CN113372384B - 磁共振造影剂及其制备方法和应用 - Google Patents
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Abstract
Description
技术领域
本发明涉及核磁共振造影剂领域,特别是涉及一种磁共振造影剂及其制备方法和应用。
背景技术
磁共振成像(Magnetic Resonance Imaging,MRI)是核磁共振技术在医学领域的应用。与其它医学影像成像方法相比,具有高软组织对比度、多参数多序列成像和无电离辐射伤害等优点。目前,临床磁共振增强扫描检查中,近50%磁共振扫描会用到基于稀土金属钆(Gd3+)的磁共振造影剂。对人体来说,钆(Gadolinium)是外源性金属。游离的Gd3+有剧毒,在体内分布于骨骼和肝脏中,并可迅速导致肝脏坏死。自1988年马根微显(Bayer)FDA获批后,钆基造影剂(Gadolinium-based contrast agents,GBCA)临床应用30年来,其肾源性系统纤维化(Nephrogenic Systemic Fibrosis,NSF)和钆离子脑沉积(Gadolinium Retation)安全性问题日益凸显。
2006年,美国食品和药品管理局(FDA)陆续发现,肾功能不全患者(肾小球滤过率<30mL/min/1.73m2)在使用某些稳定性不高的钆类造影剂后,由于不能完全及时清除体外,从而出现全身皮肤硬化的NSF症状,严重者会出现组织器官受累。目前,对NSF尚无有效的治疗方法。
近年来(自2014年起),有证据表明,一些接受过4次或更多次钆造影剂对比增强磁共振扫描的患者,在最后一次检查完毕之后很长一段时间,钆造影剂能够在大脑组织中累积。来自梅奥诊所的研究人员发现,接受过多次钆造影剂对比增强MRI检测的已故患者,脑内有钆沉积且存在剂量效应关系,而与肾功能、年龄、首次接触和死亡之间的时间间隔无关。
综上所述,钆类磁共振造影剂潜在的巨大副作用,临床Gd3+造影剂NSF和脑沉积安全性问题成为临床上亟待解决的问题。
发明内容
基于此,有必要针对钆类磁共振造影剂潜在的巨大副作用问题,提供一种具有式I结构特征的化合物或其药学上可接受的盐,式I化合物具有合适的油水分配系数,可应用于制备磁共振造影剂,为进一步制备非钆磁共振造影剂提供重要的结构基础。
在其中一个实施例中,所述药学上可接受的盐选自:具有式III或IV结构特征的化合物:
可以理解的,所述药学上可接受的盐也可采用其他形式,可根据制备过程中水解方式进行调整。
本发明一方面还提供一种具有式I结构特征的化合物的制备方法,采用以下路线合成:
S1:以环己二胺为原料,通过Kabachnik-Fields反应将多聚甲醛和亚磷酸脂缩合得到化合物1;
S2:将化合物1酸性条件下水解反应得到化合物2;
S3:将化合物2通过N-烷基化反应得到化合物3;
S4:将化合物3通过水解反应得到化合物4,即为式I化合物。
可以理解的,上述原料中环己二胺的构型可根据目标化合物选择,优选反式环己二胺(tans-CDTA),具有更佳的稳定性。
在其中一个实施例中,上述制备方法为:
S1中,以四氢呋喃为溶剂,加入环己二胺、亚磷酸二乙酯和多聚甲醛混合,回流反应,得到化合物1;
S2中,向步骤S1所得产物中加入甲醇,在高浓度酸性条件下反应,得到化合物2;上述高浓度酸性条件如2-4M的HCl,优选3M的HCl,或其它能够提供同等酸性条件(H+浓度为2-4M)的有机酸或无机酸。
S3中,将步骤S2所得产物溶于乙腈,加入KI、溴乙酸叔丁酯和碱,反应得到化合物3;优选地,所述碱选自:N,N-二异丙基乙胺(DIPEA)或三乙胺;
