CN103339136A - 尿苷二-或三-磷酸衍生物以及其用途 - Google Patents
尿苷二-或三-磷酸衍生物以及其用途 Download PDFInfo
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- CN103339136A CN103339136A CN2011800666318A CN201180066631A CN103339136A CN 103339136 A CN103339136 A CN 103339136A CN 2011800666318 A CN2011800666318 A CN 2011800666318A CN 201180066631 A CN201180066631 A CN 201180066631A CN 103339136 A CN103339136 A CN 103339136A
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- diastereomer
- alkyl
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Abstract
本发明提供了特定的尿苷二-和三-磷酸衍生物及其药物组合物。这些化合物用于治疗P2Y6受体所调节的疾病、病症和病状,尤其用于降低眼内压以及因此治疗高眼压症和/或青光眼。
Description
技术领域
本发明涉及尿苷二-和三-磷酸衍生物以及其药物组合物。所述化合物用于治疗P2Y6受体所调节的疾病、病症和病状,尤其用于降低眼内压以及因此治疗高眼压症和/或青光眼。
背景技术
活化G蛋白-偶联P2Y受体(P2YR)的胞外核苷酸由于其能够在正常的病理生理状态下调节许多组织和器官中的诸多功能而成为具有吸引力的医药标靶(Hillmann等,2009;Burnstock和Verkhratsky,2009)。胞外核苷酸和二核苷酸已被证实在眼部生理学和病理生理学中发挥作用(Crooke等,2008),并且已被建议用作干眼症、视网膜脱离和青光眼的治疗剂(Guzman-Aranguez等,2007)。
高眼压症是青光眼的最常见起因,其为降低眼内压(IOP)药剂的标靶(Pintor,2005)。当将一些核苷酸(例如,二腺苷三磷酸和二腺苷五磷酸)局部施用给新西兰白兔时,IOP增加而其它例如ATP、腺苷四磷酸和二腺苷四磷酸则降低IOP(Peral等,2009;Pintor等,2003,2004)。
已在小梁网(TM)细胞(负责引流房水的眼组织区)中鉴定出包括P2Y1、P2Y2和P2Y4在内的胞外核苷酸受体(Soto等,2005)。在这些P2YR亚型中,通过选择性激动剂2-MeS-ADP活化P2Y1R来减少牛眼的房水流出。其它研究已报道了在牛TM细胞中存在P2Y1和P2Y2受体,以及在人TM细胞系中存在P2Y1、P2Y4和P2Y11受体(Crosson等,2004)。
最近,已对P2Y6-R激动剂和拮抗剂的结构-活性关系,以及降低IOP所涉及的P2Y6-R的分子建模进行了广泛的研究(El-Tayeb等,2006;Jacobson等,2009;Costanzi等,2005;Maruoka等,2010;Besada等,2006);然而,尚未鉴定出有效的选择性P2Y6-R激动剂或拮抗剂。P2Y6-R激动剂的开发包括UDP磷酸链、核糖环、和碱基的修饰。过去几年来,已进行了不同的尿嘧啶修饰,以鉴定比内源性配体UDP更有效的激动剂(Maruoka等,2010;Ginsburg-Shmuel等,2010;Ko等,2008)。
核苷酸的治疗潜力普遍有限,尤其是对于治疗青光眼而言,因为其被胞外酶降解,降低了其效力、功效和作用时间。此外,虽然核苷酸在4-11的pH范围内化学稳定(El-Tayeb等,2006),但在较强酸性或碱性pH下快速降解。核苷酸是被外核苷酸酶的外核苷三磷酸双磷酸水解酶家族(即,e-NTPDase和碱性磷酸酶)(Nahum等.,2002),和外核苷酸焦磷酸酶/磷酸二酯酶(即,e-NPP)(Grobben等,2000;Zimmermann,2001)酶水解。因此,需要鉴定可用于开发有效的选择性P2YR激动剂的酶及化学稳定的核苷酸支架。
已报道了改善核苷酸稳定性的一些尝试(Cusack等,1987;Misiura等,2005;Kowalska等,2007),包括使用核苷酸的磷酸生物电子等排体,例如膦酸盐(Eliahu等,2009;Joseph等.,2004)、磷酰胺(Zhou等,2005)、和硼烷基磷酸类似物(Nahum等,2002;Boyle等,2005;Barral等,2006;Eliahu等,2009)。
提高潜在的P2Y-R激动剂稳定性的另一种策略是使用二核苷酸,例如,二尿苷三磷酸,其表现出比单核苷酸类似物更强的稳定性(Shaver等,2005;Yerxa等,2002)。实际上,先前已成功地将二核苷酸作为P2Y2-R激动剂来开发。因此,已分别进行了Up4U(INS365,地夸磷索(Diquafosol))和Up4dC(INS37217,地纽福索(Denufosol))治疗干眼病和囊肿性纤维化的临床试验,然而,两种化合物在3期临床试验中未表现出令人满意的结果(http://www.businesswire.com/news/home/20110103005364/en;Jacobson和Boeynaems,2010)。
Ginsburg-Shmuel等(2010)公开了作为P2Y6-受体激动剂的5-OMe-UDP。如具体所示,5-OMe-UDP采用受体偏向的反式构象,和S糖皱缩,其为P2Y6-受体并非P2Y2-或P2Y4-受体所优选的构象,因此满足P2Y6-受体的构象和H-键合需求,成为有效的P2Y6-受体激动剂(EC50=0.08μM,而UDP为0.14μM)。
US7,084,128公开了一种通过施用某些单-或二-核苷酸,优选单-或二腺苷、单-、二-、三-、四-、五-或六磷酸衍生物,或其药学上可接受的盐来降低IOP的方法。所例示的特定化合物为2’-(O)-,3’-(O)-(苄基)亚甲二氧基-腺苷-5’-三磷酸和2’-(O)-,3’-(O)-(苄基)亚甲二氧基-2”-(O)-,3”-(O)-苄基亚甲二氧基-P1,P4-二(腺苷5’-)四磷酸,如所示,在0.25mM的浓度下,这些化合物使IOP随时间降低,1-2小时后,达21-22%的最大降幅。
Eliahu等(2010)公开了某些不可水解的腺苷二-或三磷酸类似物,例如2MeS-腺苷-β,γ-CH2-5’-三磷酸和2MeS-腺苷-β,γ-CCl2-5’-三磷酸作为降低IOP的有效药剂。如此公开所述,2MeS-腺苷-β,γ-CCl2-5’-三磷酸将血压正常的兔子的IOP降低32%(EC50=95.5nM),其中作用时间约5个小时,即,发现比若干种常见的青光眼药物更有效地降低IOP,因此代表马来酸噻吗洛尔(timolol maleate)的有前景的替代药物,后者不能用于治疗罹患哮喘或心脏问题的患者。
发明概述
现已发现,根据本发明,某些5-甲氧基尿苷核苷酸在施用于眼角膜后可显著降低新西兰雄性白兔的眼内压(IOP),因此被视为通过选择性活化P2Y6受体治疗高眼压症和/或青光眼的有前景的候选物。表现出最强降压作用的特定化合物是5-甲氧基尿苷-5’-O-(α-硼烷基二磷酸)的两种非对映异构体中的一种,其将血压正常的兔子的IOP降低45%,多于目前可使用的任意药物,其中作用时间约4小时。
在一个方面,本发明提供一种具有通式I的化合物:
或其非对映异构体或非对映异构体的混合物,
其中
R是-O-(C1-C8)烷基,或-S-(C1-C8)烷基;
Y各自独立地是H或-OH;
Z1、Z2和Z3各自独立地是O-或BH3 -;
n是0或1;
m是3或4;以及
B+表示药学上可接受的阳离子,
但不包括其中Z1、Z2(如果存在)和Z3各自是O-的化合物。
在另一个方面,本发明提供了一种药物组合物,其包含具有通式I的化合物但不包括其中Z1、Z2(如果存在)和Z3各自是O-的化合物,或其非对映异构体或非对映异构体混合物,以及药学上可接受的载体或稀释剂。
在另一个方面,本发明提供了一种用于降低眼内压,更具体地讲,用于预防或治疗高眼压和/或青光眼的药物组合物,其包含具有通式I的化合物,包括其中Z1、Z2(如果存在)和Z3各自为O-的化合物,或其非对映异构体或非对映异构体混合物,以及药学上可接受的载体或稀释剂。
在另一个方面,本发明提供了一种用于降低眼内压的具有通式I的化合物,包括其中Z1、Z2(如果存在)和Z3各自为O-的化合物,或其非对映异构体或非对映异构体混合物。
在另一个方面,本发明涉及一种具有通式I的化合物,包括其中Z1、Z2(如果存在)和Z3各自为O-的化合物,或其非对映异构体或非对映异构体混合物用于制备降低眼内压的药物组合物的用途。
在另一个方面,本发明涉及一种降低有需要的个体的眼内压的方法,其包括对该个体施用治疗有效量的具有通式I的化合物,包括其中Z1、Z2(如果存在)和Z3各自为O-的化合物,或其非对映异构体或非对映异构体混合物。
附图简述
图1示出在胃液刺激状态下,在240MHz下,31P NMR所监测的13A的水解速率。不同的曲线表示在不同时刻由于水解而存在于溶液中的物种。
图2A-2B示出UDP(2A)以及11、12和13A(2B)在人血清(180μl)和RPMI-1640培养基(540μl)中在37℃下经24h的随时间变化的水解曲线,通过HPLC监测。
图3A-3D示出核苷酸13A(3A)、15(3C)、16(3B)和17(3D)、以及内源性激动剂UDP对P2Y6-R的浓度-响应曲线。自稳定表达P2Y6GFP受体的1321N1细胞获取数据,从而确定配体诱导的[Ca2+]i变化。将细胞用2μM fura-2AM预培养30分钟并检测荧光变化(ΔF340nm/F380nm)。浓度-响应曲线来自一个数据集,但为清楚起见,将其在单独的图中以常见参照的UDP响应曲线示出。
