Pyrazoles and pyrazolines as neural and inducible nitric oxide synthase (nNOS and iNOS) potential inhibitors (III)

We have previously described a series of 4,5-dihydro-1H-pyrazole as moderately potent nNOS inhibitors. As a follow up of these studies, we report here the preparation and the preliminary evaluation of a series of 1-alkyl-3-benzoyl-4,5-dihydro-1H-pyrazole and 1-alkyl-3-benzoyl-1H-pyrazole as potential inhibitors of both neuronal and inducible nitric oxide synthases (nNOS and iNOS). None of the reported compounds exhibited significant iNOS or nNOS inhibition, although the 1-benzyl-3-(2-amino-5-chlorobenzoyl)-1H-pyrazole-5-carboxylic acid ethyl ester derivative (10l), which shows an inhibition of 50% versus iNOS at a 1mM final concentration and no activity against nNOS, is potentially amenable of further optimization. The reasons for the inactivity of the reported series are discussed on the basis of docking studies.     

诱导型一氧化氮合酶和P选择素在早期自然流产中的表达及临床意义

目的　通过测定早期自然流产患者外周血和蜕膜组织中诱导型一氧化氮合酶和粘附分子 P-选择素的表达 ,探讨其与自然流产发病的关系。　方法　 (1)采用化学比色法和酶联免疫吸附法分别对 2 8例早期自然流产患者 (流产组 )和 3 0例要求终止妊娠的正常早孕妇女 (对照组 )外周血中诱导型一氧化氮合酶及 P-选择素含量进行检测。 (2 )应用免疫组织化学法检测流产组和对照组蜕膜组织中诱导型一氧化氮合酶及 P-选择素的表达。　结果　流产组中血浆诱导型一氧化氮合酶的含量为 (5.66± 2 .14 ) μmol/ L,对照组为 (1.2 3± 1.56) μm ol/ L,流产组血浆 P-选择素的含量为(157.80± 10 .0 0μg/ L ) ,对照组为 (112 .54± 5.3 8μg/ L ) ,2组比较 ,差异均有显著性 (P<0 .0 5)。流产组蜕膜组织中诱导型一氧化氮合酶及 P-选择素的表达均明显高于对照组 (P<0 .0 5)。　结论　自然流产患者外周血和蜕膜组织中诱导型一氧化氮合酶及 P-选择素均有高表达 ,在自然流产发病中具有重要意义     

结肠腺瘤组织Ezrin蛋白和诱导型一氧化氮合酶表达及其生物学意义的探讨

目的探讨Ezrin蛋白、诱导型一氧化氮合酶(iNOS)在结肠腺瘤组织中的表达及其与腺瘤癌变的关系。方法应用免疫组织化学SP法检测60例结肠管状腺瘤、40例绒毛状及绒毛管腺瘤、30例腺瘤癌变中Ezrin蛋白、iNOS和Ki-67核抗原的表达及细胞定位。结果Ezrin蛋白、iNOS在结肠管状腺瘤[58.3％(35/60)、50.0％(30/60)]、绒毛状及绒毛管腺瘤[72.5％ (29/40)、70.0％(28/40)]、腺瘤癌变[96.7％(29/30)、93.3％(28/30)]中的表达分别依次上升,差异有统计学意义(Ezrin蛋白：x2= 11.600,P= 0.005;iNOS:X2= 8.451,P= 0.015),Ezrin蛋白与iNOS表达有相关性(r=0.765,P＜0.01);Ki-67核抗原增殖指数随Ezrin蛋白、iNOS表达上升而增加。结论Ezrin蛋白和iNOS的过表达可能与结肠腺瘤的增殖、恶性转化有关。     