S4中,将步骤S3所得产物溶于酸性溶液中,加热,回流反应,脱去保护基,得到化合物4,即为式I化合物。此步骤为水解反应,可以理解的,对于酸性溶液可用酸及浓度,可根据具体反应条件调整,如4-8M的HCl,优选6M的HCl,或其它能够提供同等酸性条件(H+浓度为4-8M)的有机酸或无机酸。
在其中一个实施例中,上述制备方法具体为:
S1、将无水四氢呋喃、环己二胺、亚磷酸二乙酯和多聚甲醛混合,回流反应,反应完毕,去除四氢呋喃,加入二氯甲烷和水进行萃取,保留有机相,清洗,干燥,过滤,去除二氯甲烷,得化合物1;
S2、向步骤S1所得产物中加入甲醇和浓盐酸,浓盐酸中HCl的质量数为36%~38%,反应,反应完毕,去除甲醇,加水,加碱中和,过滤,加入二氯甲烷萃取,保留有机相,清洗,干燥,过滤,去除二氯甲烷,得化合物2;
S3、将步骤S2所得产物溶于无水乙腈,加入KI、N,N-二异丙基乙胺和溴乙酸叔丁酯,反应,反应完毕,过滤,去除乙腈,加入乙酸乙酯和水,萃取,保留有机相,清洗,干燥,过滤,去除乙酸乙酯,得化合物3;
S4、将步骤S3所得产物溶于盐酸,加热,回流反应,反应完毕,去除液体,加水溶解,加入乙腈,析出白色固体,过滤,保留固体,得化合物4,即为式I化合物。
在其中一个实施例中,所述步骤S1具体为:将无水四氢呋喃、环己二胺、亚磷酸二乙酯和部分多聚甲醛混合,在92~98℃下回流反应3~5h,回流反应期间分3~5次加入剩余多聚甲醛,反应完毕,减压旋蒸去除四氢呋喃,加入二氯甲烷和水进行萃取,保留有机相,用水或饱和食盐水清洗,有机相用无水硫酸钠干燥,过滤,减压旋蒸去除二氯甲烷,得化合物1;所述环己二胺、亚磷酸二乙酯和多聚甲醛的摩尔比为1:(1.8-2.2):(2.8-3.2)。
在其中一个实施例中,所述步骤S2具体为:向步骤S1所得产物中加入甲醇和浓盐酸,甲醇与浓盐酸的体积比为8:(2~4),在48~52℃下反应6~12h,反应完毕,减压旋蒸去除甲醇,加水,加碱中和至pH为6.8~7.2,过滤,加入二氯甲烷萃取,保留有机相,加入饱和食盐水清洗,加入无水硫酸钠干燥,过滤,减压蒸馏去除二氯甲烷,得化合物2。
在其中一个实施例中,所述步骤S3具体为:将步骤S2所得产物溶于无水乙腈,加入催化剂KI、溴乙酸叔丁酯和N,N-二异丙基乙胺或三乙胺,在68~72℃下反应5~7h,反应完毕,过滤去除沉淀,减压旋蒸去除乙腈,加入乙酸乙酯和水进行萃取,保留有机相,加入饱和食盐水进行清洗,加入无水硫酸钠进行干燥,过滤,减压旋蒸去除乙酸乙酯,过硅胶柱纯化,得化合物3。
在其中一个实施例中,所述步骤S4具体为:将步骤S3所得产物溶于5.5~6.5mol/L的盐酸中,加热,回流反应6~12h,反应完毕,减压蒸馏去除液体,加水,加热溶解残余物,溶解后冷却,加入乙腈,搅拌混合后静置,析出白色固体,过滤,烘干,得化合物4。
本发明还提供一种具有式I结构特征的化合物或其药学上可接受的盐在制备磁共振造影剂中的应用。该造影剂具有水溶性好、弛豫效率高、毒副作用低等特点。
在其中一个实施例中,式I所示化合物作为配体在制备磁共振造影剂中的应用。
本发明另一方面还提供一种具有式II结构特征的化合物或其药学上可接受的盐:
其中,
M1选自:Mn、Fe、Eu或Dy的+2价离子,或Mn、Fe、Eu或Dy的+3价离子;
M2选自:Na+、K+或葡甲胺阳离子;
当M1为+2价离子时,a=2;当M1为+3价离子时,a=3。
上述化合物或其药学上可接受的盐,以式I化合物为配体,以Mn、Fe、Eu或Dy作为配位金属离子,可用作非钆顺磁性金属配合物磁共振造影剂,具有水溶性好、弛豫效率高、毒副作用低等特点,可以用于临床的MRI造影剂。
在其中一个实施例中,M1选自:Mn的+2价离子,Fe的+3价离子;M2为Na+、K+或葡甲胺阳离子;a=2。