图4A-4C示出与对照、UDP(即,内源性P2Y6-受体配体)和市售药物相比,通过各种5-甲氧基尿苷核苷酸和二核苷酸衍生物使血压正常的兔子的眼内压(IOP)降低。4A示出经6小时测量的化合物12、13A和16A对兔子IOP的作用时程;4B示出与UDP和对照相比,通过12、13A和16A使血压正常的兔子的IOP降低;4C示出与匹鲁卡品(Pilocarpine)和对照相比,通过12、13A和16A使血压正常的兔子的IOP降低。
发明详述
本发明在一个方面提供了具有如上定义的通式I的某些尿苷核苷酸,其中尿嘧啶环中5位的碳原子被-O-烷基或-S-烷基取代,并且二-或三-磷酸中非桥接氧原子中至少一个被硼烷基取代。
如本文中所用,术语“(C1-C8)烷基”通常意指具有1-8个碳原子的直链或支链烃基,包括,例如,甲基、乙基、正丙基、异丙基、正丁基、仲丁基、异丁基、叔丁基、正戊基、2,2-二甲基丙基、正己基、正庚基、正辛基等。优选(C1-C6)烷基,更优选(C1-C4)烷基,最优选甲基和乙基。
在某些实施方案中,本发明的化合物是具有通式I的化合物,其中R是-O-(C1-C8)烷基,优选-O-(C1-C6)烷基,更优选-O-(C1-C4)烷基,最优选-OCH3或-OC2H5。
在某些实施方案中,本发明的化合物是具有通式I的化合物,其中R是-S-(C1-C8)烷基、-S-(C1-C6)烷基,更优选-S-(C1-C4)烷基,最优选-SCH3或-SC2H5。
在某些实施方案中,本发明的化合物是具有通式I的化合物,其中n是0。此类化合物可为尿苷二磷酸衍生物,即,其中Y各自为OH的化合物,以及脱氧-或二脱氧-尿苷二磷酸衍生物,即,其中Y中一个或两个分别为H的化合物。特定的此类化合物是包括(i)α位具有唯一硼烷基,即,其中Z1是BH3 -,且Z3是O-;(ii)β位具有唯一硼烷基,即,其中Z3是BH3 -,且Z1是O-;或(iii)α和β位具有两个硼烷基,即,其中Z1和Z3是BH3 -的那些化合物。
在某些实施方案中,本发明的化合物是具有通式I的化合物,其中n是1。此类化合物可为尿苷三磷酸衍生物,即,其中Y各自为OH的化合物,和脱氧-或二脱氧-尿苷三磷酸衍生物,即,其中Y中一个或两个分别为H的化合物。特定的此类化合物是包括(i)α位具有唯一硼烷基,即,其中Z1是BH3 -,且Z2和Z3是O-;β位具有唯一硼烷基,即,其中Z2是BH3 -,且Z1和Z3是O-;或γ位具有唯一硼烷基,即,其中Z3是BH3 -,且Z1和Z2是O-;(ii)α和β位具有两个硼烷基,即,其中Z1和Z2是BH3 -,且Z3是O-;α和γ位具有两个硼烷基,即,其中Z1和Z3是BH3 -,且Z2是O-;或β和γ位具有两个硼烷基,即,其中Z2和Z3是BH3 -,且Z1是O-;或(iii)α、β和γ位具有三个硼烷基,即,其中Z1至Z3是BH3 -的那些化合物。
在特定实施方案中,本发明的化合物是具有通式I的化合物,其中R是-O-(C1-C4)烷基,优选-OCH3或-OC2H5,n是0,并且(i)Z1是BH3 -,且Z3是O-;(ii)Z1是O-,且Z3是BH3 -;或(iii)Z1和Z3是BH3 -。
在其它特定实施方案中,本发明的化合物是具有通式I的化合物,其中R是-O-(C1-C4)烷基,优选-OCH3或-OC2H5,n是1,并且(i)Z1是BH3 -,且Z2和Z3是O-;(ii)Z2是BH3 -,且Z1和Z3是O-;(iii)Z3是BH3 -,且Z1和Z2是O-;(iv)Z1和Z2是BH3 -,且Z3是O-;(v)Z1和Z3是BH3 -,且Z2是O-;(vi)Z2和Z3是BH3 -,且Z1是O-;或(vii)Z1至Z3是BH3 -。
说明书中所述的具有通式I的特定尿苷核苷酸衍生物在文中用粗体标识为化合物/类似物12、13和14,而说明书中所述的通式I所未涵盖的特定尿苷二核苷酸衍生物在文中用粗体标识为化合物/类似物15、16和17。化合物12也用名称5-甲氧基尿苷二磷酸(5-OMe-UDP)标识;化合物13也用名称5-甲氧基尿苷-5’-O-(α-硼烷基二磷酸)标识;化合物14也用名称5-甲氧基尿苷-5’-O-(α-硼烷基三磷酸)标识;化合物15也用名称二-(5-OMe)-尿苷5’,5”-P1,P3,三磷酸标识;化合物16也用名称二-(5-OMe)-尿苷5’,5”-P1,P3,α-硼烷基三磷酸标识;而化合物17也用名称二-(5-OMe)-尿苷5”,5”-P1,P3,β-硼烷基三磷酸标识。万一某个类似物存在一对非对映异构体,例如在类似物13的情况下,文中将那些非对映异构体称为A和B,例如,类似物/非对映异构体13A和13B。说明书中所述的特定中间物在文中用阿拉伯数字1-7标识。尿苷-5’-O-(α-硼烷基二磷酸)在文中用阿拉伯数字11标识。所有这些化合物/类似物的化学结构在以下的附录A和/或方案1中描述。
在一个具体实施方案中,本发明的化合物是5-甲氧基尿苷-5’-O-(α-硼烷基二磷酸),即,具有通式I的化合物,其中R是-OCH3,n是0,Y各自为-OH,Z1是BH3 -,且Z3是O-(化合物13)。优选化合物的特征在于当利用半制备型反相Gemini5μ柱(C-18110A,250×10mm,5微米)和流速为5ml/min的等度洗脱[100mM三乙基乙酸铵,pH7:CH3CN,94:6]从非对映异构体混合物分离时是保留时间(Rt)为8.97min的异构体(化合物13A)。
在另一个具体实施方案中,本发明的化合物是5-甲氧基尿苷-5’-O-(α-硼烷基三磷酸),即,具有通式I的化合物,其中R是-OCH3,n是1,Y各自为-OH,Z1是BH3 -,且Z2和Z3是O-(化合物14)。
具有通式I的化合物可根据技术中已知的任意技术或步骤合成,例如,以下的实施例部分中详细叙述。本发明的化合物可具有不对称中心,例如,在Pα中,因此可存在多对非对映异构体。如果存在一对非对映异构体,则可利用技术中已知的任意技术分离并表征不同的非对映异构体,例如,采用半制备型反相柱和等度溶液,如实施例部分中所述。
具有通式I的化合物可呈药学上可接受的盐形式。
在某些实施方案中,阳离子B为碱金属的无机阳离子,例如,但不限于Na+、K+和Li+。
在其它实施方案中,阳离子B是铵(NH4 +)或衍生自式R4N+胺的有机阳离子,其中每个R独立地选自H、C1-C22,优选C1-C6烷基,例如甲基、乙基、丙基、异丙基、丁基等、苯基或杂芳基,例如吡啶基、咪唑基、嘧啶基等,或其中两个R与它们所连接的氮原子一起形成3-7元环,其任选地包含选自N、S和O的另一个杂原子,例如吡咯烷、哌啶和吗啉。
在其它实施方案中,阳离子B是阳离子脂质或阳离子脂质混合物。阳离子脂质通常在用作递送药剂前与中性脂质混合。中性脂质包括但不限于卵磷脂;磷脂酰乙醇胺;二酰基磷脂酰乙醇胺,例如二油酰基磷脂酰乙醇胺、二棕榈酰基磷脂酰乙醇胺、棕榈酰基油酰基磷脂酰乙醇胺和二硬脂酰基磷脂酰乙醇胺;磷脂酰胆碱;二酰基磷脂酰胆碱,例如二油酰基磷脂酰胆碱、二棕榈酰基磷脂酰胆碱、棕榈酰基油酰基磷脂酰胆碱和二硬脂酰基磷脂酰胆碱;脂肪酸酯;甘油酯;鞘脂类;心磷脂;脑苷脂;神经酰胺;以及其混合物。中性脂质也包括胆固醇和其它3β羟基-固醇。文中所涵盖的其它中性脂质包括磷脂酰甘油;二酰基磷脂酰甘油,例如二油酰基磷脂酰甘油、二棕榈酰基磷脂酰甘油和二硬脂酰基磷脂酰甘油;磷脂酰丝氨酸;二酰基磷脂酰丝氨酸,例如二油酰基-或二棕榈酰基磷脂酰丝氨酸;和双磷脂酰甘油。
阳离子脂质化合物的实例包括但不限于(LifeTechnologies,Burlington,Ontario)(阳离子脂质N-[l-(2,3-二油烯基氧基)丙基]-N,N,N-三甲基氯化铵与二油酰基磷脂酰乙醇胺的1:1(w/w)调配物);LipofectamineTM(Life Technologies,Burlington,Ontario)(聚阳离子脂质2,3-二油烯基氧基-N-[2(精胺-甲酰胺基)乙基]-N,N-二甲基-l-三氟乙酸丙铵与二油酰基磷脂酰乙醇胺的3:1(w/w)调配物)、Lipofectamine Plus(Life Technologies,Burlington,Ontario)(Lipofectamine and Plus试剂)、Lipofectamine2000(Life Technologies,Burlington,Ontario)(阳离子脂质)、Effectene(Qiagen,Mississauga,Ontario)(非脂质体脂质调配物)、Metafectene(Biontex,Munich,Germany)(聚阳离子脂质)、Eu-fectins(Promega Biosciences,San LuisObispo,Calif.)(乙醇阳离子脂质编号1至12:C52H106N6O4·4CF3CO2H、C88H178N8O4S2·4CF3CO2H、C40H84NO3P·CF3CO2H、C50H103N7O3·4CF3CO2H、C55H116N8O2·6CF3CO2H、C49H102N6O3·4CF3CO2H、C44H89N5O3·2CF3CO2H、C100H206N12O4S2·8CF3CO2H、C162H330N22O9·13CF3CO2H、C43H88N4O2·2CF3CO2H、C43H88N4O3·2CF3CO2H、C41H78NO8P);Cytofectene(Bio-Rad,Hercules,Calif.)(阳离子脂质与中性脂质的混合物)、(Gene Therapy Systems,San Diego,Calif.)(中性脂质(Dope)与阳离子脂质的调配物)和FuGENE6(Roche MolecularBiochemicals,Indianapolis,Ind.)(以多组分脂质为主的非脂质体试剂)。