二硫化碳对大鼠视网膜组织诱导型一氧化氮合酶及细胞凋亡的影响

目的探讨二硫化碳（CS2）对大鼠视网膜组织诱导型一氧化氮合酶（iNOS）及细胞凋亡的影响。方法将24只SD雄性大鼠随机分为对照组、低剂量CS2染毒组和高剂量CS2染毒组，染毒结束后取视网膜组织制成切片，测量内视网膜厚度比率，还原型烟酰胺腺嘌呤二核苷酸黄递酶染色（NADPH-NDP）法检测iNOS活力，原位缺口末端标记法（TUNEL）检测细胞凋亡。结果（1）染毒组的内视网膜（包括内核层、内丛状层、节细胞层、神经纤维层和内界膜）厚度减小，与对照组相比，差异有统计学意义（P〈0．05）；高剂量染毒组与低剂量染毒组之间差异亦有统计学意义（P〈0．05）。（2）染毒组视网膜iNOS表达增加，与对照组相比，差异有统计学意义（P〈0．05或P〈0．01）；高剂量染毒组与低剂量染毒组之间差异亦有统计学意义（P〈0．05）。（3）染毒组视网膜出现细胞凋亡，凋亡指数与对照组相比，差异有统计学意义（P〈0．05或P〈0．01）；高剂量染毒组与低剂量染毒组之间差异亦有统计学意义（P〈0．05）。结论CS2能激活视网膜组织iNOS表达，由此产生的过量的NO可损伤视网膜细胞。同时，视网膜细胞凋亡也是CS2所致视网膜损害的机制之一。     

三氯乙烯对大鼠视网膜的损伤及诱导型一氧化氮合酶的影响

目的探讨三氯乙烯(trichloroethylene,TCE)对大鼠视网膜组织的脂质过氧化损伤效应及诱导型一氧化氮合酶(iNOS)表达的影响。方法将32只大鼠随机分为对照组和500、1 000、2 000mg/kg TCE3个剂量染毒组,测定视网膜组织中超氧化物歧化酶(SOD)活力、丙二醛(MDA)、一氧化氮(NO)的含量,光学显微镜下观察视网膜形态学变化,免疫组织化学法测定iNOS的表达。结果与对照组比较,TCE染毒1 000、2 000mg/kg组大鼠视网膜组织中SOD活力均降低(P0.01),TCE染毒500、1 000、2 000mg/kg组大鼠视网膜组织中MDA含量均升高(P0.05或P0.01);TCE染毒的3个剂量组中,大鼠视网膜组织中NO含量均高于对照组(P0.01)。TCE染毒组大鼠视网膜组织的内核层、外核层、节细胞层出现细胞核变大、水肿、空泡等变化。TCE染毒1 000、2 000mg/kg组大鼠视网膜组织中iNOS活力高于对照组(P0.05)。结论 TCE能激活视网膜组织iNOS表达,产生过量的NO可损伤视网膜细胞;视网膜脂质过氧化作用增强也是TCE所致视网膜损害的机制之一。     

Dietary protein or arginine deficiency impairs constitutive and inducible nitric oxide synthesis by young rats.

Effects of dietary protein or arginine deficiency on constitutive and lipopolysaccharide (LPS)-induced nitric oxide (NO) synthesis were determined in young rats by quantifying urinary nitrate excretion. In Experiment 1, 30-d-old rats (n = 16) were divided randomly into two groups (n = 8/group) and pair-fed on the basis of body weight semipurified isocaloric diets containing 20 or 5% casein. In Experiment 2, 30-d-old rats (n = 24) were divided randomly into three groups (n = 8) and pair-fed on the basis of body weight purified isonitrogenous and isocaloric diets (composed of amino acids) containing 0.0, 0.3 or 1.0% L-arginine. In both experiments, daily collection of urine was initiated 10 d after the start of pair-feeding. On d 17 after the pair-feeding was initiated, LPS (1 mg/kg body wt) was injected intraperitoneally into rats, and urine was collected daily for an additional 7 d. In Experiments 3 and 4, activities of constitutive and inducible NO synthases were measured in macrophages and various tissues from protein- or arginine-deficient rats (n = 6). Body weight was lower in rats fed the 5% casein diet or the 0.0 and 0.3% arginine diets than in those fed 20% casein or 1% arginine, respectively. Dietary protein or arginine deficiency decreased serum concentrations of arginine and urinary nitrate excretion before and after LPS treatment, indicating impaired constitutive and inducible NO synthesis. Protein malnutrition reduced constitutive and inducible NO synthase activities in brain, heart, jejunum, lung, skeletal muscle and spleen, and inducible NO synthase activity in macrophages. Because NO is a mediator of the immune response and is the endothelium-dependent relaxing factor, impaired NO synthesis may help explain immunodeficiency and cardiovascular dysfunction in protein- or arginine-deficient subjects.     