锰(Manganese)是人体必需微量元素,在人体内参与了多种重要的生物化学反应。高自旋Mn2+具有高顺磁性,可以作为磁共振造影剂的顺磁金属中心。根据Mn2+配位化学特点,该新型Mn(II)T1磁共振该造影剂具有与商品钆类造影剂相当的弛豫效率,无肾源性系统性纤维化的风险,肾功能不全患者也可使用该Mn类造影剂做对比增强检查。
铁(Fe)同样是是人体必需微量元素,在体内发挥着重要的生理功能如血红蛋白输送氧气。正常情况下人体大约含有4克铁,体内存在高度协调的调控系统来保证铁元素的运输和存贮,Fe3+的长期毒性远低于Gd3+。高自旋Fe3+的配合物也具有较高的磁矩(低于Mn2+和Gd3+),考虑到钆造影剂潜在的安全性问题,本发明式II中相应的Fe3+配合物也可以作为Fe类磁共振造影剂。
本发明还提供一种具有式II结构特征的化合物的制备方法,包括以下步骤:
将具有式I结构特征的化合物或其药学上可接受的盐与含M1离子的化合物反应,加碱调节pH至6~9,即得。
在其中一个实施例中,具有式II结构特征的化合物的制备方法具体为:
将式I所示化合物与水混合,加入Mn、Fe、Eu或Dy金属化合物,所述金属化合物的金属离子为正二价或正三价,加碱调节pH至6~9,将所得溶液冻干,即得顺磁性金属配合物。
优选地,加碱调节pH至pH 7-8,更优选7.3~7.4。
在其中一个实施例中,所述碱为氢氧化钠、氢氧化钾或葡甲胺。
本发明一方面还提供一种上述具有式II结构特征的化合物在制备磁共振造影剂中的应用。
本发明一方面还提供一种磁共振造影剂,包括:具有式II结构特征的化合物,以及药学上可接受的辅料。
与现有技术相比,本发明具有以下有益效果:
本发明的具有式I结构特征的化合物或其药学上可接受的盐,作为配体具有合适的油水分配系数,可应用于制备磁共振造影剂,为进一步制备非钆磁共振造影剂提供重要的结构基础。
本发明的具有式II结构特征的化合物或其药学上可接受的盐,具有水溶性好、弛豫效率高、毒副作用低等特点,可以用于临床的MRI造影剂,减少钆类磁共振造影剂的毒副作用。
附图说明
图1为实施例中化合物4的晶体结构图;
图2为实施例中KBR0826弛豫效率测试结果;
图3为实施例中锰离子浓度的标准曲线;
图4为实施例中KBR0826药代动力学测试结果;
图5为实施例中KBR0826排泄动力学测试结果;
图6为实施例中KBR0826肝细胞毒性结果;
图7为实施例中KBR0826在大鼠体内成像图;
图8为实施例中KBR0826在大鼠体内成像信号强度图。
具体实施方式
为了便于理解本发明,以下将给出较佳实施例对本发明进行更全面的描述。但是,本发明可以以许多不同的形式来实现,并不限于本文所描述的实施例。相反地,提供这些实施例的目的是使对本发明的公开内容的理解更加透彻全面。
除非另有定义,本文所使用的所有的技术和科学术语与属于本发明的技术领域的技术人员通常理解的含义相同。本文中在本发明的说明书中所使用的术语只是为了描述具体的实施例的目的,不是旨在于限制本发明。
实施例1
制备trans-环己基二氨-二亚甲基磷酸-二乙酸配体,合成路线如下所示:
该配体的具体制备方法如下:
(1)制备化合物1
以100mL无水四氢呋喃(THF)为溶剂,加入环己二胺(10g,98%,85.8mmol),亚磷酸二乙酯(24.2g,98%,171.6mmol),多聚甲醛(总量8.31g,96%,266mmol,先加入约五分之一),加热至95℃,回流,分4次加入剩余量的多聚甲醛,回流反应4h;
反应完毕,减压旋蒸去除溶剂,残液加入二氯甲烷和水各约100mL进行萃取,水相再以二氯甲烷萃取3次(每次约20mL),合并有机相,有机相先后再以水和饱和食盐水各洗一次(每次约50mL),有机相用无水Na2SO4干燥、过滤,减压旋蒸去除二氯甲烷,得到深紫色油状化合物1粗产品(27.37g,产率为72.8%)。
化合物1的结构表征为:
1H-NMR(400MHz,CDCl3)δ:1.20(d,4H),1.35(t,12H),1.72(s,2H),1.93(d,2H),2.16(s,2H),2.71(br,2H),3.15(t,2H),3.89(s,2H),4.15(br,8H);
MS(ESI):[M+H]+计算值:427.20,测定值:427.2。
(2)制备化合物2
将(1)得到的化合物1(27.1g,63.56mmol)溶于以120mL甲醇和45mL浓盐酸兑成的溶液中,在50℃下反应过夜,反应完毕,减压旋蒸去除甲醇,残液加水至约150mL,再以NaHCO3中和,滤除多余的NaHCO3,用约100mL二氯甲烷萃取,水相再用二氯甲烷洗2次,合并有机相,有机相用饱和食盐水洗一次,经无水Na2SO4干燥、过滤后,减压旋蒸去除二氯甲烷,得酒红色化合物2粗产品(11.51g,产率为43.7%)。
化合物2的结构表征为:
1H-NMR(400MHz,CDCl3)δ:0.98(d,2H),1.18(t,2H),1.32(t,12H),1.75(d,2H),2.09(d,2H),2.22(d,2H),2.85(t,2H),3.15(t,2H),4.18(br,8H);
MS(ESI):[M+H]+计算值:415.20,测定值:415.2。
(3)制备化合物3
将(2)得到的化合物2(11.50g,27.75mmol)溶于无水乙腈(80mL)中,依次加入催化剂KI(0.46g,2.8mmol)、DIPEA(99%,8.95g,69.38mmol)、溴乙酸叔丁酯(98%,13.53g,69.38mmol),在70℃下反应6h;
反应完毕,先滤除沉淀、旋干,再用乙酸乙酯和水进行溶解和萃取,有机相再用饱和食盐水洗一次,经无水Na2SO4干燥、过滤后,减压旋蒸去除乙酸乙酯,得到化合物3粗产品,再通过硅胶柱进行分离纯化,得到化合物3(12.75g,产率71.5%)。
化合物3结构表征为:
1H-NMR(400MHz,CDCl3)δ:1.12(d,4H),1.35(t,12H),1.48(s,18H),1.69(s,2H),2.03(d,2H),2.78(s,2H),3.16(t,2H),3.42(br,2H),3.52(d,2H),3.65(d,2H),4.18(br,8H);
MS(ESI):[M+H]+计算值:643.34,测定值:643.4;[M+Na]+计算值:665.34,测定值:665.4。
(4)制备化合物4
将(3)得到的化合物(12.75g,19.84mmol)溶于6M盐酸(100mL)中,加热回流,反应过夜,反应完毕,将反应液完全旋干,加入少量水(约10mL),加热溶解残余物,完全溶解后冷却,加入约100mL乙腈,搅拌混合后静置,析出白色固体,在混合液中将固体搅拌至粉末状,过滤并用少量乙腈洗涤,得到白色粉末固体,经烘干后得到白色粉末状化合物4(7.34g,产率88.4%)。
化合物4的结构表征为:
1H-NMR(400MHz,D2O)
MS(ESI):[M+Na]+计算值:441.09,测定值:441.0;[M-H]-计算值:417.09,测定值:417.0。化合物4的晶体结构如图1所示。
实施例2
制备顺磁性金属锰配合物磁共振造影剂,步骤如下:
将实施例1中(4)得到的化合物(7.19g,17.2mmol)加入到60mL纯水中,搅拌下加入四水合氯化锰(3.33g,16.8mmol),用固体氢氧化钠调节pH至7.3~7.4,将所得溶液冻干成固体11.68g。
MS(ESI):[M+H]-计算值:470.0,测定值:469.9;[M+3H]+计算值:472.0,测定值:472.0。
实施例3
对实施例2得到的顺磁性金属锰配合物磁共振造影剂(代号:KBR0826)进行以下性能测试:
一、弛豫效率测定