如以下实施例部分中所示,类似物13A是针对在1321N1星形细胞瘤细胞中表达的P2Y6受体的有效选择性激动剂,并且明显比内源性激动剂UDP和类似物12更有效(Ginsburg-Shmuel等,2010)。通过BH3 -取代Pα的非桥接氧而将手性中心引入5-OMe-UDP中显示出相对于B-异构体而言受体对A-异构体的立体特异性。单核苷酸衍生物13的A-异构体是所测试的核苷酸中最有效的,EC50=0.008μM,比相应的B-异构体(EC50=4.3μM)高出500倍以上,比内源性激动剂UDP(EC50=0.15μM)高出24倍。虽然硼烷取代显著提高了类似物12对P2Y6-R的效力,但相应的三磷酸单核苷酸14由于受体偏向于三个磷酸根负电荷而对P2Y6-R几乎没有活性。观察到二核苷酸衍生物16的立体特异性类似于类似物13,但是不太显著,其中A-异构体的效力(EC50=0.06μM)在UDP效力的范围内,但仅高出B-异构体约40倍(EC50=2.2μM)。中间的磷酸具有BH3-取代基的非手性二核苷酸衍生物17表现出类似于标准激动剂UDP的效力(EC50=0.2μM);而未经BH3-取代的二核苷酸衍生物15表现出所测试核苷酸中最弱的效力。所测试核苷酸对1321N1细胞中P2Y2-或P2Y4-受体以及1321N1野生型细胞均无活性。
新的P2Y6-R激动剂的治疗潜力涉及其化学稳定性和其耐酶水解性。因此,在文中所述的另一个研究中,在不同条件下评价所发现的最具效力的类似物13A相对于UDP、11和12的稳定性,发现:(i)在模拟胃液酸度的条件(pH1.4和37℃)下,Pα硼烷基取代降低了13A的稳定性。具体而言,在那些条件下,类似物13A和12分别表现出16.9h和13天的半衰期,而11和UDP得到类似结果(分别16.9h和12天)。化学稳定性减弱是因为P-B键比P-O键更易于发生酸性水解(Nahum和Fischer,2004),但是即使如此,16.9h的半衰期对于候选药物而言十分令人满意;(ii)Pα硼烷基取代增加了对NPP1和NPP3降解的抗性。具体而言,在NPP1和NPP3同时存在的情况下,13A比其非-硼烷基对应物UDP和12更稳定(分别地,NPP1-15%相对于50%和51%水解,NPP3-28%相对于45%和36%水解),表明Pα引入硼烷基磷酸部分在保护UDP类似物抵抗NPP1,3水解方面发挥重要作用。实际上,虽然在Pα与Pβ之间发生NPP的酶降解,但推测类似物13A中大于母体化合物中O的BH3基团通过Pα上的必要水分子防止攻击,因此使这些类似物成为不良的NPP底物。而且,与UDP相比,尿嘧啶环仅一处修饰的类似物12被NPP3而非NPP1更缓慢地水解;(iii)Pα位置的BH3取代和尿嘧啶核苷酸C5位置的OMe增加了在人血清中的耐降解性。如具体所示,在尿嘧啶环的C5处具有甲氧基的12比UDP更稳定(半衰期11.9h对2.4h),具有BH3和甲氧基的13A表现出甚至更强的稳定性,半衰期为17h。尿苷-5’-O-(α-硼烷基二磷酸)得到类似结果(t1/2=21h)。表明甲氧基呈现出位阻,此使类似物12成为血清中多种酶的不良底物。显然,13A和11中硼烷基使核苷酸比UDP更耐酶降解。
在另一个方面,本发明因此提供了一种药物组合物,其包含具有通式I的化合物,但不包括其中Z1、Z2(如果存在)和Z3各自是O-的化合物,或其非对映异构体或非对映异构体混合物,以及药学上可接受载体或稀释剂。
在另一个方面,本发明提供了一种用于降低(即,减低)眼内压,更具体地讲,用于预防或治疗高眼压和/或青光眼的药物组合物,其包含具有通式I的化合物,包括其中Z1、Z2(如果存在)和Z3各自是O-的化合物,或其非对映异构体或非对映异构体混合物,以及药学上可接受载体或稀释剂。
在具体实施方案中,此组合物所包含的化合物是5-甲氧基尿苷二磷酸,即,具有通式I的化合物,其中R是-OCH3,n是0,Y各自是-OH,且Z1和Z3是O-(12),化合物13,更具体地为13A,或化合物14,优选化合物13A。
术语“高眼压”、“眼高压”或“眼内压”,在文中可交换使用,指患者的眼内压高于正常值并且为发展视野损失和青光眼的风险因素。
青光眼是共享某些临床特征的视神经病变的异质群体,其中视力损失是因为神经视网膜中视网膜神经节细胞的选择性死亡,其临床上通过视野的特征性变化、神经纤维层缺损、和视神经乳头(ONH)的进行性凹陷诊断。发展青光眼的主要危险因素之一是存在高眼压(眼内压升高,IOP)。IOP也似乎涉及正常眼压性青光眼的发病机理,其中患者常被认为具有正常IOP。与青光眼相关的升高的IOP是因为小梁网(TM)的房水流出阻力增加,小梁网是一种位于眼前房的虹膜-角膜角的小特化组织。TM的青光眼变化包括TM细胞的损失以及胞外碎片(包括蛋白质斑样物质)的沉积和积累。此外,青光眼ONH也发生变化。在青光眼中,ONH神经胶质细胞发生形态和流动性变化。对升高的IOP和/或短暂缺血损伤的回应,ONH胞外基质组分变化并且神经胶质细胞和视网膜神经节细胞轴突形态改变。
如本文中所用,术语“青光眼”是一种眼病,其特征是眼内压升高并造成视神经损伤。青光眼包括但不限于原发性青光眼、继发性青光眼、青少年型青光眼、先天性青光眼、假性剥脱性青光眼、急性闭角型青光眼、绝对期青光眼、慢性青光眼、窄角型青光眼、慢性开角型青光眼、单纯性青光眼和家族性青光眼,包括但不限于色素性青光眼、高眼压性青光眼、和低眼压性青光眼以及其相关疾病。
可将本发明药物组合物调配用于任何适宜的施用途径,例如,静脉内、动脉内、肌内、皮下或腹膜内施用。然而,当用于降低眼内压,更具体地讲,当用于预防或治疗高眼压和/或青光眼时,将组合物调配成眼用组合物,例如,眼用滴液、乳液、悬浮液、凝胶、软膏或膜状眼贴。
本发明眼用组合物可以多种调配物和剂量提供。这些调配物可通过传统技术制备,例如,Remington:The Science and Practice ofPharmacy,第19版,1995所述。组合物制备可例如通过使活性剂(即,具有通式I的化合物)与液体载体、微细固体载体或两者均匀地缔合,然后,如果必要,将产物成形为所需调配物。
可对旨在直接施用于眼睛的本发明眼用组合物进行调配以以便具有与眼睛相容的pH和张力。这通常需要缓冲液以使组合物pH保持在生理pH或附近,即,5-9,优选6至8,更优选6.8-7.4的范围内;并且可进一步需要张度剂以使组合物的摩尔渗透压浓度达到210-320毫渗量每千克(mOsm/kg)或附近的水平。在某些实施方案中,本发明的组合物具有50-700mOsm/kg,优选100-600mOsm/kg,更优选150-500mOsm/kg,仍更优选200-400mOsm/kg,最优选200-350mOsm/kg的摩尔渗透压浓度。
可通过任意适宜方式将本发明眼用组合物施用给受试者眼睛。在一个实施方案中,组合物呈具有通式I的化合物的液体、乳液、凝胶或悬浮液形式,以滴液、喷雾或凝胶形式施用。在另一个实施方案中,通过脂质体将活性剂(即,具有通式I的化合物)施用给眼睛。在另一个实施方案中,活性剂容纳在连续或选择性释放的装置中,例如,膜,例如,但不限于,OcusertTM System(Alza Corp.,Palo Alto,Calif.)所使用的那些。
在一个实施方案中,活性剂可容纳在放在眼睛上的隐形眼镜内,由其携带或与其接触。在其它实施方案中,活性剂容纳在药签或海绵内,或施用于眼表面的液体喷雾中。在另一个实施方案中,将活性剂直接注射到眼组织中,例如,通过结膜下、巩膜下或玻璃体内注射,或眼表面上。
除了活性剂外,本发明的眼用组合物包括生理相容载体或媒介物,眼科技术人员可利用常规标准选择。此类媒介物可选自已知的眼用媒介物,尤其包括盐溶液、水、聚醚(例如,聚乙二醇)、聚乙烯(例如聚乙烯醇和聚维酮)、纤维素衍生物(例如,甲基纤维素和羟丙基甲基纤维素)、环糊精,尤其β羟丙基环糊精、石油衍生物,例如,矿物油和白矿脂、动物脂肪(例如羊毛脂)、丙烯酸聚合物(例如,聚羧乙烯凝胶)、植物脂肪(例如花生油)、多糖(例如葡聚糖)、海藻酸盐(例如海藻酸钠,任选地包括古罗糖醛酸和/或甘露糖醛酸)、粘多糖(例如透明质酸钠)、和盐(例如氯化钠和氯化钾)。
最佳施用剂量将取决于患者状态,并由医生来确定适宜。具体而言,可每天一次、每天两次或每天3-4次施用治疗青光眼的组合物,和/或在与病状相关的症状发作后施用;持续时间与高眼压和青光眼治疗一致,例如,持续数周、数月、数年或数十年。
在另一个方面,本发明提供了用于降低眼内压的具有通式I的化合物,包括其中Z1、Z2(如果存在)和Z3各自是O-的化合物,或其非对映异构体或非对映异构体混合物。在特定实施方案中,根据本发明所使用的化合物是化合物12、13,更具体地为13A或14,优选化合物13A。
在另一个方面,本发明涉及具有通式I的化合物,包括其中Z1、Z2(如果存在)和Z3各自是O-的化合物,或其非对映异构体或非对映异构体混合物用于制备降低眼内压的药物组合物,尤其眼用组合物的用途。在特定实施方案中,根据本发明使用的化合物是化合物12、13,更具体地为13A或14,优选化合物13A。
在另一个方面,本发明涉及一种降低有需要的个体的眼内压的方法,其包括对该个体施用治疗有效量的具有通式I的化合物,包括其中Z1、Z2(如果存在)和Z3各自是O-的化合物,或其非对映异构体或非对映异构体混合物。在特定实施方案中,根据本发明方法所用的化合物是化合物12、13,更具体地为13A或14,优选化合物13A。
现将通过以下非限制实施例阐述本发明。
实施例
材料和方法
概述
