CN101115765B - 修饰的叠氮胸苷的5’-膦酸酯—潜在的抗病毒制剂 - Google Patents
修饰的叠氮胸苷的5’-膦酸酯—潜在的抗病毒制剂 Download PDFInfo
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Abstract
本发明涉及分子生物学、病毒学和医学，尤其涉及通式(I)的新3’-叠氮基-3’-脱氧胸苷膦酸酯类，其中R是烷基基团，包括含有卤素原子、羧基、羟基、烷氧基和酰氧基基团的基团以及被取代的氨基羰基基团。本发明的化合物能以抗病毒药剂的形式使用，因为它们是低毒性的、有效地抑制MT-4细胞培养物中1型免疫缺陷性病毒的复制并在哺乳动物中产生叠氮胸苷，从而确保其在血液中逐渐增加。
Description
发明领域
本发明涉及分子生物学、病毒学和医学领域，并且更具体地涉及核苷的新衍生物，也就是，取代的AZT的5’-膦酸酯。这些化合物具有抗病毒作用而且可以用于抑制人类免疫缺陷性病毒的复制。
发明背景
目前，将具有对抗HIV抗病毒活性的全部范围的化合物用于实践医学中。它们包括核苷类和非核苷类抑制剂。最常用的核苷衍生物包括3’-叠氮基-3’-脱氧胸苷(AZT，齐多夫定)，2’，3’-双去氧胞苷(ddC，扎西他宾)，2’，3’双去氧肌苷(ddI，地达诺新)，2’，3’-双去氧-2’，3’-双脱氢胸苷(d4T，司他夫定)和2’，3’-双脱氧-3’-硫杂胞苷(3TC，拉米夫定)[De Clercq，E.，2002.New development in anti-HIV chemotherapy.Biochim.Biophys.Acta，1587 258-275]。
上述化合物的作用机理包含其扩散到感染细胞中，在其中它们经历三磷酸化并特异地抑制由HIV逆转录酶催化的DNA合成。HIV的高变异性导致该病毒的耐药性毒株快速出现[Groschel，B.，Cinatl，J.H.，and Cinatl J.Jr.，1997.Viral and cellular factors for resistanceagainst antiretroviral agents.Intervirology，40，400-407；Antonelli，G，Turriziani，O.，Verri，A.，Narciso，P.，Ferri，F.，D′Offizi，G.，andDianzini，F.，1996.Long-term exposure to zidovudine affects in vitroand in vivo the efficiency of thymidine kinase.AIDS Res HumRetrovir.，12，223-228]，因此改变疗法是必要的。此外，由于细胞内转化的低功效，当前使用的药物不得不以高剂量施用，这导致显著的毒性作用。
AZT毒性引起脊髓细胞活性的抑制、肝功能损伤和肌病[Chariot，P.，Drogou，I.，De Lacroix-Szmania，I.，Eliezer-Vanerot，M.C.，Chazaud，B.，Lombes，A.，Schaeffer，A.，and Zafrani，E.S.，1999.Zidovudine-induced mitochondrial disorder with massive liversteanosis，myopathy，lactic acidosis，and mitochondrial DNA depletion.J.Hepatol.30，156-160；Kellam，P.，Boucher，C.A.，and Larder，B.A.，1992.Fifth mutations in HIV reverse transcriptase contributes to thedevelopment of high level resistance to zidovudine.Proc.Natl.Acad.Sci.U.S.A，89，1934-1938；Ren，J.，Esnouf，R.M.，Hopkins，A.L.，Jones，E.Y.，Kirby，I.，Keeling，J.，Ross，C.K.，Larder，B.A.，Stuart，D.I.，andStammers，D.K.，1998.3′-Azido-3′-deoxythymidine drug resistancemutations in HIV-1 reverse transcriptase can induce long rangeconformational changes.Proc.Natl.Acad.Sci.U.S.A，95，9518-9523]。AZT从身体中快速消除使得必需频繁地给药。此外，在用AZT长期治疗期间，该病毒的耐药性毒株产生相当快并且该治疗丧失其功效。尽管存在所有上述缺点，AZT仍然为最广泛使用的抗HIV药物。
在俄国被批准用于AIDS治疗的已知AZN(Nikavir