弛豫效率测定方法:配置梯度浓度造影剂溶液(0.054,0.109,0.224,0.451,0.906mM,in Hepes buffer,pH=7.4),利用纽迈PQ001核磁共振造影剂驰豫率测试仪(0.5±0.08T,仪器主频率:21.3MHz;32℃)测试得到不同浓度造影剂样品的T1、T2弛豫时间,以弛豫率Ri(Ri=1/Ti,i=1,2)和造影剂浓度进行线性拟合,斜率即为弛豫效率(relaxivity,ri,i=1,2)。弛豫效率反映了磁共振造影剂改变水分子弛豫时间的能力,是衡量造影剂优劣的重要参数。弛豫效率(relaxivity,ri,i=1,2)定义为单位浓度磁共振造影剂(mmol/L)对水分子弛豫率的改变(1/Ti,i=1,2)。
测试结果如图2所示,图中横坐标为Mn2+的摩尔浓度,纵坐标为1/T(S-1),在0.5T(32℃,pH=7.4),KBR0826弛豫效率为:r1=3.12mmol-1s-1,r2=3.75mmol-1s-1,r2/r1=1.2,与(r1=3.4mmol-1s-1,r2=4.0mmol-1s-1)相当。
二、药代动力学测试
药代动力学测试方法
1实验准备
8只SD大鼠,体重220-240g,雌性3只,雄性5只。3%戊巴比妥钠(50mg/kg)腹腔内注射进行麻醉,仰卧位固定。颈部脱毛,常规消毒备用。
左颈动脉置管:颈正中线(胸骨与下颌连线)左旁3mm,胸骨上7mm处作纵行切口,长度约20mm。切开皮肤,暴露皮下组织,逐层钝性分离至看见颈动脉。钝性剥离伴行的神经和静脉。分别结扎远心端及近心端,结点之间间距为2.5-3.5cm;靠近远心端结点处将留置针刺入动脉,并用止血夹固定,退出针管,松开近心段结点。注射肝素锂(150μg/kg)全身肝素化。
2血液标本采集及处理
尾静脉快速注射(<5s)KBR0826溶液(0.05mmol/mL),剂量为0.1mmol/kg。于注射前及注射后的1min、2min、3min、5min、8min、13min、20min、30min、50min采颈动脉血0.1mL,置于0.5mL EP管,置于-20℃保存。
3电感耦合等离子体质谱(ICP-MS)检测样品Mn2+浓度
3.1仪器工作条件
功率1150W,雾化气氩气流速0.95L·min-1,辅助气氩气流速1.2L·min-1,等离子气体氩气流速18L·min-1,泵转速20r·min-1,分析模式为标准模式,重复采样3次。
3.2建立标准曲线
用标准试剂(PerkinElmer,10μg/mL(1000ppb):Ag、Al、As、Cs、Cu、Sr、Se、Mn等)配制不同的浓度标准溶液:1.0、10、50、100、200、1000ppb,制作标准曲线(如图3所示),线性方程R2>0.9999。
3.3待测样品的配制及检测
取样本(全血)20μL,用含1%HNO3稀释至6~10mL,使其中Mn2+浓度在1~100ppb范围内,编号、检测,根据响应强度与标准曲线计算得出样本溶液中Mn2+浓度。
2.4绘制药-时曲线并计算药动学参数
使用PKSolver 2.0进行药代动力学相关参数计算。
测试结果如图4和表1所示,图中横坐标为时间(min),纵坐标为Mn2+浓度(ppb),结果表明KBR0826呈非室模型特征,符合相似细胞外液造影剂药代动力学特点;其血浆半衰期(t1/2=23min)与钆类磁共振造影剂接近。
表1药代动力学测试结果
三、排泄动力学测试
1实验准备
Sprague Dawley(SD)大鼠清洁级,雄性5只,雌性3只,体重250±50g,购自川北医学院动物实验中心。实验前禁食12-16h。使用3%戊巴比妥钠(40mg/kg)腹腔注射麻醉。
2尾静脉插管
用40°左右热水浸泡大鼠尾巴约1分钟,棉球蘸酒精擦拭消毒。左手大拇指与中指固定并拉伸尾部尖端,食指托平。尾静脉位于尾部两侧,呈淡青色。留置针用生理盐水排除空气,与静脉呈15°,插入后见回血平推。将留置针固定在大鼠尾端后滴注生理盐水,速度约为2mL/h。