米非司酮对早孕绒毛和蜕膜组织中诱导型一氧化氮合酶的影响

目的了解米非司酮对早孕绒毛、蜕膜组织中iNOS（iNOS）的影响，进一步探讨米非司酮早孕药物流产机制。方法采用原位杂交及免疫组化方法对40例药物流产的绒毛和蜕膜中iNOS的表达进行检测（实验组），利用计算机CMIAS系统，表达指标以数密度（N／S）及阳性单位（Pu）计算，并与40例正常人工流产的绒毛和蜕膜组织比较（对照组）。结果实验组绒毛组织iNOS原位杂交N／S、Pu分别为（0．12±0．01）、（15．3±2．6），对照组分别为（0．022±0．003）、（3．1±0．5），2组差异有统计学意义（P〈0．01）；免疫组化实验组和对照组N／S、Pu分别为（0．09±0．01）、（10．24±1．55）vs（0．016±0．002）、（1．26±0．33），差异有统计学意义。蜕膜组织中原位杂交及免疫组化iNOS表达较低，2组比较差异无统计学意义（P〉0．05）。结论米非司酮早孕药物流产与绒毛组织iNOS活性增加有关，与蜕膜组织iNOS表达无关。     

脑白质损伤新生大鼠VEGF及iNOS表达变化研究

目的 研究缺氧缺血(HI)对新生大鼠脑白质细胞血管内皮生长因子(VEGF)及诱导型一氧化氮合酶(iNOS)表达的影响,探讨VEGF及iNOS在新生大鼠脑白质损伤(WMD)发病机制中的作用。方法 将3日龄大鼠随机分为实验组及假手术组,建立新生大鼠WMD模型,分别予HI后12、24、48、72 h及7 d处死,对其脑组织取材后行HE染色观察其病理改变,并应用免疫组织化学法检测其VEGF及iNOS的表达水平。结果 在新生大鼠脑白质中,1)VEGF表达水平予HI后12 h开始明显上升,24 h后达高峰,至7 d恢复正常,与对照组比较,实验组脑室周围白质VEGF的表达在12、24、48、72 h有统计学意义。2)实验组iNOS于HI后12 h表达开始增加,72 h达高峰,7 d仍有表达,与对照组比较,差异有统计学意义。 结论 HI可导致新生大鼠脑白质VEGF及iNOS的表达水平显著增高,参与新生大鼠WMD的病理生理过程。     

铅对大鼠海马一氧化氮合酶活力及表达的影响

目的 通过研究铅对大鼠海马一氧化氮合酶（ＮＯＳ）活力及表达的影响。分析海马不同亚区ＮＯＳ活力变化与铅对海马长时程增强（ＬＴＰ）影响的关系。方法 Ｗｉｓｔａｒ大鼠采用饮水加２０、２００、２０００μｇ／ｍｌ醋酸铅（ＰｂＡｃ）方法染毒３个月后,用Ｙ－迷宫法测试大鼠神经行为的改变;用原子吸收法测定血液与海马中铅的含量;用ＮＡＤＰＨ－黄递酶（ＮＡＤＰＨ－ｄ）组化法和免疫组化法检测海马ＮＯＳ的活力及表达情况。     

