【作者】 张辉；

【导师】 张宝砚；

【作者基本信息】 东北大学 ， 高分子科学与工程， 2009， 博士

【摘要】 消化性溃疡(peptic ulcer, PU)是一种胃肠道粘膜及粘膜肌层坏死性病理损伤过程,其愈合需要上皮组织和结缔组织的重建。H2-受体拮抗剂选择性地竞争结合壁细胞膜上的H2-受体,使壁细胞内环磷腺苷(cyclic amp, cAMP)产生,胃酸分泌减少。H2-受体拮抗剂不仅对组胺刺激的胃酸分泌有抑制作用,也可部分地抑制胃泌素和乙酰胆碱刺激的胃酸分泌、促进溃疡粘膜愈合。表皮生长因子(epidermal growth factor, EGF)是-个由53个氨基酸组成的单链多肽,主要来源于颌下腺、十二指肠Brunner腺、肾小管细胞、胰腺、甲状腺及肝脏等,胃粘膜中可检测出低水平的EGF蛋白。EGF通过与其特异的受体—表皮生长因子受体(epidermal growth factor receptor, EGFR)结合发挥其在保持粘膜完整和损伤修复过程中的重要生物学作用,EGF和EGFR作用机制表达影响的研究,是在形态学水平上对药物作用机制的一种研究方法,它对我们掌握H2-受体拮抗剂药物作用靶点的规律、建立筛选抗溃疡药物模型、筛选抗溃疡药物,并成功用于H2-受体拮抗剂新药的开发研制很有指导意义。本文在总结H2-受体拮抗剂构效关系的基础上,根据第四代H2-受体拮抗剂罗沙替丁醋酸酯结构特点,运用生物等排原理,设计合成了未见他人在美国化学文摘(chemical abstracts, CA)中报道的,含有伯胺基团的苯氧丙胺类化合物：以间羟基苯甲醛为起始原料,经Leukart,双分子亲核取代,酰化反应得到中间体N-{3-[3-(1-哌啶甲基)苯氧基]丙基}氯乙酰胺,在比较温和的条件下与甲胺、正丁胺、异丙胺、环戊胺、环己胺反应得到了目标化合物。经核磁、质谱和元素分析测试,确正其化学结构。反应的平均收率为55.7%。反应时间短,产物容易分离,有比较好的收率,所用试剂基本符合环保要求。依据药物的构效关系(structure-activity relationship, SAR),在新化合物的合成设计中,选择引入环戊胺基和环己胺基设计合成化合物N-{3-[3-(1-甲基哌啶)苯氧基]丙基}环戊胺基乙酰胺5e、N-{3-[3-(1-甲基哌啶)苯氧基]丙基}环己胺基乙酰胺5f,是为了增强分子的抑制胃酸分泌的能力。选择引入直链胺烷基设计合成了化合物N-{3-[3-(1-甲基哌啶)苯氧基]丙基}甲胺基乙酰胺5a、N-{3-[3-(1-甲基哌啶)苯氧基]丙基}正丁胺基乙酰胺5b,是为了延长侧链,增强分子拮抗H2-受体的作用,进而增强分子抑制胃酸分泌的作用时间。选择引入支链胺烷基设计合成了化合物N-{3-[3-(1-甲基哌啶)苯氧基]丙基}异丙胺基乙酰胺5c、N-{3-[3-(1-甲基哌啶)苯氧基]丙基}叔丁胺基乙酰胺5d,目的是增强分子的柔性,延长化合物分子作用时间,提高抗溃疡药物的活性。对苯氧丙胺类化合物进行初步药理活性筛选：取体重350400g,雄性豚鼠(由沈阳医学院实验动物室提供),禁食12 h,击头处死,取胃,以冷浆膜液洗净胃内溶物,沿小弯剪开。将胃固定于玻璃管上。粘膜液每15 nim更换一次。取出的粘膜液用酸度计测量6个苯氧丙胺类化合物的pH值。浆膜液每小时更换一次。阳性对照药品为罗沙替丁醋酸酯草酸盐。在测定基础胃酸分泌后,移取0.30 mL于浆膜液中,终浓度为1×10-6mo1·L-1。连续测定pH值lh。初筛药理实验结果表明：化合物5a、5b离体豚鼠的胃酸分泌抑制的百分率较高,远高于阳性对照组,5e和5f的强度次之,其余较弱。通过对化合物抑制胃酸分泌过程的研究,可以初步推测胺烷基基团的几何构型对化合物的作用时间和与胃粘膜细胞结合程度的影响至关重要。根据测定的药理活性数据,我们用比较分子力场分析(comparative molecular field analysis, CoMFA)和比较分子相似性指数分析方法(comparative molecular similarity indices analysis, CoMSIA)对所合成的化合物的构效关系进行了研究,应用CoMFA技术研究了苯氧丙胺类化合物的定量构效关系(quantitative structure-activity relationship,QSAR),得到了电性效应及场效应对该类化合物活性的影响规律,在此规律基础上对能够增强活性的化合物结构给予预测,为进一步结构修饰以获得更好活性的H2-受体拮抗剂提供依据。在药理实验与计算机辅助药物设计(computer-aided drug design, CADD)研究基础上,选定了抑酸活性相对较高,稳定性较好的5b对乙酸烧灼型胃溃疡大鼠胃粘膜EGF及其EGFR表达的影响,探讨5b促进消化性溃疡愈合的作用机制：取Wistar大鼠48只,随机分成6组,每组8只,分别为：(1)正常对照组,以适量生理盐水灌胃；(2)溃疡模型对照组,以适量生理盐水灌胃；(3)-(5)5b高、中、低剂量组(28mg/kg、14mg/kg、7mg/kg);(6)雷尼替丁阳性药物对照组(27 mg/kg)。(2)-(6)组大鼠采用乙酸烧灼法制备胃溃疡模型,正常对照组大鼠不进行手术。大鼠术后连续灌胃给药15天,于末次给药后禁食24 h,自由饮水,第16天将大鼠断颈椎处死,取材。苏木素-伊红(hematoxylin-eosin, HE)染色观察5b对胃溃疡大鼠粘膜愈合情况的影响,免疫组化染色测定胃粘膜EGF、EGFR表达。试验结果表明：1.HE染色观察各组大鼠的胃溃疡形成及粘膜愈合情况正常对照组：大鼠的胃窦壁结构完整,粘膜腺体排列整齐,未见明显的渗出层出现。模型组：胃粘膜毛细血管扩张、充血,灶性出血明显,间质可见大量的渗出层出现,部分区域粘膜坏死、脱落,局部腺体有破坏。治疗组：雷尼替丁组、5b高剂量和中剂量组的粘膜溃疡均可见不同程度的愈合,溃疡灶表面有不同程度的新生上皮覆盖,渗出层明显减少,可见新生的腺体覆盖溃疡和增生的纤维结缔组织,溃疡底部有明显肉芽组织形成,胃粘膜血管轻度扩张、充血。2.大鼠胃粘膜再生粘膜厚度、粘膜肌层缺损度测定结果比较胃粘膜再生粘膜厚度的统计学分析显示,5b中剂量和高剂量组、雷尼替丁组与未治疗模型组比较,具有明显的统计学差异,p<0.01。粘膜肌层缺损度比较的统计学分析显示,5b高剂量组、雷尼替丁组与未治疗模型组比较具有明显的统计学差异,p<0.01。3.实验性胃溃疡大鼠粘膜EGF、EGFR免疫组化染色情况免疫组织化学染色显示正常对照组EGF及EGFR表达为弱阳性；5b高、中剂量组及阳性药物雷尼替丁组EGF及EGFR表达明显增高。5b小剂量组EGF及EGFR表达升高不明显。上述资料提示5b治疗溃疡过程中,剂量依赖性促进了EGF及EGFR表达,EGF及EGFR可能介导了5b促进溃疡愈合作用。研究发现5b具有增加溃疡病灶及其周围再生粘膜厚度、降低粘膜肌层缺损宽度、促进上皮组织再生能力,改善修复疲痕,从而提高了再生粘膜组织结构成熟度。5b具有剂量依赖性促EGF及EGFR表达作用,通过促进溃疡周围组织EGF及EGFR表达,从而刺激肉芽组织内微血管的形成,抑制壁细胞分泌,并刺激溃疡边缘上皮细胞的增殖、分化、移行,增加粘膜血流量,增强粘膜及腺体的再生功能,促进溃疡愈合。提示5b具有成为新的抗消化性溃疡药物潜在的可能性。更多还原