3胆总道插管
消毒后,取大鼠剑突下腹部正中作纵行切口,逐层切开皮肤、皮下组织、腹白线、腹膜进腹。镊子翻开肝脏左右叶,在其下暴露十二指肠及大网膜。于正中用镊子拉伸十二指肠横部,可见淡黄色胆道被大网膜覆盖,并由肝下延伸至十二指肠。钝性剥离出胆道,玻璃挑针挑出,于近十二指肠处插入静脉留置针,用止血夹固定,收集胆汁作为给药前样本。
4膀胱插管
于大鼠尿道口正上方0.5cm处作纵向切口,切开皮肤、钝性分离肌肉。待膀胱充盈至一定程度后,左手用镊子将膀胱提起,右手持静脉留置针沿膀胱的背外侧的输尿管开口处从下向上斜插入膀胱内,退出针管收集尿液作为给药前样本。
5给药
收集完给药前胆汁和尿液样本后,于尾静脉快速给药,剂量为0.1mmol/kg。
6胆汁和尿液的采集
收集给药后每半小时的胆汁排出量和每小时的尿液排出量;样品称重后冻存于-20℃。
7ICP-MS检测样品Mn2+浓度
7.1待测样品的配制及检测
取样本(胆汁、尿液)20μL,用含1%HNO3稀释至6-10mL,使其中Mn2+浓度在1~1000ppb范围内,编号、检测,根据响应强度与标准曲线计算得出样本溶液中Mn2+浓度。
8制作排泄曲线
使用origin9.0进行数据计算及作图。将稀释倍数、浓度测定值与尿液(胆汁)体积相乘得该时间段排泄量,各时间段排泄量之和与给药剂量相比,即得相应时间段内的药物累积排泄百分数。根据上述数据,表2列出了单位时间(小时)内KBR0826在尿液和胆汁的排泄情况:排除体积(mL)、Mn2+排泄量(mg)、累积排泄量(mg)和累积排泄率(%),并以此绘制得到肝/肾排泄曲线:时间-肝/肾排泄量曲线(图5)。
表2大鼠尿液和胆汁中锰的排泄量统计(n=7)
图5中横坐标为时间(h),纵坐标为总代谢量(mg),结果表明KBR-0826主要通过肝肾排泄,在600min内肝肾排泄约60%,肝肾各半;KBR-0826在大鼠体内同时通过尿液和胆汁排泄,前期以尿液为主,随后以胆汁为主。
四、肝细胞毒性测试
实验方法
1QSG7701细胞传代、冻存与复苏
1)传代:QSG7701细胞株(25cm2培养瓶),观察生长状态良好,标好细胞种类,日期,培养人名字置37℃、5%CO2培养箱培养。1-2天后待细胞密度符合传代要求时(细胞生长率达到80-90%),PBS清洗细胞1-2次,弃去PBS加入1mL胰酶完全覆盖细胞表面1-2分钟,在倒置显微镜下观察,90%以上细胞变大变圆,轻轻晃动能有少许脱离时,立刻加完全培养基(DMEM培养基:FBS:抗生素=100:10:1)终止消化,吹打成单细胞,转至15mL离心管,1000rpm、常温条件下离心5min,弃去上清液,转移至10MM培养皿,加入5-10mL的新鲜完全培养基继续培养。2-3天换培养基或按1:2和1:3分瓶传代。
2)冻存:待细胞处于对数生长期,生长状态良好时,选择冻存,配置冻存液(DMEM:FBS:DMSO=6:3:1)同上胰酶消化细胞后离心管收集,用冻存液悬浮细胞,调整细胞浓度(1-10)×106/mL分装到冻存管,标记好细胞种类,日期,冻存者,传代次数。放置于梯度冻存盒于-80℃冰箱。次日转移至液氮罐保存。
3)细胞复苏:从液氮罐处取出一支QSG7701冻存管放入37℃水浴中快速震摇1~2min,即可解冻。解冻后的细胞液转移至15mL离心管,在1000rpm,常温条件下离心5min,弃去上清液,加入5-10mL新鲜完全培养液,标好细胞种类、日期、培养人名字至37℃、5%CO2培养箱培养。
2KBR0826溶液的配制
取KBR0826固体粉末31.93mg,用含血清的培养基配制成12.5mmol/L,并稀释,得到浓度分别为12.5、2.5、0.5、0.1、0.02mmol/L溶液,备用;
3细胞培养及给药