锂对染铅大鼠海马胆囊收缩素和神经元型一氧化氮合酶神经元的保护作用

目的 探讨锂对染铅大鼠海马胆囊收缩素（CCK）和神经元型一氧化氮合酶（nNOS）神经元的保护作用及其与动物学习记忆功能变化的关系.方法 通过测量体重,利用反映学习记忆功能的Y迷宫法和ABC免疫组化法,研究饮用含0.2 g/L醋酸铅（PbAc）水和含不同剂量（3、30、300、3 000mg/kg）氯化锂（LiCl）饲料喂养的大鼠体格发育情况、学习记忆能力以及海马不同亚区CCK和nNOS阳性神经元数目的变化.结果铅染毒组大鼠体重增加少于对照组大鼠,差异有统计学意义（P＜0.05）,Y-迷宫学会次数多于对照组大鼠,差异有统计学意义（P＜0.05）;其海马各区CCK和nNOS阳性神经元数明显减少,差异有统计学意义（P＜0.05）.铅＋LiCl（3、30、300mg/kg）组体重增加多于对照组大鼠,差异有统计学意义（P＜0.05）;Y-迷宫学会次数少于对照组大鼠,差异有统计学意义（P＜0.05）,海马各区CCK阳性神经元数明显增加,差异有统计学意义（P＜0.05）.铅＋3 000mg/kg LiCl组大鼠Y-迷宫学会次数多于对照组大鼠,差异有统计学意义（P＜0.05）;海马各区CCK阳性神经元数明显少于对照组,差异有统计学意义（P＜0.01）;与铅染毒组比较,差异无统计学意义（P＞0.05）.结论 铅可损伤大鼠学习记忆能力,影响大脑海马CCK和nNOS阳性神经元的数目.较低剂量的锂对铅引起的学习记忆损伤和海马CCK阳性神经元的变化有一定保护作用.     

Synthesis and structure-activity of antisense peptides corresponding to the region for CaM-binding domain of the inducible nitric oxide synthase

Nitric oxide synthase (NOS) catalyses the conversion of L-arginine to nitric oxide (NO) which plays an important role in the regulation of cellular functions and intracellular communications. Three distinct isoforms of NOS have so far been identified, two constitutive and one inducible. All three mammalian isoforms of NOS contain putative CaM-binding domains with the canonical composition. In this paper we report the synthesis and the inhibitory activity on rat neuronal and lung inducible NOS of antisense peptides corresponding to the antisense strand read in 3' to 5' (CALM 1) or 5' to 3' (CALM 2) direction of the region encoding for the CaM-binding domain of the inducible NOS isoform (residues 503-522). CALM 1 inhibited, at all the concentrations tested (0.01-1 mM), both the inducible and constitutive NOS (IC(50) 98 microM and 56 microM, respectively), while CALM 2 (0.01-1 mM) was ineffective on both isoforms. The acetylation of CALM 1 at its amino terminal (CALM 8) completely abolished its inhibitory activity. We also synthesized and analysed the activity of amino terminal truncated analogues (CALM 3-7) of CALM 1, which selectively inhibited the inducible isoform, although less potently than the parent compound. The pentapeptides (CALM A-D) deriving from the cleavage of CALM 1 were ineffective, except the pentapeptide CALM C corresponding to the residues 513-517, which was as potent as the parent compound (IC(50) 65 microM).     