【Abstract】 Peptic ulcer (PU) has an experience of a gastrointestinal mucosa and submucosa muscle necrosis in the pathological injury. It has to build a healing epithelial tissue and connective tissue. H2-receptor antagonists set up the H2-receptor selectively on the membrane so that the cell wall of cyclic amp (cAMP) is generated and reduced gastric acid secretion. H2 receptor antagonist not only is inhibited to histamine-stimulated acid secretion, but also the gastrin is partially inhibited and the acid secretion which stimulated by acetylcholine is inhibited. Epidermal growth factor (EGF) is the single-chain polypeptide with a 53 amino acid. They come from the submandibular gland mainly, duodenum Brunner gland, renal tubular cells, pancreas, thyroid and liver, etc. In the gastric mucosa low levels of EGF protein can be detected. EGF combined epidermal growth factor receptor (EGFR) play a role in biology importantly to hold integrity and repair mucosal injury.The pathogenesis of digestible ulcer and the cure function for H2-Receptor antagonists were described in this paper. The pharmacological peculiarity of H2-Receptor antagonists is described in much detail. The final product was identified by comparing to the spectrum data in literature. The primary amines (cyclopentamine、cyclohexylamine、methylamine、n-butylamine、isopropylamine、tert-butyl amine) were selected to design and synthesize a series of phenoxypropylamide on the basic theory of the principle of bioisosterism. N-{3-[3-(1-Piperidinymethyl)phenoxy]propyl}chloroacetamide was synthesized by a three-step reaction from m-hydroxybenzaldehyde. Phenoxypropylamine derivatives were then synthesized by the reaction of N-{3-[3-(1-Piperidinymethyl)phenoxy]propyl}chloro acet-amide with primary amines and then with oxalic acid to obtain their corresponding salts Their chemical structures were identified by 1H-NMR, 13C-NMR, and MS. All of them were not reported by other in chemical abstracts (CA).A number of phenoxypropylamines were synthesized. With regards to possible SAR, our preliminary assumption was to synthesize the compounds N-{3-[3-(1-piperidinylmethyl) phenoxy]propyl}methylaminoacetamide 5a and N-{3-[3-(1-piperidinylmethyl)phenoxy] propyl}n-butylaminoacetamide 5b by adding primary amine straight chains in order to enhance the ability of the molecules to inhibit gastric acid. We synthesized compounds N-{3-[3-(1-piperidinylmethyl)phenoxy]propyl}isopropylaminoacetamide 5c and N-{3-[3-(1-piperidinylmethyl)phenoxy]propyl}tertiarybutylaminoacetamide 