1)取96孔板4个,每个检测时间(6h、12h、24h、48h)点用一个。每个板设对照组和5个不同浓度KBR0826的给药组,给药组每组设5个孔,共计用到30个孔。每孔加入100μL对数生长期细胞液。
2)37℃、5%CO2培养箱培养过夜(24h),观察到细胞贴壁良好后弃去旧培养基,对照组更换新的含血清培养基,其余更新为分别含有12.5、2.5、0.5、0.1、0.02mmol/LKBR0826的含血清培养基,继续培养。
3)分别于给药后的6h、12h、24h、48h,取出一个96孔板培养板,移除培养基后加入含MTT的无血清培养基(MTT 0.5mg/mL)100μL,再放入细胞培养箱培养2h。
4)最后取出96孔板,移除培养基,加入100μl DMSO后水平摇晃混匀2~3min使结晶充分溶解后,用酶标仪于595nm检测线读取OD值。
4统计方法
数据作图采用origin9.0作图软件处理,计量资料采用均数±标准差(±sd)表示;数据统计使用SPSS14.0统计软件,采用单因素分析。
5结果
正常人体肝细胞在不同浓度下的KBR0826在不同时间的存活率(595nm)结果如图6所示,加入不同浓度KBR0826进行细胞培养的统计学分析结果显示:较低浓度(0.02、0.1、0.5mmol/L)时肝细胞株QSG7701在各检测时间点(6h、12h、24h、48h)的存活率没有显著性降低;但KBR0826浓度为2.5mmol/L时肝细胞株QSG7701于24h和48h两个检测时间点的存活率有显著性降低(p<0.05),KBR0826浓度为12.5mmol/L于12h肝细胞株QSG7701的存活率即出现显著性降(p<0.05)。在24小时内,25倍临床给药剂量(2.5mmol/L),KBR0826对肝细胞株QSG7701的增殖没有明显抑制作用。
其中,*表示经过与对照组OD值比较,具有统计学差异。
五、大鼠体内成像
1、SD大鼠16只,分KBR0826造影剂组(实验组)及钆特酸葡胺(Gd-DOTA)造影剂组(对照组),每组8只。禁食12小时(自由饮水)。
2、3%戊巴比妥钠(60-70mg/kg)称重腹腔麻醉。
3、KBR0826使用剂量0.2mmol/kg。使用3.0T MRI成像仪进行SD大鼠体内成像,尾静脉注射造影剂,测定注射前及注射后不同时间大鼠心脏、肝脏、脑实质、肌肉、腹主动脉的信号强度(SI),计算信噪比(SNR),绘制时间-信号强度曲线,评价造影剂成像效果。
成像效果如图7,结果表明KBR-0826在心血管信号强度变化趋势类似Gd-DOTA,肝脏强化明显且持续;KBR-0826在大鼠器官中的成像信号强度如图8所示,横坐标为时间,纵坐标为信号强度,结果表明KBR-0826不能通过正常血-脑屏障,可经肾脏和肠道排泄。
综上所述,以上测试结果表明:本实施例的顺磁性金属锰配合物磁共振造影剂具有水溶性好、弛豫效率高、毒副作用低等特点,具有应用于临床MRI造影剂的前景。
实施例4
将实施例1中(4)得到的化合物(3.60g,8.6mmol)加入到30mL纯水中,搅拌下加入无水FeCl3(1.38g,8.5mmol),用固体氢氧化钠调节pH至7.3~7.4,将所得溶液冻干成固体5.5g。
上述配合物弛豫效率测定结果如下:r1=1.80mmol-1s-1,r2=2.10mmol-1s-1(0.5T,32℃)。
以上所述实施例的各技术特征可以进行任意的组合,为使描述简洁,未对上述实施例中的各个技术特征所有可能的组合都进行描述,然而,只要这些技术特征的组合不存在矛盾,都应当认为是本说明书记载的范围。
以上所述实施例仅表达了本发明的几种实施方式,其描述较为具体和详细,但并不能因此而理解为对发明专利范围的限制。应当指出的是,对于本领域的普通技术人员来说,在不脱离本发明构思的前提下,还可以做出若干变形和改进,这些都属于本发明的保护范围。因此,本发明专利的保护范围应以所附权利要求为准。