所有对空气和湿度敏感的反应在直火干燥、经氩气冲洗的利用橡胶隔膜密封的双颈烧瓶中进行。将对湿度敏感的反应中所有反应物在真空烘箱中干燥过夜,通过注射器引入试剂。在预涂覆的Merck硅胶板(60F-254)上利用TLC监测反应过程。通过紫外(UV)光实现可视化。在硅胶(Davisil Art.1000101501)上进行快速色谱法。所有市售试剂的使用无需进一步纯化,除非另外指明。所有磷酸化反应是在直火干燥、经氩气冲洗的利用橡胶隔膜密封的双颈烧瓶中进行。使核苷真空干燥过夜。使保持在干燥器中。使氧氯化磷蒸馏并保持在氮气下52。根据文献制备Bpi。52如前所述制备三-正-丁基焦磷酸铵溶液。53三-正-丁基铵-三-正-辛基铵和双(三辛基铵)5’-单磷酸5-OMe-尿苷盐的制备是通过在活化Dowex-H+-型柱上利用去离子水洗脱尿苷核苷酸衍生物(LC分离后获得)并洗脱到包括1当量三-正-辛基胺和1当量三-正-丁基胺的冰浴EtOH溶液中获得。四-正-丁基铵5’-二磷酸5-OMe-尿苷盐的制备是通过在先前经过量四丁基铵水溶液冲洗的CM Sephadex上洗脱尿苷核苷酸衍生物(LC分离后获得)获得。根据文献制备5-甲氧基尿嘧啶、5-甲氧基尿苷,1,和2’,3’-O-甲氧基亚甲基-5-OMe-尿苷,2(Stout和Robins,1972;Chesterfield等,1960;Niedballa和Vorbruggen,1976;Griffin等,1967)。如前所述制备5-OMe-UMP和5-OMe-UDP,12(Niedballa与Vorbruggen,1976)。利用Metrohm pH电极和Metrohm827pH实验用pH计测量pH。利用Bruker AC-200、DPX-300或DMX-600光谱计通过NMR表征化合物。在200、300或600MHz下记录1H NMR光谱。化学位移利用距离用作内标的Me4Si(TMS)的低场ppm表示。核苷酸也利用Bruker AC-200和DMX-600光谱计在D2O中通过31P NMR表征,将85%H3PO4作为外部参照。在AutoSpec Premier(Waters UK)光谱计上通过化学电离记录高分辨率质谱。在ESI(电喷雾电离)条件下通过Q-TOF微型仪器(Waters,UK)分析核苷酸。通过LC(Isco UA-6)系统,利用SephadexDEAE-A25柱对核苷酸进行初级纯化,该柱室温下泡在1M NaHCO3中1天。使用前,利用去离子水冲洗树脂。利用280nm下的UV检测来监测LC分离。如下详述应用NH4HCO3的缓冲梯度。通过HPLC(Hitachi Elite LaChrome)系统,利用半制备型反相柱(Gemini5μC-18110A,250×10.00mm,5微米,Phenomenex,Torrance,USA)对核苷酸进行最终纯化。利用分析型反相柱系统(Gemini5μC-18110A,150mm×4.60mm;5μm;Phenomenex,Torrance,CA)评价核苷酸纯度,采用两种溶剂体系:溶剂体系I,(A)100mM三乙基乙酸铵(TEAA),pH7:(B)CH3CN;溶剂体系Ⅱ,(A)10mM PBS缓冲液,pH7.4:(B)CH3CN。以下详细给出用于分离每种产物的溶剂体系条件。核苷酸纯度通常≥95%。
稳定性测定
对于化学稳定性测定,记录31P NMR光谱(240MHz的同位素频率)。由拉瓦尔大学风湿免疫科研究中心(Research in Rheumatologyand Immunology,Laval University)(加拿大魁北克)提供NPP1和3酶。人血清获自血库(Tel-Hashomer Hospital,Israel)。所有稳定性试验重复进行。
类似物13A/B、15、16和17对P2Y2/4/6受体的活性的评价
细胞培养和转染。使人类P2Y2-R、P2Y4-R和P2Y6-R的绿色荧光蛋白(GFP)构建体在1321N1星形细胞瘤细胞中表达,该细胞缺乏P2X-和P2Y-受体的内源性表达。将受体基因的响应cDNA克隆到pEGFPNl载体中。用FuGENE6转染试剂(Roche MolecularBiochemicals,Mannheim,Germany)转染后,利用0.5mg/ml G418(geneticine;Merck Chemicals,Darmstadt,Germany)选择细胞,并在补有10%胎牛血清、100U/ml青霉素和100U/ml链霉素的达尔伯克氏改良伊格尔氏培养基(DMEM)中在37℃和5%CO2下生长。通过GFP荧光的分析确定各P2Y受体的表达和细胞膜定位。在适宜的受体激动剂刺激后,通过记录[Ca2+]i的变化核实在细胞中所表达的GFP-标记受体的功能。
单细胞[Ca2+]测定。使利用铺在盖玻片(22mm直径)上并生长到约80%浓度的P2YR-GFP表达的响应质粒转染的1321N1星形细胞瘤细胞与2μM fura2/AM和0.02%普流尼克酸(pluronic acid)在Na-HBS缓冲液(Hepes缓冲盐水:145mM NaCl、5.4mM KC1、1.8mM CaCl2、1mM MgCl2、25mM葡萄糖、20mM Hepes/Tris pH7.4)中在37℃下培养30分钟。利用不同浓度的核苷酸的Na-HBS缓冲液使细胞溢出(1ml/min,37℃),在以340nm和380nm激发后,通过在510nm下检测fura2/AM的响应发射强度来测量核苷酸引起的[Ca2+]i变化(Ubl等,1998)。响应的平均最大幅度和各自标准误差通过340nm和380nm下激发的fura2/AM荧光强度的比率计算(只分析了GFP-标记细胞。)使用Microsoft Excel(Microsoft Corp.,Redmond,WA,USA)和SigmaPlot(SPSS Inc.,Chicago,IL,USA)以从至少三次独立试验得到的平均响应幅度得到浓度-响应曲线和EC50值(Ecke等,2006,2008)。数据分析只包括在激动剂脉冲施用后具有清晰GFP-信号和典型钙响应动力学的细胞。GFP-标记P2Y受体适于药理和生理研究,如先前所报道(Tulapurkar等,2004,2006;Zylberg等,2007)。
类似物14A/B对P2Y2/4/6受体的活性的评价
细胞培养。如先前所述,使稳定表达人P2Y6受体的1321N1细胞系在DMEM(5%FBS、100IU/ml青霉素、100μg/ml链霉素、1XGlutamax、10mM Hepes和0.5mg/ml G-418)中在37℃下,在含有5%CO2和95%空气的加湿氛围中生长(Gendron等,2003)。
胞质[Ca2+]测定。利用简易的胰岛素/EDTA处理收集1321N1细胞(10cm2盘中生长10×106个细胞),悬浮在完全培养基中,并通过在100×g下离心3分钟进行冲洗,然后与含Ca2+和Mg2+的4.5mlHBSS(Wisent,St.Bruno,QC)中的1μM Fluo4/AM在37℃下一起培养25分钟。通过离心冲洗细胞(100×g,3min),悬浮在含Ca2+和Mg2+的HBSS中,在37℃下培养25分钟。冲洗细胞,并悬浮在含Ca2+和Mg2+的16ml HBSS中。将细胞悬液(2ml)在石英比色皿中温和地搅拌,利用RF-5301PC Shimadzu荧光光谱计和Panormama荧光1.1软件(Man-Tech,Guelph,ON)监测[Ca2+]i。使用488nm激发波长和520nm发射波长来测量细胞内Fluo4荧光强度(F)的变化。每次记录结束时,通过将0.1%Triton X-100和50mM EDTA相继加入细胞悬液中来确定最大荧光(F最大)和最小荧光(F最小)。来自Grynkiewicz等(1985)的以下等式用于将荧光强度与Ca2+水平相关联:[Ca2+]=Kd×(F-F最 小)/(F最大-F),其中Kd是指示剂的Ca2+解离常数(345nM)。
动物
将24只雄性新西兰白兔(2.5±0.5kg)放置在各个笼中,自由获取食物和水,并控制在12h/12h的光/暗循环中。使用的所有方案遵循视觉与眼科研究协会(ARVO)关于动物在眼科和视力研究中的使用的声明,也符合欧共体理事会指令(86/609/EEC)。
眼内压测量
利用Tiolat Oy(Helsinki,Finland)提供的TonoVET回弹式眼压计测量眼内压(IOP)。将此眼压计用于动物不需使用任何麻醉剂。对于单剂量试验,将多种类似物以100μM的浓度以及10μl的固定体积单侧地施用于眼角膜。对侧眼接受相同体积的盐溶液(0.9%NaCl,媒介物)。在注入任意类似物前,取得两个IOP测量值。按照设盲设计进行试验,其中就所使用溶液性质而言,没有给实验者看得见的标识。追踪IOP达8小时,以研究作用的时程。测定1nM至100μM剂量范围内的一些类似物以产生剂量-响应曲线。对于这些试验,IOP是每种剂量的类似物所得到的最大响应测量值。通过制图给定浓度的IOP值相对于此浓度(1nM至100μM)来得到剂量-响应曲线。根据ORIGIN8.0软件,使值与剂量-响应-曲线等式拟合而得到pD2值。利用pD2值,可通过乘以-1,然后取反对数计算EC50。所得值为EC50,单位为摩尔浓度。在所有试验中,在任意的指定日,只对一个动物进行单剂量测试,该动物在施药前后清洗至少2天。商用降压剂,(拉坦前列素;0.005%)、(盐酸多佐胺(2%))、和匹鲁卡品是通过施用40μl体积来测试。
统计分析
将所有数据以平均值±标准误差表示。利用双尾斯氏t检验来确定显著性差异。利用Microcal Origin v.7.0软件(Microcal Software,U.S.A)进行剂量-响应曲线的绘制和拟合。
实施例1:合成5-OMe-尿苷-5’-O-(α-硼烷基二磷酸),13
5-甲氧基尿苷,1的合成
5-甲氧基尿苷,1,如前所述合成(Stout和Robins,1972;Chesterfield等,1960;Niedballa和Vorbruggen,1976)并用于(例如)制备5-OMe-UDP,12,如前所述(Ginsburg-Shmuel等,2010)。
2’,3’-O-甲氧基亚甲基-5-OMe-尿苷,2的合成