)的H-膦酸酯比AZT毒性更低[Intracellular metabolism and pharmacokinetics of5′-hydrohenphosphonate of 3′-azido-2′，3′-dideoxythymidine，aprodrug of 3′-azido-2′，3′-dideoxythymidine.Antiviral Research 63(2004)，107-113]。Nikavir的作用基于其释放AZT的能力，其在细胞内转化为AZT-5’-三磷酸酯之后抑制HIV的复制。根据药物动力学研究数据，Nikavir的临床优势是由于与施用适当AZT时相比血液中AZT浓度的更慢和更逐渐的增加；来自Nikavir的AZT的Cmax小于来自齐多夫定的AZT的Cmax，而且来自Nikavir的AZT的T1/2大于来自齐多夫定的AZT的T1/2[Y.Skoblov等人/Antiviral Research 63(2004)107-113]。然而，Nikavir的毒性依然相当高。另一个缺点在于对Nikavir耐药性的产生。
发明的描述
本发明解决了能渗透到细胞中并逐渐释放活性核苷(AZT)的AZT低毒性衍生物的任务。这将使得保持治疗上足够的细胞内药物浓度持续延长的时期并因此减少药物的单次剂量和/或给药频率并减轻副作用成为可能。
通过产生具有下列通式的5’-膦酰基-3’-叠氮基-3’-脱氧胸苷化合物解决了该任务：
R＝烷基基团，包括含有卤素原子、羧基-、羟基-、烷氧基-和酰氧基-基团的那些以及被取代的氨基羰基基团。
该新化合物抑制1型人类免疫缺陷性病毒在MT-4淋巴细胞系中的复制，保护细胞不受病毒的致细胞病变作用，并直到特别高的浓度没有针对宿主细胞的毒性(表1)。实验数据证实，当研究用化合物以有效浓度(50％中毒剂量为大于50％抑制剂量的2-4级)对细胞没有产生毒性作用时，研究用化合物证明高度抑制MT-4细胞培养物中的1型免疫缺陷性病毒。研究用化合物的治疗指数(计算为药物的治疗剂量与其有效剂量的比率)与AZT H-膦酸酯相当。根据以前描述的方案，进行了病毒学试验。
已证明，在犬中AZT的膦酸酯缓慢释放AZT，因此代表了AZT的潜在形式(实施例7，表2)。研究证明，对于本申请覆盖的所有膦酸酯，在动物血液中可检测的唯一代谢物是AZT。表2中包括的药物动力学参数是基于产生的AZR测定的并依赖于膦酸酯结构。
实现本发明的最佳实施例
使用下列方案产生目标膦酸酯类：
其中X＝Cl或OH Ia：R＝ClCH2
Ib：R＝ICH2
Ic：R＝HOCH2
Id：R＝CH3OCH2
Ie：R＝H2NC(O)
下面特定的实施例揭示了本发明的本质。
实施例1
5’-氯甲基膦酰基-3’-叠氮基-3’-脱氧胸苷(Ia)
将氯甲基膦酰(Cloromethyl phosphonidyl)二氯(0.92ml，9mM)加到冷却到0℃的3’-叠氮基-3’-脱氧胸苷(0.8g，3mM)的磷酸三乙酯(10ml)溶液中。该混合物在18℃搅拌18小时，用冷却的吡啶(10ml)和水(10ml)的混合物稀释，搅拌30分钟并加到水(700ml)中。将溶液注入装有DEAE纤维素的柱中，并以线性梯度的NH4HCO3(0→15mM，pH 7.5)进行洗脱。蒸发目标级分，残留物用水(3ml)稀释并另外在LiChroprep RP-18柱上使用水作为洗脱剂进行纯化。将目标级分冻干，获得1g(90％)膦酸酯(Ia)。1H NMR(D2O)：7,72q(1H，J＝0,5Hc，H-6)，6,27t(1H，J＝6Hc，H-1’)，4,55m(1H，H-3’)，4,21m(3H，H-4’，H-5’)，3,58d(2H，J＝8,5Hc，CH2-P)，2,54m(2H，H-2’)，1,95d(3H，J＝0,5Hc，CH3)。31P NMR：16,03s。
实施例2
5’-碘甲基膦酰基-3’-叠氮基-3’-脱氧胸苷(Ib)
使用关于化合物Ia所述的方法，由3’-叠氮基-3’-脱氧胸苷和碘甲基膦酸合成5’-碘甲基膦酰基-3’-叠氮基-3’-脱氧胸苷(Ib)。收率为54％。1H NMR(D2O)：7,55s(1H，H-6)，6,07t(1H，J＝6Hc，H-1’)，4,37m(1H，H-3’)，4,00m(3H，H-4’，H-5’)，2,89(2H，J＝9Hc，CH2-P)，2,34m(2H，H-2’)，1,75s(3H，CH3)。31P NMR：17,00s。
实施例3
5’-羟甲基膦酰基-3’-叠氮基-3’-脱氧胸苷(Ic)
将乙酰氧基甲基膦酸吡啶盐(1.2mM)的吡啶(3ml)溶液加到3’-叠氮基-3’-脱氧胸苷(267g，1mM)的吡啶溶液中，在搅拌的同时加入二环己基碳二亚胺(520mg，2.5mM)，反应混合物在室温搅拌10小时并用水(5ml)稀释。在搅拌30多分钟后，将沉淀物分离并蒸发溶液，残留物溶解于1M KOH(5ml)中并在室温搅拌5小时。蒸发溶液并将残留物溶解于水(100ml)中。将溶液注入装有HCO3 -形式的DEAE纤维素的柱中并以线性梯度的NH4HCO3(0→15mM，pH 7.5)进行洗脱。蒸发目标级分，并用水(5ml×3次)再蒸发，残留物用水(3ml)稀释并用水作为洗脱剂在LiChroprep RP-18柱上进行色谱分析。将目标级分冻干，获得238mg(66％)的膦酸酯(Ic)。
