环化小檗碱类似物和Sansanmycin A衍生物的设计、合成及活性研究
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摘要
在开展小檗碱(BBR)类衍生物不同生物活性研究中,基于对相关有机反应的深刻理解,巧妙地对其进行了合理的逻辑推导,在BBR分子结构中的1-和13-位骈合了一个芳香环,由此成功构建了一类全新结构骨架的化合物一环化小檗碱(CBBR),化学结构式见图1。由于其具有更好的平面性结构特征,更加容易嵌入双链DNA碱基对里,推测可能具有更好的抗肿瘤作用。生物活性实验测定显示CBBR对肝癌HepG-2细胞的IC50在1.2μM左右,活性显著强于BBR。
为了深入了解此类化合物抗肿瘤的化学以及生物作用机制,本论文以CBBR为先导化合物,针对分子中的A、C、D及E环侧链结构,开展较为系统的结构修饰与优化,累积设计合成了58个未见文献报道的CBBR类似物,通过对HepG-2细胞的活性评价,阐明此类化合物抗肿瘤活性的构效关系(SAR)。
(1)BBR分子中引入E环后增加了分子平面性,抗肿瘤活性明显增强；
(2)D环8-位合适基团取代有助于活性提高；
(3)A环上取代基以亚甲二氧环和1,2-二甲氧基活性较高；
(4)13-位苄基取代后,抗肿瘤活性升高；
(5)季铵正离子为活性必需,C环双键还原活性降低。
通过体外活性测定,化合物A-34、A-35、A-55、A-58、A-40、A-49和A-56在0.6μg/mL时对HepG-2细胞的抑制率达到85%以上,IC50在0.13到1.01μM之间,对肠癌HCT116细胞的IC50在0.09到5.61μM的范围内；尤其是A-35对阿霉素(DOX)耐药的乳腺癌MCF-7细胞(MCF-7/AdrR)具有显著的抑制作用,提示此类化合物与阿霉素不存在交叉耐药问题。
初步作用机制研究表明：细胞周期分析A-35主要阻滞HCT116细胞于G2/M期,其在15μg/mL时就对拓扑异构酶Ⅰ(TopⅠ)显示较强的抑制活性(与阳性药HCPT对比)；在同样的浓度下对Top Ⅱ的抑制活性接近依托泊苷(VP16)在100μg/mL时的抑制效果。通过分子对接软件eHiTS发现A-35与Top Ⅰ-DNA复合物、Top Ⅱ ATPase结构域具有较好的结合能力,提示CBBR类似物为一类新型Top酶抑制剂。
小鼠急性毒性试验显示A-35腹腔注射给药,其LD50大于500mg/kg,是一类有前景的新型抗肿瘤化合物,拥有对耐药肿瘤细胞有效的优势。
另外,本论文还开展了Sansanmycin A (SSA)衍生物的设计、合成和活性研究。研究所从微生物Streptomyces sp SS的发酵液中分离得到了一种新的尿苷肽类抗生素Sansanmycin A,结构式见图2,其对结核菌株H37Ra、标准株H37Rv和3株多药耐药菌株1279、2062和2199的最小抑菌浓度(MIC)为10-40μg/mL。另外,其急性毒性较低,LD50≥2400mg/kg(大鼠,静脉注射给药)。
基于此,在前期工作的基础上,本论文以SSA为先导化合物,以体外抗结核菌株H37Rv活性为导向,针对分子中的伯胺基、酚羟基、醇羟基等多个结构片段开展初步的结构修饰与优化,设计合成17个未见文献报道的SSA类似物,初步探索了此类化合物抗结核活性的SAR。构效关系表明,在SSA的伯氨基上引入异丙基的衍生物1d的体外抗H37Rv活性优于先导物,而二甲基取代物1a活性与先导物相当。另外,化合物1d抑制耐药的结核菌株与H37Rv菌株活性一样,MIC皆为8μg/mL,同样优于SSA。
In continuation of our ongoing research on different biological activities of BBR analogues, due to well understanding of a related organic reaction, a novel compound-cycloberberine (CBBR) was constructed successfully and then confirmed by MS,1H NMR and13C NMR. Because of its planar structure, which could enhance antiproliferative potency via intercalating into DNA easily, CBBR might have a significant antiproliferative activity. The biological tests showed that the IC50of CBBR against HepG2cells was1.2μM.
Taking CBBR as a lead compound because of its good activity and original structure,58new derivatives were designed and synthesized through modification of rings A, C, D and E. After measuring the inhibition rate of HepG-2cells, the structure-activity relationship (SAR) of this group of compounds were ascertained.