5d by adding a branched chain primary amine in order to enhance the flexibility of the molecules. The compounds N-{3-[3-(1-piperidinylmethyl)phenoxy]propyl}cyclopentyaminoacetamide 5e and N-{3-[3-(1-piperidinylmethyl)phenoxy]propyl}cyclohexylaminoacetamide 5f are the H2-receptor antagonists.The protocol offers several advantages such as mild reaction conditions, short reaction times, easy isolation and good yields. They showed some potential effects on the inhibition of gastric juice.In the inhibiting the gastric acid secretion of guinea pig stomach mucous membrane study, we chooses roxatidine acetate as a positive drug and found that four of the target compounds have a higher or equal activity compared with the positive drug. One of them,5b is the best. At the same time, we found that substitute on phenoxypropylamines have effect on the acting time and its binding ability with the stomach cells.Comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) methods were introduced to study the relationship between the structure and activity of the compounds. Using the compound bioactivities we studied quantitative structure-activity relationship (QSAR) regular of the phenoxypropylamines by CoMFA computation. And we got the regular how the steric factor and electrostatic factor effect on the bioactivity. And we also predicted the model structure which will have more powerful bioactivity based on the regular. This regular will benefit the work of modifying the molecular to find the better H2-receptor as inhibitor in the future.We observed that the effects of on expressions of EGF and EGFR in rat with gastric ulcer. The promotion of peptic ulcer healing mechanism was discussed:48 Wistar rats were randomly divided into six groups, each with eight, namely:(1) Control group, to appropriate saline gavage. (2) Ulcer model control group, to appropriate saline gavage. From (3) to (5), 5b is the highest, middle and the low-dose group (28 mg/kg,14 mg/kg,7 mg/kg). (6) Positive control group with drug Ranitidine (27 mg/kg). From (2) to (6), rats were burned using acetic acid to prepare gastric ulcer model, the normal control group rats was not operationed.After rats had been lavaged with doses for 15 days, continuous intragastric administration in the last administration after fasting for 24 hours, free drinking water, the first 16 days will be broken cervical rats were sacrificed, drawing. HE staining of 5b for small acetate rat mucosa of the gastric ulcer healing the impact of immunohistochemical staining of gastric EGF and EGFR expression.1. HE staining of the rats ulcer formation and healing.Normal control group:the gastric antral wall of rat has structural ntegrity, glandular mucosa is neat, and there was no obvious inflammatory cell infiltration.Model group:Gastric