三氯乙烯对人角质形成细胞一氧化氮和一氧化氮合酶的影响

目的研究三氯乙烯(TCE)对体外培养的人角质形成细胞(KC)一氧化氮(NO)合成和一氧化氮合酶(NOS)表达及活力的影响,探讨TCE的皮肤细胞毒性机制。方法无血清培养的正常人KC与0.125、0.25、0.5、1.0、2.0mmol/L的TCE共培养4h,分别于染毒后12、24、48、72h测定培养液中NO含量和NOS活力,分析其时间—剂量—效应关系,并用逆转录-聚合酶链反应(RT-PCR)法检测诱导型NOS(iNOS)mRNA的表达情况。结果低剂量组(0.125和0.25mmol/L)NO含量与iNOS活力在染毒后各时点与对照组相比均无差异;0.5mmol/L组NO含量与iNOS活力均在染毒后48h开始升高,分别为(32.19±4.75)μmol/L[对照组:(23.70±3.11)μmol/L,P<0.05]和(1.15±0.02)×103U/L[对照组:(0.77±0.06)×103U/L,P<0.05],随着染毒浓度的升高,0.5～2.0mmol/L组NO量与iNOS活力呈上升趋势,而且上升出现的时间提前,1.0和2.0mmol/L组分别于染毒后24和12h出现升高;NO释放的增加总是与iNOS活力升高一致,而结构型NOS(cNOS)活力在各剂量组的各时点均无变化;RT-PCR结果显示TCE可以诱导iNOSmRNA转录水平增高。结论TCE可以诱导人角质形成细胞NO合成增加,大量产生的NO与iNOS基因的上调有关,这一机制可能参与了TCE的皮肤细胞毒性。     

Solid-phase synthesis and pharmacological evaluation of a library of peptidomimetics as potential farnesyltransferase inhibitors: an approach to new lead compounds

Oncogenic Ras proteins whose activation is farnesylation by farnesyltransferase have been seen as important targets for novel anticancer drugs. Inhibitors of this enzyme have already been developed as potential anti-cancer drugs, particularly by rational design based on the structure of the CA(1)A(2)X carboxyl terminus of Ras. Synthesis of a peptidomimetics library via solid-phase synthesis using the Multipin method is described here. The most active hits on cellular assays were resynthesized and enzymatic activity was measured. Compounds A1, A5 and A7 present significant activity on the isolated enzyme (IC(50)=117, 57.3 and 28.5 nM) and their molecular docking in the active site of the enzyme provides details on key interactions with the protein.     

冠心病患者血浆一氧化氮及一氧化氮合酶活力的研究

目的　探讨冠心病 (CHD)患者血浆一氧化氮 (NO)和一氧化氮合酶 (NOS)的水平差异以及其可能的影响因素。方法　采用试剂盒测定血浆中NO含量和NOS的活力 ,所得数据进行多元线性回归分析和协方差分析。结果　在本研究条件下 ,冠心病患者血浆NO含量为 (2 17.0 5± 15 3.31)μmol/L,NOS活力为 (14 .0 9± 7.14 )U/ml,均明显高于对照组 [分别为 (14 0 .6 9± 90 .96 ) μmol/L、(7.75±3.79)U/ml],差异有显著性 (P <0 .0 1)。吸烟和饮酒是血浆NOS的独立影响因素。性别是血浆NO的独立影响因素。结论　血浆NO和NOS与冠心病具有密切的关系。     

Effect of copper intrauterine device on the cyclooxygenase and inducible nitric oxide synthase expression in the luteal phase endometrium

Background

To evaluate the effect of copper intrauterine device (IUD) on the expression of cyclooxygenase (COX) and inducible nitric oxide synthase (iNOS) in the luteal phase endometrium.

Study Design

A prospective clinical study was conducted on 30 women who were willing to use a copper IUD contraception. Endometrial biopsies and blood samples were taken before and 3 months after the insertion of the IUD on Day 3 and Days 20–24 of the cycle. Main outcome measures were to evaluate the effect of copper IUD on uterine artery blood flow using pulsed color Doppler ultrasonography and the relationship of bleeding abnormalities and menstrual pain level with the uterine blood flow, COX-2 and iNOS expression.

Results

Only the left uterine artery pulsatility and resistance indices decreased statistically significantly (p=.005 and p=.039, respectively). Other Doppler parameters showed no change. Cyclooxygenase-2 expression of both endometrial luminal epithelium (p=.03) and gland epithelium (p=.03) increased significantly. Inducible NOS expression of the endometrial surface epithelium decreased significantly after IUD insertion (p=.01).