(1) After the introduction of the ring E in BBR structure, with increase of the plane of the molecule, CBBR exhibited moderate activity;
(2) An appropriate volume of group substituting at the8-position of ring D increased the anti-tumor activity;
(3) The analogues substituted methylenedioxy or1,2-dimethoxy group in ring A had a significant antiproliferative activity;
(4) Introduction of benzyl at the13-position improved the inhibitory activity;
(5) The N+was essential, and removing the double bonds in ring C lead to disappearance of activity.
By screening, compounds A-34、A-35、A-55、A-58、A-40、A-49and A-56showed a increased antiproliferative activity against HepG2with inhibition rate above85%at0.6μg/mL. The IC50of these derivatives against HepG2ranged from0.13to1.01μM, while it ranged from0.09to5.61μM against HCT116cells. Moreover, compound A-35exhibited anti-tumor effect against MCF-7/ADrR cells with an IC50of3.66μg/mL. So we could draw a conclusion that cross drug-resistance between CBBR and doxorubicin might not exist.
Cell cycle analysis of the DNA profile in the HCT116verified that A-35resulted in a distinct accumulation of cells in the G2/M phase. The mechanism research revealed that A-35displayed a significant inhibitory activity against Top I and Top II at the concentration of15μg/mL. Besides, through the docking software-eHiTS, the docking models of A-35and the Top I-DNA or Top II ATPase domain were obtained. Consequently, we considered CBBR analogues to be a new class of Top I and Top II dual inhibitor.
The acute toxicity in mice was tested and the results showed the LD50was more than500mg/kg (ip).
Meanwhile, a series of sansanmycin (SSA) derivatives were designed and synthesized. SSA isolated from Streptomyces sp SS in our institute is a new uridyl-peptide antibiotic which exhibited moderate inhibitory effect against H37Ra, H37Rv and multidrug-resistant strains1279,2062,2199(MIC=10-40μg/mL). What's more, the LD50was more than2400mg/kg (iv) in rats.
However, natural SSA could not become antitubercular drug due to its weak activity. In order to improve its antitubercular effect,17derivatives were designed, semi-synthesized and evaluated for their activities with SSA as the lead compound. After measuring the MIC of drug-sensitive M. tuberculosis H37Rv, the SAR of this group of compounds were ascertained.
The compound Id substituting isopropyl group at N-terminal displayed high potency against H37Rv, while compound la bearing dimethyl group at N-terminal was equal in potency to the SSA. The analogue1d was then assayed for effect against multidrug-resistant M. tuberculosis2199. Interestingly, the compound1d exhibited better inhibitory effect against multidrug-resistant M. tuberculosis2199than the natural product.
为了深入了解此类化合物抗肿瘤的化学以及生物作用机制,本论文以CBBR为先导化合物,针对分子中的A、C、D及E环侧链结构,开展较为系统的结构修饰与优化,累积设计合成了58个未见文献报道的CBBR类似物,通过对HepG-2细胞的活性评价,阐明此类化合物抗肿瘤活性的构效关系(SAR)。
(1)BBR分子中引入E环后增加了分子平面性,抗肿瘤活性明显增强；
(2)D环8-位合适基团取代有助于活性提高；
(3)A环上取代基以亚甲二氧环和1,2-二甲氧基活性较高；
(4)13-位苄基取代后,抗肿瘤活性升高；
(5)季铵正离子为活性必需,C环双键还原活性降低。
通过体外活性测定,化合物A-34、A-35、A-55、A-58、A-40、A-49和A-56在0.6μg/mL时对HepG-2细胞的抑制率达到85%以上,IC50在0.13到1.01μM之间,对肠癌HCT116细胞的IC50在0.09到5.61μM的范围内；尤其是A-35对阿霉素(DOX)耐药的乳腺癌MCF-7细胞(MCF-7/AdrR)具有显著的抑制作用,提示此类化合物与阿霉素不存在交叉耐药问题。
初步作用机制研究表明：细胞周期分析A-35主要阻滞HCT116细胞于G2/M期,其在15μg/mL时就对拓扑异构酶Ⅰ(TopⅠ)显示较强的抑制活性(与阳性药HCPT对比)；在同样的浓度下对Top Ⅱ的抑制活性接近依托泊苷(VP16)在100μg/mL时的抑制效果。通过分子对接软件eHiTS发现A-35与Top Ⅰ-DNA复合物、Top Ⅱ ATPase结构域具有较好的结合能力,提示CBBR类似物为一类新型Top酶抑制剂。
小鼠急性毒性试验显示A-35腹腔注射给药,其LD50大于500mg/kg,是一类有前景的新型抗肿瘤化合物,拥有对耐药肿瘤细胞有效的优势。
另外,本论文还开展了Sansanmycin A (SSA)衍生物的设计、合成和活性研究。研究所从微生物Streptomyces sp SS的发酵液中分离得到了一种新的尿苷肽类抗生素Sansanmycin A,结构式见图2,其对结核菌株H37Ra、标准株H37Rv和3株多药耐药菌株1279、2062和2199的最小抑菌浓度(MIC)为10-40μg/mL。另外,其急性毒性较低,LD50≥2400mg/kg(大鼠,静脉注射给药)。
