),167.7(C-9),164.2(C-2),159.2(C-5),132.7(C-1'),129.9(C-4'),129.3(C-3',5'),127.0(C-2',6'),105.9(C-6),105.5(C-8),103.2(C-10),96.1(C-3)。为6-乙酰基-8-哌啶醇亚甲基-白杨素[6-Acetyl-5,7-dihydroxy-8-(4-hydroxy-piperidin-1-ylmethyl)-2-phenyl-chromen-4-one]。
实施例2. 6-乙酰基-8-羟乙基哌嗪亚甲基-白杨素(CH-i)
取6-乙酰基白杨素10g，加入500ml乙酸乙酯，加热回流搅拌0.5h。按摩尔比1:1.5加入N-羟乙基哌嗪和甲醛溶液，继续搅拌1.5h。将析出的结晶滤出，洗涤，60℃减压干燥，得黄色结晶7.5g。HPLC-UV峰面积归一化含量99.2％。
本品与三氯化铁溶液呈紫红色。MS(API-ESI)m/z:[M+H]+439。UV：1ml含5μg的甲醇溶液E1％ 1cm(λmax 286nm)约为1016。IR(KBr,cm-1):3486(νOH),2934(νCH3),1651(νC＝O),1624(νCH3-C＝O),1587,1446,845和773(γPh单取代)。1H-NMR(500MHz,DMSO-d6)δ:8.11(d,2H,Ph-2',6'-H),7.62(m,3H,Ph-3',4',5'-H),7.05(s,1H,3-H),3.88(s,2H,8-CH2),3.47(t,J＝6.2Hz,2H,8-CH2OH),2.68-2.66(m,4H,2”,3”,4”,5”-H),2.61(s,3H,CH3),2.40(t，J＝6.1Hz,2H,7”-H)。分子式C24H26N2O6。为6-乙酰基-8-羟乙基哌嗪亚甲基-白杨素。[6-Acetyl-5,7-dihydroxy-8-[4-(2-hydroxy-ethyl)-piperazin-1-ylmethyl]-2-phenyl-chromen-4-one]
实施例3. 6-乙酰基-8-脯氨醇(+)亚甲基-白杨素(CH-h)
取6-乙酰基白杨素10g，加入500ml乙酸乙酯，回流搅拌0.5h。按摩尔比1:2加入脯氨醇(+)和甲醛溶液，继续搅拌2.5h。将析出的结晶滤出，用少量乙酸乙酯洗涤，60℃减压干燥，得黄色结晶2.4g。重结晶，HPLC-UV峰面积归一化含量95.5％。
本品与三氯化铁溶液呈紫红色。MS(API-ESI)m/z:[M+H]+410,[M-H]-408。分子式C23H23NO6，平均分子量409.43。UV：1ml含5μg的甲醇溶液E1％ 1cm(λmax 282nm)约为968。IR(KBr,cm-1):3306(νOH),2951(νCH3),1651(νC＝O)，1625(νCH3-C＝O),1589,1451,771和686(γPh单取代)。1H-NMR(500MHz,DMSO-d6)δ:8.07(dd,Ph-2',6'-H),7.59(m,Ph-3',4',5'-H),6.82(s,1H,3-H),4.41-4.25(dd,J＝13.4Hz,2H,8-CH2,不对称同碳偶合),3.71-3.63(dd,J＝11.7,5.3Hz,2H,6”-H),3.53(H2O),3.40(m,1H,5”-H),3.19(m,1H,2”-H),2.95(m,1H,2”-H),2.46(s,3H,COCH3),2.06(m,1H,4”-H),1.88(m,1H,3”-H),1.75(m,2H,3”,4”-H)。分子式C23H23NO6.H2O。为6-乙酰基-8-脯氨醇(+)亚甲基-白杨素一水合物。[6-Acetyl-5,7-dihydroxy-8-(2-hydroxymethyl-pyrrolidin-1-ylmethyl)-2-phenyl-chrom en-4-one]
实施例4. 6-乙酰基-8-吗啉亚甲基-白杨素(CH-d)
取6-乙酰基白杨素10g，加入200ml二氧六环，95℃加热搅拌0.5h。按摩尔比1:1.5加入吗啉和甲醛溶液，继续搅拌2.5h。将析出的结晶滤出，洗涤，60℃减压干燥，得橙黄色结晶9.2g。重结晶，HPLC-UV峰面积归一化含量99.8％。