受保护的核苷,2,如前所述般合成(Griffin等,1967)。具体而言,在直火干燥、经氮气冲洗的双颈圆底烧瓶中制备5-OMe-尿苷,1(300mg,1.09mmol)和p-TsOH(催化量)在原甲酸三甲酯(1.09ml,9.85mmol,9当量)中的悬浮液,在室温(RT)下搅拌。24小时后,溶液变得差不多澄清,TLC(CHCl3:MeOH8:2)显示出两个弱极性点并且起始物完全消失。添加Dowex(弱碱)(0.31gr,1.09mmol,1当量),并将混合物在RT下搅拌3小时。倾倒出液体,利用MeOH冲洗。使溶液蒸发以得到油状残余物。与醚共蒸发,得到白色固体(两种非对映异构体的混合物:331.8mg,96.3%)。
2的表征:1H-NMR(DMSO-d6,300MHz):δ11.54(bs,1H,NH),7.46,7.44(2s,1H,H-6),6.101,6.01(2s,1H,CH-OCH3),6.00,5.86(2d,J=2.9Hz,和J=2.7Hz,H-l',1H),5.21,5.17(2m,1H,OH-5'),4.99-5.05(m,2H,H-2'),4.88,4.82(2dd,J=6.6,3.4Hz和J=7.6,3.8Hz,1H,H-3'),4.18和4.09(2m,1H,H-4’),3.58-3.66(m,2H,H-5'和H-5"),3.606(s,3H,C-OCH3),3.30,3.21(2s,3H,CH-OCH3)ppm。Cl2H16N2Na1O8的HRMALDI(正离子)计算值339.080,实测值339.080。
类似物13的合成
如以下方案1中所述,在直火干燥、经氩气冲洗的双颈烧瓶中制备2',3'-O-甲氧基亚甲基5-OMe-尿苷,2(327.1mg,1.03mmol)在无水DMF(2ml)中的溶液。添加无水吡啶(0.42ml,5.17mmol,5当量)和2-C1-1,3,2-苯并二氧杂磷杂苯-4-酮(230.4mg,1.14mmol,1.1当量)在无水二氧六环(2ml)中的溶液,使溶液在RT下搅拌10分钟。然后,添加1M(Bu3NH+)2P2O7H2 -2在DMF(1.55ml.1.55mml,1.5当量)和Bu3N(0.99ml,4.14mmol,4当量)中的混合物,溶液变浑浊,然后再次澄清。然后,添加2M BH3-SMe2络合物的THF(5.17ml,10.34mmol,10当量)溶液。15分钟后,添加乙二胺(0.35ml,5.17mmol,5当量),形成白色沉淀。在RT下一小时后,利用蒸馏水(1.4ml)使反应猝灭,使澄清溶液蒸发并冷冻干燥。粗产物的TLC(异丙醇:25%NH4OH:H2O11:2:7)显示出主要的极性产物(Rf=0.35)。通过酸性水解将甲氧基亚甲基保护基团移除(添加10%HCL溶液,直至得到pH2.3)。在RT下3小时后,通过添加24%NH4OH溶液(pH11)使pH迅速地升到9,使溶液在RT下搅拌45分钟,然后冷冻干燥。使冷冻干燥后得到的半固体在活化Sephadex DEAE-A25柱上层析。利用去离子水冲洗树脂,并使其负载溶于最少量水的粗反应残余物。利用280nm下的UV检测(ISCO,UA-6)来监测分离。应用0-0.2M NH4HCO3的缓冲梯度(每种溶液200ml),然后应用0.2-0.4M NH4HCO3的第二种缓冲梯度(每种溶液200ml)。将不同的馏分汇集在一起,冷冻干燥三次,得到白色固体。通过HPLC系统,利用半制备型反相柱,在下述条件下,进行非对映异构体的最终分离和相关馏分的纯化。通过分析型反相柱系统,利用下述的两种溶剂体系评价核苷酸的纯度。最后,使产物的水溶液通过Dowex50WX8-200离子交换树脂Na +-型柱,利用去离子水洗脱产物以在冷冻干燥后得到相应的钠盐。
非对映异构体13A和13B的分离
利用半制备型反相Gemini5μ柱分离类似物13非对映异构体,13A和13B,94:6(A)100mM TEAA,pH7:(B)CH3CN等度洗脱,流速为5ml/min。收集含纯化异构体[Rt=8.97min(13A);13.45min(13B异构体)]的馏分并冷冻干燥。重复冷冻干燥循环使过量缓冲液移除,每次将固体残余物溶于去离子水中。LC分离后,以50.9%总收率(253.5mg)得到非对映异构体13A和13B。
13A的表征
1H NMR(D2O;600MHz):δ7.41(s,1H,H-6),6.00(d,J=5.6,1H,H-l'),4.47(t,J=5.5,1H,H-3'),4.42(t,J=4.7,1H,H-3'),4.32(m,1H,H-4'),4.28(m,1H,H-5’),4.10(m,1H,H-5"),3.83(s,3H,CH3),0.39(m,3H,BH3)ppm。31P NMR(240MHz,D2O)δ:80.43(m,1P,Pα-BH3),-7.16(d,J=29.48Hz,1P,Pβ)ppm。C10H18B1N2O12P2的HR MALDI(负离子)计算值431.042,实测值431.043。通过分析型柱得到纯度数据:保留时间:3.88min(94.48%纯度),利用溶剂体系I,95:5A:B等度洗脱10分钟,然后95:5至85:15梯度洗脱2分钟,流速为1ml/min。保留时间:2.71min(95.31%纯度),97.5:2.5A:B等度洗脱8分钟,然后97.5:2.5至85:15梯度洗脱2分钟,流速为1ml/min。
13B的表征
1H NMR(D2O;600MHz):δ7.41(s,1H,H-6),6.02(d,J=5.8,1H,H-l'),4.44(m,2H,H-3’,H-2'),4.27(m,2H,H-4',H-5'),4.11(m,1H,H-5'),3.83(s,3H,CH3),0.39(m,3H,BH3)ppm。31P NMR(240MHz,D2O)δ:80.83(m,1P,Pα-BH3),-7.51(d,J=32.4Hz,1P,Pβ)ppm。C10H18B1N2O12P2的HR MALDI(负离子)计算值431.042,实测值431.043。通过分析型柱得到纯度数据:保留时间:6.06min(95.40%纯度),利用溶剂体系I,95:5A:B等度洗脱10分钟,然后95:5至85:15梯度洗脱2分钟,流速为1ml/min。保留时间:4.21min(95.08%纯度),99.5:0.5A:B等度洗脱8分钟,然后99.5:0.5至85:15梯度洗脱2分钟,流速为1ml/min。
实施例2:5-OMe-尿苷-5’-O-(α-硼烷基三磷酸),14的合成
从方案1所示的13的合成中得到作为副产物的5-OMe-尿苷-5’-O-(α-硼烷基三磷酸),14。LC分离后,将相关馏分汇集在一起,冷冻干燥三次,得到白色固体。通过HPLC系统,利用半制备型反相柱,在下述条件下,进行非对映异构体的最终分离和相关馏分的纯化。通过分析型反相柱系统,利用下述的两种溶剂体系评价核苷酸的纯度。最后,使产物水溶液通过Dowex50WX8-200离子交换树脂Na+-型柱,利用去离子水洗脱产物以在冷冻干燥后得到相应的钠盐。
非对映异构体14A和14B的分离
利用半制备型反相Gemini5μ柱分离类似物14非对映异构体,14A和14B,93:7(A)100mM TEAA,pH7:(B)CH3CN等度洗脱,流速为5mL/min。收集含纯化异构体[Rt6.15min(14A);9.22min(14B异构体)]的馏分,并冷冻干燥。重复冷冻干燥循环使过量缓冲液移除,每次将固体残余物溶于去离子水中。LC分离后,以8.66%总收率(51.7mg)得到非对映异构体14A和14B。
14A的表征
1H NMR(D2O;200MHz):δ7.32(s,1H,H-6),6.00(d,J=5.5,1H,H-l'),4.38(m,2H,H-2',H-3'),4.26(m,2H,H-4',H-5'),4.08(m,1H,H-5"),3.78(s,3H,CH3),0.39(m,3H,BH3)ppm.31P NMR(81MHz,D2O)δ:84.51(m,1P,Pα-BH3),-10.33(d,J=19.8Hz,1P,Pγ),-22.48(dd,J=29.4,19.8Hz,1P,Pβ)ppm。C10H19BN2O15P3的HR MALDI(负离子)计算值511.009,实测值511.008。通过分析型柱得到纯度数据:保留时间:4.43min(94.32%纯度),利用溶剂体系I,93:7A:B等度洗脱10分钟,然后93:7至85:15梯度洗脱2分钟,流速为1ml/min。保留时间:3.27min(94.11%纯度),97.5:2.5A:B等度洗脱8分钟,然后97.5:2.5至85:15梯度洗脱2分钟,流速为1ml/min。
14B的表征
1H NMR(D2O;200MHz):δ7.32(s,1H,H-6),6.00(d,J=6.1,1H,H-l'),4.38(m,2H,H-2',H-3'),4.24(m,2H,H-4',H-5'),4.10(m,1H,H-5"),3.78(s,3H,CH3),0.37(m,3H,BH3)ppm.31P NMR(81MHz,D2O)δ:84.58(m,1P,Pα-BH3),-10.20(d,J=19.5Hz,1P,Pγ),-22.46(dd,J=33.3,19.5Hz,1P,Pβ)ppm。通过分析型柱得到纯度数据:保留时间:6.75min(96.85%纯度),利用溶剂体系I,93:7A:B等度洗脱10分钟,然后93:7至85:15梯度洗脱2分钟,流速为1ml/min。保留时间:4.82min(95.33%纯度),97.5:2.5A:B等度洗脱8分钟,然后97.5:2.5至85:15梯度洗脱2分钟,流速为1ml/min。
实施例3:二-(5-OMe)-尿苷5’,5"-P1,P3,三磷酸,15的合成