Conclusions

Although COX-2 expression increased 3 months after copper IUD insertion, iNOS expression of the luminal epithelium decreased. Local hypoxia caused by copper and vasoconstrictor prostanoids may play a role in IUD-related menstrual abnormalities.     

Effect of copper intrauterine device on the cyclooxygenase and inducible nitric oxide synthase expression in the luteal phase endometrium

BackgroundTo evaluate the effect of copper intrauterine device (IUD) on the expression of cyclooxygenase (COX) and inducible nitric oxide synthase (iNOS) in the luteal phase endometrium.Study DesignA prospective clinical study was conducted on 30 women who were willing to use a copper IUD contraception. Endometrial biopsies and blood samples were taken before and 3 months after the insertion of the IUD on Day 3 and Days 20–24 of the cycle. Main outcome measures were to evaluate the effect of copper IUD on uterine artery blood flow using pulsed color Doppler ultrasonography and the relationship of bleeding abnormalities and menstrual pain level with the uterine blood flow, COX-2 and iNOS expression.ResultsOnly the left uterine artery pulsatility and resistance indices decreased statistically significantly (p=.005 and p=.039, respectively). Other Doppler parameters showed no change. Cyclooxygenase-2 expression of both endometrial luminal epithelium (p=.03) and gland epithelium (p=.03) increased significantly. Inducible NOS expression of the endometrial surface epithelium decreased significantly after IUD insertion (p=.01).ConclusionsAlthough COX-2 expression increased 3 months after copper IUD insertion, iNOS expression of the luminal epithelium decreased. Local hypoxia caused by copper and vasoconstrictor prostanoids may play a role in IUD-related menstrual abnormalities.     

Associations of patella lead with polymorphisms in the vitamin D receptor, delta-aminolevulinic acid dehydratase and endothelial nitric oxide synthase genes

A cross-sectional analysis was performed to evaluate associations of polymorphisms in the vitamin D receptor (VDR), delta-aminolevulinic acid dehydratase (ALAD), and endothelial nitric oxide synthase (eNOS) genes with patella lead concentrations in 652 lead workers in the Republic of Korea. There was a wide range of patella lead (from below detection limit to 946 microg Pb/g bone mineral), with a mean (standard deviation) of 75.2 (101.0). There were no associations of ALAD or eNOS genotypes with patella lead, but workers with the VDR B allele had significantly (P value < 0.05) higher patella lead (on average, 25% or approximately 6.6 microg Pb/g bone mineral) than lead workers with the VDR bb genotype. There was evidence that the relation between age and patella lead was modified by both the VDR and eNOS genotypes.     

Total antioxidant status in the blood serum of rats exposed to N-nitroso compounds and nitric oxide synthase inhibitors

In this study, the total antioxidant status (TAS) was assayed in the blood serum of rats pretreated per os with either N-nitrosodiethylamine (NDEA) (0.1 mg/kg b.w./day) or N-methyl-N-nitrosourea (NMU) (0.1 mg/kg b.w./day) for 30 days. The animals were also dosed per os with spermidine (SPR) (10 mg/kg b.w./day) for a first 21 day period, and Nomega)-nitro-L-arginine methyl ester (L-NAME) (10 mg/kg b.w./day) given to animals for 3 days (days 22-24), respectively. Nitric oxide synthase (NOS) inhibitor, L-NAME was found to mitigate TAS levels in the blood serum of rats pretreated with NDEA and NMU. No such changes were found in animals dosed with L-NAME only nor even with L-NAME and spermidine, respectively. Since spermidine, also known as an inhibitor of iNOS synthesis, elevated TAS levels in rats dosed with L-NAME and NDEA/NMU, the polyamine was suggested to modify the NOS/NO origin to serve the physiological level of the total anti-oxidant status in rat blood serum.