肉桂酰天冬氨酸类氨肽酶N抑制剂的合成和初步活性研究

目的 以氨肽酶N（APN／CD13）为药物作用靶点,设计合成一系列新型的肉桂酰天冬氨酸类衍生物,并对其进行抑制活性评价。方法 以肉桂酸和L-天冬氨酸甲酯为基本原料经缩合、水解、酸化、环合反应得到关键中间体环状酸酐,再经氨解反应合成了一系列新型的肉桂酰天冬氨酸类衍生物;采用体外抑酶试验测定化合物对氨肽酶N的抑制活性。 结果与结论 合成了24个未见文献报道的肉桂酰天冬氨酸类衍生物, 其结构经过1H-NMR、MS谱确证。其中化合物6d,6g,6k, 6l显示出较好的抑制氨肽酶N活性(IC50=12~28 μmol.L-1),有进一步研究的价值。     

新型查耳酮衍生物的合成及其初步抗蛋白酪氨酸 激酶(PTKs)活性研究

目的 设计合成一系列全新的查耳酮类衍生物,并初步测试其蛋白酪氨酸激酶（PTKs）抑制活性。方法 以间二甲苯为原料,经硝化、还原、水解、甲氧甲基保护等反应得到中间体取代苯乙酮,该中间体再与取代苯甲醛发生羟醛缩合反应后脱保护基得到目标化合物。采用酶联免疫吸附法（ELISA）,以金雀异黄素为阳性对照,对目标化合物进行体外 PTKs 抑制活性检测。 结果与结论 合成了 10 个查耳酮衍生物,其中 9 个是未见报道的新化合物,10 个化合物的结构经核磁共振氢谱和质谱确证。化合物 6a 及新化合物 6b、6c 和 6d 对 PTKs 具有良好的抑制活性。     

灯盏乙素衍生物的合成及其体外抗凝血活性研究

目的 对灯盏乙素葡萄糖醛酸羧基进行结构修饰,合成葡萄糖醛酸羧基酯类衍生物,并测定这些衍生物的体外抗凝血活性。方法 通过将灯盏乙素葡萄糖醛酸羧基先成盐后酯化的方法合成6个灯盏乙素酯类衍生物,其中灯盏乙素乙酯及苄酯为已知化合物,其余4个为新化合物,目标化合物的抗凝血活性采用家兔的贫血小板血浆（PPP）进行凝血酶原时间（PT）、凝血酶时间(TT)、活化部分凝血活酶时间（APTT）及纤维蛋白原（FIB）的测定,以80% EtOH作溶剂,以生理盐水作为阳性对照。结果与结论 合成了6个灯盏乙素酯类衍生物Ⅱa~Ⅱf。药理实验表明,该类衍生物的抗凝血活性没有灯盏乙素强,灯盏乙素羧酸基改造后对凝血活性影响不大。     

金刚烷胺衍生物的设计、合成及其抗禽流感病毒活性

目的 设计合成金刚烷胺衍生物并对其进行抗禽流感病毒活性测试。方法 以金刚烷甲酰氯和氨基酸甲酯盐酸盐为起始原料经酰胺化、水解、成盐等反应依次得到3个系列金刚烷胺衍生物。以金刚烷胺为阳性对照,采用幼犬肾（MDCK）细胞系噬斑形成实验测定目标化合物的抗禽流感病毒活性。结果与结论 共合成了20 个未见报道的新化合物,目标化合物的结构均经¹H-NMR、IR、MS 谱确证;仅有化合物 A₅ 显示出较好的抗禽流感病毒活性。     

苯甲酰胺类组蛋白去乙酰化酶抑制剂的合成及 抗肿瘤细胞增殖活性

目的 针对组蛋白去乙酰化酶（HDAC）的结构特点,设计合成一系列化合物并进行体外抗肿瘤活性评价,为进一步优化设计提供指导。方法 以 2-羟基-4-硝基苯甲醛为原料,经环合、还原、与芳醛衍生物反应、水解后,在N,N-碳酰二咪唑存在下经缩合反应合成目标化合物。结果与结论 共合成了11个新苯甲酰胺类衍生物,化合物的结构经质谱、核磁共振氢谱确证;体外抗肿瘤活性评价结果表明,其中 4 个化合物（4b、4c、4d、4h）对肿瘤细胞具有显著的增殖抑制活性,化合物4h的活性最好,它对 HL60、MCF-7、A549 肿瘤细胞的 IC₅₀值分别为2.81、＞50、4.79 μmol·L^-1。     