1H NMR(D2O)：7,68q(1H，J＝0,5Hc，H-6)，6,22t(1H，J＝6Hc，H-1’)，4,48m(1H，H-3’)，4,16m(3H，H-4’，H-5’)，3,77d(2H，J＝7Hc，CH2-P)，2,51t(2H，J＝6Hc，H-2’)，1,93d(3H，J＝0,5Hc，CH3)。31PNMR：16.03s。
实施例4
5’-甲氧甲基膦酰基-3’-叠氮基-3’-脱氧胸苷(Id)
使用关于化合物Ic所述的方法，由3’-叠氮基-3’-脱氧胸苷和甲氧甲基膦酸合成5’-甲氧甲基膦酰基-3’-叠氮基-3’-脱氧胸苷。1H NMR(D2O)：7,66q(1H，J＝0,5Hc，H-6)，6,21t(1H，J＝6Hc，H-1’)，4,48m(1H，H-3’)，4,15m(3H，H-4’，H-5’)，3,68q(2H，J＝8Hc，CH2-P)，2,52t(2H，J＝6Hc，H-2’)，1,94d(3H，J＝0.5Hc，CH3)。31P NMR：16,03s。
实施例5
5’-氨基羰基膦酰基-3’-叠氮基-3’-脱氧胸苷(Ie)
使用关于化合物Ic所述的方法，由3’-叠氮基-3’-脱氧胸苷和氨基羰基膦酸合成5’-氨基羰基膦酰基-3’-叠氮基-3’-脱氧胸苷(Ie)，获得70mg(94％)的化合物Ie。1H NMR(DMSO-d6)：7,82s(1H，H6)，7,17s，7,13s(2H，NH2)，6,12t(1H，J 6,9，H1′)，4,5m(1H，H3′)，3,95m(3H，H4′，H5′)，2,30m(2H，H2′)，1,81s(3H，CH3)。31P NMR：(DMSO-d6)：-1,56s。质谱：m/e 374,3[M+]。
实施例6
通过在一个传代(即4天)周期，在研究用化合物(浓度为每1ml培养基含0.001-100μg)存在下，培养MT-4细胞系的预先感染的淋巴样细胞，研究了HIV复制的抑制。
通过在培养的第4天与使用溴化3-(4，5-二甲基噻唑-2-基)-2，5-二苯基四钠(MTT)的对照进行比较时，在所述药物的存在下p24病毒特异性蛋白质累积的减少(根据免疫酶测定法)以及细胞生存力的增加，来评价在敏感细胞培养物中HIV复制的抑制。
化合物的细胞毒性的评价
通过将其在无血清RPMI-1640培养基中的稀释物添加到MT-4细胞悬浮液(在96-孔板(Cel-Cult，UK)的孔中的起始浓度到终浓度为0.001-100μg/ml(每剂量3孔))并在37℃培养4天，来评价药物细胞毒性。使用的接种浓度为0.5×106细胞/ml。在不含药物的相同体积无血清培养基中的细胞用作对照。在培养的第4天，使用formasan方法(活细胞的MTT染色)对活细胞进行计数。不同剂量药物的毒性通过与对照比较细胞生存力进行评价，结果用于标绘剂量依赖性曲线并确定降低50％细胞生存力的浓度(CD50)。研究用化合物的有效浓度对MT-4细胞不产生毒性作用。应该注意到50％中毒剂量比对抗HIV-1的有效剂量高5-6级(表1)。
使用已知方法研究了研究用化合物对MT-4细胞培养物中HIV-1复制的作用。
将治疗指数，或选择性指数(IS)计算为化合物的50％毒性浓度与其50％有效剂量的比率(表1中提供了该结果)。基于这些定量抑制指数，根据本申请判断所述化合物的抗病毒功效是可能的，其表现出可与Nikavir相比的对MT-4细胞培养物中HIV-1复制的高度抑制作用。
实施例7
给一条重12kg的狗口服250mg研究用化合物(与凝乳混合)。以确定的间隔从股静脉取血样(1ml)。将样品离心(2000rpm 10分钟)并分离上清液。将Oxetan(内标，0.25μg)和甲醇(0.75ml)加到上清液的等分部分(0.25ml)中。得到的混合物以5000rpm离心3分钟。分离上清液并在40℃在空气流中蒸发，加水(1ml)到残留物中。将等分部分(20μl)在Gynkotec色谱仪(德国)中，使用UltrasphereODC Beckman分析柱(美国)，通过HPLC进行分析。洗脱剂：0.1％H3PO4(pH 2.1)中的6％乙腈，存在0.15％三乙胺；在30℃，λmax 265nm进行检测。表2中提供了通过分析数据结果获得的药物动力学参数。
表1.AZT膦酸酯类抗GKV-4046HIV-1的抗病毒活性
| 化合物 | CD50，μM | ID50，μM | IS |
| Ia | 300 | 0.05-0.1 | ＞3000 |
| Ib | ＞500 | 1-5 | ＞100 |
| Ic | ＞500 | 0.08-0.3 | ＞1600 |
| Id | ＞500 | 1-5 | ＞100 |
| Ie | ＞300 | 0.05-0.1 | ＞3000 |
| Nikavir | 260 | 0.13 | 2015 |