将三-正-丁基铵-三-正-辛基铵5-OMe-尿苷单磷酸盐(160.8mg,0.18mmol)溶于无水DMF(0.7ml)中,并加入含有CDI(145.8mg,0.9mmol,5当量)的直火干燥、经氮气冲洗的双颈圆底烧瓶中。将反应在RT下搅拌。2小时后,TLC(异丙醇:25%NH4OH:H2O11:2:7)显示出存在弱极性产物(Rf=0.62)并且起始物完全消失(Rf=0.35)。添加MeOH(0.06ml,1.62mmol,9当量)以破坏CDI剩余物,10分钟后,添加四-正-丁基铵5-OMe-尿苷二磷酸,12,(0.18mmol,1当量)在无水DMF(0.5ml)中的溶液和MgCl2(68.4mg,0.72mmol,4当量)。RT下搅拌溶液,24小时后,TLC监测表明存在较强极性的产物(Rf=0.39)并且中间物完全消失。加水后,使溶液冷冻干燥。使冷冻干燥后得到的半固体在活化Sephadex DEAE-A25柱上层析。利用去离子水冲洗树脂,并使其负载溶于最少量水中的粗反应残余物。利用280nm下的UV检测(ISCO,UA-6)来监测分离。应用0-0.2M NH4HCO3的缓冲梯度(每种溶液250ml),然后应用0.2-0.4M NH4HCO3的第二种缓冲梯度(每种溶液300ml)。将不同馏分汇集在一起,冷冻干燥三次,得到白色固体。通过HPLC系统,利用半制备型反相柱,在下述条件下进行相关馏分的最终纯化。通过分析型反相柱系统,利用下述的两种溶剂体系评价核苷酸的纯度。最后,使产物的水溶液通过Dowex50WX8-200离子交换树脂Na+-型柱,利用去离子水洗脱产物以在冷冻干燥后得到相应钠盐。
15的纯化
利用半制备型反相Gemini5μ柱纯化类似物15,96:4(A)100mMTEAA,pH7:(B)CH3CN等度洗脱,流速为5ml/min。收集含有纯化类似物(Rt=11.3min)的馏分,并冷冻干燥。重复冷冻干燥循环使过量缓冲液移除,每次将固体残余物溶于去离子水中。LC分离后,以51.8%总收率(76.6mg)得到类似物15。
15的表征
1H NMR(D2O;600MHz):δ7.31(s,2H,H-6),5.90(d,J=5.2,2H,H-l’),4.38-4.40(m,4H,H-2,H-3),4.21-4.25(m,6H,H-4'H-5',H-5"),3.79(s,6H,CH3)ppm.31P NMR(240MIIz,D2O)δ:-0.89(d,J=18.0Hz,1P,Pα),-22.26(dd,J=18.0,J=18.3Hz,1P,Pβ)ppm。C20H28N4O22P3的HR MALDI(负离子)计算值769.041,实测值769.045。通过分析型柱得到纯度数据:保留时间:3.72min(97.21%纯度),利用溶剂体系I,96:4A:B等度洗脱10分钟,然后96:4至85:15梯度洗脱2分钟,流速为1ml/min。保留时间:2.47min(97.35%纯度),99.5:0.5A:B等度洗脱8分钟,然后99.5:0.5至85:15梯度洗脱2分钟,流速为1ml/min。
实施例4:二-(5-OMe)-尿苷5',5"-P1,P3,α-硼烷基三磷酸,16的合成
将三-正-丁基铵-三-正-辛基铵5-OMe-尿苷单磷酸盐(0.201mmol)溶于无水DMF(0.4ml)中,并加入含有CDI(162.8mg,1.005mmol,5当量)的直火干燥、经氮气冲洗的双颈圆底烧瓶中。将反应在RT下搅拌。2h后,TLC(NH4OH:H2O:2-丙醇2:7:11)表现出存在弱极性产物(Rf=0.62)并且起始物完全消失(Rf=0.35)。加入MeOH(0.07ml,1.809mmol,9当量)以破环CDI剩余物,10分钟后,添加13(0.201mmol,1当量)在无水DMF(1ml)中的溶液以及MgCl2(76mg,0.804mmol,4当量)。使溶液在RT下搅拌,24小时后,TLC监测表现出存在较强极性的产物(Rf=0.41)并且中间物完全消失。加水后,使溶液冷冻干燥。使冷冻干燥后得到的半固体在活化Sephadex DEAE-A25柱上层析。利用去离子水冲洗树脂,并使其负载溶于最少量水中的粗反应残余物。利用280nm下的UV检测(ISCO,UA-6)来监测分离。应用0-0.2MNH4HCO3的缓冲梯度(每种溶液200ml),然后应用0.2-0.4MNH4HCO3的第二种缓冲梯度(每种溶液250ml)。将不同馏分汇集在一起,冷冻干燥三次,得到白色固体。通过HPLC系统,利用半制备型反相柱,在下述条件下进行非对映异构体的最终分离和相关馏分的纯化。通过分析型反相柱系统,利用下述的两种溶剂体系评价核苷酸的纯度。最后,使产物的水溶液通过Dowex50WX8-200离子交换树脂Na+-型柱,利用去离子水洗脱产物以在冷冻干燥后得到相应的钠盐。
非对映异构体16A和16B的分离
利用半制备型反相Gemini5μ柱分离类似物16非对映异构体,16A和16B,93:7(A)100mM TEAA,pH7:(B)CH3CN等度洗脱,流速为5mL/min。收集含纯化异构体[Rt=6.60min(16A);10.87min(16B异构体)]的馏分,并冷冻干燥。重复冷冻干燥循环使过量缓冲液移除,每次将固体残余物溶于去离子水中。LC分离后,以45.2%总收率(74.4mg)得到非对映异构体16A和16B。
16A的表征
1H NMR(D2O;600MHz):δ7.33(s,1H,H-6A),7.32(s,1H,H-6B),6.02(d,J=6.3,1H,H-l'A),6.02(d,J=5.6,1H,H-1'B),4.37-4.44(m,4H,H-2'A,H-2'B,H-3'A,H-3'B),4.22-4.29(m,4H,H-4'A,H-4'B,H-5'A,H-5'B),4.26(m,1H,H-5"A),4.13(m,1H,H-5"B),3.83(s,6H,CH3A,CH3B),0.49(m,3H,BH3)ppm.31P NMR(240MHz,D2O)δ:84.29(m,1P,Pα-BH3),-11.03(d,J=18.3Hz,1P,Pγ),-22.52(dd,J=27.8,J=18.3Hz,1P,Pβ)ppm。C20H31B1N4O21P3的HR MALDI(负离子)计算值767.078,实测值767.079。通过分析型柱得到纯度数据:保留时间:3.76min(96.84%纯度),利用溶剂体系I,94:6A:B等度洗脱10分钟,然后94:6至85:15梯度洗脱2分钟,流速为1ml/min。保留时间:2.43min(95.14%纯度),97.5:2.5A:B等度洗脱8分钟,然后97.5:2.5至85:15梯度洗脱2分钟,流速为1ml/min。
16B的表征
1H NMR(D2O;600MHz):δ7.33(s,1H,H-6A),7.31(s,1H,H-6B),6.02(m,1H,H-1'A,H-1'B),4.35-4.41(m,4H,H-2'A,H-2'B,H-3'A,H-3'B),4.24-4.32(m,5H,H-4'A,H-4'B,H-5'A,H-5'B,H-5"A),4.11(m,1H,H-5"B),3.80(s,6H,CH3A,CH3B),0.43(m,3H,BH3)ppm.31P NMR(240MHz,D2O)δ:84.05(m,1P,Pα-BH3),-11.09(d,J=18.3Hz,1P,Pγ),-22.55(dd,J=31.4,J=18.3Hz,1P,Pβ)ppm。C20H31B1N4O21P3的HR MALDI(负离子)计算值767.078,实测值767.079。通过分析型柱得到纯度数据:保留时间:7.01min(97.31%纯度),利用溶剂体系I,94:6A:B等度洗脱10分钟,然后94:6至85:15梯度洗脱2分钟,流速为1ml/min。
保留时间:5.08min(95.92%纯度),97.5:2.5A:B等度洗脱8分钟,然后97.5:2.5至85:15梯度洗脱2分钟,流速为1ml/min。
实施例5:二-(5-OMe)-尿苷5',5"-P1,P3,β-硼烷基三磷酸,17的合成
将三-正-丁基铵-三-正-辛基铵5-OMe-尿苷单磷酸盐(401.9mg,0.45mmol)溶于无水DMF(2ml)中,并加入含有CDI(364.5mg,2.25mmol,5当量)的直火干燥、经氮气冲洗的双颈圆底烧瓶中。将反应在RT下搅拌。2h后,TLC(异丙醇:25%NH4OH:H2O11:2:7)表现出存在弱极性产物(Rf=0.62)并且起始物完全消失(Rf=0.35)。加入MeOH(0.09ml,2.25mmol,5当量)以破环CDI剩余物,10分钟后,添加BPi(523.7mg,1.125mmol,2.5当量)在无水DMF(0.5ml)中的溶液以及MgCl2(342.7mg,3.6mmol,8当量)。使溶液在RT下搅拌,24小时后,TLC监测表现出存在较强极性的产物并且中间物完全消失。加水后,使溶液冷冻干燥。使冷冻干燥后得到的半固体在活化SephadexDEAE-A25柱上层析。利用去离子水冲洗树脂,并使其负载溶于最少量水中的粗反应残余物。利用280nm下的UV检测(ISCO,UA-6)来监测分离。应用0-0.2M NH4HCO3的缓冲梯度(每种溶液200ml),然后应用0.2-0.4M NH4HCO3的第二种缓冲梯度(每种溶液300ml)。将不同馏分汇集在一起,冷冻干燥三次,得到白色固体。通过HPLC系统,利用半制备型反相柱,在下述条件下进行相关馏分的最终纯化。通过分析型反相柱系统,利用下述的两种溶剂体系评价核苷酸的纯度。最后,使产物的水溶液通过Dowex50WX8-200离子交换Na+-型树脂柱,利用去离子水洗脱产物以在冷冻干燥后得到相应的钠盐。
17的纯化
利用半制备型反相Gemini5μ柱纯化类似物17,96:4(A)100mMTEAA,pH7:(B)CH3CN等度洗脱,流速为5ml/min。收集含有纯化类似物(Rt=6.13min)的馏分,并冷冻干燥。重复冷冻干燥循环使过量缓冲液移除,每次将固体残余物溶于去离子水中。LC分离后,以8%总收率(30.8mg)得到类似物17。
17的表征