头孢类化合物的设计、合成与生物活性

目的 设计合成具有抗菌活性的头孢类化合物。方法 以GCLE和其衍生物为起始原料,经Mannich反应、加成反应等步骤得到一系列目标化合物;以头孢唑啉为对照品,通过微量稀释法测定了8个目标化合物对标准金黄色葡萄球菌和标准肺炎克雷白杆菌的最小抑菌浓度（MIC）。结果 合成了8个新化合物,并利用1H-NMR验证了结构。结论 初步生物活性试验显示,化合物5b对标准金黄色葡萄球菌的抗菌活性较好, 化合物4a和化合物4b对β-内酰胺酶的结合能力较强。     

具有手性吡咯烷侧环的噁唑烷酮类化合物的合成及抗菌活性研究

目的 设计合成具有手性吡咯烷侧环的噁唑烷酮类化合物,并考察其体外抗菌活性。方法 以手性脯氨酸和3,4-二氟硝基苯为原料,通过多步反应合成目标化合物;采用微量液体稀释法检测目标化合物的抗菌活性。结果与结论 合成了 8 个新化合物,其结构经 ¹H-NMR、MS 谱确证。体外活性试验表明：由（R）-脯氨酸衍生的化合物7b 对革兰阳性菌[金黄色葡萄球菌、表皮葡萄球菌以及耐甲氧西林金黄色葡萄球菌（MRSA）具有良好的抑制活性,对革兰阴性菌具有较弱的抑制活性。由（S）-脯氨酸衍生的化合物 7a 的抗菌活性明显低于 7b。研究结果表明,吡咯烷侧环的手性因素对抗菌活性具有显著影响。     

13-酰胺基取代苦参碱衍生物的合成及抗肿瘤活性研究

目的 合成13-酰胺基取代苦参碱衍生物及研究该类化合物的体外抗肿瘤活性。方法 以槐果碱为原料,通过迈克尔加成（Michael addition）,叠氮还原酰化反应,制得系列13-位酰胺取代的衍生物,所有化合物结构均经¹H NMR等谱确证;选取人肝癌细胞（BEL-7404）和小鼠黑色素瘤细胞（K111）对所合成的目标化合物进行体外抗肿瘤药理活性筛选。结果 设计合成了9个新化合物,大多数化合物对两株肿瘤细胞都具有较强的抑制活性。结论 化合物4b和4e对人肝癌细胞（BEL-7404）有较强的抑制活性。     

β-榄香烯胺类衍生物的合成及抗癌活性研究

目的 合成β-榄香烯胺类衍生物,并对其体外抗癌活性进行初步的评价。方法 β-榄香烯经氯代、胺化制备目标化合物。以SRB法测试目标化合物对HL-60、HeLa、SGC-7901细胞的增殖抑制活性。结果与结论 合成的12个新化合物经IR、MS和1H-NMR确证结构。初步药理结果显示大部分目标化合物的体外抗癌活性明显强于β-榄香烯。     

2,2-二甲基苯并吡喃类衍生物的合成

目的 设计合成一系列苯并吡喃类衍生物。方法 以2-甲基-丁-3-炔-2-醇为起始原料,经氯代、Williamson 成醚、环合、Aldol 缩合4步反应得到目标化合物。结果与结论 合成了16个未见文献报道的新化合物,其结构经¹H-NMR 和 MS 谱确定。所有目标化合物的抗肿瘤活性测试正在进行中。     