Claims (10)
3.根据权利要求2所述的应用,其特征在于:
S1中,以四氢呋喃为溶剂,加入环己二胺、亚磷酸二乙酯和多聚甲醛混合,回流反应,得到化合物1;
S2中,向步骤S1所得产物中加入甲醇,在H+浓度为2-4M的有机酸或无机酸条件下反应,得到化合物2;
S3中,将步骤S2所得产物溶于乙腈,加入KI、溴乙酸叔丁酯和碱,反应得到化合物3;
S4中,将步骤S3所得产物溶于酸性溶液中,加热,回流反应,脱去保护基,得到化合物4,即为式I化合物。
4.根据权利要求1所述的应用,其特征在于,式I所示化合物作为配体在制备磁共振造影剂中的应用。
6.根据权利要求5所述的具有式II结构特征的化合物或其药学上可接受的盐,其特征在于,M1选自:Mn的+2价离子,Fe的+3价离子;M2选自:Na+。
8.根据权利要求7所述的制备方法,其特征在于,所述碱选自:氢氧化钠、氢氧化钾或葡甲胺。
9.一种如权利要求5或6所述的具有式II结构特征的化合物在制备磁共振造影剂中的应用。
10.一种磁共振造影剂,其特征在于,包括:如权利要求5或6所述的具有式II结构特征的化合物,以及药学上可接受的辅料。
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- 2021-02-23 JP JP2022549168A patent/JP2023515413A/ja active Pending
- 2021-02-23 US US17/801,817 patent/US20230098403A1/en active Pending
- 2021-02-23 EP EP21760873.6A patent/EP4112627A4/en not_active Withdrawn
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WO2000024751A1 (en) * | 1998-10-23 | 2000-05-04 | I.N.S.E.R.M. (Institut National De La Sante Et De La Recherche Medicale) | Chelating agents for radioimmunotherapy |
CN105492449A (zh) * | 2013-01-07 | 2016-04-13 | 通用医疗公司 | 锰基磁共振造影剂 |
CN108779082A (zh) * | 2016-04-13 | 2018-11-09 | 伯拉考成像股份公司 | 造影剂 |
CN106187800A (zh) * | 2016-07-05 | 2016-12-07 | 川北医学院 | 一种含有邻二酚羟基的类edta配体和非钆磁共振造影剂及其制备方法 |
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Publication number | Publication date |
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EP4112627A1 (en) | 2023-01-04 |
US20230098403A1 (en) | 2023-03-30 |
WO2021169934A1 (zh) | 2021-09-02 |
JP2023515413A (ja) | 2023-04-13 |
EP4112627A4 (en) | 2023-07-19 |
CN113372384A (zh) | 2021-09-10 |
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