1H NMR(D2O;600MHz):δ7.32(s,1H,H-6),6.02(m,2H,H-l'),4.41(m,4H,H-2',H-3'),4.22(m,6H,H-4',H-5',H-5"),3.80(s,6H,CH3),0.50(m,3H,BH3)ppm.31P NMR(240MHz,D2O)δ:80.43(m,1P,Pα-BH3),-7.16(d,J=29.48Hz,1P,Pβ)ppm。C20H31B1N4O21P3的HRMALDI(负离子)计算值767.078,实测值767.079。通过分析型柱得到纯度数据:保留时间:6.09min(96.33%纯度),利用溶剂体系I,96:4A:B等度洗脱10分钟,然后96:4至85:15梯度洗脱2分钟,流速为1ml/min。保留时间:5.34min(96.12%纯度),99.5:0.5A:B等度洗脱8分钟,然后99.5:0.5至85:15梯度洗脱2分钟,流速为1ml/min。
实施例6:UDP、11、12和13A的化学稳定性
利用31P NMR在37℃下评价UDP、11、12和13A在缓冲溶液(pH1.4)中的稳定性,以监测可能的脱磷产物(磷酸水解产物的信号在~0ppm,而硼烷基磷酸类似物在~85ppm)。通过具有31P NMR探针(同位素频率为240MHz)的Bruker DMX-600光谱计记录NMR光谱,将85%H3PO4作为外部参照。
将UDP、11、12和13A的钠盐溶于0.45ml KC1/HC1缓冲液(pH1.4)中,并加入D2O(0.05ml)。将最终pH调节到pH1.4。利用MetrohmpH电极和Metrohm827pH仪测量pH。在37℃下,以15min、1h或24h的时间间隔记录光谱。对于持续几天的试验,使溶液置于37℃油浴中,并以约24h的时间间隔记录光谱。通过测定起始物的一种磷酸信号积分随时间的变化确定磷酸酯水解速率。
通过测定一种磷酸信号积分随时间的变化确定13A的磷酸酯水解速率,并拟合拟一级反应模型,如图1所示。具体而言,在13A水解期间,我们首先观察到在0.62ppm处出现的无机磷酸信号,以及在96.57ppm处剩余的5-OMe-UMP硼烷基磷酸(α-B)信号。同时,13A的Pβ(-10.69ppm)和Pα(86.55ppm)信号减少,表明在这些条件下,终端磷酸快速消失。其次,在7.31和4.42ppm处出现了两个其它信号,表明分别形成了5-OMe-尿苷-H-膦酸和无机H-膦酸部分。确定13的速率常数为1.31×10-5s-1(t1/2=16.9h),而UDP、11和12分别为6.66×10-7s-1(t1/2=~12天)、1.14×10-5s-1(t1/2=16.8h)和6.25×10-7s-1(t1/2=~13天)。
实施例7:UDP、12和13A对NPP1和NPP3降解的抗性
在此研究中,确定在适宜缓冲液中在37℃下培养后人NPP1和NPP3对类似物13A(与UDP和类似物12相比)的水解速率。
分别将56.15μg或57.78μg人NPP1或NPP3提取物加入0.575ml培养混合物(1mM CaCl3、200mM NaCl、10mM KC1和100mMTris,pH8.5)中,并在37℃下预培养3分钟。通过添加0.015ml4mMUDP、12或13A使反应开始。分别与NPP1或NPP3一起培养2h或3h后,将0.1ml反应混合物等分试样转入0.350ml冰冷的1M高氯酸中,使反应停止。使这些样品在10000×g下离心1分钟。利用1M4℃KOH中和上清液,并在10000×g下离心1分钟。将反应混合物过滤,并冷冻干燥。通过以与对照相比较的方式测定每种类似物的HPLC峰积分随时间的变化确定类似物UDP、12和13A的NPP1或NPP3的水解速率。以与对照相比较的方式计算化合物降解百分比,以考虑因添加酸而停止酶反应的化合物降解。因此,将每种样品与转入酸中但未加入酶的对照比较。核苷单磷酸峰曲线下面积减去对照后得到降解百分比,对照百分比是因为化学酸性水解而形成的核苷单磷酸峰值。
表1:NPP1和NPP3对UDP、12和13A的水解百分比
*在缓冲液(1mM CaCl3、200mM NaCl、10mM KC1和100mMTris,pH8.5)中分别与NPP1或NPP3一起在37℃下培养2或3h后。值表示两组试验的平均值±标准偏差(p<0.05)。
如表1所汇总,在NPP1存在下,UDP与酶一起培养2h后,50%水解为UMP。类似物12类似地水解为5-OMe-UMP(49%)。然而,在相同的培养时间后,仅15%类似物13A水解。与类似物UDP和12相比较,类似物13A也对NPP3水解表现出相对的稳定性,分别水解了28%、45%和36%,培养3h后,生成相应的核苷5'-单磷酸。
实施例8:UDP、11、12和13A在人血清中的稳定性
血清含有去磷酸化的酶,因此提供了评价核苷酸类似物的体内稳定性的良好模型系统。在此研究中,确定类似物13A在人血清中的与UDP、11和12相比的半衰期。
将包含0.1mg每种类似物的去离子水(4.5μl)、人血清(180μl)和RPMI-1640培养基(540μl)的测定混合物(Eliahu等,2010a)在37℃下培养0-24h。0.5-12h后,将每种样品加热到80℃并保持30min,利用CM Sephadex(1-2mg)处理,振荡2h并离心6min(12000rpm),收集水层并利用氯仿(2×500μ1)萃取。使水层冷冻干燥,然后溶于去离子水(100μl)中。将样品加载到活性Starta X-AW弱阴离子交换柱上,利用H2O(1ml)冲洗,并依序利用MeOH:H2O(1:1,1ml)和NH4OH:MeOH:H2O(2:25:73,1ml)洗脱,然后冷冻干燥。在Gemini分析柱(5μC-18557110A;150mm×4.60mm)上通过HPLC分析所得的残余物,利用溶剂体系I(对于UDP和12而言-A:B96:4,10min)或II(对于尿苷-5'-O-(α-硼烷基二磷酸)和13A而言-A:B97.5:2.5,10min)梯度洗脱,流速为1ml/min。通过以与对照相比较的方式测定每种类似物的HPLC峰积分随时间的变化确定类似物UDP、尿苷-5’-O-(α-硼烷基二磷酸),12和13A的血清水解速率。计算化合物相对于对照的降解百分比,将归因于酶反应后的后处理的化合物降解考虑在内。因此,将每种样品与通过相同后处理但未添加血清的对照进行。根据核苷单磷酸峰曲线下面积计算出降解百分比,减去对照后,其为由于化学水解而形成的核苷单磷酸峰值。
如图2A-2B所示,UDP被水解为UMP,然后进一步降解为尿苷,半衰期为2.4h(2A)。然而,类似物12被水解为相应的核苷5’-单磷酸和核苷,半衰期为11.9h,并且11表现出21h的半衰期。13A被水解为5-OMe-UMP(α-B),然后水解为5-OMe-尿苷,半衰期为17h(2B)。
实施例9:类似物13-17对P2Y2/4/6-受体的活性
如Ginsburg-Shmuel等(2010)所述,发现5-甲氧基尿苷三磷酸(5-OMe-UTP)和5-甲氧基尿苷二磷酸(5-OMe-UDP),12均为P2Y6-受体的激动剂,EC50分别为0.9和0.08μM。
在此研究中,如材料和方法中所述,根据经各自质粒转染的1321N1星形细胞瘤细胞中细胞内钙浓度的增量,测定类似物13-17对P2Y2,4,6-受体的效力和选择性,结果汇总在表2中。
表2:在1321N1星形细胞瘤细胞中类似物13-17对P2Y2/4/6-受体的效力(EC50,μM)
n.d.r.-对于高达100μM的核苷酸浓度检测不到响应
a-对于高达100μM的核苷酸浓度,不能计算出EC50值,因为未达到[Ca2+]i的上升坪值。
如所示,13A是针对在1321N1星形细胞瘤细胞中表现的P2Y6受体的有效的选择性激动剂,并比标准激动剂UDP更有效。通过BH3-取代Pα的非桥接氧而将手性中心引入5-OMe-UDP中显示出相对于B-异构体而言受体对A-异构体的立体-选择性偏向。单核苷酸衍生物13A的A-异构体(Rp异构体)是所测试的核苷酸中最有效的激动剂,EC50=0.008μM,比相应的B-异构体13B(EC50=4.3μM,图3A)高出500倍以上,比内源性激动剂UDP(EC50=0.15μM)高出19倍。对于5-OMe-UDP的二-核苷酸衍生物,16A和16B,发现了类似的立体-选择性,但是不显著,其中A-异构体(Rp异构体)的效力(EC50=0.06μM)类似于UDP(图3B),但仅高出相应的B-异构体37倍左右(EC50=2.2μM)。
以前没有发现P2Y6-R对Rp异构体的偏向。然而,我们描述了P2Y1-和P2Y11-受体的非对映选择性,其分别表现出对硼烷基磷酸和硫代磷酸腺嘌呤核苷酸的Rp和Sp异构体的偏向(Major等,2004;Ecke等,2006)。根据我们对P2Y1-R的计算研究,我们先前指出结合至受体内核苷酸的硬质Mg2+离子优先结合ATP-α-B类似物中Pα氧原子,而非硼烷基团。因此,在ATP-α-B(Sp)异构体中,Pα氧并不处在与Mg2+离子配合的位置,在此情况下,可通过Pβ,γ配合。因此,此异构体失去可能与Mg2+离子的紧密相互作用,最终得到更高的EC50值(Major等,2004)。同样,我们认为13B不及13A有效,因为13B缺少P2Y6-R与Pα的结合相互作用。
虽然硼烷基取代显著提高了12对P2Y6-R的效力,但相应的三磷酸单核苷酸14因为受体偏向于三个磷酸负电荷而几乎对P2Y6-R没有活性(Jacobson等,2009;Shaver等,2005)。同样,14对P2Y2-R完全无活性,即使其类似于受体的内源性激动剂UTP。对于P2Y2-R,14无活性而5-OMe-UTP具有活性(EC50=2μM,而UTP,EC50=0.1μM)(Ginsburg-Shmuel等,2010),表明P2Y2-R不耐受Pα-硼烷基。此前已经对ATP(α-B)的进行了观察,其对P2Y2受体诱发极弱的响应(与内源性配体ATP和UTP相比)(Tulapurkar等,2004)。