大黄酸对慢性移植肾肾病大鼠肾组织抗纤维化因子表达的影响

目的:观察慢性移植肾肾病(CAN)大鼠肾组织中肝细胞生长因子(HGF)、骨形态发生蛋白(BMP7)的表达及大黄酸对其影响。方法:以F344及Lewis大鼠分别作为供受体,建立CAN大鼠动物模型。将移植后30只大鼠分为治疗组(n=16)及模型组(n=14),另以5只Lewis正常大鼠作为对照组。治疗组予以大黄酸100mg.kg-1.d-1灌胃,模型组及对照组给予等量的0.5%羧甲基纤维素钠溶液。于移植后1个月、2个月及4个月分别收集大鼠的血和尿样,检测肾功能及尿蛋白含量。并于移植后2个月及4个月分别宰杀大鼠,观察各组大鼠移植肾组织病理学变化。并应用免疫组化以及实时荧光定量PCR方法分别检测CAN大鼠肾组织HGF、BMP7以及转化生长因子β1(TGF-β1)的蛋白质表达水平以及mRNA表达水平。结果:(1)与模型组比较,大黄酸治疗组大鼠肾功能改善(P<0.05);肾组织病理检查示肾间质纤维化及间质炎症明显改善(P<0.05);(2)与模型组比较,大黄酸治疗组大鼠肾组织TGF-β1无明显差异,但抗纤维化因子BMP7及HGF明显升高(P<0.05)。结论:(1)大黄酸能改善CAN大鼠肾功能及肾脏慢性病变;(2)大黄酸有可能通过诱导肾组织抗纤维化因子HGF、BMP7表达,抑制肾组织炎细胞浸润,发挥抗肾组织纤维化的作用,从而延缓CAN的进展。     

大黄酸分散体的制备及其对糖尿病肾病大鼠肾功能的影响

目的以聚乙烯吡咯烷酮(PVP)为载体,将大黄酸制成固体分散体。观察大黄酸固体分散体对糖尿病大鼠肾功能的影响。方法采用溶剂法制备大黄酸固体分散体;对制备的固体分散体进行差热分析和红外光谱分析。采用链脲佐菌素(STZ)诱导大鼠糖尿病模型,观察大黄酸固体分散体对糖尿病肾病大鼠肾功能的影响。结果 12周后,糖尿病大鼠血中尿素氮(BUN)、内生肌酐清除率(Ccr)、24 h尿蛋白排泄量(UPE)明显升高(P<0.01)。肾组织ET含量、24 h尿ET排泄量(UET)显著升高(P<0.01)。给予糖尿病肾病大鼠大黄酸固体分散体干预后,糖尿病大鼠的BUN、Ccr、UPE减少,UET和肾组织中ET含量降低。结论大黄酸固体分散体能改善糖尿病大鼠的肾脏功能。     

大黄酸对人肾小球系膜细胞功能的影响

目的探讨大黄酸防治糖尿病肾病的作用机制。方法用细胞培养、ELISA、明胶酶谱及免疫沉淀、Western blot等技术,研究了大黄酸对肾小球系膜细胞转化生长因子(TGFβ₁)、基质金属蛋白酶-2,-9(MMP-2和MMP-9)及p38活化丝裂原活化蛋白激酶(p38MAPK)活性的作用。结果大黄酸可显著抑制肾小球系膜细胞的增殖,对抗高糖诱导肾小球系膜细胞TGFβ₁活性升高的作用;对高糖诱导肾小球系膜细胞MMP-2,MMP-9,pro-MMP-2及pro-MMP-9的活性增加无明显影响,但可明显对抗高糖引起的肾小球系膜细胞p38MAPK活性增加。结论大黄酸减少FN的分泌可能与其降低p38MAPK及TGFβ₁活性有关。     

Rhein reverses the diabetic phenotype of mesangial cells over-expressing the glucose transporter (GLUT1) by inhibiting the hexosamine pathway

BACKGROUND AND PURPOSE: Rhein, an anthraquinone compound isolated from rhubarb, has been proved effective in treatment of experimental diabetic nephropathy (DN). To explore the mechanism of its therapeutic effect on DN, rhein was tested for its effect on the hexosamine pathway. EXPERIMENTAL APPROACH: The influence of rhein on cellular hypertrophy, fibronectin synthesis, glucose uptake, glutamine: fructose 6-phosphate aminotransferase (GFAT) activity, UDP-N-acetylglucosamine (UDP-GlcNAc) level and TGF-beta1 and p21 expression was evaluated in MCGT1 cells, a GLUT1 transgenic rat mesangial cell line. GFAT activity in normal rat mesangial cells in high glucose concentrations and in vitro was also measured. KEY RESULTS: Significantly increased fibronectin synthesis, cellular hypertrophy, much higher GFAT activity and UDP-GlcNAc level and increased TGF-beta1 and p21 expression were found in MCGT1 cells cultured in normal glucose concentration. Rhein treatment decreased all these features of MCGT1 cells but did not exert a direct effect on GFAT enzymatic activity. CONCLUSIONS AND IMPLICATIONS: There was over-activity of the hexosamine pathway in MCGT1 cells, which may explain the higher expression of TGF-beta1 and p21, the cellular hypertrophy and the increased expression of extracellular matrix (ECM) components in the cells. By inhibiting the increased activity the hexosamine pathway, rhein decreased TGF-beta1 and p21 expression and thus contributed to the decreased cellular hypertrophy and ECM synthesis. Inhibition of the hexosamine pathway may be one of the mechanism through which rhein exerts its therapeutic role in diabetic nephropathy.     