基于此,在前期工作的基础上,本论文以SSA为先导化合物,以体外抗结核菌株H37Rv活性为导向,针对分子中的伯胺基、酚羟基、醇羟基等多个结构片段开展初步的结构修饰与优化,设计合成17个未见文献报道的SSA类似物,初步探索了此类化合物抗结核活性的SAR。构效关系表明,在SSA的伯氨基上引入异丙基的衍生物1d的体外抗H37Rv活性优于先导物,而二甲基取代物1a活性与先导物相当。另外,化合物1d抑制耐药的结核菌株与H37Rv菌株活性一样,MIC皆为8μg/mL,同样优于SSA。
In continuation of our ongoing research on different biological activities of BBR analogues, due to well understanding of a related organic reaction, a novel compound-cycloberberine (CBBR) was constructed successfully and then confirmed by MS,1H NMR and13C NMR. Because of its planar structure, which could enhance antiproliferative potency via intercalating into DNA easily, CBBR might have a significant antiproliferative activity. The biological tests showed that the IC50of CBBR against HepG2cells was1.2μM.
Taking CBBR as a lead compound because of its good activity and original structure,58new derivatives were designed and synthesized through modification of rings A, C, D and E. After measuring the inhibition rate of HepG-2cells, the structure-activity relationship (SAR) of this group of compounds were ascertained.
(1) After the introduction of the ring E in BBR structure, with increase of the plane of the molecule, CBBR exhibited moderate activity;
(2) An appropriate volume of group substituting at the8-position of ring D increased the anti-tumor activity;
(3) The analogues substituted methylenedioxy or1,2-dimethoxy group in ring A had a significant antiproliferative activity;
(4) Introduction of benzyl at the13-position improved the inhibitory activity;
(5) The N+was essential, and removing the double bonds in ring C lead to disappearance of activity.
By screening, compounds A-34、A-35、A-55、A-58、A-40、A-49and A-56showed a increased antiproliferative activity against HepG2with inhibition rate above85%at0.6μg/mL. The IC50of these derivatives against HepG2ranged from0.13to1.01μM, while it ranged from0.09to5.61μM against HCT116cells. Moreover, compound A-35exhibited anti-tumor effect against MCF-7/ADrR cells with an IC50of3.66μg/mL. So we could draw a conclusion that cross drug-resistance between CBBR and doxorubicin might not exist.
Cell cycle analysis of the DNA profile in the HCT116verified that A-35resulted in a distinct accumulation of cells in the G2/M phase. The mechanism research revealed that A-35displayed a significant inhibitory activity against Top I and Top II at the concentration of15μg/mL. Besides, through the docking software-eHiTS, the docking models of A-35and the Top I-DNA or Top II ATPase domain were obtained. Consequently, we considered CBBR analogues to be a new class of Top I and Top II dual inhibitor.
The acute toxicity in mice was tested and the results showed the LD50was more than500mg/kg (ip).
Meanwhile, a series of sansanmycin (SSA) derivatives were designed and synthesized. SSA isolated from Streptomyces sp SS in our institute is a new uridyl-peptide antibiotic which exhibited moderate inhibitory effect against H37Ra, H37Rv and multidrug-resistant strains1279,2062,2199(MIC=10-40μg/mL). What's more, the LD50was more than2400mg/kg (iv) in rats.
However, natural SSA could not become antitubercular drug due to its weak activity. In order to improve its antitubercular effect,17derivatives were designed, semi-synthesized and evaluated for their activities with SSA as the lead compound. After measuring the MIC of drug-sensitive M. tuberculosis H37Rv, the SAR of this group of compounds were ascertained.
The compound Id substituting isopropyl group at N-terminal displayed high potency against H37Rv, while compound la bearing dimethyl group at N-terminal was equal in potency to the SSA. The analogue1d was then assayed for effect against multidrug-resistant M. tuberculosis2199. Interestingly, the compound1d exhibited better inhibitory effect against multidrug-resistant M. tuberculosis2199than the natural product.
引文
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