telangiectasia, congestive, obvious focal bleeding. There are a large number of the interstitial infiltration of inflammatory cells, and some regional mucosal necrosis, exfoliated, local glands was damaged.Treatment group:ranitidine,5b small acetate for high-dose and the dose of mucosal ulceration were observed varying degrees of healing ulcer lesions have varying degrees of the surface epithelium new coverage, inflammatory cells decreased significantly, we can see that the nascent gland hyperplasia coverage ulcers and fibrous connective tissue, obviously at the bottom of ulcer formation of granulation tissue, vascular mild gastric expansion, congestive.2. Renewable rat gastric mucosal thickness, the muscularis mucosa defects of the results of comparison:Renewable thickness of the gastric mucosa statistical analysis shows that for 5b in small doses and high dose group, ranitidine group and the untreated group model has obvious significant difference, p<0.01.Comparing the muscularis mucosa defects of the statistical analysis showed that butylamine roxatidine derivate(BRD) small acetate for the high dose group, ranitidine group and group therapy model has obvious significant difference, p<0.01.3. Experimental gastric ulcer rat mucosa EGF and EGFR immunohistochemical staining: Immunohistochemical staining showed that the normal control group EGF and EGFR expression was weakly positive for small 5b high dose group and the positive drug Ranitidine group EGF and EGFR expression was significantly increased.5b for the small-dose group increased expression of EGF and EGFR not obvious. These data suggest that n-butylamine Jiluosha acetate for the treatment of small ulcer in the process, for the dose-dependent expression of EGF and EGFR, EGF and EGFR may be mediated by 5b for small acetate promote ulcer healing role.The rat model with gastric ulcer model is established successfully. The study for 5b found that for a small increase in acetate ulcer mucosal lesions and the surrounding renewable thickness, width decreased muscularis mucosa defects, and promote epithelial tissue regeneration capabilities, and improved weary scar repair. From the study of 5b we found that for a small increase in acetate ulcer mucosal lesions and the surrounding renewable thickness, width decreased muscularis mucosa defects, and promote epithelial tissue regeneration capabilities, and improved weary scar repair, Granulation tissue, there by stimulating the formation of microvascular, inhibit secretion of parietal cells, and stimulate the ulcer edge epithelial cell proliferation, differentiation, migration and increased mucosal blood flow, and enhance the mucosal regeneration glands function, and promote ulcer healing. We have concluded that 5b would be a kind of antiulcer drugs probably and potentially.更多还原