大黄素及其衍生物：合成与药理作用研究进展

大黄酸是大黄的主要成分之一,其应用广泛,可用于抗肿瘤、抗病毒、糖尿病肾病、抑菌、抗炎等,具有不错的临床应用价值。本文主要综述近年大黄酸及其衍生物的合成与药理活性方面的研究进展,以期为大黄酸及其衍生物的开发应用提供参考依据。     

大黄酸抑制转化生长因子β1诱导的肾小管上皮细胞肥大及细胞外基质积聚

目的：观察大黄酸对TGFβ1诱导的肾近端小管上皮细胞肥大及细胞外基质的影响。方法：在体外以TGFβ1（2μg/L）刺激LLC－PK1细胞,诱导细胞肥大和细胞外基质合成的增加。同时,以不同浓度的大黄酸处理细胞,检测细胞体积、蛋白质含量以及[^3H]亮氨酸掺入以观察细胞肥大的变化。此外,检测细胞培养上清液中的纤维素增生（FN）含量。[^3H]脯氨酸掺入以及细胞胶原IV及FN mRNA的表达以观察大黄酸对细胞外基质的影响。结果：TGFβ1（2μg/L）刺激可以导致LLC－PK1细胞出现细胞肥大,表现为细胞体积、细胞内蛋白量及[^3H]亮氨酸掺入量明显增加,大黄酸治疗后细胞体积及细胞内蛋白量降低。TGFβ1也能明显增加LLC－PK1细胞[^3H]脯氨酸掺入量,培养上清液中FN含量,以及细胞内胶原IV和FN mRNA的表达。大黄酸则能抑制上述细胞外基质合成的影响,明显降低细胞内胶原IV和FN mRNA表达水平。结论：大黄酸可以逆转TGFβ1诱导的近端肾小管上皮细胞肥大,抑制TGFβ1刺激的细胞外基质合成,这可能是大黄酸预防或改善糖尿病肾脏病变、延缓糖尿病肾病进展的作用机制之一。     

Rhein attenuates renal inflammatory injury of uric acid nephropathy via lincRNA-Cox2/miR-150-5p/STAT1 axis

Rhein has protective effect on uric acid nephropathy (UAN). This article aims to demystify the mechanism of function of rhein in UAN. Mouse kidney epithelial cell line (TCMK-1) was incubated with uric acid (UA) to induce inflammatory injury. Then, the TCMK-1 cells were treated with rhein. The relationships among lincRNA-Cox2, miR-150-5p and STAT1 were evaluated by luciferase reporter assay. CCK8 and flow cytometry were performed to detect cell proliferation and apoptosis. The levels of IL-6, IL-1β and TNF-α were investigated by enzyme linked immunosorbent assay. Western blot and quantitative real-time PCR were performed to examine the expression of genes and proteins. We found that UA suppressed proliferation and enhanced apoptosis and the levels of IL-6, IL-1β and TNF-α of TCMK-1 cells, which was effectively improved by rhein treatment. Furthermore, lincRNA-Cox2 overexpression caused an increase of apoptosis and inflammatory factors in the rhein-treated TCMK-1 cells. LincRNA-Cox2 regulated STAT1 expression by sponging miR-150-5p. And lincRNA-Cox2 promoted apoptosis and inflammatory injury of TCMK-1 cells by regulating miR-150-5p/STAT1 axis. In summary, our studies demonstrate that rhein has a protective effect against UAN by inhibiting renal inflammatory injury via lincRNA-Cox2/miR-150-5p/STAT1 axis.     