本品与三氯化铁溶液呈紫红色。MS(API-ESI)m/z:[M+H]+396。UV：1ml含5μg的甲醇溶液E1％1cm(λmax 286nm)约为1129。IR(KBr,cm-1):3307(νOH),2953(νCH3),1647(νC＝O),1630(νCH3-C＝O),1587,1451,771和694(γPh单取代)。1H-NMR(500MHz,DMSO-d6)δ:8.14(d,Ph-2',6'-H),7.64(m,Ph-3',4',5'-H),7.15(s,1H,3-H),3.79(s,2H,8-CH2),3.57(m,2H,3”,5”-H),2.69(s,3H,COCH3),2.55(m,2H，2”,6”-H)。分子式C22H21NO6。为6-乙酰基-8-吗啉亚甲基-白杨素。[6-Acetyl-5,7-dihydroxy-8-morpholin-4-ylmethyl-2-phenyl-chromen-4-one]
实施例5. 6-乙酰基-8-硫吗啉亚甲基-白杨素(CH-e)
取6-乙酰基白杨素10g，加入200ml乙酰丙酮，95℃加热搅拌0.5h。按1：2M加入硫吗啉和甲醛溶液，继续搅拌2.5h。将析出的结晶滤出，洗涤，60℃减压干燥，得橙黄色结晶10.3g。HPLC-UV峰面积归一化含量95.4％。
本品与三氯化铁溶液呈紫红色。MS(API-ESI)m/z:[M+H]+412。UV：1ml含5μg的甲醇溶液E1％ 1cm(λmax 286nm)约为1133。IR(KBr,cm-1):3467(νOH)，2908(νCH3),1647(νC＝O),1626(νCH3-C＝O),1587,1441,777和688(γPh单取代)。1H-NMR(500MHz,DMSO-d6)δ:8.13(m,Ph-2',6'-H,),7.64(m,Ph-3',4',5'-H),7.14(s,1H,3-H),3.82(s,2H,8-CH2),2.82(m,2H,2”,6”-H),2.68(s,3H,CH3),2.62(m,2H,3”,5”-H)。分子式C22H21NO5S。为6-乙酰基-8-硫吗啉亚甲基-白杨素。[6-Acetyl-5,7-dihydroxy-2-phenyl-8-thiomorpholin-4-ylmethyl-chromen-4-one]
实施例6. 6-乙酰基-8-甲基哌嗪亚甲基-白杨素(CH-f)
取6-乙酰基白杨素10g，加入500ml乙酸乙酯，加热回流搅拌0.5h。按1：1.5M加入N-甲基哌嗪和甲醛溶液，继续搅拌1.5h。将析出的结晶滤出，洗涤，60℃减压干燥，得黄色针结晶7.1g。HPLC-UV峰面积归一化含量99.8％。
本品与三氯化铁溶液呈紫红色。MS(API-ESI)m/z:[M+H]+409,[M-H]-407。本品在乙酸乙酯中与2M甲磺酸成盐，80℃减压干燥，得黄色结晶。1H-NMR(500MHz,DMSO-d6)δ:8.21(m,Ph-2',6'-H),7.66(m，Ph-3',4'，5'-H)，7.31(s,1H,3-H),4.47(s,2H,8-CH2),3.65-3.24(m,8H,CH22”,3”,4”,5”-H),2.86(s,3H,N-CH3),2.78(s,3H,COCH3),2.35(s,6H,2MCH3SO3H)。为6-乙酰基-8-甲基哌嗪亚甲基-白杨素[6-Acetyl-5,7-dihydroxy-8-((4-methylpiperazin-1-yl)methyl)-2-phenyl-4H-chromen-4-one]。
实施例7. 6-乙酰基-8-哌啶亚甲基-白杨素(CH-c)
取6-乙酰基白杨素10g，加入500ml乙酸乙酯，加热回流搅拌0.5h。按1：1.5M加入哌啶和甲醛溶液，继续回流搅拌1.5h。将析出的结晶滤出，洗涤，60℃减压干燥，得黄色结晶9.2g。HPLC-UV含量99.5％。