表2：经口施用250mg物质AZT、Nikavir，以及相当于250mgAZT量的化合物Ia和Ie之后的叠氮胸苷的药物动力学参数
| 化合物 | T1/2小时 | AUC，mg×h/L | MRT，小时 | CL，升/小时 | Tmax，小时 | Cmax，mg/L |
| Ie | 9.6 | 9.24 | 13.9 | 27.0 | 5.0 | 0.74 |
| Nikavir | 7.2 | 16.6 | 10.4 | 15.0 | 4.0 | 1.89 |
| AZT | 5.2 | 58.8 | 7.5 | 4.2 | 2.5 | 9.77 |
因此，已经证明本申请中包括的所述化合物具有低毒性并且能有效地抑制MT-4细胞培养物中1型免疫缺陷性病毒的复制，并在哺乳动物中产生AZT，确保其在血液中的浓度逐渐增加。
Claims (1)
1.具有下列通式的AZT的5’-膦酸酯在制备抗HIV药物中的用途：
其中R＝NH2-C(O)-、ClCH2、ICH2、HOCH2或CH3OCH2。
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| RU2004134388/04A RU2322450C2 (ru) | 2004-11-25 | 2004-11-25 | Модифицированные 5'-фосфонаты азт в качестве активных компонентов для потенциальных противовирусных препаратов |
| RU2004134388 | 2004-11-25 | ||
| PCT/RU2005/000249 WO2006062434A1 (en) | 2004-11-25 | 2005-05-06 | Modified 5'- phosphonate azidothymidine-potential anti-viral preparations |
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| SU1548182A1 (ru) * | 1987-12-29 | 1990-03-07 | Институт молекулярной биологии АН СССР | 5 @ -Фосфонаты 3 @ -азидо-2 @ ,3 @ -дидезоксинуклеозидов, вл ющиес специфическими ингибиторами вируса СПИД в культуре лимфоцитов человека Н9/ШВ |
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| Title |
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| Orlov V. M. |
| Orlov, V. M., et al..Investigation of nucleoside and nucleotide derivatives by plasma desorption mass spectrometry.《Molekulyarnaya Biologiya (Moscow)》.1994,第28卷(第3期),708-713. * |
| Shirokova E.A. et al..Uncharged AZT and D4T Derivatives of Phosphonoformic Phosphonoformic and Phosphonoacetic Acids as Anti-HIV Pronucleosides.《J. Med. Chem.》.2004,第47卷(第14期),3606-3614. * |
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| KR20070116580A (ko) | 2007-12-10 |
| CN101115765A (zh) | 2008-01-30 |
| ES2550136T3 (es) | 2015-11-04 |
| EP1829885B1 (en) | 2015-07-15 |
| EP1829885A4 (en) | 2013-07-10 |
| WO2006062434A1 (en) | 2006-06-15 |
| WO2006062434A8 (fr) | 2007-11-01 |
| US20090111979A1 (en) | 2009-04-30 |
| RU2322450C2 (ru) | 2008-04-20 |
| EP1829885A1 (en) | 2007-09-05 |
| KR101323698B1 (ko) | 2013-10-30 |
| RU2004134388A (ru) | 2006-05-20 |
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