Toll-like receptor 4 is involved in a protective effect of rhein on immunoglobulin A nephropathy

Objectives:The objective was to investigate the protective effects of rhein on renal histology change and the effects of rhein on renal tissue toll-like receptor (TLR) 4, TLR9, transforming growth factor-β1 (TGF-β1) expression in immunoglobulin A nephropathy (IgAN) rats.Results:The biochemical parameters results of IgAN model rats showed that the 24-h UP excretion and ALT concentration were much higher, and TP concentration was much lower than those of the control group (P < 0.05). Granule-like or mass-like IgA depositions in the mesangial area, glomerular hypercellularity, hyperplasia of mesangial matrix, and tubulointerstitial fibrosis were found in IgAN group. Rhein-prevented and rhein-treated both improved the biochemical parameters and relieved renal pathological injury. The expressions of renal tissue TLR4, TGF-β1, but not TLR9 were significantly elevated in IgAN model rats (P < 0.05). Rhein-prevented and rhein-treated both inhibited TLR4 and TGF-β1 expressions.Conclusion:Rhein significantly improved the serum and urine biochemical parameters, and attenuated the glomerular pathological changes and tubulointerstitial fibrosis in IgAN rats. The mechanism may involve inhibition of renal TLR4 and TGF-β1 secretion.KEY WORDS: Immunoglobulin A nephropathy, rhein, toll-like receptor, transforming growth factor-β1     

Protection of rhein on IgA nephropathy mediated by inhibition of fibronectin expression in rats

Objective:

To investigate the protective effects of rhein on IgA nephropathy (IgAN) in the rat model.

Materials and Methods:

Twenty-eight female sprague dawley rats were divided randomly into four groups, namely control, IgAN, rhein-prevented and rhein-treated. The pathologic changes on renal tissue were observed by the H and E, staining and the amount of urinary red blood cells and 24-h urinary protein excretion were measured. The glomerular deposition of immune globulin A (IgA) was measured by immunofluorescence staining. Fibronectin (FN) and α-smooth muscle actin (α-SMA) expression on renal tissue were measured via immunohistochemistry.

Results:

The model of IgAN was established according to Bovine serum albumin-Lipopolysaccharide-Carbon tetrachloride protocol, which was evidenced by histological structural lesions of glomeruli, IgA deposition and urinary measurement. Histological examination of kidney sections from both rhein-prevented group and rhein-treated group showed that glomerular hypertrophy, mesangial expansion, excessive extracellular matrix, and renal capsule dilation were markedly ameliorated compared with IgAN group. Moreover, rhein treatment significantly reduced IgA deposition in glomerulus, the volume of urinary red blood cells and 24-h urinary protein excretion. More importantly, increased FN expression in IgAN was back to normal level in rhein-prevented and rhein-treated group, which was along with the reduction of α-SMA expression in renal tissues.

Conclusions:

These findings indicate that rhein prevents the development of glomerulosclerosis and halts the progression of IgAN via inhibition of FN and α-SMA expression.KEY WORDS: IgA nephropathy, rat, rhein     

Potential role of ATP-binding cassette transporters in the intestinal transport of rhein

Rhein, a lipophilic anthraquinone, exhibits anti-inflammatory and anti-tumor activities; however, it is hepatotoxic. ATP-binding cassette transporters, including P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and multidrug resistance-associated protein 2 (MRP2), can pump toxicants from gut epithelial cells back into the intestinal lumen to prevent poisoning. We investigated their roles in rhein transport using a rat intestinal perfusion model and Caco-2, MDCKII-MDR1 (high expression of P-gp), MDCKII-BCRP (high expression of BCRP) and MDCKII-MRP2 (high expression of MRP2) cell models. The permeability of rhein in the duodenum significantly increased with increasing perfused concentration of rhein in the rat model, suggesting that efflux transporters were involved in rhein transport. In the Caco-2 cells, the permeability of rhein from the basolateral (B) to the apical (A) was significantly higher than that from A to B. In the presence of BCRP or MRP2 inhibitor, the permeability of rhein significantly decreased from B to A direction. In the MDCKII-BCRP cells, rhein was more permeable in B to A side than that in the opposite side. However, no significant differences of rhein permeability were observed in two directions in both MDCKII-MDR1 and MDCKII-MRP2 cells. Taken together, these results suggested that only BCRP was involved in rhein transport.