本品与三氯化铁溶液呈紫红色。MS(API-ESI)m/z:[M+H]+394,[M-H]-392。1H-NMR(DMSO-d6,500MHz)δ:8.07(d,2H,Ph-2',6'-H),7.60(m,3H,Ph-3',4',5'-H),6.89(s,1H,3-H),4.14,(s,2H,C8-CH2),2.95(s,2H,COCH3),1.64-1.23(m,10H,2”,3”,4”，5”,6”-CH2)。为6-乙酰基-8-哌啶亚甲基-白杨素[6-Acetyl-5,7-dihydroxy-2-phenyl-8-piperidin-1-ylmethyl-chromen-4-one]。
实施例8. 8-乙酰基-6-哌啶醇亚甲基-白杨素(CH8-j)
将8-乙酰基白杨素13g(含量60％)，加入乙酸乙酯500ml，加热回流搅拌溶解成红色溶液。按1：1.5M加入哌啶和甲醛溶液，继续回流搅拌1h。将析出的结晶分离，洗涤，60℃减压干燥，得黄色结晶性粉末5.3g。(含量82.5％)。多次重结晶，得到浅黄色结晶，HPLC-UV含量97.0％。
本品与三氯化铁溶液呈三氯化铁溶液呈橙红色。25℃甲醇溶液比旋度为+2450。MS(API-ESI)m/z:[M+H]+410,[M+H-101]+309,[M-H]-408。HR-MS(API-ESI)m/z:[M-H]-408.1444(计算值408.1447),分子式C23H23NO6，平均分子量409.43。UV：1ml含5μg的甲醇溶液E1％ 1cm(λmax 255nm)为878。IR(KBr,cm-1)：3349(宽强νOH),2953(νCH3),1646(宽强νC＝O，CH3-C＝O)，1599,1578,1549,769和682(γPh单取代)。1H-NMR(DMSO-d6,500MHz)δ:8.12(d,2H,Ph-2',6'-H),7.56(m,3H,Ph-3',4',5'-H),6.88(s,1H,3-H),4.07,(s,2H,C6-CH2),3.77(m,1H，CH-OH),2.99-3.25(m,4H,2”,6”-CH2),2.57(s,3H,COCH3),1.65-1.95(m,4H,3”,5”-CH2)。13C-NMR(DMSO-d6,400MHz)δ:198.1(C＝O),180.3(C-4),175.6(C-7),161.3(C-5),161.2(C-2),157.0(C-9),131.5(C-1'),131.0(C-4'),128.9(C-3',5'),126.2(C-2',6'),109.4(C-8),104.4(C-6),102.3(C-3),98.6(C-10)。
UVλmax 255nm比CH-j紫移28nm；13C-NMR 104.4(C-6)和109.4(C-8)，而CH-j 105.9(C-6)和105.5(C-8)。因此，为化合物CH8-j，即8-乙酰基-6-哌啶醇亚甲基白杨素。[8-Acetyl-5,7-dihydroxy-6-(4-hydroxy-piperidin-1-ylmethyl)-2-phenyl-chromen-4-one]
实施例9. 6-乙酰基白杨素(CH)
取2，4，6-三羟基苯乙酮一水合物150g，按文献法合成乙酰基白杨素[CN200410008987.8]，得到浅橙黄色针状结晶155g(含量95.3％)。用DMF重结晶，得浅黄色丝状结晶，HPLC-UV含量99.3％。
本品与三氯化铁溶液呈紫红色。MS(API-ESI)m/z:[M+H]+297，[M-H]-295。UV：1ml含5μg的甲醇溶液E1％ 1cm(λmax 283nm)为1532。IR(KBr,cm-1)：3063(νCH3)，1646(C＝O)，1586(νCH3-C＝O)，1452，1379，1178，767和682(γPh单取代)。1H-NMR(DMSO-d6,500MHz)δ:8.03(d,2H,Ar-2',6'-H),7.62(m,3H,Ar-3',4',5'-H),7.10(s,1H,3-H),6.63(s,1H,8-H)，2.74(s,CH3)。分子式C17H12O5，平均分子量296.27。为6-乙酰基白杨素。[6-Acetyl-5,7-dihydroxy-2-phenyl-4H-chromen-4-one]