硼烷基-二核苷酸衍生物16A效力比其单-核苷酸对应物13A的效力低9倍。类似物17,5-OMe-UDP的Pβ-硼烷基二核苷酸衍生物等效于UDP(EC50=0.2μM)(图3D)。显然,16A相对于13A的活性的降低是因为需要末端磷酸以供P2Y6-R的分子识别。即使这样,16A仍比15或17更具活性,因为其结构与13A更相似。活性最低的P2Y6-R激动剂是15,其是具有两个甲氧基取代基的二核苷酸,每个尿嘧啶环上一个。意外的是,与先前报道的二核苷酸三磷酸Up3U相比,此类似物对P2Y6-R活性较低。显然,二核苷酸15的双5-OMe取代显著降低了对P2Y6受体的效力(图3C)。对于高达100μM的核苷酸浓度,未达到细胞内钙响应的坪值。然而,类似于单核苷酸,硼烷基明显有利于提高对P2Y6-R的效力:所有具有硼烷基的核苷酸,无论Pα处或Pβ处,远比其非-硼烷基对应物更有活性-13A(相对于12),以及16A和17(相对于15)。
测试的所有核苷酸对1321N1细胞中P2Y2-受体和P2Y4-受体以及1321N1野生型细胞无活性,证实具有特异性。
实施例10:类似物12和13A降低血压正常的兔子的IOP
利用各种5-甲氧基尿苷核苷酸和二核苷酸衍生物降低血压正常的兔子的眼内压(IOP),并与作为对照的UDP比较(UDP为内源性P2Y6-受体配体)以及与某些市售药物比较,如图4所示,类似物12将IOP降低31%,而类似物13A将IOP降低45%,超出任一种市售药物,例如,和匹鲁卡品。作用时间约4h。
附录
方案1:类似物13和14的合成
反应条件
a)(1)HC(OMe)3,p-TsOH,RT,过夜;以及(2)Dowex(弱碱),RT,3h,96.3%;b)2-Cl-1,3,2-苯并二氧杂磷杂苯-4-酮,无水DMF,无水二氧六环,RT,10min;c)1M P2O7H2 2-(Bu3N+H)2的无水DMF,Bu3N,RT,5min;以及d)2M BH3·SMe2的THF,RT,15min;e)乙二胺,RT,10min;以及f)(1)10%HCl,pH2.3,RT,3h;以及(2)24%NH4OH,pH9,RT,45min。得到化合物6和7的混合物。分别以50.9%和9%的收率得到化合物13和14,每种为两种非对映异构体的混合物。
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Claims (21)
2.根据权利要求1所述的化合物,其中R是-O-(C1-C8)烷基,优选-O-(C1-C4)烷基,更优选-OCH3或-OC2H5。
3.根据权利要求1所述的化合物,其中R是-S-(C1-C8)烷基,优选-S-(C1-C4)烷基,更优选-SCH3或-SC2H5。
4.根据权利要求1所述的化合物,其中n是0,并且Z1和Z3中至少一者是BH3 -;或n是1,并且Z1至Z3中至少一者是BH3 -。
5.根据权利要求4所述的化合物,其中n是0,并且(i)Z1是BH3 -,且Z3是O-;(ii)Z3是BH3 -,且Z1是O-;或(iii)Z1和Z3是BH3 -。
6.根据权利要求4所述的化合物,其中n是1,并且(i)Z1是BH3 -,且Z2和Z3是O-;(ii)Z2是BH3 -,且Z1和Z3是O-;(iii)Z3是BH3 -,且Z1和Z2是O-;(iv)Z1和Z2是BH3 -,且Z3是O-;(v)Z1和Z3是BH3 -,且Z2是O-;(vi)Z2和Z3是BH3 -,且Z1是O-;或(vii)Z1至Z3是BH3 -。
7.根据权利要求1至6中任一项所述的化合物,其中R是-O-(C1-C4)烷基,优选-OCH3或-OC2H5,n是0,并且(i)Z1是BH3 -,且Z3是O-;(ii)Z1是O-,且Z3是BH3 -;或(iii)Z1和Z3是BH3 -。
8.根据权利要求7所述的化合物,其中R是-OCH3,Y各自是-OH,n是0,Z1是BH3 -,并且Z3是O-(在本文中标识为化合物13)。
9.根据权利要求8所述的化合物,其特征在于当利用半制备型反相Gemini5μ柱(C-18110A,250×10mm,5微米)和流速为5ml/min的等度洗脱[100mM三乙基乙酸铵,pH7:CH3CN,94:6]从非对映异构体混合物分离时是保留时间(Rt)为8.97min的异构体(在本文中标识为化合物13A)。
10.根据权利要求7所述的化合物,其中R是-OCH3,Y各自是-OH,n是1,Z1是BH3 -,并且Z2和Z3是O-(在本文中标识为化合物14)。
11.根据权利要求1至6中任一项所述的化合物,其中R是-O-(C1-C4)烷基,优选-OCH3或-OC2H5,n是1,并且(i)Z1是BH3 -,且Z2和Z3是O-;(ii)Z2是BH3 -,且Z1和Z3是O-;(iii)Z3是BH3 -,且Z1和Z2是O-;(iv)Z1和Z2是BH3 -,且Z3是O-;(v)Z1和Z3是BH3 -,且Z2是O-;(vi)Z2和Z3是BH3 -,且Z1是O-;或(vii)Z1至Z3是BH3 -。
12.根据权利要求1至11中任一项所述的化合物,其中B是碱金属阳离子NH4 +、式R4N+的有机阳离子,其中每个R独立地是H或C1-C22,优选C1-C6烷基、阳离子脂质或阳离子脂质的混合物。
13.一种药物组合物,其包含根据权利要求1所述的具有通式I的化合物,或其非对映异构体或非对映异构体的混合物,以及药学上可接受的载体或稀释剂。
14.一种用于降低眼内压的药物组合物,其包含根据权利要求1所述的具有通式I的化合物,或其非对映异构体或非对映异构体的混合物,以及药学上可接受的载体或稀释剂。
15.根据权利要求14所述的药物组合物,其包含化合物13,优选13A,或14,或具有通式I的化合物,其中R是-OCH3,n是0,Y各自是-OH,并且Z1和Z3是O-(化合物12)。
16.根据权利要求14所述的药物组合物,其用于预防或治疗高眼压和/或青光眼。
17.根据权利要求16所述的药物组合物,其中所述青光眼是原发性开角型青光眼、正常眼压性青光眼、急性闭角型青光眼、绝对期青光眼、慢性青光眼、先天性青光眼、青少年型青光眼、窄角型青光眼、慢性开角型青光眼或单纯性青光眼。
18.根据权利要求14至17中任一项所述的药物组合物,其可调配成眼用滴液、乳液、悬浮液、凝胶、软膏或膜状眼贴。
19.根据权利要求1所述的具有通式I的化合物,或其非对映异构体或非对映异构体的混合物,其用于降低眼内压。
20.根据权利要求1所述的具有通式I的化合物,或其非对映异构体或非对映异构体的混合物的用途,其用于制备降低眼内压的药物组合物。
21.一种降低有需要的个体的眼内压的方法,其包括对所述个体施用治疗有效量的根据权利要求1所述的具有通式I的化合物,或其非对映异构体或非对映异构体的混合物。
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US7084128B2 (en) | 2002-01-18 | 2006-08-01 | Inspire Pharmaceuticals, Inc. | Method for reducing intraocular pressure |
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- 2011-12-01 CN CN2011800666318A patent/CN103339136A/zh active Pending
- 2011-12-01 EP EP11808361.7A patent/EP2646449B1/en not_active Not-in-force
- 2011-12-01 US US13/990,491 patent/US9221868B2/en not_active Expired - Fee Related
- 2011-12-01 WO PCT/IL2011/000913 patent/WO2012073237A1/en active Application Filing
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US20060287271A1 (en) * | 2005-06-15 | 2006-12-21 | Bar-Ilan University | Dinucleoside poly(borano)phosphate derivatives and uses thereof |
WO2009066298A1 (en) * | 2007-11-23 | 2009-05-28 | Bar-Ilan University | Non-hydrolyzable nucleoside di- or tri-phosphate derivatives and uses thereof |
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CN105407898A (zh) * | 2013-03-13 | 2016-03-16 | 塔夫斯大学 | 尿苷核苷衍生物、组合物及使用方法 |
CN110804081A (zh) * | 2019-12-09 | 2020-02-18 | 美亚药业海安有限公司 | 一种地夸磷索杂质的合成方法 |
Also Published As
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EP2646449A1 (en) | 2013-10-09 |
EP2646449B1 (en) | 2015-08-19 |
IL226615A (en) | 2017-04-30 |
US20130324495A1 (en) | 2013-12-05 |
US9221868B2 (en) | 2015-12-29 |
WO2012073237A1 (en) | 2012-06-07 |
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