实施例10. 8-乙酰基白杨素(CH8)
将分离6-乙酰基白杨素后的洗液，蒸馏回收部分溶剂，将析出的结晶滤出，得到浅橙黄色结晶(含8-乙酰基白杨素60％)。以石油醚-乙酸乙酯-氯仿(40：15:3)为流动相，经硅胶闪色谱分离，得到类白色结晶，HPLC-UV含量95.0％。
本品与三氯化铁溶液呈橙红色。MS(API-ESI)m/z:[M+H]+297，[M-H]-295。UV：甲醇溶液λmax 276nm。IR(KBr,cm-1)：1H-NMR(DMSO-d6,500MHz)δ:8.08(d,Ar-2H,2',6'-H),7.63(m,Ar-3H,3',4',5'-H),7.13(s,1H,3-H),6.36(s,1H,6-H),2.77(s,CH3)。分子式C17H12O5，平均分子量296.27。为8-乙酰基白杨素。[8-Acetyl-5,7-dihydroxy-2-phenyl-4H-chromen-4-one]
。
Claims (10)
1.乙酰基白杨素Mannich碱衍生物，具有通式I的结构：
通式I中，R1和R2各自独立地选自乙酰基和有机仲胺亚甲基，R1与R2中有且只有一个是乙酰基。
2.根据权利要求1所述的乙酰基白杨素Mannich碱衍生物，其特征在于，所述的有机仲胺亚甲基选自二甲胺基亚甲基，吡咯烷亚甲基，哌啶亚甲基，吗啉亚甲基，硫吗啉亚甲基，4`-甲基哌嗪亚甲基，哌嗪亚甲基，4`-羟乙基哌嗪亚甲基，脯氨醇(+)亚甲基，4`-哌啶醇亚甲基，4`-哌啶酮亚甲基，噻吩并[3，2-c]吡咯烷亚甲基和脯氨醇(-)亚甲基。
3.根据权利要求1所述的乙酰基白杨素Mannich碱衍生物，其特征在于，所述的有机仲胺亚甲基选自吗啉亚甲基，硫吗啉亚甲基，4`-甲基哌嗪亚甲基，4`-羟乙基哌嗪亚甲基，脯氨醇(+)亚甲基和4`-哌啶醇亚甲基。
4.根据权利要求1所述的乙酰基白杨素Mannich碱衍生物，其特征在于，所述的有机仲胺亚甲基是4`-哌啶醇亚甲基。
5.根据权利要求1-4中任意权利要求所述的乙酰基白杨素Mannich碱衍生物，其特征在于，所述的R1是乙酰基。
6.权利要求1所述的乙酰基白杨素Mannich碱衍生物的合成方法，是以乙酰基白杨素为先导化合物，与有机仲胺类化合物和甲醛溶液在有机溶剂中经Mannich热缩合反应得到的。
7.根据权利要求6所述的合成方法，其特征在于，其中所述的有机仲胺类化合物选自二甲胺，吡咯烷，哌啶，吗啉，硫吗啉，4-甲基哌嗪，哌嗪，4-羟乙基哌嗪，脯氨醇(+)，4-羟基哌啶，4`-哌啶酮，噻吩并[3，2-c]吡咯烷和脯氨醇(-)。
8.根据权利要求6或7所述的合成方法，其特征在于，所述的有机溶剂选自乙醇，乙腈，四氢呋喃，二氧六环，乙酸乙酯，乙酸丁酯，乙酰丙酮，乙酰乙酸乙酯和甲基异丁酮。
9.权利要求1所述的乙酰基白杨素Mannich碱衍生物在制备抗肿瘤药物中的应用。
10.根据权利要求8所述的应用，其特征在于，所述的抗肿瘤药物是抗实体瘤药物。
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| WO2017215538A1 (zh) * | 2016-06-15 | 2017-12-21 | 张帆 | 乙酰基白杨素Mannich碱衍生物及其用途 |
| CN113549044A (zh) * | 2021-07-23 | 2021-10-26 | 中国药科大学 | 8-氮杂环取代色酮类衍生物及其制备方法与制药用途 |
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| WO2017215538A1 (zh) * | 2016-06-15 | 2017-12-21 | 张帆 | 乙酰基白杨素Mannich碱衍生物及其用途 |
| CN113549044A (zh) * | 2021-07-23 | 2021-10-26 | 中国药科大学 | 8-氮杂环取代色酮类衍生物及其制备方法与制药用途 |
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